Cardiac myofibrillar contractile properties during the progression from hypertension to decompensated heart failure.

PubMed

Hanft, Laurin M; Emter, Craig A; McDonald, Kerry S

2017-07-01

Heart failure arises, in part, from a constellation of changes in cardiac myocytes including remodeling, energetics, Ca 2+ handling, and myofibrillar function. However, little is known about the changes in myofibrillar contractile properties during the progression from hypertension to decompensated heart failure. The aim of the present study was to provide a comprehensive assessment of myofibrillar functional properties from health to heart disease. A rodent model of uncontrolled hypertension was used to test the hypothesis that myocytes in compensated hearts exhibit increased force, higher rates of force development, faster loaded shortening, and greater power output; however, with progression to overt heart failure, we predicted marked depression in these contractile properties. We assessed contractile properties in skinned cardiac myocyte preparations from left ventricles of Wistar-Kyoto control rats and spontaneous hypertensive heart failure (SHHF) rats at ~3, ~12, and >20 mo of age to evaluate the time course of myofilament properties associated with normal aging processes compared with myofilaments from rats with a predisposition to heart failure. In control rats, the myofilament contractile properties were virtually unchanged throughout the aging process. Conversely, in SHHF rats, the rate of force development, loaded shortening velocity, and power all increased at ~12 mo and then significantly fell at the >20-mo time point, which coincided with a decrease in left ventricular fractional shortening. Furthermore, these changes occurred independent of changes in β-myosin heavy chain but were associated with depressed phosphorylation of myofibrillar proteins, and the fall in loaded shortening and peak power output corresponded with the onset of clinical signs of heart failure. NEW & NOTEWORTHY This novel study systematically examined the power-generating capacity of cardiac myofilaments during the progression from hypertension to heart disease. Previously undiscovered changes in myofibrillar power output were found and were associated with alterations in myofilament proteins, providing potential new targets to exploit for improved ventricular pump function in heart failure. Copyright © 2017 the American Physiological Society.

Ecstasy produces left ventricular dysfunction and oxidative stress in rats

PubMed Central

Shenouda, Sylvia K.; Lord, Kevin C.; McIlwain, Elizabeth; Lucchesi, Pamela A.; Varner, Kurt J.

2008-01-01

Aims Our aim was to determine whether the repeated, binge administration of 3,4-methylenedioxymethamphetamine (ecstasy; MDMA) produces structural and/or functional changes in the myocardium that are associated with oxidative stress. Methods and results Echocardiography and pressure–volume conductance catheters were used to assess left ventricular (LV) structure and function in rats subjected to four ecstasy binges (9 mg/kg i.v. for 4 days, separated by a 10 day drug-free period). Hearts from treated and control rats were used for either biochemical and proteomic analysis or the isolation of adult LV myocytes. After the fourth binge, treated hearts showed eccentric LV dilation and diastolic dysfunction. Systolic function was not altered in vivo; however, the magnitude of the contractile responses to electrical stimulation was significantly smaller in myocytes from rats treated in vivo with ecstasy compared with myocytes from control rats. The magnitude of the peak increase in intracellular calcium (measured by Fura-2) was also significantly smaller in myocytes from ecstasy-treated vs. control rats. The relaxation kinetics of the intracellular calcium transients were significantly longer in myocytes from ecstasy-treated rats. Ecstasy significantly increased nitrotyrosine content in the left ventricle. Proteomic analysis revealed increased nitration of contractile proteins (troponin-T, tropomyosin alpha-1 chain, myosin light polypeptide, and myosin regulatory light chain), mitochondrial proteins (Ub-cytochrome-c reductase and ATP synthase), and sarcoplasmic reticulum calcium ATPase. Conclusion The repeated binge administration of ecstasy produces eccentric LV dilation and dysfunction that is accompanied by oxidative stress. These functional responses may result from the redox modification of proteins involved in excitation-contraction coupling and/or mitochondrial energy production. Together, these results indicate that ecstasy has the potential to produce serious cardiac toxicity and ventricular dysfunction. PMID:18495670

Glucose-6-phosphate dehydrogenase and NADPH redox regulates cardiac myocyte L-type calcium channel activity and myocardial contractile function.

PubMed

Rawat, Dhwajbahadur K; Hecker, Peter; Watanabe, Makino; Chettimada, Sukrutha; Levy, Richard J; Okada, Takao; Edwards, John G; Gupte, Sachin A

2012-01-01

We recently demonstrated that a 17-ketosteroid, epiandrosterone, attenuates L-type Ca(2+) currents (I(Ca-L)) in cardiac myocytes and inhibits myocardial contractility. Because 17-ketosteroids are known to inhibit glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme in the pentose phosphate pathway, and to reduce intracellular NADPH levels, we hypothesized that inhibition of G6PD could be a novel signaling mechanism which inhibit I(Ca-L) and, therefore, cardiac contractile function. We tested this idea by examining myocardial function in isolated hearts and Ca(2+) channel activity in isolated cardiac myocytes. Myocardial function was tested in Langendorff perfused hearts and I(Ca-L) were recorded in the whole-cell patch configuration by applying double pulses from a holding potential of -80 mV and then normalized to the peak amplitudes of control currents. 6-Aminonicotinamide, a competitive inhibitor of G6PD, increased pCO(2) and decreased pH. Additionally, 6-aminonicotinamide inhibited G6PD activity, reduced NADPH levels, attenuated peak I(Ca-L) amplitudes, and decreased left ventricular developed pressure and ±dp/dt. Finally, dialyzing NADPH into cells from the patch pipette solution attenuated the suppression of I(Ca-L) by 6-aminonicotinamide. Likewise, in G6PD-deficient mice, G6PD insufficiency in the heart decreased GSH-to-GSSG ratio, superoxide, cholesterol and acetyl CoA. In these mice, M-mode echocardiographic findings showed increased diastolic volume and end-diastolic diameter without changes in the fraction shortening. Taken together, these findings suggest that inhibiting G6PD activity and reducing NADPH levels alters metabolism and leads to inhibition of L-type Ca(2+) channel activity. Notably, this pathway may be involved in modulating myocardial contractility under physiological and pathophysiological conditions during which the pentose phosphate pathway-derived NADPH redox is modulated (e.g., ischemia-reperfusion and heart failure).

Is depressed myocyte contractility centrally involved in heart failure?

PubMed

Houser, Steven R; Margulies, Kenneth B

2003-03-07

This review examines the evidence for and against the hypothesis that abnormalities in cardiac contractility initiate the heart failure syndrome and drive its progression. There is substantial evidence that the contractility of failing human hearts is depressed and that abnormalities of basal Ca2+ regulation and adrenergic regulation of Ca2+ signaling are responsible. The cellular and molecular defects that cause depressed myocyte contractility are not well established but seem to culminate in abnormal sarcoplasmic reticulum uptake, storage, and release. There are also strong links between Ca2+ regulation, Ca2+ signaling pathways, hypertrophy, and heart failure that need to be more clearly delineated. There is not substantial direct evidence for a causative role for depressed contractility in the initiation and progression of human heart failure, and some studies show that heart failure can occur without depressed myocyte contractility. Stronger support for a causal role for depressed contractility in the initiation of heart failure comes from animal studies where maintaining or improving contractility can prevent heart failure. Recent clinical studies in humans also support the idea that beneficial heart failure treatments, such as beta-adrenergic antagonists, involve improved contractility. Current or previously used heart failure treatments that increase contractility, primarily by increasing cAMP, have generally increased mortality. Novel heart failure therapies that increase or maintain contractility or adrenergic signaling by selectively modulating specific molecules have produced promising results in animal experiments. How to reliably implement these potentially beneficial inotropic therapies in humans without introducing negative side effects is the major unanswered question in this field.

Comparative Effects of Urocortins and Stresscopin on Cardiac Myocyte Contractility

PubMed Central

Makarewich, Catherine A.; Troupes, Constantine D.; Schumacher, Sarah M.; Gross, Polina; Koch, Walter J.; Crandall, David L.; Houser, Steven R.

2015-01-01

Rationale There is a current need for development of new therapies for patients with heart failure. Objective To test the effects of members of the Corticotropin-Releasing Factor (CRF) family of peptides on myocyte contractility to validate them as potential heart failure therapeutics. Methods and Results Adult feline left ventricular myocytes (AFMs) were isolated and contractility was assessed in the presence and absence of CRF peptides Urocortin 2 (UCN2), Urocortin 3 (UCN3), Stresscopin (SCP), and the β-adrenergic agonist isoproterenol (Iso). An increase in fractional shortening and peak Ca2+ transient amplitude was seen in the presence of all CRF peptides. A decrease in Ca2+ decay rate (Tau) was also observed at all concentrations tested. cAMP generation was measured by ELISA in isolated AFMs in response to the CRF peptides and Iso and significant production was seen at all concentrations and time points tested. Conclusions The CRF family of peptides effectively increases cardiac contractility and should be evaluated as potential novel therapeutics for heart failure patients. PMID:26231084

TRPC3 contributes to regulation of cardiac contractility and arrhythmogenesis by dynamic interaction with NCX1

PubMed Central

Doleschal, Bernhard; Primessnig, Uwe; Wölkart, Gerald; Wolf, Stefan; Schernthaner, Michaela; Lichtenegger, Michaela; Glasnov, Toma N.; Kappe, C. Oliver; Mayer, Bernd; Antoons, Gudrun; Heinzel, Frank; Poteser, Michael; Groschner, Klaus

2015-01-01

Aim TRPC3 is a non-selective cation channel, which forms a Ca2+ entry pathway involved in cardiac remodelling. Our aim was to analyse acute electrophysiological and contractile consequences of TRPC3 activation in the heart. Methods and results We used a murine model of cardiac TRPC3 overexpression and a novel TRPC3 agonist, GSK1702934A, to uncover (patho)physiological functions of TRPC3. GSK1702934A induced a transient, non-selective conductance and prolonged action potentials in TRPC3-overexpressing myocytes but lacked significant electrophysiological effects in wild-type myocytes. GSK1702934A transiently enhanced contractility and evoked arrhythmias in isolated Langendorff hearts from TRPC3-overexpressing but not wild-type mice. Interestingly, pro-arrhythmic effects outlasted TRPC3 current activation, were prevented by enhanced intracellular Ca2+ buffering, and suppressed by the NCX inhibitor 3′,4′-dichlorobenzamil hydrochloride. GSK1702934A substantially promoted NCX currents in TRPC3-overexpressing myocytes. The TRPC3-dependent electrophysiologic, pro-arrhythmic, and inotropic actions of GSK1702934A were mimicked by angiotensin II (AngII). Immunocytochemistry demonstrated colocalization of TRPC3 with NCX1 and disruption of local interaction upon channel activation by either GSK1702934A or AngII. Conclusion Cardiac TRPC3 mediates Ca2+ and Na+ entry in proximity of NCX1, thereby elevating cellular Ca2+ levels and contractility. Excessive activation of TRPC3 is associated with transient cellular Ca2+ overload, spatial uncoupling between TRPC3 and NCX1, and arrhythmogenesis. We propose TRPC3-NCX micro/nanodomain communication as determinant of cardiac contractility and susceptibility to arrhythmogenic stimuli. PMID:25631581

Effects of cannabidiol on contractions and calcium signaling in rat ventricular myocytes.

PubMed

Ali, Ramez M; Al Kury, Lina T; Yang, Keun-Hang Susan; Qureshi, Anwar; Rajesh, Mohanraj; Galadari, Sehamuddin; Shuba, Yaroslav M; Howarth, Frank Christopher; Oz, Murat

2015-04-01

Cannabidiol (CBD), a major nonpsychotropic cannabinoid found in Cannabis plant, has been shown to influence cardiovascular functions under various physiological and pathological conditions. In the present study, the effects of CBD on contractility and electrophysiological properties of rat ventricular myocytes were investigated. Video edge detection was used to measure myocyte shortening. Intracellular Ca(2+) was measured in cells loaded with the Ca(2+) sensitive fluorescent indicator fura-2 AM. Whole-cell patch clamp was used to measure action potential and Ca(2+) currents. Radioligand binding was employed to study pharmacological characteristics of CBD binding. CBD (1μM) caused a significant decrease in the amplitudes of electrically evoked myocyte shortening and Ca(2+) transients. However, the amplitudes of caffeine-evoked Ca(2+) transients and the rate of recovery of electrically evoked Ca(2+) transients following caffeine application were not altered. CBD (1μM) significantly decreased the duration of APs. Further studies on L-type Ca(2+) channels indicated that CBD inhibits these channels with IC50 of 0.1μM in a voltage-independent manner. Radioligand studies indicated that the specific binding of [(3)H]Isradipine, was not altered significantly by CBD. The results suggest that CBD depresses myocyte contractility by suppressing L-type Ca(2+) channels at a site different than dihydropyridine binding site and inhibits excitation-contraction coupling in cardiomyocytes. Copyright © 2015 Elsevier Ltd. All rights reserved.

The evolutionary origin of bilaterian smooth and striated myocytes

PubMed Central

Brunet, Thibaut; Fischer, Antje HL; Steinmetz, Patrick RH; Lauri, Antonella; Bertucci, Paola; Arendt, Detlev

2016-01-01

The dichotomy between smooth and striated myocytes is fundamental for bilaterian musculature, but its evolutionary origin is unsolved. In particular, interrelationships of visceral smooth muscles remain unclear. Absent in fly and nematode, they have not yet been characterized molecularly outside vertebrates. Here, we characterize expression profile, ultrastructure, contractility and innervation of the musculature in the marine annelid Platynereis dumerilii and identify smooth muscles around the midgut, hindgut and heart that resemble their vertebrate counterparts in molecular fingerprint, contraction speed and nervous control. Our data suggest that both visceral smooth and somatic striated myocytes were present in the protostome-deuterostome ancestor and that smooth myocytes later co-opted the striated contractile module repeatedly – for example, in vertebrate heart evolution. During these smooth-to-striated myocyte conversions, the core regulatory complex of transcription factors conveying myocyte identity remained unchanged, reflecting a general principle in cell type evolution. DOI: http://dx.doi.org/10.7554/eLife.19607.001 PMID:27906129

Heart repair by reprogramming non-myocytes with cardiac transcription factors

PubMed Central

Song, Kunhua; Nam, Young-Jae; Luo, Xiang; Qi, Xiaoxia; Tan, Wei; Huang, Guo N.; Acharya, Asha; Smith, Christopher L.; Tallquist, Michelle D.; Neilson, Eric G.; Hill, Joseph A.; Bassel-Duby, Rhonda; Olson, Eric N.

2012-01-01

The adult mammalian heart possesses little regenerative potential following injury. Fibrosis due to activation of cardiac fibroblasts impedes cardiac regeneration and contributes to loss of contractile function, pathological remodeling and susceptibility to arrhythmias. Cardiac fibroblasts account for a majority of cells in the heart and represent a potential cellular source for restoration of cardiac function following injury through phenotypic reprogramming to a myocardial cell fate. Here we show that four transcription factors, GATA4, Hand2, MEF2C and Tbx5 can cooperatively reprogram adult mouse tail-tip and cardiac fibroblasts into beating cardiac-like myocytes in vitro. Forced expression of these factors in dividing non-cardiomyocytes in mice reprograms these cells into functional cardiac-like myocytes, improves cardiac function and reduces adverse ventricular remodeling following myocardial infarction. Our results suggest a strategy for cardiac repair through reprogramming fibroblasts resident in the heart with cardiogenic transcription factors or other molecules. PMID:22660318

Beta1-adrenoceptor antagonist, metoprolol attenuates cardiac myocyte Ca2+ handling dysfunction in rats with pulmonary artery hypertension.

PubMed

Fowler, Ewan D; Drinkhill, Mark J; Norman, Ruth; Pervolaraki, Eleftheria; Stones, Rachel; Steer, Emma; Benoist, David; Steele, Derek S; Calaghan, Sarah C; White, Ed

2018-07-01

Right heart failure is the major cause of death in Pulmonary Artery Hypertension (PAH) patients but is not a current, specific therapeutic target. Pre-clinical studies have shown that adrenoceptor blockade can improve cardiac function but the mechanisms of action within right ventricular (RV) myocytes are unknown. We tested whether the β 1 -adrenoceptor blocker metoprolol could improve RV myocyte function in an animal model of PAH, by attenuating adverse excitation-contraction coupling remodeling. PAH with RV failure was induced in rats by monocrotaline injection. When PAH was established, animals were given 10 mg/kg/day metoprolol (MCT + BB) or vehicle (MCT). The median time to the onset of heart failure signs was delayed from 23 days (MCT), to 31 days (MCT + BB). At 23 ± 1 days post-injection, MCT + BB showed improved in vivo cardiac function, measured by echocardiography. RV hypertrophy was reduced despite persistent elevated afterload. RV myocyte contractility during field stimulation was improved at higher pacing frequencies in MCT + BB. Preserved t-tubule structure, more uniform evoked Ca 2+ release, increased SERCA2a expression and faster ventricular repolarization (measured in vivo by telemetry) may account for the improved contractile function. Sarcoplasmic reticulum Ca 2+ overload was prevented in MCT + BB myocytes resulting in fewer spontaneous Ca 2+ waves, with a lower pro-arrhythmic potential. Our novel finding of attenuation of defects in excitation contraction coupling by β 1 -adrenoceptor blockade with delays in the onset of HF, identifies the RV as a promising therapeutic target in PAH. Moreover, our data suggest existing therapies for left ventricular failure may also be beneficial in PAH induced RV failure. Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.

Self-organization of muscle cell structure and function.

PubMed

Grosberg, Anna; Kuo, Po-Ling; Guo, Chin-Lin; Geisse, Nicholas A; Bray, Mark-Anthony; Adams, William J; Sheehy, Sean P; Parker, Kevin Kit

2011-02-01

The organization of muscle is the product of functional adaptation over several length scales spanning from the sarcomere to the muscle bundle. One possible strategy for solving this multiscale coupling problem is to physically constrain the muscle cells in microenvironments that potentiate the organization of their intracellular space. We hypothesized that boundary conditions in the extracellular space potentiate the organization of cytoskeletal scaffolds for directed sarcomeregenesis. We developed a quantitative model of how the cytoskeleton of neonatal rat ventricular myocytes organizes with respect to geometric cues in the extracellular matrix. Numerical results and in vitro assays to control myocyte shape indicated that distinct cytoskeletal architectures arise from two temporally-ordered, organizational processes: the interaction between actin fibers, premyofibrils and focal adhesions, as well as cooperative alignment and parallel bundling of nascent myofibrils. Our results suggest that a hierarchy of mechanisms regulate the self-organization of the contractile cytoskeleton and that a positive feedback loop is responsible for initiating the break in symmetry, potentiated by extracellular boundary conditions, is required to polarize the contractile cytoskeleton.

Overexpression Myocardial Inducible Nitric Oxide Synthase Exacerbates Cardiac Dysfunction and Beta-Adrenergic Desensitization in Experimental Hypothyroidism☆,☆☆

PubMed Central

Shao, Qun; Cheng, Heng-Jie; Callahan, Michael F.; Kitzman, Dalane W; Li, Wei-Min; Cheng, Che Ping

2015-01-01

Background Altered nitric oxide synthase (NOS) has been implicated in the pathophysiology of heart failure (HF). Recent evidence links hypothyroidism to the pathology of HF. However, the precise mechanisms are incompletely understood. The alterations and functional effects of cardiac NOS in hypothyroidism are unknown. We tested the hypothesis that hypothyroidism increases cadiomyocyte inducible NOS (iNOS) expression, which plays an important role in hypothyroidism-induced depression of cardiomyocyte contractile properties, [Ca2+]i transient ([Ca2+]iT), and β-adrenergic hyporesponsiveness. Methods and Results We simultaneously evaluated LV functional performance and compared myocyte three NOS, β-adrenergic receptors (AR) and SERCA2a expressions and assessed cardiomyocyte contractile and [Ca2+]iT responses to β-AR stimulation with and without pretreatment of iNOS inhibitor (1400W, 10−5 mol/L) in 26 controls and 26 rats with hypothyroidism induced by methimazole (~30 mg/kg/day for 8 weeks in the drinking water). Compared with controls, in hypothyroidism, total serum T3 and T4 were significantly reduced followed by significantly decreased LV contractility (EES) with increased LV time constant of relaxation. These LV abnormalities were accompanied by concomitant significant decreases in myocyte contraction (dL/dtmax), relaxation (dR/dtmax), and [Ca2+]iT. In hypothyroidism, isoproterenol (10−8 M) produced significantly smaller increases in dL/dtmax, dR/dtmax and [Ca2+]iT. These changes were associated with decreased β1-AR and SERCA2a, but significantly increased iNOS. Moreover, only in hypothyroidism, pretreatment with iNOS inhibitor significantly improved basal and isoproterenol-stimulated myocyte contraction, relaxation and [Ca2+]iT. Conclusions Hypothyroidism produces intrinsic defects of LV myocyte force-generating capacity and relaxation with β-AR desensitization. Up-regulation of cadiomyocyte iNOS may promote progressive cardiac dysfunction in hypothyroidism. PMID:26681542

Overexpression myocardial inducible nitric oxide synthase exacerbates cardiac dysfunction and beta-adrenergic desensitization in experimental hypothyroidism.

PubMed

Shao, Qun; Cheng, Heng-Jie; Callahan, Michael F; Kitzman, Dalane W; Li, Wei-Min; Cheng, Che Ping

2016-02-01

Altered nitric oxide synthase (NOS) has been implicated in the pathophysiology of heart failure (HF). Recent evidence links hypothyroidism to the pathology of HF. However, the precise mechanisms are incompletely understood. The alterations and functional effects of cardiac NOS in hypothyroidism are unknown. We tested the hypothesis that hypothyroidism increases cardiomyocyte inducible NOS (iNOS) expression, which plays an important role in hypothyroidism-induced depression of cardiomyocyte contractile properties, [Ca(2+)]i transient ([Ca(2+)]iT), and β-adrenergic hyporesponsiveness. We simultaneously evaluated LV functional performance and compared myocyte three NOS, β-adrenergic receptors (AR) and SERCA2a expressions and assessed cardiomyocyte contractile and [Ca(2+)]iT responses to β-AR stimulation with and without pretreatment of iNOS inhibitor (1400 W, 10(-5)mol/L) in 26 controls and 26 rats with hypothyroidism induced by methimazole (~30 mg/kg/day for 8 weeks in the drinking water). Compared with controls, in hypothyroidism, total serum T3 and T4 were significantly reduced followed by significantly decreased LV contractility (EES) with increased LV time constant of relaxation. These LV abnormalities were accompanied by concomitant significant decreases in myocyte contraction (dL/dtmax), relaxation (dR/dtmax), and [Ca(2+)]iT. In hypothyroidism, isoproterenol (10(-8)M) produced significantly smaller increases in dL/dtmax, dR/dtmax and [Ca(2+)]iT. These changes were associated with decreased β1-AR and SERCA2a, but significantly increased iNOS. Moreover, only in hypothyroidism, pretreatment with iNOS inhibitor significantly improved basal and isoproterenol-stimulated myocyte contraction, relaxation and [Ca(2+)]iT. Hypothyroidism produces intrinsic defects of LV myocyte force-generating capacity and relaxation with β-AR desensitization. Up-regulation of cardiomyocyte iNOS may promote progressive cardiac dysfunction in hypothyroidism. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Standard and Strain Measurements by Echocardiography Detect Early Overloaded Right Ventricular Dysfunction: Validation against Hemodynamic and Myocyte Contractility Changes in a Large Animal Model.

PubMed

Hodzic, Amir; Bobin, Pierre; Mika, Delphine; Ly, Mohamed; Lefebvre, Florence; Lechêne, Patrick; Le Bret, Emmanuel; Gouadon, Elodie; Coblence, Mathieu; Vandecasteele, Grégoire; Capderou, André; Leroy, Jérôme; Rucker-Martin, Catherine; Lambert, Virginie

2017-11-01

Early detection of right ventricular (RV) failure is required to improve the management of patients with congenital heart diseases. The aim of this study was to validate echocardiography for the early detection of overloaded RV dysfunction, compared with hemodynamic and myocyte contractility assessment. Using a porcine model reproducing repaired tetralogy of Fallot, RV function was evaluated over 4 months using standard echocardiography and speckle-tracking compared with hemodynamic parameters (conductance catheter). Sarcomere shortening and calcium transients were recorded in RV isolated myocytes. Contractile reserve (ΔE max ) was assessed by β-adrenergic stimulation in vivo (dobutamine 5 μg/kg) and ex vivo (isoproterenol 100 nM). Six operated animals were compared with four age- and sex-matched controls. In the operated group, hemodynamic RV efficient ejection fraction was significantly decreased (29.7% [26.2%-34%] vs 42.9% [40.7%-48.6%], P < .01), and inotropic responses to dobutamine were attenuated (ΔE max was 51% vs 193%, P < .05). Echocardiographic measurements of fraction of area change, tricuspid annular plane systolic excursion, tricuspid annular peak systolic velocity (S') and RV free wall longitudinal systolic strain and strain rate were significantly decreased. Strain rate, S', and tricuspid annular plane systolic excursion were correlated with ΔE max (r = 0.75, r = 0.78, and r = 0.65, respectively, P < .05). These alterations were associated in RV isolated myocytes with the decrease of sarcomere shortening in response to isoproterenol and perturbations of calcium homeostasis assessed by the increase of spontaneous calcium waves. In this porcine model, both standard and strain echocardiographic parameters detected early impairments of RV function and cardiac reserve, which were associated with cardiomyocyte excitation-contraction coupling alterations. Copyright © 2017 American Society of Echocardiography. Published by Elsevier Inc. All rights reserved.

NADPH oxidase-2 inhibition restores contractility and intracellular calcium handling and reduces arrhythmogenicity in dystrophic cardiomyopathy

PubMed Central

Gonzalez, Daniel R.; Treuer, Adriana V.; Lamirault, Guillaume; Mayo, Vera; Cao, Yenong; Dulce, Raul A.

2014-01-01

Duchenne muscular dystrophy may affect cardiac muscle, producing a dystrophic cardiomyopathy in humans and the mdx mouse. We tested the hypothesis that oxidative stress participates in disrupting calcium handling and contractility in the mdx mouse with established cardiomyopathy. We found increased expression (fivefold) of the NADPH oxidase (NOX) 2 in the mdx hearts compared with wild type, along with increased superoxide production. Next, we tested the impact of NOX2 inhibition on contractility and calcium handling in isolated cardiomyocytes. Contractility was decreased in mdx myocytes compared with wild type, and this was restored toward normal by pretreating with apocynin. In addition, the amplitude of evoked intracellular Ca2+ concentration transients that was diminished in mdx myocytes was also restored with NOX2 inhibition. Total sarcoplasmic reticulum (SR) Ca2+ content was reduced in mdx hearts and normalized by apocynin treatment. Additionally, NOX2 inhibition decreased the production of spontaneous diastolic calcium release events and decreased the SR calcium leak in mdx myocytes. In addition, nitric oxide (NO) synthase 1 (NOS-1) expression was increased eightfold in mdx hearts compared with wild type. Nevertheless, cardiac NO production was reduced. To test whether this paradox implied NOS-1 uncoupling, we treated cardiac myocytes with exogenous tetrahydrobioterin, along with the NOX inhibitor VAS2870. These agents restored NO production and phospholamban phosphorylation in mdx toward normal. Together, these results demonstrate that, in mdx hearts, NOX2 inhibition improves the SR calcium handling and contractility, partially by recoupling NOS-1. These findings reveal a new layer of nitroso-redox imbalance in dystrophic cardiomyopathy. PMID:25015966

Myocyte repolarization modulates myocardial function in aging dogs

PubMed Central

Sorrentino, Andrea; Signore, Sergio; Borghetti, Giulia; Meo, Marianna; Cannata, Antonio; Zhou, Yu; Wybieralska, Ewa; Luciani, Marco; Kannappan, Ramaswamy; Zhang, Eric; Matsuda, Alex; Webster, Andrew; Cimini, Maria; Kertowidjojo, Elizabeth; D'Alessandro, David A.; Wunimenghe, Oriyanhan; Michler, Robert E.; Royer, Christopher; Goichberg, Polina; Leri, Annarosa; Barrett, Edward G.; Anversa, Piero; Hintze, Thomas H.

2016-01-01

Studies of myocardial aging are complex and the mechanisms involved in the deterioration of ventricular performance and decreased functional reserve of the old heart remain to be properly defined. We have studied a colony of beagle dogs from 3 to 14 yr of age kept under a highly regulated environment to define the effects of aging on the myocardium. Ventricular, myocardial, and myocyte function, together with anatomical and structural properties of the organ and cardiomyocytes, were evaluated. Ventricular hypertrophy was not observed with aging and the structural composition of the myocardium was modestly affected. Alterations in the myocyte compartment were identified in aged dogs, and these factors negatively interfere with the contractile reserve typical of the young heart. The duration of the action potential is prolonged in old cardiomyocytes contributing to the slower electrical recovery of the myocardium. Also, the remodeled repolarization of cardiomyocytes with aging provides inotropic support to the senescent muscle but compromises its contractile reserve, rendering the old heart ineffective under conditions of high hemodynamic demand. The defects in the electrical and mechanical properties of cardiomyocytes with aging suggest that this cell population is an important determinant of the cardiac senescent phenotype. Collectively, the delayed electrical repolarization of aging cardiomyocytes may be viewed as a critical variable of the aging myopathy and its propensity to evolve into ventricular decompensation under stressful conditions. PMID:26801307

Patterns of sarcomere activation, temperature dependence, and effect of ryanodine in chemically skinned cardiac fibers

PubMed Central

1986-01-01

Functionally skinned and electrochemically shunted myocytes were prepared by perfusing rat hearts with collagenase in order to obtain a technically improved measurement of sarcomere dynamics and to evaluate the role of sarcoplasmic reticulum in situ with respect to contractile activation. In the presence of micromolar calcium, the myocytes exhibited phasic and propagated contraction waves beginning at one end and proceeding along the myocyte. Beating rates, the propagation velocity of the activation wave, and single sarcomere shortening and relaxation velocities were obtained by manual or automated analysis of 16-mm film recorded at 170 frames/s from a camera attached to a microscope that was equipped with a temperature-controlled stage. In parallel experiments, calcium accumulation by the sarcoplasmic reticulum of the myocytes in situ was measured by direct isotopic tracer methods. The frequency (10-38 min-1) of spontaneous contractions, the velocity (1.9-7.4 microns . s-1) of sarcomere shortening, and the velocity (1.7-6.8 microns . s-1) of sarcomere relaxation displayed identical temperature dependences (Q10 = 2.2), which are similar to that of the calcium pump of sarcoplasmic reticulum and are consistent with a rate limit imposed by enzyme-catalyzed mechanisms on all these parameters. On the other hand, the velocity (77- 159 microns . s-1) of sequential sarcomere activation displayed a lower temperature dependence (Q10 = 1.5), which is consistent with a diffusion-limited and self-propagating release of calcium from one sarcomere to the other. The phasic contractile activity of the dissociated myocytes was inhibited by 10(-8)-10(6) M ryanodine (and not by myolemmal calcium blockers) under conditions in which calcium accumulation by sarcoplasmic reticulum in situ was demonstrated to proceed optimally. The effect of ryanodine is attributed to an interaction of this drug with sarcotubular structures, producing inhibition of calcium release from the sarcoplasmic reticulum. The consequent lack of sarcomere activation underlines the role of sarcoplasmic reticulum uptake and release in the phasic contractile activation of the electrochemically shunted myocytes. PMID:3522803

Noncontact minimally invasive technique for the assessment of mechanical properties of single cardiac myocyte via magnetic field loading

NASA Astrophysics Data System (ADS)

Yin, Shizhuo; Zhang, Xueqian; Cheung, Joseph; Wu, Juntao; Zhan, Chun; Xue, Jinchao

2004-07-01

In this paper, a unique non-contact, minimum invasive technique for the assessment of mechanical properties of single cardiac myocyte is presented. The assessment process includes following major steps: (1) attach a micro magnetic bead to the cell to be measured, (2) measure the contractile performance of the cell under the different magnetic field loading, (3) calculate mechanical loading force, and (4) derive the contractile force from the measured contraction data under different magnetic field loading.

Cell biology of sarcomeric protein engineering: disease modeling and therapeutic potential.

PubMed

Thompson, Brian R; Metzger, Joseph M

2014-09-01

The cardiac sarcomere is the functional unit for myocyte contraction. Ordered arrays of sarcomeric proteins, held in stoichiometric balance with each other, respond to calcium to coordinate contraction and relaxation of the heart. Altered sarcomeric structure-function underlies the primary basis of disease in multiple acquired and inherited heart disease states. Hypertrophic and restrictive cardiomyopathies are caused by inherited mutations in sarcomeric genes and result in altered contractility. Ischemia-mediated acidosis directly alters sarcomere function resulting in decreased contractility. In this review, we highlight the use of acute genetic engineering of adult cardiac myocytes through stoichiometric replacement of sarcomeric proteins in these disease states with particular focus on cardiac troponin I. Stoichiometric replacement of disease causing mutations has been instrumental in defining the molecular mechanisms of hypertrophic and restrictive cardiomyopathy in a cellular context. In addition, taking advantage of stoichiometric replacement through gene therapy is discussed, highlighting the ischemia-resistant histidine-button, A164H cTnI. Stoichiometric replacement of sarcomeric proteins offers a potential gene therapy avenue to replace mutant proteins, alter sarcomeric responses to pathophysiologic insults, or neutralize altered sarcomeric function in disease. © 2014 Wiley Periodicals, Inc.

Novel role of transient receptor potential vanilloid 2 in the regulation of cardiac performance

PubMed Central

Lasko, Valerie M.; Koch, Sheryl E.; Singh, Vivek P.; Carreira, Vinicius; Robbins, Nathan; Patel, Amit R.; Jiang, Min; Bidwell, Philip; Kranias, Evangelia G.; Jones, W. Keith; Lorenz, John N.

2013-01-01

Transient receptor potential cation channels have been implicated in the regulation of cardiovascular function, but only recently has our laboratory described the vanilloid-2 subtype (TRPV2) in the cardiomyocyte, though its exact mechanism of action has not yet been established. This study tests the hypothesis that TRPV2 plays an important role in regulating myocyte contractility under physiological conditions. Therefore, we measured cardiac and vascular function in wild-type and TRPV2−/− mice in vitro and in vivo and found that TRPV2 deletion resulted in a decrease in basal systolic and diastolic function without affecting loading conditions or vascular tone. TRPV2 stimulation with probenecid, a relatively selective TRPV2 agonist, caused an increase in both inotropy and lusitropy in wild-type mice that was blunted in TRPV2−/− mice. We examined the mechanism of TRPV2 inotropy/lusitropy in isolated myocytes and found that it modulates Ca2+ transients and sarcoplasmic reticulum Ca2+ loading. We show that the activity of this channel is necessary for normal cardiac function and that there is increased contractility in response to agonism of TRPV2 with probenecid. PMID:24322617

PDE1C deficiency antagonizes pathological cardiac remodeling and dysfunction

PubMed Central

Knight, Walter E.; Chen, Si; Zhang, Yishuai; Oikawa, Masayoshi; Wu, Meiping; Zhou, Qian; Miller, Clint L.; Cai, Yujun; Mickelsen, Deanne M.; Moravec, Christine; Small, Eric M.; Abe, Junichi; Yan, Chen

2016-01-01

Cyclic nucleotide phosphodiesterase 1C (PDE1C) represents a major phosphodiesterase activity in human myocardium, but its function in the heart remains unknown. Using genetic and pharmacological approaches, we studied the expression, regulation, function, and underlying mechanisms of PDE1C in the pathogenesis of cardiac remodeling and dysfunction. PDE1C expression is up-regulated in mouse and human failing hearts and is highly expressed in cardiac myocytes but not in fibroblasts. In adult mouse cardiac myocytes, PDE1C deficiency or inhibition attenuated myocyte death and apoptosis, which was largely dependent on cyclic AMP/PKA and PI3K/AKT signaling. PDE1C deficiency also attenuated cardiac myocyte hypertrophy in a PKA-dependent manner. Conditioned medium taken from PDE1C-deficient cardiac myocytes attenuated TGF-β–stimulated cardiac fibroblast activation through a mechanism involving the crosstalk between cardiac myocytes and fibroblasts. In vivo, cardiac remodeling and dysfunction induced by transverse aortic constriction, including myocardial hypertrophy, apoptosis, cardiac fibrosis, and loss of contractile function, were significantly attenuated in PDE1C-knockout mice relative to wild-type mice. These results indicate that PDE1C activation plays a causative role in pathological cardiac remodeling and dysfunction. Given the continued development of highly specific PDE1 inhibitors and the high expression level of PDE1C in the human heart, our findings could have considerable therapeutic significance. PMID:27791092

Calcium release-dependent inactivation precedes formation of the tubular system in developing rat cardiac myocytes.

PubMed

Macková, Katarina; Zahradníková, Alexandra; Hoťka, Matej; Hoffmannová, Barbora; Zahradník, Ivan; Zahradníková, Alexandra

2017-12-01

Developing cardiac myocytes undergo substantial structural and functional changes transforming the mechanism of excitation-contraction coupling from the embryonic form, based on calcium influx through sarcolemmal DHPR calcium channels, to the adult form, relying on local calcium release through RYR calcium channels of sarcoplasmic reticulum stimulated by calcium influx. We characterized day-by-day the postnatal development of the structure of sarcolemma, using techniques of confocal fluorescence microscopy, and the development of the calcium current, measured by the whole-cell patch-clamp in isolated rat ventricular myocytes. We characterized the appearance and expansion of the t-tubule system and compared it with the appearance and progress of the calcium current inactivation induced by the release of calcium ions from sarcoplasmic reticulum as structural and functional measures of direct DHPR-RYR interaction. The release-dependent inactivation of calcium current preceded the development of the t-tubular system by several days, indicating formation of the first DHPR-RYR couplons at the surface sarcolemma and their later spreading close to contractile myofibrils with the growing t-tubules. Large variability of both of the measured parameters among individual myocytes indicates uneven maturation of myocytes within the growing myocardium.

Osmolality- and Na+ -dependent effects of hyperosmotic NaCl solution on contractile activity and Ca2+ cycling in rat ventricular myocytes.

PubMed

Ricardo, Rafael A; Bassani, Rosana A; Bassani, José W M

2008-01-01

Hypertonic NaCl solutions have been used for small-volume resuscitation from hypovolemic shock. We sought to identify osmolality- and Na(+)-dependent components of the effects of the hyperosmotic NaCl solution (85 mOsm/kg increment) on contraction and cytosolic Ca(2+) concentration ([Ca(2+)](i)) in isolated rat ventricular myocytes. The biphasic change in contraction and Ca(2+) transient amplitude (decrease followed by recovery) was accompanied by qualitatively similar changes in sarcoplasmic reticulum (SR) Ca(2+) content and fractional release and was mimicked by isosmotic, equimolar increase in extracellular [Na(+)] ([Na(+)](o)). Raising osmolality with sucrose, however, augmented systolic [Ca(2+)](i) monotonically without change in SR parameters and markedly decreased contraction amplitude and diastolic cell length. Functional SR inhibition with thapsigargin abolished hyperosmolality effects on [Ca(2+)](i). After 15-min perfusion, both hyperosmotic solutions slowed mechanical relaxation during twitches and [Ca(2+)](i) decline during caffeine-evoked transients, raised diastolic and systolic [Ca(2+)](i), and depressed systolic contractile activity. These effects were greater with sucrose solution, and were not observed after isosmotic [Na(+)](o) increase. We conclude that under the present experimental conditions, transmembrane Na(+) redistribution apparently plays an important role in determining changes in SR Ca(2+) mobilization, which markedly affect contractile response to hyperosmotic NaCl solutions and attenuate the osmotically induced depression of contractile activity.

A mathematical model for active contraction in healthy and failing myocytes and left ventricles.

PubMed

Cai, Li; Wang, Yongheng; Gao, Hao; Li, Yiqiang; Luo, Xiaoyu

2017-01-01

Cardiovascular disease is one of the leading causes of death worldwide, in particular myocardial dysfunction, which may lead to heart failure eventually. Understanding the electro-mechanics of the heart will help in developing more effective clinical treatments. In this paper, we present a multi-scale electro-mechanics model of the left ventricle (LV). The Holzapfel-Ogden constitutive law was used to describe the passive myocardial response in tissue level, a modified Grandi-Pasqualini-Bers model was adopted to model calcium dynamics in individual myocytes, and the active tension was described using the Niederer-Hunter-Smith myofilament model. We first studied the electro-mechanics coupling in a single myocyte in the healthy and diseased left ventricle, and then the single cell model was embedded in a dynamic LV model to investigate the compensation mechanism of LV pump function due to myocardial dysfunction caused by abnormality in cellular calcium dynamics. The multi-scale LV model was solved using an in-house developed hybrid immersed boundary method with finite element extension. The predictions of the healthy LV model agreed well with the clinical measurements and other studies, and likewise, the results in the failing states were also consistent with clinical observations. In particular, we found that a low level of intracellular Ca2+ transient in myocytes can result in LV pump function failure even with increased myocardial contractility, decreased systolic blood pressure, and increased diastolic filling pressure, even though they will increase LV stroke volume. Our work suggested that treatments targeted at increased contractility and lowering the systolic blood pressure alone are not sufficient in preventing LV pump dysfunction, restoring a balanced physiological Ca2+ handling mechanism is necessary.

Targeted GLUT-4 deficiency in the heart induces cardiomyocyte hypertrophy and impaired contractility linked with Ca(2+) and proton flux dysregulation.

PubMed

Domenighetti, Andrea A; Danes, Vennetia R; Curl, Claire L; Favaloro, Jennifer M; Proietto, Joseph; Delbridge, Lea M D

2010-04-01

There is clinical evidence to suggest that impaired myocardial glucose uptake contributes to the pathogenesis of hypertrophic, insulin-resistant cardiomyopathy. The goal of this study was to determine whether cardiac deficiency of the insulin-sensitive glucose transporter, GLUT4, has deleterious effect on cardiomyocyte excitation-contraction coupling. Cre-Lox mouse models of cardiac GLUT4 knockdown (KD, 85% reduction) and knockout (KO, >95% reduction), which exhibit similar systemic hyperinsulinemic and hyperglycemic states, were investigated. The Ca(2+) current (I(Ca)) and Na(+)-Ca(2+) exchanger (NCX) fluxes, Na(+)-H(+) exchanger (NHE) activity, and contractile performance of GLUT4-deficient myocytes was examined using whole-cell patch-clamp, epifluorescence, and imaging techniques. GLUT4-KO exhibited significant cardiac enlargement characterized by cardiomyocyte hypertrophy (40% increase in cell area) and fibrosis. GLUT4-KO myocyte contractility was significantly diminished, with reduced mean maximum shortening (5.0+/-0.4% vs. 6.2+/-0.6%, 5 Hz). Maximal rates of shortening and relaxation were also reduced (20-25%), and latency was delayed. In GLUT4-KO myocytes, the I(Ca) density was decreased (-2.80+/-0.29 vs. -5.30+/-0.70 pA/pF), and mean I(NCX) was significantly increased in both outward (by 60%) and inward (by 100%) directions. GLUT4-KO expression levels of SERCA2 and RyR2 were reduced by approximately 50%. NHE-mediated H(+) flux in response to NH(4)Cl acid loading was markedly elevated GLUT4-KO myocytes, associated with doubled expression of NHE1. These findings demonstrate that, independent of systemic endocrinological disturbance, cardiac GLUT4 deficiency per se provides a lesion sufficient to induce profound alterations in cardiomyocyte Ca(2+) and pH homeostasis. Our investigation identifies the cardiac GLUT4 as a potential primary molecular therapeutic target in ameliorating the functional deficits associated with insulin-resistant cardiomyopathy. Copyright (c) 2009 Elsevier Ltd. All rights reserved.

Intravital imaging of cardiac function at the single-cell level.

PubMed

Aguirre, Aaron D; Vinegoni, Claudio; Sebas, Matt; Weissleder, Ralph

2014-08-05

Knowledge of cardiomyocyte biology is limited by the lack of methods to interrogate single-cell physiology in vivo. Here we show that contracting myocytes can indeed be imaged with optical microscopy at high temporal and spatial resolution in the beating murine heart, allowing visualization of individual sarcomeres and measurement of the single cardiomyocyte contractile cycle. Collectively, this has been enabled by efficient tissue stabilization, a prospective real-time cardiac gating approach, an image processing algorithm for motion-artifact-free imaging throughout the cardiac cycle, and a fluorescent membrane staining protocol. Quantification of cardiomyocyte contractile function in vivo opens many possibilities for investigating myocardial disease and therapeutic intervention at the cellular level.

Cell and gene therapy for severe heart failure patients: The time and place for Pim-1 Kinase

PubMed Central

Siddiqi, Sailay; Sussman, Mark A

2014-01-01

Regenerative therapy in severe heart failure patients presents a challenging set of circumstances including a damaged myocardial environment that accelerates senescence in myocytes and cardiac progenitor cells. Failing myocardium suffers from deterioration of contractile function coupled with impaired regenerative potential that drives the heart toward decompensation. Efficacious regenerative cell therapy for severe heart failure requires disruption of this vicious circle that can be accomplished by alteration of the compromised myocyte phenotype and rejuvenation of progenitor cells. This review focuses upon potential for Pim-1 kinase to mitigate chronic heart failure by improving myocyte quality through preservation of mitochondrial integrity, prevention of hypertrophy and inhibition of apoptosis. In addition, cardiac progenitors engineered with Pim-1 possess enhanced regenerative potential, making Pim-1 an important player in future treatment of severe heart failure. PMID:23984924

Angiotensin II reduces the surface abundance of KV 1.5 channels in arterial myocytes to stimulate vasoconstriction.

PubMed

Kidd, Michael W; Bulley, Simon; Jaggar, Jonathan H

2017-03-01

Several different voltage-dependent K + (K V ) channel isoforms are expressed in arterial smooth muscle cells (myocytes). Vasoconstrictors inhibit K V currents, but the isoform selectivity and mechanisms involved are unclear. We show that angiotensin II (Ang II), a vasoconstrictor, stimulates degradation of K V 1.5, but not K V 2.1, channels through a protein kinase C- and lysosome-dependent mechanism, reducing abundance at the surface of mesenteric artery myocytes. The Ang II-induced decrease in cell surface K V 1.5 channels reduces whole-cell K V 1.5 currents and attenuates K V 1.5 function in pressurized arteries. We describe a mechanism by which Ang II stimulates protein kinase C-dependent K V 1.5 channel degradation, reducing the abundance of functional channels at the myocyte surface. Smooth muscle cells (myocytes) of resistance-size arteries express several different voltage-dependent K + (K V ) channels, including K V 1.5 and K V 2.1, which regulate contractility. Myocyte K V currents are inhibited by vasoconstrictors, including angiotensin II (Ang II), but the mechanisms involved are unclear. Here, we tested the hypothesis that Ang II inhibits K V currents by reducing the plasma membrane abundance of K V channels in myocytes. Angiotensin II (applied for 2 h) reduced surface and total K V 1.5 protein in rat mesenteric arteries. In contrast, Ang II did not alter total or surface K V 2.1, or K V 1.5 or K V 2.1 cellular distribution, measured as the percentage of total protein at the surface. Bisindolylmaleimide (BIM; a protein kinase C blocker), a protein kinase C inhibitory peptide or bafilomycin A (a lysosomal degradation inhibitor) each blocked the Ang II-induced decrease in total and surface K V 1.5. Immunofluorescence also suggested that Ang II reduced surface K V 1.5 protein in isolated myocytes; an effect inhibited by BIM. Arteries were exposed to Ang II or Ang II plus BIM (for 2 h), after which these agents were removed and contractility measurements performed or myocytes isolated for patch-clamp electrophysiology. Angiotensin II reduced both whole-cell K V currents and currents inhibited by Psora-4, a K V 1.5 channel blocker. Angiotensin II also reduced vasoconstriction stimulated by Psora-4 or 4-aminopyridine, another K V channel inhibitor. These data indicate that Ang II activates protein kinase C, which stimulates K V 1.5 channel degradation, leading to a decrease in surface K V 1.5, a reduction in whole-cell K V 1.5 currents and a loss of functional K V 1.5 channels in myocytes of pressurized arteries. © 2016 The Authors. The Journal of Physiology © 2016 The Physiological Society.

Rapid Estrogen Receptor-Mediated Mechanisms Determine the Sexually Dimorphic Sensitivity of Ventricular Myocytes to 17β-Estradiol and the Environmental Endocrine Disruptor Bisphenol A

PubMed Central

Belcher, Scott M.; Chen, Yamei; Yan, Sujuan

2012-01-01

Previously we showed that 17β-estradiol (E2) and/or the xenoestrogen bisphenol A (BPA) alter ventricular myocyte Ca2+ handing, resulting in increased cardiac arrhythmias in a female-specific manner. In the present study, the roles of estrogen receptors (ER) in mediating the rapid contractile and arrhythmogenic effects of estrogens were examined. Contractility was used as an index to assess the impact of E2 or BPA on Ca2+ handling in rodent ventricular myocytes. The concentration-response curve for the stimulatory effects of BPA and E2 on female myocyte was inverted-U shaped. Detectable effects for each compound were observed at 10−12 m, and the most efficacious concentrations for each were at 10−9 m. Sensitivity to E2 and BPA was not observed in male myocytes and was abolished in myocytes from ovariectomized females. Analysis using protein-conjugated E2 suggests that these rapid actions are induced by membrane-associated receptors. Analysis using selective ER agonists and antagonists and a genetic ERβ knockout mouse model showed that ERα and ERβ have opposing actions in myocytes and that the balance between ERβ and ERα signaling is the prime regulator of the sex-specific sensitivity toward estrogens. The response of female myocytes to E2 and BPA is dominated by the stimulatory ERβ-mediated signaling, and the absence of BPA and E2 responsiveness in males is due to a counterbalancing-suppressive action of ERα. We conclude that the sex-specific sensitivity of myocytes to estrogens and the rapid arrhythmogenic effects of BPA and estradiol in the female heart are regulated by the balance between ERα and ERβ signaling. PMID:22166976

Longstanding Hyperthyroidism Is Associated with Normal or Enhanced Intrinsic Cardiomyocyte Function despite Decline in Global Cardiac Function

PubMed Central

Redetzke, Rebecca A.; Gerdes, A. Martin

2012-01-01

Thyroid hormones (THs) play a pivotal role in cardiac homeostasis. TH imbalances alter cardiac performance and ultimately cause cardiac dysfunction. Although short-term hyperthyroidism typically leads to heightened left ventricular (LV) contractility and improved hemodynamic parameters, chronic hyperthyroidism is associated with deleterious cardiac consequences including increased risk of arrhythmia, impaired cardiac reserve and exercise capacity, myocardial remodeling, and occasionally heart failure. To evaluate the long-term consequences of chronic hyperthyroidism on LV remodeling and function, we examined LV isolated myocyte function, chamber function, and whole tissue remodeling in a hamster model. Three-month-old F1b hamsters were randomized to control or 10 months TH treatment (0.1% grade I desiccated TH). LV chamber remodeling and function was assessed by echocardiography at 1, 2, 4, 6, 8, and 10 months of treatment. After 10 months, terminal cardiac function was assessed by echocardiography and LV hemodynamics. Hyperthyroid hamsters exhibited significant cardiac hypertrophy and deleterious cardiac remodeling characterized by myocyte lengthening, chamber dilatation, decreased relative wall thickness, increased wall stress, and increased LV interstitial fibrotic deposition. Importantly, hyperthyroid hamsters demonstrated significant LV systolic and diastolic dysfunction. Despite the aforementioned remodeling and global cardiac decline, individual isolated cardiac myocytes from chronically hyperthyroid hamsters had enhanced function when compared with myocytes from untreated age-matched controls. Thus, it appears that long-term hyperthyroidism may impair global LV function, at least in part by increasing interstitial ventricular fibrosis, in spite of normal or enhanced intrinsic cardiomyocyte function. PMID:23056390

Three-dimensional contractile muscle tissue consisting of human skeletal myocyte cell line.

PubMed

Shima, Ai; Morimoto, Yuya; Sweeney, H Lee; Takeuchi, Shoji

2018-06-18

This paper describes a method to construct three-dimensional (3D) contractile human skeletal muscle tissues from a cell line. The 3D tissue was fabricated as a fiber-based structure and cultured for two weeks under tension by anchoring its both ends. While myotubes from the immortalized human skeletal myocytes used in this study never contracted in the conventional two-dimensional (2D) monolayer culture, myotubes in the 3D tissue showed spontaneous contraction at a high frequency and also reacted to the electrical stimulation. Immunofluorescence revealed that the myotubes in the 3D tissues had sarcomeres and expressed ryanodine receptor (RyR) and sarco/endoplasmic reticulum Ca 2+ -ATPase (SERCA). In addition, intracellular calcium oscillations in the myotubes in the 3D tissue were observed. These results indicated that the 3D culture enabled the myocyte cell line to reach a more highly matured state compared to 2D culture. Since contraction is the most significant feature of skeletal muscle, we believe that our 3D human muscle tissue with the contractile ability would be a useful tool for both basic biology research and drug discovery as one of the muscle-on-chips. Copyright © 2018. Published by Elsevier Inc.

Cardiac Overexpression of Antioxidant Catalase Attenuates Aging-Induced Cardiomyocyte Relaxation Dysfunction

PubMed Central

Ren, Jun; Li, Qun; Wu, Shan; Li, Shi-Yan; Babcock, Sara A.

2007-01-01

Catalase, an enzyme which detoxifies H2O2, may interfere with cardiac aging. To test this hypothesis, contractile and intracellular Ca2+ properties were evaluated in cardiomyocytes from young (3–4 mo) and old (26–28 mo) FVB and transgenic mice with cardiac overexpression of catalase. Contractile indices analyzed included peak shortening (PS), time-to-90% PS (TPS90), time-to-90% relengthening (TR90), half-width duration (HWD), maximal velocity of shortening/relengthening (± dL/dt) and intracellular Ca2+ levels or decay rate. Levels of advanced glycation endproduct (AGE), Na+/Ca2+ exchanger (NCX), sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA2a), phospholamban (PLB), myosin heavy chain (MHC), membrane Ca2+ and K+ channels were measured by western blot. Catalase transgene prolonged survival while did not alter myocyte function by itself. Aging depressed ± dL/dt, prolonged HWD, TR90 and intracellular Ca2+ decay without affecting other indices in FVB myocytes. Aged FVB myocytes exhibited a stepper decline in PS in response to elevated stimulus or a dampened rise in PS in response to elevated extracellular Ca2+ levels. Interestingly, aging-induced defects were nullified or significantly attenuated by catalase. AGE level was elevated by 5-fold in aged FVB compared with young FVB mice, which was reduced by catalase. Expression of SERCA2a, NCX and Kv1.2 K+ channel was significantly reduced although levels of PLB, L-type Ca2+ channel dihydropyridine receptor and β-MHC isozyme remained unchanged in aged FVB hearts. Catalase restored NCX and Kv1.2 K+ channel but not SERCA2a level in aged mice. In summary, our data suggested that catalase protects cardiomyocytes from aging-induced contractile defect possibly via improved intracellular Ca2+ handling. PMID:17250874

Hypotonic swelling promotes nitric oxide release in cardiac ventricular myocytes: impact on swelling-induced negative inotropic effect

PubMed Central

Gonano, Luis Alberto; Morell, Malena; Burgos, Juan Ignacio; Dulce, Raul Ariel; De Giusti, Verónica Celeste; Aiello, Ernesto Alejandro; Hare, Joshua Michael; Vila Petroff, Martin

2014-01-01

Aims Cardiomyocyte swelling occurs in multiple pathological situations and has been associated with contractile dysfunction, cell death, and enhanced propensity to arrhythmias. We investigate whether hypotonic swelling promotes nitric oxide (NO) release in cardiomyocytes, and whether it impacts on swelling-induced contractile dysfunction. Methods and results Superfusing rat cardiomyocytes with a hypotonic solution (HS; 217 mOsm), increased cell volume, reduced myocyte contraction and Ca2+ transient, and increased NO-sensitive 4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate (DAF-FM) fluorescence. When cells were exposed to HS + 2.5 mM of the NO synthase inhibitor l-NAME, cell swelling occurred in the absence of NO release. Swelling-induced NO release was also prevented by the nitric oxide synthase 1 (NOS1) inhibitor, nitroguanidine, and significantly reduced in NOS1 knockout mice. Additionally, colchicine (inhibitor of microtubule polymerization) prevented the increase in DAF-FM fluorescence induced by HS, indicating that microtubule integrity is necessary for swelling-induced NO release. The swelling-induced negative inotropic effect was exacerbated in the presence of either l-NAME, nitroguandine, the guanylate cyclase inhibitor, ODQ, or the PKG inhibitor, KT5823, suggesting that NOS1-derived NO provides contractile support via a cGMP/PKG-dependent mechanism. Indeed, ODQ reduced Ca2+ wave velocity and both ODQ and KT5823 reduced the HS-induced increment in ryanodine receptor (RyR2, Ser2808) phosphorylation, suggesting that in this context, cGMP/PKG may contribute to preserve contractile function by enhancing sarcoplasmic reticulum Ca2+ release. Conclusions Our findings suggest a novel mechanism for NO release in cardiomyocytes with putative pathophysiological relevance determined, at least in part, by its capability to reduce the extent of contractile dysfunction associated with hypotonic swelling. PMID:25344365

Putative β4-adrenoceptors in rat ventricle mediate increases in contractile force and cell Ca2+: comparison with atrial receptors and relationship to (−)-[3H]-CGP 12177 binding

PubMed Central

Sarsero, Doreen; Molenaar, Peter; Kaumann, Alberto J; Freestone, Nicholas S

1999-01-01

We identified putative β4-adrenoceptors by radioligand binding, measured increases in ventricular contractile force by (−)-CGP 12177 and (±)-cyanopindolol and demonstrated increased Ca2+ transients by (−)-CGP 12177 in rat cardiomyocytes.(−)-[3H]-CGP 12177 labelled 13–22 fmol mg−1 protein ventricular β1, β2-adrenoceptors (pKD ∼9.0) and 50–90 fmol mg−1 protein putative β4-adrenoceptors (pKD ∼7.3). The affinity values (pKi) for (β1,β2-) and putative β4-adrenoceptors, estimated from binding inhibition, were (−)-propranolol 8.4, 5.7; (−)-bupranolol 9.7, 5.8; (±)-cyanopindolol 10.0,7.4.In left ventricular papillary muscle, in the presence of 30 μM 3-isobutyl-1-methylxanthine, (−)-CGP 12177 and (±)-cyanopindolol caused positive inotropic effects, (pEC50, (−)-CGP 12177, 7.6; (±)-cyanopindolol, 7.0) which were antagonized by (−)-bupranolol (pKB 6.7–7.0) and (−)-CGP 20712A (pKB 6.3–6.6). The cardiostimulant effects of (−)-CGP 12177 in papillary muscle, left and right atrium were antagonized by (±)-cyanopindolol (pKP 7.0–7.4).(−)-CGP 12177 (1 μM) in the presence of 200 nM (−)-propranolol increased Ca2+ transient amplitude by 56% in atrial myocytes, but only caused a marginal increase in ventricular myocytes. In the presence of 1 μM 3-isobutyl-1-methylxanthine and 200 nM (−)-propranolol, 1 μM (−)-CGP 12177 caused a 73% increase in Ca2+ transient amplitude in ventricular myocytes. (−)-CGP 12177 elicited arrhythmic transients in some atrial and ventricular myocytes.Probably by preventing cyclic AMP hydrolysis, 3-isobutyl-1-methylxanthine facilitates the inotropic function of ventricular putative β4-adrenoceptors, suggesting coupling to Gs protein-adenylyl cyclase. The receptor-mediated increases in contractile force are related to increases of Ca2+ in atrial and ventricular myocytes. The agreement of binding affinities of agonists with cardiostimulant potencies is consistent with mediation through putative β4-adrenoceptors labelled with (−)-[3H]-CGP 12177. PMID:10602323

Cardiac-specific deletion of protein phosphatase 1β promotes increased myofilament protein phosphorylation and contractile alterations

PubMed Central

Liu, Ruijie; Correll, Robert N.; Davis, Jennifer; Vagnozzi, Ronald J.; York, Allen J.; Sargent, Michelle A.; Nairn, Angus C.; Molkentin, Jeffery D.

2015-01-01

There are 3 protein phosphatase 1 (PP1) catalytic isoforms (α, β and γ) encoded within the mammalian genome. These 3 gene products share ~90% amino acid homology within their catalytic domains but each has unique N- and C-termini that likely underlie distinctive subcellular localization or functionality. In this study, we assessed the effect associated with loss of each PP1 isoform in the heart using a conditional Cre-loxP targeting approach in mice. Ppp1ca-loxP, Ppp1cb-loxP and Ppp1cc-oxP alleles were crossed with either an Nkx2.5-Cre knock-in containing allele for early embryonic deletion or a tamoxifen inducible α-myosin heavy chain (αMHC)-MerCreMer transgene for adult and cardiac-specific deletion. We determined that while deletion of Ppp1ca (PP1α) or Ppp1cc (PP1γ) had little effect on the whole heart, deletion of Ppp1cb (PP1β) resulted in concentric remodeling of the heart, interstitial fibrosis and contractile dysregulation, using either the embryonic or adult-specific Cre-expressing alleles. However, myocytes isolated from Ppp1cb deleted hearts surprisingly showed enhanced contractility. Mechanistically we found that deletion of any of the 3 PP1 gene-encoding isoforms had no effect on phosphorylation of phospholamban, nor were Ca2+ handling dynamics altered in adult myocytes from Ppp1cb deleted hearts. However, loss of Ppp1cb from the heart, but not Ppp1ca or Ppp1cc, resulted in elevated phosphorylation of myofilament proteins such as myosin light chain 2 and cardiac myosin binding protein C, consistent with an enriched localization profile of this isoform to the sarcomeres. These results suggest a unique functional role for the PP1β isoform in affecting cardiac contractile function. PMID:26334248

Loss of functional K+ channels encoded by ether-à-go-go-related genes in mouse myometrium prior to labour onset

PubMed Central

Greenwood, I A; Yeung, S Y; Tribe, R M; Ohya, S

2009-01-01

There is a growing appreciation that ion channels encoded by the ether-à-go-go-related gene family have a functional impact in smooth muscle in addition to their accepted role in cardiac myocytes and neurones. This study aimed to assess the expression of ERG1–3 (KCNH1–3) genes in the murine myometrium (smooth muscle layer of the uterus) and determine the functional impact of the ion channels encoded by these genes in pregnant and non-pregnant animals. Quantitative RT-PCR did not detect message for ERG2 and 3 in whole myometrial tissue extracts. In contrast, message for two isoforms of mERG1 were readily detected with mERG1a more abundant than mERG1b. In isometric tension studies of non-pregnant myometrium, the ERG channel blockers dofetilide (1 μm), E4031 (1 μm) and Be-KM1 (100 nm) increased spontaneous contractility and ERG activators (PD118057 and NS1643) inhibited spontaneous contractility. In contrast, neither ERG blockade nor activation had any effect on the inherent contractility in myometrium from late pregnant (19 days gestation) animals. Moreover, dofetilide-sensitive K+ currents with distinctive ‘hooked’ kinetics were considerably smaller in uterine myocytes from late pregnant compared to non-pregnant animals. Expression of mERG1 isoforms did not alter throughout gestation or upon delivery, but the expression of genes encoding auxillary subunits (KCNE) were up-regulated considerably. This study provides the first evidence for a regulation of ERG-encoded K+ channels as a precursor to late pregnancy physiological activity. PMID:19332483

Modeling beta-adrenergic control of cardiac myocyte contractility in silico.

PubMed

Saucerman, Jeffrey J; Brunton, Laurence L; Michailova, Anushka P; McCulloch, Andrew D

2003-11-28

The beta-adrenergic signaling pathway regulates cardiac myocyte contractility through a combination of feedforward and feedback mechanisms. We used systems analysis to investigate how the components and topology of this signaling network permit neurohormonal control of excitation-contraction coupling in the rat ventricular myocyte. A kinetic model integrating beta-adrenergic signaling with excitation-contraction coupling was formulated, and each subsystem was validated with independent biochemical and physiological measurements. Model analysis was used to investigate quantitatively the effects of specific molecular perturbations. 3-Fold overexpression of adenylyl cyclase in the model allowed an 85% higher rate of cyclic AMP synthesis than an equivalent overexpression of beta 1-adrenergic receptor, and manipulating the affinity of Gs alpha for adenylyl cyclase was a more potent regulator of cyclic AMP production. The model predicted that less than 40% of adenylyl cyclase molecules may be stimulated under maximal receptor activation, and an experimental protocol is suggested for validating this prediction. The model also predicted that the endogenous heat-stable protein kinase inhibitor may enhance basal cyclic AMP buffering by 68% and increasing the apparent Hill coefficient of protein kinase A activation from 1.0 to 2.0. Finally, phosphorylation of the L-type calcium channel and phospholamban were found sufficient to predict the dominant changes in myocyte contractility, including a 2.6x increase in systolic calcium (inotropy) and a 28% decrease in calcium half-relaxation time (lusitropy). By performing systems analysis, the consequences of molecular perturbations in the beta-adrenergic signaling network may be understood within the context of integrative cellular physiology.

Modeling beta-adrenergic control of cardiac myocyte contractility in silico

NASA Technical Reports Server (NTRS)

Saucerman, Jeffrey J.; Brunton, Laurence L.; Michailova, Anushka P.; McCulloch, Andrew D.; McCullough, A. D. (Principal Investigator)

2003-01-01

The beta-adrenergic signaling pathway regulates cardiac myocyte contractility through a combination of feedforward and feedback mechanisms. We used systems analysis to investigate how the components and topology of this signaling network permit neurohormonal control of excitation-contraction coupling in the rat ventricular myocyte. A kinetic model integrating beta-adrenergic signaling with excitation-contraction coupling was formulated, and each subsystem was validated with independent biochemical and physiological measurements. Model analysis was used to investigate quantitatively the effects of specific molecular perturbations. 3-Fold overexpression of adenylyl cyclase in the model allowed an 85% higher rate of cyclic AMP synthesis than an equivalent overexpression of beta 1-adrenergic receptor, and manipulating the affinity of Gs alpha for adenylyl cyclase was a more potent regulator of cyclic AMP production. The model predicted that less than 40% of adenylyl cyclase molecules may be stimulated under maximal receptor activation, and an experimental protocol is suggested for validating this prediction. The model also predicted that the endogenous heat-stable protein kinase inhibitor may enhance basal cyclic AMP buffering by 68% and increasing the apparent Hill coefficient of protein kinase A activation from 1.0 to 2.0. Finally, phosphorylation of the L-type calcium channel and phospholamban were found sufficient to predict the dominant changes in myocyte contractility, including a 2.6x increase in systolic calcium (inotropy) and a 28% decrease in calcium half-relaxation time (lusitropy). By performing systems analysis, the consequences of molecular perturbations in the beta-adrenergic signaling network may be understood within the context of integrative cellular physiology.

Role of STIM1 (Stromal Interaction Molecule 1) in Hypertrophy-Related Contractile Dysfunction.

PubMed

Troupes, Constantine D; Wallner, Markus; Borghetti, Giulia; Zhang, Chen; Mohsin, Sadia; von Lewinski, Dirk; Berretta, Remus M; Kubo, Hajime; Chen, Xiongwen; Soboloff, Jonathan; Houser, Steven

2017-07-07

Pathological increases in cardiac afterload result in myocyte hypertrophy with changes in myocyte electrical and mechanical phenotype. Remodeling of contractile and signaling Ca 2+ occurs in pathological hypertrophy and is central to myocyte remodeling. STIM1 (stromal interaction molecule 1) regulates Ca 2+ signaling in many cell types by sensing low endoplasmic reticular Ca 2+ levels and then coupling to plasma membrane Orai channels to induce a Ca 2+ influx pathway. Previous reports suggest that STIM1 may play a role in cardiac hypertrophy, but its role in electrical and mechanical phenotypic alterations is not well understood. To define the contributions of STIM1-mediated Ca 2+ influx on electrical and mechanical properties of normal and diseased myocytes, and to determine whether Orai channels are obligatory partners for STIM1 in these processes using a clinically relevant large animal model of hypertrophy. Cardiac hypertrophy was induced by slow progressive pressure overload in adult cats. Hypertrophied myocytes had increased STIM1 expression and activity, which correlated with altered Ca 2 + -handling and action potential (AP) prolongation. Exposure of hypertrophied myocytes to the Orai channel blocker BTP2 caused a reduction of AP duration and reduced diastolic Ca 2+ spark rate. BTP2 had no effect on normal myocytes. Forced expression of STIM1 in cultured adult feline ventricular myocytes increased diastolic spark rate and prolonged AP duration. STIM1 expression produced an increase in the amount of Ca 2+ stored within the sarcoplasmic reticulum and activated Ca 2+ /calmodulin-dependent protein kinase II. STIM1 expression also increased spark rates and induced spontaneous APs. STIM1 effects were eliminated by either BTP2 or by coexpression of a dominant negative Orai construct. STIM1 can associate with Orai in cardiac myocytes to produce a Ca 2+ influx pathway that can prolong the AP duration and load the sarcoplasmic reticulum and likely contributes to the altered electromechanical properties of the hypertrophied heart. © 2017 American Heart Association, Inc.

Micromolded gelatin hydrogels for extended culture of engineered cardiac tissues.

PubMed

McCain, Megan L; Agarwal, Ashutosh; Nesmith, Haley W; Nesmith, Alexander P; Parker, Kevin Kit

2014-07-01

Defining the chronic cardiotoxic effects of drugs during preclinical screening is hindered by the relatively short lifetime of functional cardiac tissues in vitro, which are traditionally cultured on synthetic materials that do not recapitulate the cardiac microenvironment. Because collagen is the primary extracellular matrix protein in the heart, we hypothesized that micromolded gelatin hydrogel substrates tuned to mimic the elastic modulus of the heart would extend the lifetime of engineered cardiac tissues by better matching the native chemical and mechanical microenvironment. To measure tissue stress, we used tape casting, micromolding, and laser engraving to fabricate gelatin hydrogel muscular thin film cantilevers. Neonatal rat cardiac myocytes adhered to gelatin hydrogels and formed aligned tissues as defined by the microgrooves. Cardiac tissues could be cultured for over three weeks without declines in contractile stress. Myocytes on gelatin had higher spare respiratory capacity compared to those on fibronectin-coated PDMS, suggesting that improved metabolic function could be contributing to extended culture lifetime. Lastly, human induced pluripotent stem cell-derived cardiac myocytes adhered to micromolded gelatin surfaces and formed aligned tissues that remained functional for four weeks, highlighting their potential for human-relevant chronic studies. Copyright © 2014 Elsevier Ltd. All rights reserved.

Strategies for regeneration of heart muscle.

PubMed

Guyette, Jacques P; Cohen, Ira S; Gaudette, Glenn R

2010-01-01

Regenerative medicine has emerged to the forefront of cardiac research, marrying discoveries in both basic science and engineering to develop viable therapeutic approaches for treating the diseased heart. Signifi cant advancements in gene therapy, stem cell biology, and cardiomyoplasty provide new optimism for regenerating damaged myocardium. Exciting new strategies for endogenous and exogenous regeneration have been proposed. However, questions remain as to whether these approaches can provide enough new myocyte mass to sufficiently restore mechanical function to the heart. In this article, we consider the mechanisms of endogenous cardiomyocyte regeneration and exogenous cell differentiation (with respect to myoblasts, stem cells, and induced pluripotent cells being researched for cell therapies). We begin by reviewing some of the cues that are being harnessed in strategies of gene/cell therapy for regenerating myocardium. We also consider some of the technical challenges that remain in determining new myocyte generation, tracking delivered cells in vivo, and correlating new myocyte contractility with cardiac function. Strategies for regenerating the heart are being realized as both animal and clinical trials suggest that these new approaches provide short-term improvement of cardiac function. However, a more complete understanding of the underlying mechanisms and applications is necessary to sustain longer-term therapeutic success.

Micromolded Gelatin Hydrogels for Extended Culture of Engineered Cardiac Tissues

PubMed Central

McCain, Megan L.; Agarwal, Ashutosh; Nesmith, Haley W.; Nesmith, Alexander P.; Parker, Kevin Kit

2014-01-01

Defining the chronic cardiotoxic effects of drugs during preclinical screening is hindered by the relatively short lifetime of functional cardiac tissues in vitro, which are traditionally cultured on synthetic materials that do not recapitulate the cardiac microenvironment. Because collagen is the primary extracellular matrix protein in the heart, we hypothesized that micromolded gelatin hydrogel substrates tuned to mimic the elastic modulus of the heart would extend the lifetime of engineered cardiac tissues by better matching the native chemical and mechanical microenvironment. To measure tissue stress, we used tape casting, micromolding, and laser engraving to fabricate gelatin hydrogel muscular thin film cantilevers. Neonatal rat cardiac myocytes adhered to gelatin hydrogels and formed aligned tissues as defined by the microgrooves. Cardiac tissues could be cultured for over three weeks without declines in contractile stress. Myocytes on gelatin had higher spare respiratory capacity compared to those on fibronectin-coated PDMS, suggesting that improved metabolic function could be contributing to extended culture lifetime. Lastly, human induced pluripotent stem cell-derived cardiac myocytes adhered to micromolded gelatin surfaces and formed aligned tissues that remained functional for four weeks, highlighting their potential for human-relevant chronic studies. PMID:24731714

Hydralazine and Organic Nitrates Restore Impaired Excitation-Contraction Coupling by Reducing Calcium Leak Associated with Nitroso-Redox Imbalance*

PubMed Central

Dulce, Raul A.; Yiginer, Omer; Gonzalez, Daniel R.; Goss, Garrett; Feng, Ning; Zheng, Meizi; Hare, Joshua M.

2013-01-01

Although the combined use of hydralazine and isosorbide dinitrate confers important clinical benefits in patients with heart failure, the underlying mechanism of action is still controversial. We used two models of nitroso-redox imbalance, neuronal NO synthase-deficient (NOS1−/−) mice and spontaneously hypertensive heart failure rats, to test the hypothesis that hydralazine (HYD) alone or in combination with nitroglycerin (NTG) or isosorbide dinitrate restores Ca2+ cycling and contractile performance and controls superoxide production in isolated cardiomyocytes. The response to increased pacing frequency was depressed in NOS1−/− compared with wild type myocytes. Both sarcomere length shortening and intracellular Ca2+ transient (Δ[Ca2+]i) responses in NOS1−/− cardiomyocytes were augmented by HYD in a dose-dependent manner. NTG alone did not affect myocyte shortening but reduced Δ[Ca2+]i across the range of pacing frequencies and increased myofilament Ca2+ sensitivity thereby enhancing contractile efficiency. Similar results were seen in failing myocytes from the heart failure rat model. HYD alone or in combination with NTG reduced sarcoplasmic reticulum (SR) leak, improved SR Ca2+ reuptake, and restored SR Ca2+ content. HYD and NTG at low concentrations (1 μm), scavenged superoxide in isolated cardiomyocytes, whereas in cardiac homogenates, NTG inhibited xanthine oxidoreductase activity and scavenged NADPH oxidase-dependent superoxide more efficiently than HYD. Together, these results revealed that by reducing SR Ca2+ leak, HYD improves Ca2+ cycling and contractility impaired by nitroso-redox imbalance, and NTG enhanced contractile efficiency, restoring cardiac excitation-contraction coupling. PMID:23319593

Caveolin Contributes to the Modulation of Basal and β-Adrenoceptor Stimulated Function of the Adult Rat Ventricular Myocyte by Simvastatin: A Novel Pleiotropic Effect

PubMed Central

Agarwal, Shailesh R.; Harvey, Robert D.; Porter, Karen E.; Calaghan, Sarah

2014-01-01

The number of people taking statins is increasing across the globe, highlighting the importance of fully understanding statins' effects on the cardiovascular system. The beneficial impact of statins extends well beyond regression of atherosclerosis to include direct effects on tissues of the cardiovascular system (‘pleiotropic effects’). Pleiotropic effects on the cardiac myocyte are often overlooked. Here we consider the contribution of the caveolin protein, whose expression and cellular distribution is dependent on cholesterol, to statin effects on the cardiac myocyte. Caveolin is a structural and regulatory component of caveolae, and is a key regulator of cardiac contractile function and adrenergic responsiveness. We employed an experimental model in which inhibition of myocyte HMG CoA reductase could be studied in the absence of paracrine influences from non-myocyte cells. Adult rat ventricular myocytes were treated with 10 µM simvastatin for 2 days. Simvastatin treatment reduced myocyte cholesterol, caveolin 3 and caveolar density. Negative inotropic and positive lusitropic effects (with corresponding changes in [Ca2+]i) were seen in statin-treated cells. Simvastatin significantly potentiated the inotropic response to β2-, but not β1-, adrenoceptor stimulation. Under conditions of β2-adrenoceptor stimulation, phosphorylation of phospholamban at Ser16 and troponin I at Ser23/24 was enhanced with statin treatment. Simvastatin increased NO production without significant effects on eNOS expression or phosphorylation (Ser1177), consistent with the reduced expression of caveolin 3, its constitutive inhibitor. In conclusion, statin treatment can reduce caveolin 3 expression, with functional consequences consistent with the known role of caveolae in the cardiac cell. These data are likely to be of significance, particularly during the early phases of statin treatment, and in patients with heart failure who have altered β-adrenoceptor signalling. In addition, as caveolin is ubiquitously expressed and has myriad tissue-specific functions, the impact of statin-dependent changes in caveolin is likely to have many other functional sequelae. PMID:25211146

Characterization of muscle contraction with second harmonic generation microscopy

NASA Astrophysics Data System (ADS)

Prent, Nicole

Muscle cells have the ability to change length and generate force due to orchestrated action of myosin nanomotors that cause sliding of actin filaments along myosin filaments in the sarcomeres, the fundamental contractile units, of myocytes. The correlated action of hundreds of sarcomeres is needed to produce the myocyte contractions. This study probes the molecular structure of the myofilaments and investigates the movement correlations between sarcomeres during contraction. In this study, second harmonic generation (SHG) microscopy is employed for imaging striated myocytes. Myosin filaments in striated myocytes inherently have a nonzero second-order susceptibility, [special characters omitted] and therefore generate efficient SHG. Employing polarization-in polarization-out (PIPO) SHG microscopy allows for the accurate determination of the characteristic ratio, [special characters omitted] in birefringent myocytes, which describes the structure of the myosin filament. Analysis shows that the b value at the centre of the myosin filament, where the nonlinear dipoles are better aligned, is slightly lower than the value at the edges of the filament, where there is more disorder in orientation of the nonlinear dipoles from the myosin heads. Forced stretching of myocytes resulted in an SHG intensity increase with the elongation of the sarcomere. SHG microscopy captured individual sarcomeres during contraction, allowing for the measurement of sarcomere length (SL) and SHG intensity (SI) fluctuations. The fluctuations also revealed higher SHG intensity in elongated sarcomeres. The sarcomere synchronization model (SSM) for contracting and quiescent myocytes was developed, and experimentally verified for three cases (isolated cardiomyocyte, embryonic chicken cardiomyocyte, and larva myocyte). During contraction, the action of SLs and SIs between neighbouring sarcomeres partially correlated, whereas in quiescent myocytes the SLs show an anti-correlation and the SIs have no correlations. The characteristic correlation coefficients and amplitudes were obtained for each case, allowing for the characterization of the synchronization of sarcomere movement during muscle contraction. These investigations constitute the basis for studying the structure and physiology of muscle cell contractions with polarization SHG microscopy. Live dynamic imaging of myocytes is applicable for contractility research, drug discovery, and as a diagnostic tool for monitoring muscular diseases.

CALIX[4]ARENE C-99 INHIBITS MYOSIN ATPase ACTIVITY AND CHANGES THE ORGANIZATION OF CONTRACTILE FILAMENTS OF MYOMETRIUM.

PubMed

Labyntseva, R D; Bevza, A A; Lul'ko, A O; Cherenok, S O; Kalchenko, V I; Kosterin, S O

2015-01-01

Calix[4]arenes are cup-like macrocyclic (polyphenolic) compounds, they are regarded as promising molecular "platforms" for the design of new physiologically active compounds. We have earlier found that calix[4]arene C-99 inhibits the ATPase activity of actomyosin and myosin subfragment-1 of pig uterus in vitro. The aim of this study was to investigate the interaction of calix[4]arene C-99 with myosin from rat uterine myocytes. It was found that the ATPase activity of myosin prepared from pre-incubated with 100 mM of calix[4]arene C-99 myocytes was almost 50% lower than in control. Additionally, we have revealed the effect of calix[4]arene C-99 on the subcellular distribution of actin and myosin in uterus myocytes by the method of confocal microscopy. This effect can be caused by reorganization of the structure of the contractile smooth muscle cell proteins due to their interaction with calix[4]arene. The obtained results demonstrate the ability of calix[4]arene C-99 to penetrate into the uterus muscle cells and affect not only the myosin ATPase activity, but also the structure of the actin and myosin filaments in the myometrial cells. Demonstrated ability of calix[4]arene C-99 can be used for development of new pharmacological agents for efficient normalization of myometrial contractile hyperfunction.

Mitochondria-Targeted Antioxidant Prevents Cardiac Dysfunction Induced by Tafazzin Gene Knockdown in Cardiac Myocytes

PubMed Central

He, Quan; Harris, Nicole; Ren, Jun; Han, Xianlin

2014-01-01

Tafazzin, a mitochondrial acyltransferase, plays an important role in cardiolipin side chain remodeling. Previous studies have shown that dysfunction of tafazzin reduces cardiolipin content, impairs mitochondrial function, and causes dilated cardiomyopathy in Barth syndrome. Reactive oxygen species (ROS) have been implicated in the development of cardiomyopathy and are also the obligated byproducts of mitochondria. We hypothesized that tafazzin knockdown increases ROS production from mitochondria, and a mitochondria-targeted antioxidant prevents tafazzin knockdown induced mitochondrial and cardiac dysfunction. We employed cardiac myocytes transduced with an adenovirus containing tafazzin shRNA as a model to investigate the effects of the mitochondrial antioxidant, mito-Tempo. Knocking down tafazzin decreased steady state levels of cardiolipin and increased mitochondrial ROS. Treatment of cardiac myocytes with mito-Tempo normalized tafazzin knockdown enhanced mitochondrial ROS production and cellular ATP decline. Mito-Tempo also significantly abrogated tafazzin knockdown induced cardiac hypertrophy, contractile dysfunction, and cell death. We conclude that mitochondria-targeted antioxidant prevents cardiac dysfunction induced by tafazzin gene knockdown in cardiac myocytes and suggest mito-Tempo as a potential therapeutic for Barth syndrome and other dilated cardiomyopathies resulting from mitochondrial oxidative stress. PMID:25247053

Hypotension Due to Kir6.1 Gain‐of‐Function in Vascular Smooth Muscle

PubMed Central

Li, Anlong; Knutsen, Russell H.; Zhang, Haixia; Osei‐Owusu, Patrick; Moreno‐Dominguez, Alex; Harter, Theresa M.; Uchida, Keita; Remedi, Maria S.; Dietrich, Hans H.; Bernal‐Mizrachi, Carlos; Blumer, Kendall J.; Mecham, Robert P.; Koster, Joseph C.; Nichols, Colin G.

2013-01-01

Background KATP channels, assembled from pore‐forming (Kir6.1 or Kir6.2) and regulatory (SUR1 or SUR2) subunits, link metabolism to excitability. Loss of Kir6.2 results in hypoglycemia and hyperinsulinemia, whereas loss of Kir6.1 causes Prinzmetal angina–like symptoms in mice. Conversely, overactivity of Kir6.2 induces neonatal diabetes in mice and humans, but consequences of Kir6.1 overactivity are unknown. Methods and Results We generated transgenic mice expressing wild‐type (WT), ATP‐insensitive Kir6.1 [Gly343Asp] (GD), and ATP‐insensitive Kir6.1 [Gly343Asp,Gln53Arg] (GD‐QR) subunits, under Cre‐recombinase control. Expression was induced in smooth muscle cells by crossing with smooth muscle myosin heavy chain promoter–driven tamoxifen‐inducible Cre‐recombinase (SMMHC‐Cre‐ER) mice. Three weeks after tamoxifen induction, we assessed blood pressure in anesthetized and conscious animals, as well as contractility of mesenteric artery smooth muscle and KATP currents in isolated mesenteric artery myocytes. Both systolic and diastolic blood pressures were significantly reduced in GD and GD‐QR mice but normal in mice expressing the WT transgene and elevated in Kir6.1 knockout mice as well as in mice expressing dominant‐negative Kir6.1 [AAA] in smooth muscle. Contractile response of isolated GD‐QR mesenteric arteries was blunted relative to WT controls, but nitroprusside relaxation was unaffected. Basal KATP conductance and pinacidil‐activated conductance were elevated in GD but not in WT myocytes. Conclusions KATP overactivity in vascular muscle can lead directly to reduced vascular contractility and lower blood pressure. We predict that gain of vascular KATP function in humans would lead to a chronic vasodilatory phenotype, as indeed has recently been demonstrated in Cantu syndrome. PMID:23974906

Reading tarot cards.

PubMed

Edmunds, L Henry

2004-02-01

In some patients acute myocardial infarction and/or infarct expansion induces progressive left ventricular dilatation that eventually leads to heart failure and death. The five year mortality after onset of heart failure is 50%. Chronically stretched viable myocardium adjacent to or remote from an expanding infarction initiates a myopathic process that leads to progressive myocyte apoptosis and adverse postinfarction remodeling. Revascularization of stunned or hibernating myocardium restores contractility and benefits patients in heart failure; however, revascularization does not restore contractility to myopathic, remodeling myocardium. Contemporary operations for heart failure temporarily reduce ventricular wall stress, but fail to reverse stretch induced myocyte apoptosis, which may not be reversible. Logically, prevention of this myopathic process after acute infarction seems required to extend survival. It follows that surgeons should operate before adverse postinfarction left ventricular remodeling occurs, using new operations, rather than afterwards.

The impact of age and frailty on ventricular structure and function in C57BL/6J mice

PubMed Central

Feridooni, H. A.; Kane, A. E.; Ayaz, O.; Boroumandi, A.; Polidovitch, N.; Tsushima, R. G.; Rose, R. A.

2017-01-01

Key points Heart size increases with age (called hypertrophy), and its ability to contract declines. However, these reflect average changes that may not be present, or present to the same extent, in all older individuals.That aging happens at different rates is well accepted clinically. People who are aging rapidly are frail and frailty is measured with a ‘frailty index’.We quantified frailty with a validated mouse frailty index tool and evaluated the impacts of age and frailty on cardiac hypertrophy and contractile dysfunction.Hypertrophy increased with age, while contractions, calcium currents and calcium transients declined; these changes were graded by frailty scores.Overall health status, quantified as frailty, may promote maladaptive changes associated with cardiac aging and facilitate the development of diseases such as heart failure.To understand age‐related changes in heart structure and function, it is essential to know both chronological age and the health status of the animal. Abstract On average, cardiac hypertrophy and contractile dysfunction increase with age. Still, individuals age at different rates and their health status varies from fit to frail. We investigated the influence of frailty on age‐dependent ventricular remodelling. Frailty was quantified as deficit accumulation in adult (≈7 months) and aged (≈27 months) C57BL/6J mice by adapting a validated frailty index (FI) tool. Hypertrophy and contractile function were evaluated in Langendorff‐perfused hearts; cellular correlates/mechanisms were investigated in ventricular myocytes. FI scores increased with age. Mean cardiac hypertrophy increased with age, but values in the adult and aged groups overlapped. When plotted as a function of frailty, hypertrophy was graded by FI score (r = 0.67–0.55, P < 0.0003). Myocyte area also correlated positively with FI (r = 0.34, P = 0.03). Left ventricular developed pressure (LVDP) plus rates of pressure development (+dP/dt) and decay (−dP/dt) declined with age and this was graded by frailty (r = −0.51, P = 0.0007; r = −0.48, P = 0.002; r = −0.56, P = 0.0002 for LVDP, +dP/dt and −dP/dt). Smaller, slower contractions graded by FI score were also seen in ventricular myocytes. Contractile dysfunction in cardiomyocytes isolated from frail mice was attributable to parallel changes in underlying Ca2+ transients. These changes were not due to reduced sarcoplasmic reticulum stores, but were graded by smaller Ca2+ currents (r = −0.40, P = 0.008), lower gain (r = −0.37, P = 0.02) and reduced expression of Cav1.2 protein (r = −0.68, P = 0.003). These results show that cardiac hypertrophy and contractile dysfunction in naturally aging mice are graded by overall health and suggest that frailty, in addition to chronological age, can help explain heterogeneity in cardiac aging. PMID:28502095

Characterization of the adenosine receptor in cultured embryonic chick atrial myocytes: Coupling to modulation of contractility and adenylate cyclase activity and identification by direct radioligand binding

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liang, B.T.

1989-06-01

Adenosine receptors in a spontaneously contracting atrial myocyte culture from 14-day chick embryos were characterized by radioligand binding studies and by examining the involvement of G-protein in coupling these receptors to a high-affinity state and to the adenylate cyclase and the myocyte contractility. Binding of the antagonist radioligand (3H)-8-cyclopentyl-1,3-diproylxanthine ((3H)CPX) was rapid, reversible and saturable and was to a homogeneous population of sites with a Kd value of 2.1 +/- 0.2 nM and an apparent maximum binding of 26.2 +/- 3 fmol/mg of protein (n = 10, +/- S.E.). Guanyl-5-yl-(beta, gamma-imido)diphosphate had no effect on either the Kd or themore » maximum binding and CPX reversed the N6-R-phenyl-2-propyladenosine-induced inhibition of adenylate cyclase activity and contractility, indicating that (3H) CPX is an antagonist radioligand. Competition curves for (3H) CPX binding by a series of reference adenosine agonists were consistent with labeling of an A1 adenosine receptor and were better fit by a two-site model than by a one-site model. ADP-ribosylation of the G-protein by the endogenous NAD+ in the presence of pertussis toxin shifted the competition curves from bi to monophasic with Ki values similar to those of the KL observed in the absence of prior pertussis intoxication. The adenosine agonists were capable of inhibiting both the adenylate cyclase activity and myocyte contractility in either the absence or the presence of isoproterenol. The A1 adenosine receptor-selective antagonist CPX reversed these agonist effects. The order of ability of the reference adenosine receptor agonists in causing these inhibitory effects was similar to the order of potency of the same agonists in inhibiting the specific (3H)CPX binding (N6-R-phenyl-2-propyladenosine greater than N6-S-phenyl-2-propyladenosine or N-ethyladenosine-5'-uronic acid).« less

American ginseng acutely regulates contractile function of rat heart.

PubMed

Jiang, Mao; Murias, Juan M; Chrones, Tom; Sims, Stephen M; Lui, Edmund; Noble, Earl G

2014-01-01

Chronic ginseng treatments have been purported to improve cardiac performance. However reports of acute administration of ginseng on cardiovascular function remain controversial and potential mechanisms are not clear. In this study, we examined the effects of acute North American ginseng (Panax quinquefolius) administration on rat cardiac contractile function by using electrocardiogram (ECG), non-invasive blood pressure (BP) measurement, and Langendorff isolated, spontaneously beating, perfused heart measurements (LP). Eight-week old male Sprague-Dawley rats (n = 8 per group) were gavaged with a single dose of water-soluble American ginseng at 300 mg/kg body weight. Heart rate (HR) and BP were measured prior to and at 1 and 24 h after gavaging (ECG and BP). Additional groups were used for each time point for Langendorff measurements. HR was significantly decreased (ECG: 1 h: 6 ± 0.2%, 24 h: 8 ± 0.3%; BP: 1 h: 8.8 ± 0.2%, 24 h: 13 ± 0.4% and LP: 1 h: 22 ± 0.4%, 24 h: 19 ± 0.4%) in rats treated with water-soluble ginseng compared with pre or control measures. An initial marked decrease in left ventricular developed pressure was observed in LP hearts but BP changes were not observed in BP group. A direct inhibitory effect of North American ginseng was observed on cardiac contractile function in LP rats and on fluorescence measurement of intracellular calcium transient in freshly isolated cardiac myocytes when exposed to ginseng (1 and 10 μg/ml). Collectively these data present evidence of depressed cardiac contractile function by acute administration of North American ginseng in rat. This acute reduction in cardiac contractile function appears to be intrinsic to the myocardium.

American ginseng acutely regulates contractile function of rat heart

PubMed Central

Jiang, Mao; Murias, Juan M.; Chrones, Tom; Sims, Stephen M.; Lui, Edmund; Noble, Earl G.

2014-01-01

Chronic ginseng treatments have been purported to improve cardiac performance. However reports of acute administration of ginseng on cardiovascular function remain controversial and potential mechanisms are not clear. In this study, we examined the effects of acute North American ginseng (Panax quinquefolius) administration on rat cardiac contractile function by using electrocardiogram (ECG), non-invasive blood pressure (BP) measurement, and Langendorff isolated, spontaneously beating, perfused heart measurements (LP). Eight-week old male Sprague–Dawley rats (n = 8 per group) were gavaged with a single dose of water-soluble American ginseng at 300 mg/kg body weight. Heart rate (HR) and BP were measured prior to and at 1 and 24 h after gavaging (ECG and BP). Additional groups were used for each time point for Langendorff measurements. HR was significantly decreased (ECG: 1 h: 6 ± 0.2%, 24 h: 8 ± 0.3%; BP: 1 h: 8.8 ± 0.2%, 24 h: 13 ± 0.4% and LP: 1 h: 22 ± 0.4%, 24 h: 19 ± 0.4%) in rats treated with water-soluble ginseng compared with pre or control measures. An initial marked decrease in left ventricular developed pressure was observed in LP hearts but BP changes were not observed in BP group. A direct inhibitory effect of North American ginseng was observed on cardiac contractile function in LP rats and on fluorescence measurement of intracellular calcium transient in freshly isolated cardiac myocytes when exposed to ginseng (1 and 10 μg/ml). Collectively these data present evidence of depressed cardiac contractile function by acute administration of North American ginseng in rat. This acute reduction in cardiac contractile function appears to be intrinsic to the myocardium. PMID:24672484

Swimming exercise reverses aging-related contractile abnormalities of female heart by improving structural alterations.

PubMed

Ozturk, Nihal; Olgar, Yusuf; Er, Hakan; Kucuk, Murathan; Ozdemir, Semir

2017-01-01

The objective of this study was to examine the effect of swimming exercise on aging-related Ca2+ handling alterations and structural abnormalities of female rat heart. For this purpose, 4-month and 24-month old female rats were used and divided into three following groups: sedentary young (SY), sedentary old (SO), and exercised old (Ex-O). Swimming exercise was performed for 8 weeks (60 min/day, 5 days/week). Myocyte shortening, L-type Ca2+ currents and associated Ca2+ transients were measured from ventricular myocytes at 36 ± 1°C. NOX-4 levels, aconitase activity, glutathione measurements and ultrastructural examination by electron microscopy were conducted in heart tissue. Swimming exercise reversed the reduced shortening and slowed kinetics of aged cardiomyocytes. Although the current density was similar for all groups, Ca2+ transients were higher in SO and Ex-O myocytes with respect to the SY group. Caffeine-induced Ca2+ transients and the integrated NCX current were lower in cardiomyocytes of SY rats compared with other groups, suggesting an increased sarcoplasmic reticulum Ca2+ content in an aged heart. Aging led to upregulated cardiac NOX-4 along with declined aconitase activity. Although it did not reverse these oxidative parameters, swimming exercise achieved a significant increase in glutathione levels and improved structural alterations of old rats' hearts. We conclude that swimming exercise upregulates antioxidant defense capacity and improves structural abnormalities of senescent female rat heart, although it does not change Ca2+ handling alterations further. Thereby, it improves contractile function of aged myocardium by mitigating detrimental effects of oxidative stress.

Negative Inotropic Effects of High Mobility Box Group 1 Protein in Isolated Contracting Cardiac Myocytes

PubMed Central

Tzeng, Huei-Ping; Fan, Jinping; Vallejo, Jesus G.; Dong, Jian Wen; Chen, Xiongwen; Houser, Steven R.; Mann, Douglas L.

2013-01-01

HMGB1 released from necrotic cells or macrophages functions as a late inflammatory mediator, and has been shown to induce cardiovascular collapse during sepsis. Thus far, however, the effect(s) of HMGB1 in the heart are not known. We determined the effects of HMGB1 on isolated feline cardiac myocytes by measuring sarcomere shortening in contracting cardiac myocytes, intracellular Ca2+ transients using fluo-3, and L-type calcium currents using whole cell perforate configuration of the patch clamp technique. Treatment of isolated myocytes with HMGB1 (100 ng/ml) resulted in a 70% decrease in sarcomere shortening and a 50% decrease in the height of the peak Ca++ transient within 5 min (p <0.01). The immediate negative inotropic effects HMGB1 on cell contractility and calcium homeostasis were partially reversible upon washout of HMGB1. A significant inhibition of the inward L-type calcium currents also was documented by the patch clamp technique. HMGB1 induced the PKCε translocation and a PKC inhibitor significantly attenuated the negative inotropic effects of HMGB1. These studies show for the first time that HMGB1 impairs sarcomere shortening by decreasing calcium availability in cardiac myocytes through modulating membrane calcium influx, and suggest that HMGB1 maybe act as a novel myocardial depressant factor during cardiac injury. PMID:18223193

OxLDL enhances L-type Ca2+ currents via lysophosphatidylcholine-induced mitochondrial reactive oxygen species (ROS) production.

PubMed

Fearon, Ian M

2006-03-01

To examine the mechanisms underlying oxidised LDL- (oxLDL)-induced alterations in Ca(2+) currents, an effect which underlies altered vascular contractility and cardiac myocyte function. Ca(2+) currents (I(Ca)) were recorded by whole-cell patch-clamp in HEK293 cells expressing L-type Ca(2+) channel alpha(1C) subunits or isolated rat ventricular myocytes. oxLDL (but not native LDL) significantly enhanced recombinant I(Ca), an effect mimicked by 1 microM lysophosphatidylcholine (LPC). LPC failed to enhance I(Ca) either in mitochondrial electron transport chain-depleted rho(0) cells, or in the presence of rotenone (1 microM), or MPP(+) (10 microM). The LPC response was similarly ablated by ascorbate (200 microM) or TROLOX (500 microM) and by the mitochondria-targeted antioxidant, MitoQ (250 nM). In myocytes, enhancement of I(Ca) due to LPC was similarly abrogated with rotenone and MitoQ. These data suggest that LPC enhanced recombinant Ca(2+) currents due to increased mitochondrial ROS production. In support with this, LPC enhanced fluorescence in HEK293 cells and cardiac myocytes loaded with a ROS-sensitive mitochondrial dye, reduced mitotracker red. LPC up-regulates L-type Ca(2+) currents due to altered mitochondrial ROS production, an effect which mediates the response of the native I(Ca) in cardiac myocytes to oxLDL.

PKA catalytic subunit compartmentation regulates contractile and hypertrophic responses to β-adrenergic signaling

PubMed Central

Yang, Jason H.; Polanowska-Grabowska, Renata K.; Smith, Jeffrey S.; Shields, Charles W.; Saucerman, Jeffrey J.

2014-01-01

β-adrenergic signaling is spatiotemporally heterogeneous in the cardiac myocyte, conferring exquisite control to sympathetic stimulation. Such heterogeneity drives the formation of protein kinase A (PKA) signaling microdomains, which regulate Ca2+ handling and contractility. Here, we test the hypothesis that the nucleus independently comprises a PKA signaling microdomain regulating myocyte hypertrophy. Spatially-targeted FRET reporters for PKA activity identified slower PKA activation and lower isoproterenol sensitivity in the nucleus (t50 = 10.60±0.68 min; EC50 = 89.00 nmol/L) than in the cytosol (t50 = 3.71±0.25 min; EC50 = 1.22 nmol/L). These differences were not explained by cAMP or AKAP-based compartmentation. A computational model of cytosolic and nuclear PKA activity was developed and predicted that differences in nuclear PKA dynamics and magnitude are regulated by slow PKA catalytic subunit diffusion, while differences in isoproterenol sensitivity are regulated by nuclear expression of protein kinase inhibitor (PKI). These were validated by FRET and immunofluorescence. The model also predicted differential phosphorylation of PKA substrates regulating cell contractility and hypertrophy. Ca2+ and cell hypertrophy measurements validated these predictions and identified higher isoproterenol sensitivity for contractile enhancements (EC50 = 1.84 nmol/L) over cell hypertrophy (EC50 = 85.88 nmol/L). Over-expression of spatially targeted PKA catalytic subunit to the cytosol or nucleus enhanced contractile and hypertrophic responses, respectively. We conclude that restricted PKA catalytic subunit diffusion is an important PKA compartmentation mechanism and the nucleus comprises a novel PKA signaling microdomain, insulating hypertrophic from contractile β-adrenergic signaling responses. PMID:24225179

Sprint training shortens prolonged action potential duration in postinfarction rat myocyte: mechanisms.

PubMed

Zhang, X Q; Zhang, L Q; Palmer, B M; Ng, Y C; Musch, T I; Moore, R L; Cheung, J Y

2001-05-01

Two electrophysiological manifestations of myocardial infarction (MI)-induced myocyte hypertrophy are prolongation of action potential duration (APD) and reduction of transient outward current (I(to)) density. Because high-intensity sprint training (HIST) ameliorated myocyte hypertrophy and improved myocyte Ca(2+) homeostasis and contractility after MI, the present study evaluated whether 6-8 wk of HIST would shorten the prolonged APD and improve the depressed I(to) in post-MI myocytes. There were no differences in resting membrane potential and action potential amplitude (APA) measured in myocytes isolated from sham-sedentary (Sed), MI-Sed, and MI-HIST groups. Times required for repolarization to 50 and 90% APA were significantly (P < 0.001) prolonged in MI-Sed myocytes. HIST reduced times required for repolarization to 50 and 90% APA to values observed in Sham-Sed myocytes. The fast and slow components of I(to) were significantly (P < 0.0001) reduced in MI-Sed myocytes. HIST significantly (P < 0.001) enhanced the fast and slow components of I(to) in MI myocytes, although not to levels observed in Sham-Sed myocytes. There were no significant differences in steady-state I(to) inactivation and activation parameters among Sham-Sed, MI-Sed, and MI-HIST myocytes. Likewise, recovery from time-dependent inactivation was also similar among the three groups. We suggest that normalization of APD after MI by HIST may be mediated by restoration of I(to) toward normal levels.

Bioengineering Human Myocardium on Native Extracellular Matrix

PubMed Central

Guyette, Jacques P.; Charest, Jonathan M; Mills, Robert W; Jank, Bernhard J.; Moser, Philipp T.; Gilpin, Sarah E.; Gershlak, Joshua R.; Okamoto, Tatsuya; Gonzalez, Gabriel; Milan, David J.; Gaudette, Glenn R.; Ott, Harald C.

2015-01-01

Rationale More than 25 million individuals suffer from heart failure worldwide, with nearly 4,000 patients currently awaiting heart transplantation in the United States. Donor organ shortage and allograft rejection remain major limitations with only about 2,500 hearts transplanted each year. As a theoretical alternative to allotransplantation, patient-derived bioartificial myocardium could provide functional support and ultimately impact the treatment of heart failure. Objective The objective of this study is to translate previous work to human scale and clinically relevant cells, for the bioengineering of functional myocardial tissue based on the combination of human cardiac matrix and human iPS-derived cardiac myocytes. Methods and Results To provide a clinically relevant tissue scaffold, we translated perfusion-decellularization to human scale and obtained biocompatible human acellular cardiac scaffolds with preserved extracellular matrix composition, architecture, and perfusable coronary vasculature. We then repopulated this native human cardiac matrix with cardiac myocytes derived from non-transgenic human induced pluripotent stem cells (iPSCs) and generated tissues of increasing three-dimensional complexity. We maintained such cardiac tissue constructs in culture for 120 days to demonstrate definitive sarcomeric structure, cell and matrix deformation, contractile force, and electrical conduction. To show that functional myocardial tissue of human scale can be built on this platform, we then partially recellularized human whole heart scaffolds with human iPSC-derived cardiac myocytes. Under biomimetic culture, the seeded constructs developed force-generating human myocardial tissue, showed electrical conductivity, left ventricular pressure development, and metabolic function. Conclusions Native cardiac extracellular matrix scaffolds maintain matrix components and structure to support the seeding and engraftment of human iPS-derived cardiac myocytes, and enable the bioengineering of functional human myocardial-like tissue of multiple complexities. PMID:26503464

A simplified method for identification of human cardiac myosin heavy-chain isoforms.

PubMed

Piao, Shengfu; Yu, Fushun; Mihm, Michael J; Reiser, Peter J; McCarthy, Patrick M; Van Wagoner, David R; Bauer, John Anthony

2003-02-01

Cardiac myosin is a central participant in the cross-bridge cycling that mediates myocyte contraction and consists of multiple subunits that mediate both hydrolysis of ATP and mechanical production of contractile force Two isoforms of myosin heavy chain (MHC- alpha and MHC- beta ) are known to exist in mammalian cardiac tissue, and it is within this myosin subunit that ATPase activity resides. These isoforms differ by less than 0.2% in total molecular mass and amino acid sequence, but, strikingly, influence the rate and efficiency of energy utilization for generation of contractile force. Changes in the MHC- alpha /MHC- beta ratio has been classically viewed as an adaptation of a failing myocyte in both animal models and humans; however, their measurement has traditionally required specialized preparations and materials for sufficient resolution. Here we describe a greatly simplified method for routine assessments of myosin isoform composition in human cardiac tissues. The primary advantages of our approach include higher throughput and reduced supply costs with no apparent loss of statistical power, reproducibility or achieved results. Use of this more convenient method may provide enhanced access to an otherwise specialized technique and could provide additional opportunity for investigation of cardiac myocyte adaptive changes.

Ginsenosides Rb1 and Re decrease cardiac contraction in adult rat ventricular myocytes: role of nitric oxide

PubMed Central

Scott, Glenda I; Colligan, Peter B; Ren, Bonnie H; Ren, Jun

2001-01-01

Panax ginseng is used to enhance stamina and relieve fatigue as well as physical stress. Ginsenoside, the effective component of ginseng, regulates cardiovascular function. This study was to examine the effect of ginsenosides Rb1 and Re on cardiac contractile function at the cellular level. Ventricular myocytes were isolated from adult rat hearts and were stimulated to contract at 0.5 Hz. Contractile properties analysed included: peak shortening (PS), time-to-90%PS (TPS), time-to-90% relengthening (TR90), and fluorescence intensity change (ΔFFI). Nitric oxide synthase (NOS) activity was determined by the 3H-arginine to 3H-citrulline conversion assay. Both Rb1 and Re exhibited dose-dependent (1 – 1000 nM) inhibition in PS and ΔFFI, with maximal inhibitions between 20 – 25%. Concurrent application Rb1 and Re did not produce any additive inhibition on peak shortening amplitude (with a maximal inhibition of 24.9±6.1%), compared to Rb1 or Re alone. Pretreatment with the NOS inhibitor Nω-nitro-L-arginine methyl ester (L-NAME, 100 μM) abolished the effect of Rb1 and Re. Both Rb1 and Re significantly (P<0.05) stimulated NOS activity concentration-dependently. This study demonstrated a direct depressant action of ginsenosides on cardiomyocyte contraction, which may be mediated in part through increased NO production. PMID:11704635

Traction force microscopy of engineered cardiac tissues.

PubMed

Pasqualini, Francesco Silvio; Agarwal, Ashutosh; O'Connor, Blakely Bussie; Liu, Qihan; Sheehy, Sean P; Parker, Kevin Kit

2018-01-01

Cardiac tissue development and pathology have been shown to depend sensitively on microenvironmental mechanical factors, such as extracellular matrix stiffness, in both in vivo and in vitro systems. We present a novel quantitative approach to assess cardiac structure and function by extending the classical traction force microscopy technique to tissue-level preparations. Using this system, we investigated the relationship between contractile proficiency and metabolism in neonate rat ventricular myocytes (NRVM) cultured on gels with stiffness mimicking soft immature (1 kPa), normal healthy (13 kPa), and stiff diseased (90 kPa) cardiac microenvironments. We found that tissues engineered on the softest gels generated the least amount of stress and had the smallest work output. Conversely, cardiomyocytes in tissues engineered on healthy- and disease-mimicking gels generated significantly higher stresses, with the maximal contractile work measured in NRVM engineered on gels of normal stiffness. Interestingly, although tissues on soft gels exhibited poor stress generation and work production, their basal metabolic respiration rate was significantly more elevated than in other groups, suggesting a highly ineffective coupling between energy production and contractile work output. Our novel platform can thus be utilized to quantitatively assess the mechanotransduction pathways that initiate tissue-level structural and functional remodeling in response to substrate stiffness.

Redox signaling in cardiac myocytes

PubMed Central

Santos, Celio X.C.; Anilkumar, Narayana; Zhang, Min; Brewer, Alison C.; Shah, Ajay M.

2011-01-01

The heart has complex mechanisms that facilitate the maintenance of an oxygen supply–demand balance necessary for its contractile function in response to physiological fluctuations in workload as well as in response to chronic stresses such as hypoxia, ischemia, and overload. Redox-sensitive signaling pathways are centrally involved in many of these homeostatic and stress-response mechanisms. Here, we review the main redox-regulated pathways that are involved in cardiac myocyte excitation–contraction coupling, differentiation, hypertrophy, and stress responses. We discuss specific sources of endogenously generated reactive oxygen species (e.g., mitochondria and NADPH oxidases of the Nox family), the particular pathways and processes that they affect, the role of modulators such as thioredoxin, and the specific molecular mechanisms that are involved—where this knowledge is available. A better understanding of this complex regulatory system may allow the development of more specific therapeutic strategies for heart diseases. PMID:21236334

TMEM16A Channels Contribute to the Myogenic Response in Cerebral Arteries

PubMed Central

Bulley, Simon; Neeb, Zachary P.; Burris, Sarah K.; Bannister, John P.; Thomas-Gatewood, Candice M.; Jangsangthong, Wanchana; Jaggar, Jonathan H.

2013-01-01

Rationale Pressure-induced arterial depolarization and constriction (the myogenic response), is a smooth muscle cell (myocyte)-specific mechanism that controls regional organ blood flow and systemic blood pressure. Several different non-selective cation channels contribute to pressure-induced depolarization, but signaling mechanisms involved are unclear. Similarly uncertain is the contribution of anion channels to the myogenic response and physiological functions and mechanisms of regulation of recently discovered transmembrane 16A (TMEM16A) chloride (Cl−) channels in arterial myocytes. Objective Investigate the hypothesis that myocyte TMEM16A channels control membrane potential and contractility and contribute to the myogenic response in cerebral arteries. Methods and Results Cell swelling induced by hyposmotic bath solution stimulated Cl− currents in arterial myocytes that were blocked by TMEM16A channel inhibitory antibodies, RNAi-mediated selective TMEM16A channel knockdown, removal of extracellular calcium (Ca2+), replacement of intracellular EGTA with BAPTA, a fast Ca2+ chelator, and Gd3+ and SKF-96365, non-selective cation channel blockers. In contrast, nimodipine, a voltage-dependent Ca2+ channel inhibitor, or thapsigargin, which depletes intracellular Ca2+ stores, did not alter swelling-activated TMEM16A currents. Pressure (−40 mmHg)-induced membrane stretch activated ion channels in arterial myocyte cell-attached patches that were inhibited by TMEM16A antibodies and were of similar amplitude to recombinant TMEM16A channels. TMEM16A knockdown reduced intravascular pressure-induced depolarization and vasoconstriction, but did not alter depolarization (60 mmol/L K+)-induced vasoconstriction. Conclusions Membrane stretch activates arterial myocyte TMEM16A channels, leading to membrane depolarization and vasoconstriction. Data also provide a mechanism by which a local Ca2+ signal generated by non-selective cation channels stimulates TMEM16A channels to induce myogenic constriction. PMID:22872152

Pre-treatment of synthetic elastomeric scaffolds by cardiac fibroblasts improves engineered heart tissue

PubMed Central

Radisic, Milica; Park, Hyoungshin; Martens, Timothy P.; Salazar-Lazaro, Johanna E.; Geng, Wenliang; Wang, Yadong; Langer, Robert; Freed, Lisa E.; Vunjak-Novakovic, Gordana

2009-01-01

Native myocardium consists of several cell types, of which approximately one-third are myocytes and most of the nonmyocytes are fibroblasts. By analogy with monolayer culture in which fibroblasts were removed to prevent overgrowth, early attempts to engineer myocardium utilized cell populations enriched for cardiac myocytes (CMs; ~80–90% of total cells). We hypothesized that the pre-treatment of synthetic elastomeric scaffolds with cardiac fibroblasts (CFs) will enhance the functional assembly of the engineered cardiac constructs by creating an environment supportive of cardiomyocyte attachment and function. Cells isolated from neonatal rat ventricles were prepared to form three distinct populations: rapidly plating cells identified as CFs, slowly plating cells identified as CMs, and unseparated initial population of cells (US). The cell fractions (3 × 106 cells total) were seeded into poly(glycerol sebacate) scaffolds (highly porous discs, 5 mm in diameter × 2-mm thick) using Matrigel™, either separately (CM or CF), concurrently (US), or sequentially (CF pre-treatment followed by CM culture, CF + CM), and cultured in spinner flasks. The CF + CM group had the highest amplitude of contraction and the lowest excitation threshold, superior DNA content, and higher glucose consumption rate. The CF + CM group exhibited compact 100- to 200-μm thick layers of elongated myocytes aligned in parallel over layers of collagen-producing fibroblasts, while US and CM groups exhibited scattered and poorly elongated myocytes. The sequential co-culture of CF and CM on a synthetic elastomer scaffold thus created an environment supportive of cardiomyocyte attachment, differentiation, and contractile function, presumably due to scaffold conditioning by cultured fibroblasts. When implanted over the infarcted myocardium in a nude rat model, cell-free poly(glycerol sebacate) remained at the ventricular wall after 2 weeks of in vivo, and was vascularized. PMID:18041719

Moderate Continuous Aerobic Exercise Training Improves Cardiomyocyte Contractility in Β1 Adrenergic Receptor Knockout Mice.

PubMed

Rodrigues, Aurora Corrêa; Natali, Antônio José; Cunha, Daise Nunes Queiroz da; Costa, Alexandre Jayme Lopes Dantas; Moura, Anselmo Gomes de; Araújo Carneiro-Júnior, Miguel; Félix, Leonardo Bonato; Brum, Patrícia Chakur; Prímola-Gomes, Thales Nicolau

2018-03-01

The lack of cardiac β1-adrenergic receptors (β1-AR) negatively affects the regulation of both cardiac inotropy and lusitropy, leading, in the long term, to heart failure (HF). Moderate-intensity aerobic exercise (MCAE) is recommended as an adjunctive therapy for patients with HF. We tested the effects of MCAE on the contractile properties of left ventricular (LV) myocytes from β1 adrenergic receptor knockout (β1ARKO) mice. Four- to five-month-old male wild type (WT) and β1ARKO mice were divided into groups: WT control (WTc) and trained (WTt); and β1ARKO control (β1ARKOc) and trained (β1ARKOt). Animals from trained groups were submitted to a MCAE regimen (60 min/day; 60% of maximal speed, 5 days/week) on a treadmill, for 8 weeks. P ≤ 0.05 was considered significant in all comparisons. The β1ARKO and exercised mice exhibited a higher (p < 0.05) running capacity than WT and sedentary ones, respectively. The β1ARKO mice showed higher body (BW), heart (HW) and left ventricle (LVW) weights, as well as the HW/BW and LVW/BW than WT mice. However, the MCAE did not affect these parameters. Left ventricular myocytes from β1ARKO mice showed increased (p < 0.05) amplitude and velocities of contraction and relaxation than those from WT. In addition, MCAE increased (p < 0.05) amplitude and velocities of contraction and relaxation in β1ARKO mice. MCAE improves myocyte contractility in the left ventricle of β1ARKO mice. This is evidence to support the therapeutic value of this type of exercise training in the treatment of heart diseases involving β1-AR desensitization or reduction.

Modulation of contraction by intracellular Na+ via Na(+)-Ca2+ exchange in single shark (Squalus acanthias) ventricular myocytes.

PubMed Central

Näbauer, M; Morad, M

1992-01-01

1. The effect of direct alteration of intracellular Na+ concentration on contractile properties of whole-cell clamped shark ventricular myocytes was studied using an array of 256 photodiodes to monitor the length of the isolated myocytes. 2. In myocytes dialysed with Na(+)-free solution, the voltage dependence of Ca2+ current (ICa) and contraction were similar and bell shaped. Contractions activated at all voltages were completely suppressed by nifedipine (5 microM), and failed to show significant tonic components, suggesting dependence of the contraction on Ca2+ influx through the L-type Ca2+ channel. 3. In myocytes dialysed with 60 mM Na+, a ICa-dependent and a ICa-independent component of contraction could be identified. The Ca2+ current-dependent component was prominent in voltages between -30 to +10 mV. The ICa-independent contractions were maintained for the duration of depolarization, increased with increasing depolarization between +10 to +100 mV, and were insensitive to nifedipine. 4. In such myocytes, repolarization produced slowly decaying inward tail currents closely related to the time course of relaxation and the degree of shortening prior to repolarization. 5. With 60 mM Na+ in the pipette solution, positive clamp potentials activated decaying outward currents which correlated to the size of contraction. These outward currents appeared to be generated by the Na(+)-Ca(2+)-exchanger since they depended on the presence of intracellular Na+, and were neither suppressed by nifedipine nor by K+ channel blockers. 6. The results suggest that in shark (Squalus acanthias) ventricular myocytes, which lack functionally relevant Ca2+ release pools, both Ca2+ channel and the Na(+)-Ca2+ exchanger deliver sufficient Ca2+ to activate contraction, though the effectiveness of the latter mechanism was highly dependent on the [Na+]i. PMID:1338467

Ventricular action potential adaptation to regular exercise: role of β-adrenergic and KATP channel function.

PubMed

Wang, Xinrui; Fitts, Robert H

2017-08-01

Regular exercise training is known to affect the action potential duration (APD) and improve heart function, but involvement of β-adrenergic receptor (β-AR) subtypes and/or the ATP-sensitive K + (K ATP ) channel is unknown. To address this, female and male Sprague-Dawley rats were randomly assigned to voluntary wheel-running or control groups; they were anesthetized after 6-8 wk of training, and myocytes were isolated. Exercise training significantly increased APD of apex and base myocytes at 1 Hz and decreased APD at 10 Hz. Ca 2+ transient durations reflected the changes in APD, while Ca 2+ transient amplitudes were unaffected by wheel running. The nonselective β-AR agonist isoproterenol shortened the myocyte APD, an effect reduced by wheel running. The isoproterenol-induced shortening of APD was largely reversed by the selective β 1 -AR blocker atenolol, but not the β 2 -AR blocker ICI 118,551, providing evidence that wheel running reduced the sensitivity of the β 1 -AR. At 10 Hz, the K ATP channel inhibitor glibenclamide prolonged the myocyte APD more in exercise-trained than control rats, implicating a role for this channel in the exercise-induced APD shortening at 10 Hz. A novel finding of this work was the dual importance of altered β 1 -AR responsiveness and K ATP channel function in the training-induced regulation of APD. Of physiological importance to the beating heart, the reduced response to adrenergic agonists would enhance cardiac contractility at resting rates, where sympathetic drive is low, by prolonging APD and Ca 2+ influx; during exercise, an increase in K ATP channel activity would shorten APD and, thus, protect the heart against Ca 2+ overload or inadequate filling. NEW & NOTEWORTHY Our data demonstrated that regular exercise prolonged the action potential and Ca 2+ transient durations in myocytes isolated from apex and base regions at 1-Hz and shortened both at 10-Hz stimulation. Novel findings were that wheel running shifted the β-adrenergic receptor agonist dose-response curve rightward compared with controls by reducing β 1 -adrenergic receptor responsiveness and that, at the high activation rate, myocytes from trained animals showed higher K ATP channel function. Copyright © 2017 the American Physiological Society.

Testosterone deficiency prevents left ventricular contractility dysfunction after myocardial infarction.

PubMed

Ribeiro Júnior, R F; Ronconi, K S; Jesus, I C G; Almeida, P W M; Forechi, L; Vassallo, D V; Guatimosim, S; Stefanon, I; Fernandes, A A

2018-01-15

Testosterone may affect myocardial contractility since its deficiency decreases the contraction and relaxation of the heart. Meanwhile, testosterone replacement therapy has raised concerns because it may worsen cardiac dysfunction and remodeling after myocardial infarction (MI). In this study, we evaluate cardiac contractility 60 days after MI in rats with suppressed testosterone. Male Wistar rats underwent bilateral orchidectomy one week before the ligation of the anterior descending left coronary artery. The animals were divided into orchidectomized (OCT); MI; orchidectomized + MI (OCT + MI); orchidectomized + MI + testosterone (OCT + MI + T) and control (Sham) groups. Eight weeks after MI, papillary muscle contractility was analyzed under increasing calcium (0.62, 1.25, 2.5 and 3.75 mM) and isoproterenol (10 -8 to 10 -2  M) concentrations. Ventricular myocytes were isolated for intracellular calcium measurements and assessment of Ca 2+ handling proteins. Contractility was preserved in the orchidectomized animals after myocardial infarction and was reduced when testosterone was replaced (Ca 2+ 3.75 mM: Sham: 608 ± 70 (n = 11); OCT: 590 ± 37 (n = 16); MI: 311 ± 33* (n = 9); OCT + MI: 594 ± 76 (n = 7); OCT + MI + T: 433 ± 38* (n=4), g/g *p < 0.05 vs Sham). Orchidectomy also increased the Ca 2+ transient amplitude of the ventricular myocytes and SERCA-2a protein expression levels. PLB phosphorylation levels at Thr 17 were not different in the orchidectomized animals compared to the Sham animals but were reduced after testosterone replacement. CAMKII phosphorylation and protein nitrosylation increased in the orchidectomized animals. Our results support the view that testosterone deficiency prevents MI contractility dysfunction by altering the key proteins involved in Ca 2+ handling. Copyright © 2017 Elsevier B.V. All rights reserved.

Bladder smooth muscle organ culture preparation maintains the contractile phenotype

PubMed Central

Wang, Tanchun; Kendig, Derek M.; Chang, Shaohua; Trappanese, Danielle M.; Chacko, Samuel

2012-01-01

Smooth muscle cells, when subjected to culture, modulate from a contractile to a secretory phenotype. This has hampered the use of cell culture for molecular techniques to study the regulation of smooth muscle biology. The goal of this study was to develop a new organ culture model of bladder smooth muscle (BSM) that would maintain the contractile phenotype and aid in the study of BSM biology. Our results showed that strips of BSM subjected to up to 9 days of organ culture maintained their contractile phenotype, including the ability to achieve near-control levels of force with a temporal profile similar to that of noncultured tissues. The technical aspects of our organ culture preparation that were responsible, in part, for the maintenance of the contractile phenotype were a slight longitudinal stretch during culture and subjection of the strips to daily contraction-relaxation. The tissues contained viable cells throughout the cross section of the strips. There was an increase in extracellular collagenous matrix, resulting in a leftward shift in the passive length-tension relationship. There were no significant changes in the content of smooth muscle-specific α-actin, calponin, h-caldesmon, total myosin heavy chain, protein kinase G, Rho kinase-I, or the ratio of SM1 to SM2 myosin isoforms. Moreover the organ cultured tissues maintained functional voltage-gated calcium channels and large-conductance calcium-activated potassium channels. Therefore, we propose that this novel BSM organ culture model maintains the contractile phenotype and will be a valuable tool for the use in cellular/molecular biology studies of bladder myocytes. PMID:22896042

PGC-1{alpha} accelerates cytosolic Ca{sup 2+} clearance without disturbing Ca{sup 2+} homeostasis in cardiac myocytes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, Min, E-mail: chenminyx@gmail.com; Yunnan Centers for Diseases Prevention and Control, Kunming 650022; Wang, Yanru

2010-06-11

Energy metabolism and Ca{sup 2+} handling serve critical roles in cardiac physiology and pathophysiology. Peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1{alpha}) is a multi-functional coactivator that is involved in the regulation of cardiac mitochondrial functional capacity and cellular energy metabolism. However, the regulation of PGC-1{alpha} in cardiac Ca{sup 2+} signaling has not been fully elucidated. To address this issue, we combined confocal line-scan imaging with off-line imaging processing to characterize calcium signaling in cultured adult rat ventricular myocytes expressing PGC-1{alpha} via adenoviral transduction. Our data shows that overexpressing PGC-1{alpha} improved myocyte contractility without increasing the amplitude of Ca{sup 2+}more » transients, suggesting that myofilament sensitivity to Ca{sup 2+} increased. Interestingly, the decay kinetics of global Ca{sup 2+} transients and Ca{sup 2+} waves accelerated in PGC-1{alpha}-expressing cells, but the decay rate of caffeine-elicited Ca{sup 2+} transients showed no significant change. This suggests that sarcoplasmic reticulum (SR) Ca{sup 2+}-ATPase (SERCA2a), but not Na{sup +}/Ca{sup 2+} exchange (NCX) contribute to PGC-1{alpha}-induced cytosolic Ca{sup 2+} clearance. Furthermore, PGC-1{alpha} induced the expression of SERCA2a in cultured cardiac myocytes. Importantly, overexpressing PGC-1{alpha} did not disturb cardiac Ca{sup 2+} homeostasis, because SR Ca{sup 2+} load and the propensity for Ca{sup 2+} waves remained unchanged. These data suggest that PGC-1{alpha} can ameliorate cardiac Ca{sup 2+} cycling and improve cardiac work output in response to physiological stress. Unraveling the PGC-1{alpha}-calcium handing pathway sheds new light on the role of PGC-1{alpha} in the therapy of cardiac diseases.« less

Sarcoplasmic reticulum Ca(2+) atpase (SERCA) 1a structurally substitutes for SERCA2a in the cardiac sarcoplasmic reticulum and increases cardiac Ca(2+) handling capacity.

PubMed

Lalli, M J; Yong, J; Prasad, V; Hashimoto, K; Plank, D; Babu, G J; Kirkpatrick, D; Walsh, R A; Sussman, M; Yatani, A; Marbán, E; Periasamy, M

2001-07-20

Ectopic expression of the sarcoplasmic reticulum (SR) Ca(2+) ATPase (SERCA) 1a pump in the mouse heart results in a 2.5-fold increase in total SERCA pump level. SERCA1a hearts show increased rates of contraction/relaxation and enhanced Ca(2+) transients; however, the cellular mechanisms underlying altered Ca(2+) handling in SERCA1a transgenic (TG) hearts are unknown. In this study, using confocal microscopy, we demonstrate that SERCA1a protein traffics to the cardiac SR and structurally substitutes for the endogenous SERCA2a isoform. SR Ca(2+) load measurements revealed that TG myocytes have significantly enhanced SR Ca(2+) load. Confocal line-scan images of field-stimulated SR Ca(2+) release showed an increased rate of Ca(2+) removal in TG myocytes. On the other hand, ryanodine receptor binding activity was decreased by approximately 30%. However, TG myocytes had a greater rate of spontaneous ryanodine receptor opening as measured by spark frequency. Whole-cell L-type Ca(2+) current density was reduced by approximately 50%, whereas the time course of inactivation was unchanged in TG myocytes. These studies provide important evidence that SERCA1a can substitute both structurally and functionally for SERCA2a in the heart and that SERCA1a overexpression can be used to enhance SR Ca(2+) transport and cardiac contractility.

Stimulation of Single Isolated Adult Ventricular Myocytes within a Low Volume Using a Planar Microelectrode Array

PubMed Central

Klauke, Norbert; Smith, Godfrey L.; Cooper, Jon

2003-01-01

Microchannels (40-μm wide, 10-μm high, 10-mm long, 70-μm pitch) were patterned in the silicone elastomer, polydimethylsiloxane on a microscope coverslip base. Integrated within each microchamber were individually addressable stimulation electrodes (40-μm wide, 20-μm long, 100-nm thick) and a common central pseudo-reference electrode (60-μm wide, 500-μm long, 100-nm thick). Isolated rabbit ventricular myocytes were introduced into the chamber by micropipetting and subsequently capped with a layer of mineral oil, thus creating limited volumes of saline around individual myocytes that could be varied from 5 nL to 100 pL. Excitation contraction coupling was studied by monitoring myocyte shortening and intracellular Ca2+ transients (using Fluo-3 fluorescence) . The amplitude of stimulated myocyte shortening and Ca2+ transients remained constant for 90 min in the larger volume (5 nL) configuration, although the shortening (but not the Ca2+ transient) amplitude gradually decreased to 20% of control within 60 min in the low volume (100 pL) arrangement. These studies indicate a lower limit for the extracellular volume required to stimulate isolated adult cardiac myocytes. Whereas this arrangement could be used to create a screening assay for drugs, individual microchannels (100 pL) can also be used to study the effects of limited extracellular volume on the contractility of single cardiac myocytes. PMID:12944291

A Simplified, Langendorff-Free Method for Concomitant Isolation of Viable Cardiac Myocytes and Nonmyocytes From the Adult Mouse Heart

PubMed Central

Ackers-Johnson, Matthew; Li, Peter Yiqing; Holmes, Andrew P.; O’Brien, Sian-Marie; Pavlovic, Davor; Foo, Roger S.

2018-01-01

Rationale Cardiovascular disease represents a global pandemic. The advent of and recent advances in mouse genomics, epigenomics, and transgenics offer ever-greater potential for powerful avenues of research. However, progress is often constrained by unique complexities associated with the isolation of viable myocytes from the adult mouse heart. Current protocols rely on retrograde aortic perfusion using specialized Langendorff apparatus, which poses considerable logistical and technical barriers to researchers and demands extensive training investment. Objective To identify and optimize a convenient, alternative approach, allowing the robust isolation and culture of adult mouse cardiac myocytes using only common surgical and laboratory equipment. Methods and Results Cardiac myocytes were isolated with yields comparable to those in published Langendorff-based methods, using direct needle perfusion of the LV ex vivo and without requirement for heparin injection. Isolated myocytes can be cultured antibiotic free, with retained organized contractile and mitochondrial morphology, transcriptional signatures, calcium handling, responses to hypoxia, neurohormonal stimulation, and electric pacing, and are amenable to patch clamp and adenoviral gene transfer techniques. Furthermore, the methodology permits concurrent isolation, separation, and coculture of myocyte and nonmyocyte cardiac populations. Conclusions We present a novel, simplified method, demonstrating concomitant isolation of viable cardiac myocytes and nonmyocytes from the same adult mouse heart. We anticipate that this new approach will expand and accelerate innovative research in the field of cardiac biology. PMID:27502479

A device for rapid and quantitative measurement of cardiac myocyte contractility

NASA Astrophysics Data System (ADS)

Gaitas, Angelo; Malhotra, Ricky; Li, Tao; Herron, Todd; Jalife, José

2015-03-01

Cardiac contractility is the hallmark of cardiac function and is a predictor of healthy or diseased cardiac muscle. Despite advancements over the last two decades, the techniques and tools available to cardiovascular scientists are limited in their utility to accurately and reliably measure the amplitude and frequency of cardiomyocyte contractions. Isometric force measurements in the past have entailed cumbersome attachment of isolated and permeabilized cardiomyocytes to a force transducer followed by measurements of sarcomere lengths under conditions of submaximal and maximal Ca2+ activation. These techniques have the inherent disadvantages of being labor intensive and costly. We have engineered a micro-machined cantilever sensor with an embedded deflection-sensing element that, in preliminary experiments, has demonstrated to reliably measure cardiac cell contractions in real-time. Here, we describe this new bioengineering tool with applicability in the cardiovascular research field to effectively and reliably measure cardiac cell contractility in a quantitative manner. We measured contractility in both primary neonatal rat heart cardiomyocyte monolayers that demonstrated a beat frequency of 3 Hz as well as human embryonic stem cell-derived cardiomyocytes with a contractile frequency of about 1 Hz. We also employed the β-adrenergic agonist isoproterenol (100 nmol l-1) and observed that our cantilever demonstrated high sensitivity in detecting subtle changes in both chronotropic and inotropic responses of monolayers. This report describes the utility of our micro-device in both basic cardiovascular research as well as in small molecule drug discovery to monitor cardiac cell contractions.

Enhanced functional expression of transient outward current in hypertrophied feline myocytes.

PubMed

Ten Eick, R E; Zhang, K; Harvey, R D; Bassett, A L

1993-08-01

Cardiac hypertrophy can decrease myocardial contractility and alter the electrophysiological activity of the heart. It is well documented that action potentials recorded from hypertrophied feline ventricular cells can exhibit depressed plateau voltages and prolonged durations. Similar findings have been made by others in rabbit, rat, guinea pig, and human heart. Whole-cell patch voltage-clamp studies designed to explain these changes in the action potential suggest that the only component of the membrane current recorded from feline right ventricular (RV) myocytes found to be substantially different from normal is the 4-amino-pyridine-sensitive transient outward current (I(to)). However, it was not clear if the change in I(to) could explain the changes in the action potential of hypertrophied cardiocytes, nor was it clear if these changes reflect an alteration in the electrophysiological character of the channels underlying I(to). A kinetic comparison of I(to) elicited by hypertrophied RV myocytes with that elicited by comparable normal RV myocytes previously revealed no differences, suggesting that the increased magnitude of the peak I(to) recorded from hypertrophied myocytes arises because the current density increases and not because of any alteration in the kinetic parameters governing the current. This finding suggests that in hypertrophy additional normal channels are expressed rather than a kinetically different channel subtype emerging. Investigations designed to determine if enhancement of I(to) could explain the hypertrophy-induced changes in plateau voltage and action potential duration suggest that a change in I(to) density can indeed explain the entire effect of hypertrophy on RV action potentials. If this notion is correct, the likelihood of "sudden death" in patients with myocardial hypertrophy might be decreased by a blocker selective for cardiac I(to).

Role of ROS signaling in differential hypoxic Ca2+ and contractile responses in pulmonary and systemic vascular smooth muscle cells.

PubMed

Wang, Yong-Xiao; Zheng, Yun-Min

2010-12-31

Hypoxia causes a large increase in [Ca2+]i and attendant contraction in pulmonary artery smooth muscle cells (PASMCs), but not in systemic artery SMCs. The different responses meet the respective functional needs in these two distinct vascular myocytes; however, the underlying molecular mechanisms are not well known. We and other investigators have provided extensive evidence to reveal that voltage-dependent K+ (KV) channels, canonical transient receptor potential (TRPC) channels, ryanodine receptor Ca2+ release channels (RyRs), cyclic adenosine diphosphate-ribose, FK506 binding protein 12.6, protein kinase C, NADPH oxidase and reactive oxygen species (ROS) are the essential effectors and signaling intermediates in the hypoxic increase in [Ca2+]i in PASMCs and HPV, but they may not primarily underlie the diverse cellular responses in pulmonary and systemic vascular myocytes. Hypoxia significantly increases mitochondrial ROS generation in PASMCs, which can induce intracellular Ca2+ release by opening RyRs, and may also cause extracellular Ca2+ influx by inhibiting KV channels and activating TRPC channels, leading to a large increase in [Ca2+]i in PASMCs and HPV. In contrast, hypoxia has no or a minor effect on mitochondrial ROS generation in systemic SMCs, thereby causing no change or a negligible increase in [Ca2+]i and contraction. Further preliminary work indicates that Rieske iron-sulfur protein in the mitochondrial complex III may perhaps serve as a key initial molecular determinant for the hypoxic increase in [Ca2+]i in PASMCs and HPV, suggesting its potential important role in different cellular changes to respond to hypoxic stimulation in pulmonary and systemic artery myocytes. All these findings have greatly improved our understanding of the molecular processes for the differential hypoxic Ca2+ and contractile responses in vascular SMCs from distinct pulmonary and systemic circulation systems. Copyright © 2010 Elsevier B.V. All rights reserved.

Hypertrophic gene expression induced by chronic stretch of excised mouse heart muscle.

PubMed

Raskin, Anna M; Hoshijima, Masahiko; Swanson, Eric; McCulloch, Andrew D; Omens, Jeffrey H

2009-09-01

Altered mechanical stress and strain in cardiac myocytes induce modifications in gene expression that affects cardiac remodeling and myocyte contractile function. To study the mechanisms of mechanotransduction in cardiomyocytes, probing alterations in mechanics and gene expression has been an effective strategy. However, previous studies are self-limited due to the general use of isolated neonatal rodent myocytes or intact animals. The main goal of this study was to develop a novel tissue culture chamber system for mouse myocardium that facilitates loading of cardiac tissue, while measuring tissue stress and deformation within a physiological environment. Intact mouse right ventricular papillary muscles were cultured in controlled conditions with superfusate at 95% O2/ 5% CO2, and 34 degrees C, such that cell to extracellular matrix adhesions as well as cell to cell adhesions were undisturbed and both passive and active mechanical properties were maintained without significant changes. The system was able to measure the induction of hypertrophic markers (BNP, ANP) in tissue after 2 hrs and 5 hrs of stretch. ANP induction was highly correlated with the diastolic load of the muscle but not with developed systolic load. Load induced ANP expression was blunted in muscles from muscle-LIM protein knockout mice, in which defective mechanotransduction pathways have been predicted.

Ionizing radiation regulates cardiac Ca handling via increased ROS and activated CaMKII.

PubMed

Sag, Can M; Wolff, Hendrik A; Neumann, Kay; Opiela, Marie-Kristin; Zhang, Juqian; Steuer, Felicia; Sowa, Thomas; Gupta, Shamindra; Schirmer, Markus; Hünlich, Mark; Rave-Fränk, Margret; Hess, Clemens F; Anderson, Mark E; Shah, Ajay M; Christiansen, Hans; Maier, Lars S

2013-11-01

Ionizing radiation (IR) is an integral part of modern multimodal anti-cancer therapies. IR involves the formation of reactive oxygen species (ROS) in targeted tissues. This is associated with subsequent cardiac dysfunction when applied during chest radiotherapy. We hypothesized that IR (i.e., ROS)-dependently impaired cardiac myocytes' Ca handling might contribute to IR-dependent cardiocellular dysfunction. Isolated ventricular mouse myocytes and the mediastinal area of anaesthetized mice (that included the heart) were exposed to graded doses of irradiation (sham 4 and 20 Gy) and investigated acutely (after ~1 h) as well as chronically (after ~1 week). IR induced a dose-dependent effect on myocytes' systolic function with acutely increased, but chronically decreased Ca transient amplitudes, which was associated with an acutely unaltered but chronically decreased sarcoplasmic reticulum (SR) Ca load. Likewise, in vivo echocardiography of anaesthetized mice revealed acutely enhanced left ventricular contractility (strain analysis) that declined after 1 week. Irradiated myocytes showed persistently increased diastolic SR Ca leakage, which was acutely compensated by an increase in SR Ca reuptake. This was reversed in the chronic setting in the face of slowed relaxation kinetics. As underlying cause, acutely increased ROS levels were identified to activate Ca/calmodulin-dependent protein kinase II (CaMKII). Accordingly, CaMKII-, but not PKA-dependent phosphorylation sites of the SR Ca release channels (RyR2, at Ser-2814) and phospholamban (at Thr-17) were found to be hyperphosphorylated following IR. Conversely, ROS-scavenging as well as CaMKII-inhibition significantly attenuated CaMKII-activation, disturbed Ca handling, and subsequent cellular dysfunction upon irradiation. Targeted cardiac irradiation induces a biphasic effect on cardiac myocytes Ca handling that is associated with chronic cardiocellular dysfunction. This appears to be mediated by increased oxidative stress and persistently activated CaMKII. Our findings suggest impaired cardiac myocytes Ca handling as a so far unknown mediator of IR-dependent cardiac damage that might be of relevance for radiation-induced cardiac dysfunction.

Underlying mechanism of the contractile dysfunction in atrophied ventricular myocytes from a murine model of hypothyroidism.

PubMed

Montalvo, Dolores; Pérez-Treviño, Perla; Madrazo-Aguirre, Katheryne; González-Mondellini, Fabio A; Miranda-Roblero, Hipólito O; Ramonfaur-Gracia, Diego; Jacobo-Antonio, Mariana; Mayorga-Luna, Maritza; Gómez-Víquez, Norma L; García, Noemí; Altamirano, Julio

2018-06-01

Hypothyroidism (Hypo) is a risk factor for cardiovascular diseases, including heart failure. Hypo rapidly induces Ca 2+ mishandling and contractile dysfunction (CD), as well as atrophy and ventricular myocytes (VM) remodeling. Hypo decreases SERCA-to-phospholamban ratio (SERCA/PLB), and thereby contributes to CD. Nevertheless, detailed spatial and temporal Ca 2+ cycling characterization in VM is missing, and contribution of other structural and functional changes to the mechanism underlying Ca 2+ mishandling and CD, as transverse tubules (T-T) remodeling, mitochondrial density (D mit ) and energy availability, is unclear. Therefore, in a rat model of Hypo, we aimed to characterize systolic and diastolic Ca 2+ signaling, T-T remodeling, D mit , citrate synthase (CS) activity and high-energy phosphate metabolites (ATP and phosphocreatine). We confirmed a decrease in SERCA/PLB (59%), which slowed SERCA activity (48%), reduced SR Ca 2+ (19%) and blunted Ca 2+ transient amplitude (41%). Moreover, assessing the rate of SR Ca 2+ release (dRel/dt), we found that early and maximum dRel/dt decreased, and this correlated with staggered Ca 2+ transients. However, dRel/dt persisted during Ca 2+ transient relaxation due to abundant late Ca 2+ sparks. Isoproterenol significantly up-regulated systolic Ca 2+ cycling. T-T were unchanged, hence, cannot explain staggered Ca 2+ transients and altered dRel/dt. Therefore, we suggest that these might be caused by RyR2 clusters desynchronization, due to diminished Ca 2+ -dependent sensitivity of RyR2, which also caused a decrease in diastolic SR Ca 2+ leak. Furthermore, D mit was unchanged and CS activity slightly decreased (14%), however, the ratio phosphocreatine/ATP did not change, therefore, energy deficiency cannot account for Ca 2+ and contractility dysregulation. We conclude that decreased SR Ca 2+ , due to slower SERCA, disrupts systolic RyR2 synchronization, and this underlies CD. Copyright © 2018 Elsevier Ltd. All rights reserved.

Cathepsin K knockout alleviates aging-induced cardiac dysfunction

PubMed Central

Hua, Yinan; Robinson, Timothy J; Cao, Yongtao; Shi, Guo-Ping; Ren, Jun; Nair, Sreejayan

2015-01-01

Aging is a major risk factor for cardiovascular disease. It has previously been shown that protein levels of cathepsin K, a lysosomal cysteine protease, are elevated in the failing heart and that genetic ablation of cathepsin K protects against pressure overload-induced cardiac hypertrophy and contractile dysfunction. Here we test the hypothesis that cathepsin K knockout alleviates age-dependent decline in cardiac function. Cardiac geometry, contractile function, intracellular Ca2+ properties, and cardiomyocyte apoptosis were evaluated using echocardiography, fura-2 technique, immunohistochemistry, Western blot and TUNEL staining, respectively. Aged (24-month-old) mice exhibited significant cardiac remodeling (enlarged chamber size, wall thickness, myocyte cross-sectional area, and fibrosis), decreased cardiac contractility, prolonged relengthening along with compromised intracellular Ca2+ release compared to young (6-month-old) mice, which were attenuated in the cathepsin K knockout mice. Cellular markers of senescence, including cardiac lipofuscin, p21 and p16, were lower in the aged-cathepsin K knockout mice compared to their wild-type counterpart. Mechanistically, cathepsin K knockout mice attenuated an age-induced increase in cardiomyocyte apoptosis and nuclear translocation of mitochondrial apoptosis-inducing factor (AIF). In cultured H9c2 cells, doxorubicin stimulated premature senescence and apoptosis. Silencing of cathepsin K blocked the doxorubicin-induced translocation of AIF from the mitochondria to the nuclei. Collectively, these results suggest that cathepsin K knockout attenuates age-related decline in cardiac function via suppressing caspase-dependent and caspase-independent apoptosis. PMID:25692548

High-fat diet induces protein kinase A and G-protein receptor kinase phosphorylation of β2 -adrenergic receptor and impairs cardiac adrenergic reserve in animal hearts.

PubMed

Fu, Qin; Hu, Yuting; Wang, Qingtong; Liu, Yongming; Li, Ning; Xu, Bing; Kim, Sungjin; Chiamvimonvat, Nipavan; Xiang, Yang K

2017-03-15

Patients with diabetes show a blunted cardiac inotropic response to β-adrenergic stimulation despite normal cardiac contractile reserve. Acute insulin stimulation impairs β-adrenergically induced contractile function in isolated cardiomyocytes and Langendorff-perfused hearts. In this study, we aimed to examine the potential effects of hyperinsulinaemia associated with high-fat diet (HFD) feeding on the cardiac β 2 -adrenergic receptor signalling and the impacts on cardiac contractile function. We showed that 8 weeks of HFD feeding leads to reductions in cardiac functional reserve in response to β-adrenergic stimulation without significant alteration of cardiac structure and function, which is associated with significant changes in β 2 -adrenergic receptor phosphorylation at protein kinase A and G-protein receptor kinase sites in the myocardium. The results suggest that clinical intervention might be applied to subjects in early diabetes without cardiac symptoms to prevent further cardiac complications. Patients with diabetes display reduced exercise capability and impaired cardiac contractile reserve in response to adrenergic stimulation. We have recently uncovered an insulin receptor and adrenergic receptor signal network in the heart. The aim of this study was to understand the impacts of high-fat diet (HFD) on the insulin-adrenergic receptor signal network in hearts. After 8 weeks of HFD feeding, mice exhibited diabetes, with elevated insulin and glucose concentrations associated with body weight gain. Mice fed an HFD had normal cardiac structure and function. However, the HFD-fed mice displayed a significant elevation of phosphorylation of the β 2 -adrenergic receptor (β 2 AR) at both the protein kinase A site serine 261/262 and the G-protein-coupled receptor kinase site serine 355/356 and impaired adrenergic reserve when compared with mice fed on normal chow. Isolated myocytes from HFD-fed mice also displayed a reduced contractile response to adrenergic stimulation when compared with those of control mice fed normal chow. Genetic deletion of the β 2 AR led to a normalized adrenergic response and preserved cardiac contractile reserve in HFD-fed mice. Together, these data indicate that HFD promotes phosphorylation of the β 2 AR, contributing to impairment of cardiac contractile reserve before cardiac structural and functional remodelling, suggesting that early intervention in the insulin-adrenergic signalling network might be effective in prevention of cardiac complications in diabetes. © 2016 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.

High‐fat diet induces protein kinase A and G‐protein receptor kinase phosphorylation of β2‐adrenergic receptor and impairs cardiac adrenergic reserve in animal hearts

PubMed Central

Hu, Yuting; Wang, Qingtong; Liu, Yongming; Li, Ning; Xu, Bing; Kim, Sungjin; Chiamvimonvat, Nipavan

2017-01-01

Key points Patients with diabetes show a blunted cardiac inotropic response to β‐adrenergic stimulation despite normal cardiac contractile reserve.Acute insulin stimulation impairs β‐adrenergically induced contractile function in isolated cardiomyocytes and Langendorff‐perfused hearts.In this study, we aimed to examine the potential effects of hyperinsulinaemia associated with high‐fat diet (HFD) feeding on the cardiac β2‐adrenergic receptor signalling and the impacts on cardiac contractile function.We showed that 8 weeks of HFD feeding leads to reductions in cardiac functional reserve in response to β‐adrenergic stimulation without significant alteration of cardiac structure and function, which is associated with significant changes in β2‐adrenergic receptor phosphorylation at protein kinase A and G‐protein receptor kinase sites in the myocardium.The results suggest that clinical intervention might be applied to subjects in early diabetes without cardiac symptoms to prevent further cardiac complications. Abstract Patients with diabetes display reduced exercise capability and impaired cardiac contractile reserve in response to adrenergic stimulation. We have recently uncovered an insulin receptor and adrenergic receptor signal network in the heart. The aim of this study was to understand the impacts of high‐fat diet (HFD) on the insulin–adrenergic receptor signal network in hearts. After 8 weeks of HFD feeding, mice exhibited diabetes, with elevated insulin and glucose concentrations associated with body weight gain. Mice fed an HFD had normal cardiac structure and function. However, the HFD‐fed mice displayed a significant elevation of phosphorylation of the β2‐adrenergic receptor (β2AR) at both the protein kinase A site serine 261/262 and the G‐protein‐coupled receptor kinase site serine 355/356 and impaired adrenergic reserve when compared with mice fed on normal chow. Isolated myocytes from HFD‐fed mice also displayed a reduced contractile response to adrenergic stimulation when compared with those of control mice fed normal chow. Genetic deletion of the β2AR led to a normalized adrenergic response and preserved cardiac contractile reserve in HFD‐fed mice. Together, these data indicate that HFD promotes phosphorylation of the β2AR, contributing to impairment of cardiac contractile reserve before cardiac structural and functional remodelling, suggesting that early intervention in the insulin–adrenergic signalling network might be effective in prevention of cardiac complications in diabetes. PMID:27983752

Insulin-Like Growth Factor I (IGF-1) Deficiency Ameliorates Sex Difference in Cardiac Contractile Function and Intracellular Ca2+ Homeostasis

PubMed Central

Ceylan-Isik, Asli F.; Li, Qun; Ren, Jun

2011-01-01

Sex difference in cardiac contractile function exists which may contribute to the different prevalence in cardiovascular diseases between genders. However, the precise mechanisms of action behind sex difference in cardiac function are still elusive. Given that sex difference exists in insulin-like growth factor I (IGF-1) cascade, this study is designed to evaluate the impact of severe liver IGF-1 deficiency (LID) on sex difference in cardiac function. Echocardiographic, cardiomyocyte contractile and intracellular Ca2+ properties were evaluated including ventricular geometry, fractional shortening, peak shortening, maximal velocity of shortening/relengthening (± dL/dt), time-to-peak shortening (TPS), time-to-90% relengthening (TR90), fura-fluorescence intensity (FFI) and intracellular Ca2+ clearance. Female C57 mice exhibited significantly higher plasma IGF-1 levels than their male counterpart. LID mice possessed comparably low IGF-1 levels in both sexes. Female C57 and LID mice displayed lower body, heart and liver weights compared to male counterparts. Echocardiographic analysis revealed larger LV mass in female C57 but not LID mice without sex difference in other cardiac geometric indices. Myocytes from female C57 mice exhibited reduced peak shortening, ± dL/dt, longer TPS, TR90 and intracellular Ca2+ clearance compared with males. Interestingly, this sex difference was greatly attenuated or abolished by IGF-1 deficiency. Female C57 mice displayed significantly decreased mRNA and protein levels of Na+-Ca2+ exchanger, SERCA2a and phosphorylated phospholamban as well as SERCA activity compared with male C57 mice. These sex differences in Ca2+ regulatory proteins were abolished or overtly attenuated by IGF-1 deficiency. In summary, our data suggested that IGF-1 deficiency may significantly attenuated or mitigate the sex difference in cardiomyocyte contractile function associated with intracellular Ca2+ regulation. PMID:21763763

Insulin-like growth factor I (IGF-1) deficiency ameliorates sex difference in cardiac contractile function and intracellular Ca(2+) homeostasis.

PubMed

Ceylan-Isik, Asli F; Li, Qun; Ren, Jun

2011-10-10

Sex difference in cardiac contractile function exists which may contribute to the different prevalence in cardiovascular diseases between genders. However, the precise mechanisms of action behind sex difference in cardiac function are still elusive. Given that sex difference exists in insulin-like growth factor I (IGF-1) cascade, this study is designed to evaluate the impact of severe liver IGF-1 deficiency (LID) on sex difference in cardiac function. Echocardiographic, cardiomyocyte contractile and intracellular Ca(2+) properties were evaluated including ventricular geometry, fractional shortening, peak shortening, maximal velocity of shortening/relengthening (±dL/dt), time-to-peak shortening (TPS), time-to-90% relengthening (TR(90)), fura-fluorescence intensity (FFI) and intracellular Ca(2+) clearance. Female C57 mice exhibited significantly higher plasma IGF-1 levels than their male counterpart. LID mice possessed comparably low IGF-1 levels in both sexes. Female C57 and LID mice displayed lower body, heart and liver weights compared to male counterparts. Echocardiographic analysis revealed larger LV mass in female C57 but not LID mice without sex difference in other cardiac geometric indices. Myocytes from female C57 mice exhibited reduced peak shortening, ±dL/dt, longer TPS, TR(90) and intracellular Ca(2+) clearance compared with males. Interestingly, this sex difference was greatly attenuated or abolished by IGF-1 deficiency. Female C57 mice displayed significantly decreased mRNA and protein levels of Na(+)-Ca(2+) exchanger, SERCA2a and phosphorylated phospholamban as well as SERCA activity compared with male C57 mice. These sex differences in Ca(2+) regulatory proteins were abolished or overtly attenuated by IGF-1 deficiency. In summary, our data suggested that IGF-1 deficiency may significantly attenuated or mitigate the sex difference in cardiomyocyte contractile function associated with intracellular Ca(2+) regulation. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

The coordinated increased expression of biliverdin reductase and heme oxygenase-2 promotes cardiomyocyte survival: a reductase-based peptide counters β-adrenergic receptor ligand-mediated cardiac dysfunction

PubMed Central

Ding, Bo; Gibbs, Peter E. M.; Brookes, Paul S.; Maines, Mahin D.

2011-01-01

HO-2 oxidizes heme to CO and biliverdin; the latter is reduced to bilirubin by biliverdin reductase (BVR). In addition, HO-2 is a redox-sensitive K/Ca2-associated protein, and BVR is an S/T/Y kinase. The two enzymes are components of cellular defense mechanisms. This is the first reporting of regulation of HO-2 by BVR and that their coordinated increase in isolated myocytes and intact heart protects against cardiotoxicity of β-adrenergic receptor activation by isoproterenol (ISO). The induction of BVR mRNA, protein, and activity and HO-2 protein was maintained for ≥96 h; increase in HO-1 was modest and transient. In isolated cardiomyocytes, experiments with cycloheximide, proteasome inhibitor MG-132, and siBVR suggested BVR-mediated stabilization of HO-2. In both models, activation of BVR offered protection against the ligand's stimulation of apoptosis. Two human BVR-based peptides known to inhibit and activate the reductase, KKRILHC281 and KYCCSRK296, respectively, were tested in the intact heart. Perfusion of the heart with the inhibitory peptide blocked ISO-mediated BVR activation and augmented apoptosis; conversely, perfusion with the activating peptide inhibited apoptosis. At the functional level, peptide-mediated inhibition of BVR was accompanied by dysfunction of the left ventricle and decrease in HO-2 protein levels. Perfusion of the organ with the activating peptide preserved the left ventricular contractile function and was accompanied by increased levels of HO-2 protein. Finding that BVR and HO-2 levels, myocyte apoptosis, and contractile function of the heart can be modulated by small human BVR-based peptides offers a promising therapeutic approach for treatment of cardiac dysfunctions.—Ding, B., Gibbs, P. E. M., Brookes, P. S., Maines, M. D. The coordinated increased expression of biliverdin reductase and heme oxygenase-2 promotes cardiomyocyte survival; a reductase-based peptide counters β-adrenergic receptor ligand-mediated cardiac dysfunction. PMID:20876213

A novel SOD mimic with a redox-modulating mn (II) complex, ML1 attenuates high glucose-induced abnormalities in intracellular Ca2+ transients and prevents cardiac cell death through restoration of mitochondrial function.

PubMed

Kain, Vasundhara; Sawant, Mithila A; Dasgupta, Aparajita; Jaiswal, Gaurav; Vyas, Alok; Padhye, Subhash; Sitasawad, Sandhya L

2016-03-01

A key contributor to the pathophysiology of diabetic cardiomyopathy, mitochondrial superoxide can be adequately countered by Mn-superoxide dismutase, which constitutes the first line of defense against mitochondrial oxidative stress. Our group has recently synthesized low molecular weight SOD mimics, demonstrating superior protection against oxidative damages to kidney cells. In the current study, we sought to evaluate the protective effect of the SOD mimic ML1 against high glucose induced cardiomyopathy in diabetes. Mechanistic studies using rat cardiac myoblast H9c2 showed that ML1 markedly inhibited High Glucose (HG) induced cytotoxicity. This was associated with increased Mn-SOD expression along with decreased mitochondrial [Formula: see text], ONOO- and Ca 2+ accumulation, unveiling its anti-oxidant potentials. ML1 also attenuated HG-induced loss of mitochondrial membrane potential (Δ Ψ m ) and release of cytochrome c, suggesting that ML1 effectuates its cytoprotective action via the preservation of mitochondrial function. In an ex-vivo model normal adult rat ventricular myocytes (ARVMs) were isolated and cultured in either normal glucose (5.5 mmol/l glucose) or HG (25.5 mmol/l glucose) conditions and the efficiency of ML-1 was analyzed by studying contractile function and calcium indices. Mechanical properties were assessed using a high-speed video-edge detection system, and intracellular Ca 2+ transients were recorded in fura-2-loaded myocytes. Pretreatment of myocytes with ML1 (10 nM) ameliorated HG induced abnormalities in relaxation including depressed peak shortening, prolonged time to 90% relenghthening, and slower Ca 2+ transient decay. Thus, ML1 exhibits significant cardio protection against oxidative damage, perhaps through its potent antioxidant action via activation of Mn-SOD.

Massive palmitoylation-dependent endocytosis during reoxygenation of anoxic cardiac muscle

PubMed Central

Lin, Mei-Jung; Fine, Michael; Lu, Jui-Yun; Hofmann, Sandra L; Frazier, Gary; Hilgemann, Donald W

2013-01-01

In fibroblasts, large Ca transients activate massive endocytosis (MEND) that involves membrane protein palmitoylation subsequent to mitochondrial permeability transition pore (PTP) openings. Here, we characterize this pathway in cardiac muscle. Myocytes with increased expression of the acyl transferase, DHHC5, have decreased Na/K pump activity. In DHHC5-deficient myocytes, Na/K pump activity and surface area/volume ratios are increased, the palmitoylated regulatory protein, phospholemman (PLM), and the cardiac Na/Ca exchanger (NCX1) show greater surface membrane localization, and MEND is inhibited in four protocols. Both electrical and optical methods demonstrate that PTP-dependent MEND occurs during reoxygenation of anoxic hearts. Post-anoxia MEND is ablated in DHHC5-deficient hearts, inhibited by cyclosporine A (CsA) and adenosine, promoted by staurosporine (STS), reduced in hearts lacking PLM, and correlates with impaired post-anoxia contractile function. Thus, the MEND pathway appears to be deleterious in severe oxidative stress but may constitutively contribute to cardiac sarcolemma turnover in dependence on metabolic stress. DOI: http://dx.doi.org/10.7554/eLife.01295.001 PMID:24282237

The lethal effects of cytokine-induced nitric oxide on cardiac myocytes are blocked by nitric oxide synthase antagonism or transforming growth factor beta.

PubMed Central

Pinsky, D J; Cai, B; Yang, X; Rodriguez, C; Sciacca, R R; Cannon, P J

1995-01-01

Inducible nitric oxide (NO) produced by macrophages is cytotoxic to invading organisms and has an important role in host defense. Recent studies have demonstrated inducible NO production within the heart, and that cytokine-induced NO mediates alterations in cardiac contractility, but the cytotoxic potential of nitric oxide with respect to the heart has not been defined. To evaluate the role of inducible nitric oxide synthase (iNOS) on cardiac myocyte cytotoxicity, we exposed adult rat cardiac myocytes to either cytokines alone or to activated J774 macrophages in coculture. Increased expression of both iNOS message and protein was seen in J774 macrophages treated with IFN gamma and LPS and cardiac myocytes treated with TNF-alpha, IL-1 beta, and IFN gamma. Increased NO synthesis was confirmed in both the coculture and isolated myocyte preparations by increased nitrite production. Increased NO synthesis was associated with a parallel increase in myocyte death as measured by CPK release into the culture medium as well as by loss of membrane integrity, visualized by trypan blue staining. Addition of the competitive NO synthase inhibitor L-NMMA to the culture medium prevented both the increased nitrite production and the cytotoxicity observed after cytokine treatment in both the isolated myocyte and the coculture experiments. Because transforming growth-factor beta modulates iNOS expression in other cell types, we evaluated its effects on cardiac myocyte iNOS expression and NO-mediated myocyte cytotoxicity. TGF-beta reduced expression of cardiac myocyte iNOS message and protein, reduced nitrite production, and reduced NO-mediated cytotoxicity in parallel. Taken together, these experiments show the cytotoxic potential of endogenous NO production within the heart, and suggest a role for TGF-beta or NO synthase antagonists to mute these lethal effects. These findings may help explain the cardiac response to sepsis or allograft rejection, as well as the progression of dilated cardiomyopathies of diverse etiologies. Images PMID:7532189

Loss of β-adrenergic-stimulated phosphorylation of CaV1.2 channels on Ser1700 leads to heart failure.

PubMed

Yang, Linghai; Dai, Dao-Fu; Yuan, Can; Westenbroek, Ruth E; Yu, Haijie; West, Nastassya; de la Iglesia, Horacio O; Catterall, William A

2016-12-06

L-type Ca 2+ currents conducted by voltage-gated calcium channel 1.2 (Ca V 1.2) initiate excitation-contraction coupling in the heart, and altered expression of Ca V 1.2 causes heart failure in mice. Here we show unexpectedly that reducing β-adrenergic regulation of Ca V 1.2 channels by mutation of a single PKA site, Ser1700, in the proximal C-terminal domain causes reduced contractile function, cardiac hypertrophy, and heart failure without changes in expression, localization, or function of the Ca V 1.2 protein in the mutant mice (SA mice). These deficits were aggravated with aging. Dual mutation of Ser1700 and a nearby casein-kinase II site (Thr1704) caused accelerated hypertrophy, heart failure, and death in mice with these mutations (STAA mice). Cardiac hypertrophy was increased by voluntary exercise and by persistent β-adrenergic stimulation. PKA expression was increased, and PKA sites Ser2808 in ryanodine receptor type-2, Ser16 in phospholamban, and Ser23/24 in troponin-I were hyperphosphorylated in SA mice, whereas phosphorylation of substrates for calcium/calmodulin-dependent protein kinase II was unchanged. The Ca 2+ pool in the sarcoplasmic reticulum was increased, the activity of calcineurin was elevated, and calcineurin inhibitors improved contractility and ameliorated cardiac hypertrophy. Cardio-specific expression of the SA mutation also caused reduced contractility and hypertrophy. These results suggest engagement of compensatory mechanisms, which initially may enhance the contractility of individual myocytes but eventually contribute to an increased sensitivity to cardiovascular stress and to heart failure in vivo. Our results demonstrate that normal regulation of Ca V 1.2 channels by phosphorylation of Ser1700 in cardiomyocytes is required for cardiovascular homeostasis and normal physiological regulation in vivo.

Protein kinase-dependent oxidative regulation of the cardiac Na+–K+ pump: evidence from in vivo and in vitro modulation of cell signalling

PubMed Central

Galougahi, Keyvan Karimi; Liu, Chia-Chi; Garcia, Alvaro; Fry, Natasha A S; Hamilton, Elisha J; Rasmussen, Helge H; Figtree, Gemma A

2013-01-01

The widely reported stimulation of the cardiac Na+–K+ pump by protein kinase A (PKA) should oppose other effects of PKA to increase contractility of the normal heart. It should also reduce harmful raised myocyte Na+ levels in heart failure, yet blockade of the β1 adrenergic receptor (AR), coupled to PKA signalling, is beneficial. We treated rabbits with the β1 AR antagonist metoprolol to modulate PKA activity and studied cardiac myocytes ex vivo. Metoprolol increased electrogenic pump current (Ip) in voltage clamped myocytes and reduced glutathionylation of the β1 pump subunit, an oxidative modification causally related to pump inhibition. Activation of adenylyl cyclase with forskolin to enhance cAMP synthesis or inclusion of the catalytic subunit of PKA in patch pipette solutions abolished the increase in Ip in voltage clamped myocytes induced by treatment with metoprolol, supporting cAMP/PKA-mediated pump inhibition. Metoprolol reduced myocardial PKA and protein kinase C (PKC) activities, reduced coimmunoprecipitation of cytosolic p47phox and membranous p22phox NADPH oxidase subunits and reduced myocardial O2•−-sensitive dihydroethidium fluorescence. Treatment also enhanced coimmunoprecipitation of the β1 pump subunit with glutaredoxin 1 that catalyses de-glutathionylation. Since angiotensin II induces PKC-dependent activation of NADPH oxidase, we examined the effects of angiotensin-converting enzyme inhibition with captopril. This treatment had no effect on PKA activity but reduced the activity of PKC, reduced β1 subunit glutathionylation and increased Ip. The PKA-induced Na+–K+ pump inhibition we report should act with other mechanisms that enhance contractility of the normal heart but accentuate the harmful effects of raised cytosolic Na+ in the failing heart. This scheme is consistent with the efficacy of β1 AR blockade in the treatment of heart failure. PMID:23587884

Mathematical modeling physiological effects of the overexpression of β2-adrenoceptors in mouse ventricular myocytes.

PubMed

Rozier, Kelvin; Bondarenko, Vladimir E

2018-03-01

Transgenic (TG) mice overexpressing β 2 -adrenergic receptors (β 2 -ARs) demonstrate enhanced myocardial function, which manifests in increased basal adenylyl cyclase activity, enhanced atrial contractility, and increased left ventricular function in vivo. To gain insights into the mechanisms of these effects, we developed a comprehensive mathematical model of the mouse ventricular myocyte overexpressing β 2 -ARs. We found that most of the β 2 -ARs are active in control conditions in TG mice. The simulations describe the dynamics of major signaling molecules in different subcellular compartments, increased basal adenylyl cyclase activity, modifications of action potential shape and duration, and the effects on L-type Ca 2+ current and intracellular Ca 2+ concentration ([Ca 2+ ] i ) transients upon stimulation of β 2 -ARs in control, after the application of pertussis toxin, upon stimulation with a specific β 2 -AR agonist zinterol, and upon stimulation with zinterol in the presence of pertussis toxin. The model also describes the effects of the β 2 -AR inverse agonist ICI-118,551 on adenylyl cyclase activity, action potential, and [Ca 2+ ] i transients. The simulation results were compared with experimental data obtained in ventricular myocytes from TG mice overexpressing β 2 -ARs and with simulation data on wild-type mice. In conclusion, a new comprehensive mathematical model was developed that describes multiple experimental data on TG mice overexpressing β 2 -ARs and can be used to test numerous hypotheses. As an example, using the developed model, we proved the hypothesis of the major contribution of L-type Ca 2+ current to the changes in the action potential and [Ca 2+ ] i transient upon stimulation of β 2 -ARs with zinterol. NEW & NOTEWORTHY We developed a new mathematical model for transgenic mouse ventricular myocytes overexpressing β 2 -adrenoceptors that describes the experimental findings in transgenic mice. The model reveals mechanisms of the differential effects of stimulation of β 2 -adrenoceptors in wild-type and transgenic mice overexpressing β 2 -adrenoceptors.

Functional assembly of engineered myocardium by electrical stimulation of cardiac myocytes cultured on scaffolds.

PubMed

Radisic, Milica; Park, Hyoungshin; Shing, Helen; Consi, Thomas; Schoen, Frederick J; Langer, Robert; Freed, Lisa E; Vunjak-Novakovic, Gordana

2004-12-28

The major challenge of tissue engineering is directing the cells to establish the physiological structure and function of the tissue being replaced across different hierarchical scales. To engineer myocardium, biophysical regulation of the cells needs to recapitulate multiple signals present in the native heart. We hypothesized that excitation-contraction coupling, critical for the development and function of a normal heart, determines the development and function of engineered myocardium. To induce synchronous contractions of cultured cardiac constructs, we applied electrical signals designed to mimic those in the native heart. Over only 8 days in vitro, electrical field stimulation induced cell alignment and coupling, increased the amplitude of synchronous construct contractions by a factor of 7, and resulted in a remarkable level of ultrastructural organization. Development of conductive and contractile properties of cardiac constructs was concurrent, with strong dependence on the initiation and duration of electrical stimulation.

MicroRNA-1 and -133 Increase Arrhythmogenesis in Heart Failure by Dissociating Phosphatase Activity from RyR2 Complex

PubMed Central

Belevych, Andriy E.; Sansom, Sarah E.; Terentyeva, Radmila; Ho, Hsiang-Ting; Nishijima, Yoshinori; Martin, Mickey M.; Jindal, Hitesh K.; Rochira, Jennifer A.; Kunitomo, Yukiko; Abdellatif, Maha; Carnes, Cynthia A.; Elton, Terry S.; Györke, Sandor; Terentyev, Dmitry

2011-01-01

In heart failure (HF), arrhythmogenic spontaneous sarcoplasmic reticulum (SR) Ca2+ release and afterdepolarizations in cardiac myocytes have been linked to abnormally high activity of ryanodine receptors (RyR2s) associated with enhanced phosphorylation of the channel. However, the specific molecular mechanisms underlying RyR2 hyperphosphorylation in HF remain poorly understood. The objective of the current study was to test the hypothesis that the enhanced expression of muscle-specific microRNAs (miRNAs) underlies the HF-related alterations in RyR2 phosphorylation in ventricular myocytes by targeting phosphatase activity localized to the RyR2. We studied hearts isolated from canines with chronic HF exhibiting increased left ventricular (LV) dimensions and decreased LV contractility. qRT-PCR revealed that the levels of miR-1 and miR-133, the most abundant muscle-specific miRNAs, were significantly increased in HF myocytes compared with controls (2- and 1.6-fold, respectively). Western blot analyses demonstrated that expression levels of the protein phosphatase 2A (PP2A) catalytic and regulatory subunits, which are putative targets of miR-133 and miR-1, were decreased in HF cells. PP2A catalytic subunit mRNAs were validated as targets of miR-133 by using luciferase reporter assays. Pharmacological inhibition of phosphatase activity increased the frequency of diastolic Ca2+ waves and afterdepolarizations in control myocytes. The decreased PP2A activity observed in HF was accompanied by enhanced Ca2+/calmodulin-dependent protein kinase (CaMKII)-mediated phosphorylation of RyR2 at sites Ser-2814 and Ser-2030 and increased frequency of diastolic Ca2+ waves and afterdepolarizations in HF myocytes compared with controls. In HF myocytes, CaMKII inhibitory peptide normalized the frequency of pro-arrhythmic spontaneous diastolic Ca2+ waves. These findings suggest that altered levels of major muscle-specific miRNAs contribute to abnormal RyR2 function in HF by depressing phosphatase activity localized to the channel, which in turn, leads to the excessive phosphorylation of RyR2s, abnormal Ca2+ cycling, and increased propensity to arrhythmogenesis. PMID:22163007

BAG3 regulates contractility and Ca(2+) homeostasis in adult mouse ventricular myocytes.

PubMed

Feldman, Arthur M; Gordon, Jennifer; Wang, JuFang; Song, Jianliang; Zhang, Xue-Qian; Myers, Valerie D; Tilley, Douglas G; Gao, Erhe; Hoffman, Nicholas E; Tomar, Dhanendra; Madesh, Muniswamy; Rabinowitz, Joseph; Koch, Walter J; Su, Feifei; Khalili, Kamel; Cheung, Joseph Y

2016-03-01

Bcl2-associated athanogene 3 (BAG3) is a 575 amino acid anti-apoptotic protein that is constitutively expressed in the heart. BAG3 mutations, including mutations leading to loss of protein, are associated with familial cardiomyopathy. Furthermore, BAG3 levels have been found to be reduced in end-stage non-familial failing myocardium. In contrast to neonatal myocytes in which BAG3 is found in the cytoplasm and involved in protein quality control and apoptosis, in adult mouse left ventricular (LV) myocytes BAG3 co-localized with Na(+)-K(+)-ATPase and L-type Ca(2+) channels in the sarcolemma and t-tubules. BAG3 co-immunoprecipitated with β1-adrenergic receptor, L-type Ca(2+) channels and phospholemman. To simulate decreased BAG3 protein levels observed in human heart failure, we targeted BAG3 by shRNA (shBAG3) in adult LV myocytes. Reducing BAG3 by 55% resulted in reduced contraction and [Ca(2+)]i transient amplitudes in LV myocytes stimulated with isoproterenol. L-type Ca(2+) current (ICa) and sarcoplasmic reticulum (SR) Ca(2+) content but not Na(+)/Ca(2+) exchange current (INaCa) or SR Ca(2+) uptake were reduced in isoproterenol-treated shBAG3 myocytes. Forskolin or dibutyryl cAMP restored ICa amplitude in shBAG3 myocytes to that observed in WT myocytes, consistent with BAG3 having effects upstream and at the level of the receptor. Resting membrane potential and action potential amplitude were unaffected but APD50 and APD90 were prolonged in shBAG3 myocytes. Protein levels of Ca(2+) entry molecules and other important excitation-contraction proteins were unchanged in myocytes with lower BAG3. Our findings that BAG3 is localized at the sarcolemma and t-tubules while modulating myocyte contraction and action potential duration through specific interaction with the β1-adrenergic receptor and L-type Ca(2+) channel provide novel insight into the role of BAG3 in cardiomyopathies and increased arrhythmia risks in heart failure. Copyright © 2016 Elsevier Ltd. All rights reserved.

BAG3 regulates contractility and Ca2+ homeostasis in adult mouse ventricular myocytes

PubMed Central

Feldman, Arthur M.; Gordon, Jennifer; Wang, JuFang; Song, Jianliang; Zhang, Xue-Qian; Myers, Valerie D.; Tilley, Douglas G.; Gao, Erhe; Hoffman, Nicholas E.; Tomar, Dhanendra; Madesh, Muniswamy; Rabinowitz, Joseph; Koch, Walter J.; Su, Feifei; Khalili, Kamel; Cheung, Joseph Y.

2016-01-01

Bcl2-associated athanogene 3 (BAG3) is a 575 amino acid anti-apoptotic protein that is constitutively expressed in the heart. BAG3 mutations, including mutations leading to loss of protein, are associated with familial cardiomyopathy. Furthermore, BAG3 levels have been found to be reduced in end-stage non-familial failing myocardium. In contrast to neonatal myocytes in which BAG3 is found in the cytoplasm and involved in protein quality control and apoptosis, in adult mouse left ventricular (LV) myocytes BAG3 co-localized with Na+-K+-ATPase and L-type Ca2+ channels in the sarcolemma and t-tubules. BAG3 co-immunoprecipitated with β1-adrenergic receptor, L-type Ca2+ channels and phospholemman. To simulate decreased BAG3 protein levels observed in human heart failure, we targeted BAG3 by shRNA (shBAG3) in adult LV myocytes. Reducing BAG3 by 55% resulted in reduced contraction and [Ca2+]i transient amplitudes in LV myocytes stimulated with isoproterenol. L-type Ca2+ current (ICa) and sarcoplasmic reticulum (SR) Ca2+ content but not Na+/Ca2+ exchange current (INaCa) or SR Ca2+ uptake were reduced in isoproterenol-treated shBAG3 myocytes. Forskolin or dibutyrl cAMP restored ICa amplitude in shBAG3 myocytes to that observed in WT myocytes, consistent with BAG3 having effects upstream and at the level of the receptor. Resting membrane potential and action potential amplitude were unaffected but APD50 and APD90 were prolonged in shBAG3 myocytes. Protein levels of Ca2+ entry molecules and other important excitation-contraction proteins were unchanged in myocytes with lower BAG3. Our findings that BAG3 is localized at the sarcolemma and t-tubules while modulating myocyte contraction and action potential duration through specific interaction with the β1-adrenergic receptor and L-type Ca2+ channel provide novel insight into the role of BAG3 in cardiomyopathies and increased arrhythmia risks in heart failure. PMID:26796036

Protective effect of hydroalcoholic extract of Andrographis paniculata on ischaemia-reperfusion induced myocardial injury in rats.

PubMed

Ojha, Shreesh Kumar; Bharti, Saurabh; Joshi, Sujata; Kumari, Santosh; Arya, Dharamvir Singh

2012-03-01

Protecting myocardium from ischaemia-reperfusion (I-R) injury is important to reduce the complication of myocardial infarction (MI) and interventional revascularization procedures. In the present study, the cardioprotective potential of hydroalcoholic extract of Andrographis paniculata was evaluated against left anterior descending coronary artery (LADCA) ligation-induced I-R injury of myocardium in rats. MI was induced in rats by LADCA ligation for 45 min followed by reperfusion for 60 min. The rats were divided into five experimental groups viz., sham (saline treated, but LADCA was not ligated), I-R control (saline treated + I-R), benazepril (30 mg/kg + I-R), A. paniculata (200 mg/kg per se) and A. paniculata (200 mg/kg + I-R). A. paniculata was administered orally for 31 days. On day 31, rats were subjected to the I-R and cardiac function parameters were recorded. Further, rats were sacrificed and heart was excised for biochemical and histopathological studies. In I-R control group, LADCA ligation resulted in significant cardiac dysfunction evidenced by reduced haemodynamic parameters; mean arterial pressure (MAP) and heart rate (HR). The left ventricular contractile function was also altered. In I-R control group, I-R caused decline in superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and reduced glutathione (GSH) as well as leakage of myocytes injury marker enzymes, creatine phosphokinase-MB (CK-MB) isoenzyme and lactate dehydrogenase (LDH), and enhanced lipid peroxidation product, malonaldialdehyde (MDA). However, rats pretreated with A. paniculata 200 mg/kg showed favourable modulation of haemodynamic and left ventricular contractile function parameters, restoration of the myocardial antioxidants and prevention of depletion of myocytes injury marker enzymes along with inhibition of lipid peroxidation. Histopathological observations confirmed the protective effects of A. paniculata. The cardioprotective effects of A. paniculata were found comparable to that of benazepril treatment. Our results showed the cardioprotective effects of A. paniculata against I-R injury likely result from the suppression of oxidative stress and preserved histoarchitecture of myofibrils along with improved haemodynamic and ventricular functions.

Protective effect of hydroalcoholic extract of Andrographis paniculata on ischaemia-reperfusion induced myocardial injury in rats

PubMed Central

Ojha, Shreesh Kumar; Bharti, Saurabh; Joshi, Sujata; Kumari, Santosh; Arya, Dharamvir Singh

2012-01-01

Background & objectives: Protecting myocardium from ischaemia-reperfusion (I-R) injury is important to reduce the complication of myocardial infarction (MI) and interventional revascularization procedures. In the present study, the cardioprotective potential of hydroalcoholic extract of Andrographis paniculata was evaluated against left anterior descending coronary artery (LADCA) ligation-induced I-R injury of myocardium in rats. Methods: MI was induced in rats by LADCA ligation for 45 min followed by reperfusion for 60 min. The rats were divided into five experimental groups viz., sham (saline treated, but LADCA was not ligated), I-R control (saline treated + I-R), benazepril (30 mg/kg + I-R), A. paniculata (200 mg/kg per se) and A. paniculata (200 mg/kg + I-R). A. paniculata was administered orally for 31 days. On day 31, rats were subjected to the I-R and cardiac function parameters were recorded. Further, rats were sacrificed and heart was excised for biochemical and histopathological studies. Results: In I-R control group, LADCA ligation resulted in significant cardiac dysfunction evidenced by reduced haemodynamic parameters; mean arterial pressure (MAP) and heart rate (HR). The left ventricular contractile function was also altered. In I-R control group, I-R caused decline in superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and reduced glutathione (GSH) as well as leakage of myocytes injury marker enzymes, creatine phosphokinase-MB (CK-MB) isoenzyme and lactate dehydrogenase (LDH), and enhanced lipid peroxidation product, malonaldialdehyde (MDA). However, rats pretreated with A. paniculata 200 mg/kg showed favourable modulation of haemodynamic and left ventricular contractile function parameters, restoration of the myocardial antioxidants and prevention of depletion of myocytes injury marker enzymes along with inhibition of lipid peroxidation. Histopathological observations confirmed the protective effects of A. paniculata. The cardioprotective effects of A. paniculata were found comparable to that of benazepril treatment. Interpretation & Conclusions: Our results showed the cardioprotective effects of A. paniculata against I-R injury likely result from the suppression of oxidative stress and preserved histoarchitecture of myofibrils along with improved haemodynamic and ventricular functions. PMID:22561631

Effects of phloretin and phloridzin on Ca2+ handling, the action potential, and ion currents in rat ventricular myocytes.

PubMed

Olson, Marnie L; Kargacin, Margaret E; Ward, Christopher A; Kargacin, Gary J

2007-06-01

The effects of the phytoestrogens phloretin and phloridzin on Ca(2+) handling, cell shortening, the action potential, and Ca(2+) and K(+) currents in freshly isolated cardiac myocytes from rat ventricle were examined. Phloretin increased the amplitude and area and decreased the rate of decline of electrically evoked Ca(2+) transients in the myocytes. These effects were accompanied by an increase in the Ca(2+) load of the sarcoplasmic reticulum, as determined by the area of caffeine-evoked Ca(2+) transients. An increase in the extent of shortening of the myocytes in response to electrically evoked action potentials was also observed in the presence of phloretin. To further examine possible mechanisms contributing to the observed changes in Ca(2+) handling and contractility, the effects of phloretin on the cardiac action potential and plasma membrane Ca(2+) and K(+) currents were examined. Phloretin markedly increased the action potential duration in the myocytes, and it inhibited the Ca(2+)-independent transient outward K(+) current (I(to)). The inwardly rectifying K(+) current, the sustained outward delayed rectifier K(+) current, and L-type Ca(2+) currents were not significantly different in the presence and absence of phloretin, nor was there any evidence that the Na(+)/Ca(2+) exchanger was affected. The effects of phloretin on Ca(2+) handling in the myocytes are consistent with its effects on I(to). Phloridzin did not significantly alter the amplitude or area of electrically evoked Ca(2+) transients in the myocytes, nor did it have detectable effects on the sarcoplasmic reticulum Ca(2+) load, cell shortening, or the action potential.

Autocrine A2 in the T-System of Ventricular Myocytes Creates Transmural Gradients in Ion Transport: A Mechanism to Match Contraction with Load?

PubMed Central

Gao, Junyuan; Sun, Xiurong; Potapova, Irina A.; Cohen, Ira S.; Mathias, Richard T.; Kim, Jeremy H.

2014-01-01

Transmural heterogeneities in Na/K pump current (IP), transient outward K+-current (Ito), and Ca2+-current (ICaL) play an important role in regulating electrical and contractile activities in the ventricular myocardium. Prior studies indicated angiotensin II (A2) may determine the transmural gradient in Ito, but the effects of A2 on IP and ICaL were unknown. In this study, myocytes were isolated from five muscle layers between epicardium and endocardium. We found a monotonic gradient in both Ip and Ito, with the lowest currents in ENDO. When AT1Rs were inhibited, EPI currents were unaffected, but ENDO currents increased, suggesting endogenous extracellular A2 inhibits both currents in ENDO. IP- and Ito-inhibition by A2 yielded essentially the same K0.5 values, so they may both be regulated by the same mechanism. A2/AT1R-mediated inhibition of IP or Ito or stimulation of ICaL persisted for hours in isolated myocytes, suggesting continuous autocrine secretion of A2 into a restricted diffusion compartment, like the T-system. Detubulation brought EPI IP to its low ENDO value and eliminated A2 sensitivity, so the T-system lumen may indeed be the restricted diffusion compartment. These studies showed that 33–50% of IP, 57–65% of Ito, and a significant fraction of ICaL reside in T-tubule membranes where they are transmurally regulated by autocrine secretion of A2 into the T-system lumen and activation of AT1Rs. Increased AT1R activation regulates each of these currents in a direction expected to increase contractility. Endogenous A2 activation of AT1Rs increases monotonically from EPI to ENDO in a manner similar to reported increases in passive tension when the ventricular chamber fills with blood. We therefore hypothesize load is the signal that regulates A2-activation of AT1Rs, which create a contractile gradient that matches the gradient in load. PMID:24896115

Cardiac Alpha1-Adrenergic Receptors: Novel Aspects of Expression, Signaling Mechanisms, Physiologic Function, and Clinical Importance

PubMed Central

O’Connell, Timothy D.; Jensen, Brian C.; Baker, Anthony J.

2014-01-01

Adrenergic receptors (AR) are G-protein-coupled receptors (GPCRs) that have a crucial role in cardiac physiology in health and disease. Alpha1-ARs signal through Gαq, and signaling through Gq, for example, by endothelin and angiotensin receptors, is thought to be detrimental to the heart. In contrast, cardiac alpha1-ARs mediate important protective and adaptive functions in the heart, although alpha1-ARs are only a minor fraction of total cardiac ARs. Cardiac alpha1-ARs activate pleiotropic downstream signaling to prevent pathologic remodeling in heart failure. Mechanisms defined in animal and cell models include activation of adaptive hypertrophy, prevention of cardiac myocyte death, augmentation of contractility, and induction of ischemic preconditioning. Surprisingly, at the molecular level, alpha1-ARs localize to and signal at the nucleus in cardiac myocytes, and, unlike most GPCRs, activate “inside-out” signaling to cause cardioprotection. Contrary to past opinion, human cardiac alpha1-AR expression is similar to that in the mouse, where alpha1-AR effects are seen most convincingly in knockout models. Human clinical studies show that alpha1-blockade worsens heart failure in hypertension and does not improve outcomes in heart failure, implying a cardioprotective role for human alpha1-ARs. In summary, these findings identify novel functional and mechanistic aspects of cardiac alpha1-AR function and suggest that activation of cardiac alpha1-AR might be a viable therapeutic strategy in heart failure. PMID:24368739

Heterogeneity of transverse-axial tubule system in mouse atria: Remodeling in atrial-specific Na+-Ca2+ exchanger knockout mice.

PubMed

Yue, Xin; Zhang, Rui; Kim, Brian; Ma, Aiqun; Philipson, Kenneth D; Goldhaber, Joshua I

2017-07-01

Transverse-axial tubules (TATs) are commonly assumed to be sparse or absent in atrial myocytes from small animals. Atrial myocytes from rats, cats and rabbits lack TATs, which results in a characteristic "V"-shaped Ca release pattern in confocal line-scan recordings due to the delayed rise of Ca in the center of the cell. To examine TAT expression in isolated mouse atrial myocytes, we loaded them with the membrane dye Di-4-ANEPPS to label TATs. We found that >80% of atrial myocytes had identifiable TATs. Atria from male mice had a higher TAT density than female mice, and TAT density correlated with cell width. Using the fluorescent Ca indicator Fluo-4-AM and confocal imaging, we found that wild type (WT) mouse atrial myocytes generate near-synchronous Ca transients, in contrast to the "V"-shaped pattern typically reported in other small animals such as rat. In atrial-specific Na-Ca exchanger (NCX) knockout (KO) mice, which develop sinus node dysfunction and atrial hypertrophy with dilation, we found a substantial loss of atrial TATs in isolated atrial myocytes. There was a greater loss of transverse tubules compared to axial tubules, resulting in a dominance of axial tubules. Consistent with the overall loss of TATs, NCX KO atrial myocytes displayed a "V"-shaped Ca transient with slower and reduced central (CT) Ca release and uptake in comparison to subsarcolemmal (SS) Ca release. We compared chemically detubulated (DT) WT cells to KO, and found similar slowing of CT Ca release and uptake. However, SS Ca transients in the WT DT cells had faster uptake kinetics than KO cells, consistent with the presence of NCX and normal sarcolemmal Ca efflux in the WT DT cells. We conclude that the remodeling of NCX KO atrial myocytes is accompanied by a loss of TATs leading to abnormal Ca release and uptake that could impact atrial contractility and rhythm. Copyright © 2017 Elsevier Ltd. All rights reserved.

Establishing the framework to support bioartificial heart fabrication using fibrin-based three-dimensional artificial heart muscle.

PubMed

Hogan, Matthew; Mohamed, Mohamed; Tao, Ze-Wei; Gutierrez, Laura; Birla, Ravi

2015-02-01

Only 3000 heart transplants are performed in the USA every year, leaving some 30 000-70 000 Americans without proper care. Current treatment modalities for heart failure have saved many lives yet still do not correct the underlying problems of congestive heart failure. Tissue engineering represents a potential field of study wherein a combination of cells, scaffolds, and/or bioreactors can be utilized to create constructs to mimic, replace, and/or repair defective tissue. The focus of this study was to generate a bioartificial heart (BAH) model using artificial heart muscle (AHM), composed of fibrin gel and neonatal rat cardiac myocytes, and a decellularized scaffold, formed by subjecting an adult rat heart to a series of decellularization solutions. By suturing the AHM around the outside of the decellularized heart and culturing while suspended in media, we were able to retain functional cardiac cells on the scaffold as evinced by visible contractility. Observed contractility rate was correlated with biopotential measurements to confirm essential functionality of cardiac constructs. Cross-sections of the BAH show successful decellularization of the scaffold and contiguous cell-rich AHM around the perimeter of the heart. Copyright © 2014 International Center for Artificial Organs and Transplantation and Wiley Periodicals, Inc.

Cav1.2 channel current block by the PKA inhibitor H-89 in rat tail artery myocytes via a PKA-independent mechanism: Electrophysiological, functional, and molecular docking studies.

PubMed

Fusi, Fabio; Trezza, Alfonso; Spiga, Ottavia; Sgaragli, Giampietro; Bova, Sergio

2017-09-15

To characterize the role of cAMP-dependent protein kinase (PKA) in regulating vascular Ca 2+ current through Ca v 1.2 channels [I Ca1.2 ], we have documented a marked capacity of the isoquinoline H-89, widely used as a PKA inhibitor, to reduce current amplitude. We hypothesized that the I Ca1.2 inhibitory activity of H-89 was mediated by mechanisms unrelated to PKA inhibition. To support this, an in-depth analysis of H-89 vascular effects on both I Ca1.2 and contractility was undertaken by performing whole-cell patch-clamp recordings and functional experiments in rat tail main artery single myocytes and rings, respectively. H-89 inhibited I Ca1.2 with a pIC 50 (M) value of about 5.5, even under conditions where PKA activity was either abolished by both the PKA antagonists KT5720 and protein kinase inhibitor fragment 6-22 amide or enhanced by the PKA stimulators 6-Bnz-cAMP and 8-Br-cAMP. Inhibition of I Ca1.2 by H-89 appeared almost irreversible upon washout, was charge carrier- and voltage-dependent, and antagonised by the Ca v 1.2 channel agonist (S)-(-)-Bay K 8644. H-89 did not alter both potency and efficacy of verapamil, did not affect current kinetics or voltage-dependent activation, while shifting to the left the 50% voltage of inactivation in a concentration-dependent manner. H-89 docked at the α 1C subunit in a pocket region close to that of (S)-(-)-Bay K 8644 docking, forming a hydrogen bond with the same, key amino acid residue Tyr-1489. Finally, both high K + - and (S)-(-)-Bay K 8644-induced contractions of rings were fully reverted by H-89. In conclusion, these results indicate that H-89 inhibited vascular I Ca1.2 and, consequently, the contractile function through a PKA-independent mechanism. Therefore, caution is recommended when interpreting experiments where H-89 is used to inhibit vascular smooth muscle PKA. Copyright © 2017 Elsevier Inc. All rights reserved.

Ranolazine improves abnormal repolarization and contraction in left ventricular myocytes of dogs with heart failure by inhibiting late sodium current

PubMed Central

Undrovinas, Albertas I.; Belardinelli, Luiz; Undrovinas, Nidas A.; Sabbah, Hani N.

2005-01-01

Background Ventricular repolarization and contractile function are frequently abnormal in ventricular myocytes from human failing hearts as well as canine hearts with experimentally induced heart failure (HF). These abnormalities have been attributed to dysfunction involving various steps of the excitation-contraction coupling process, leading to impaired intracellular sodium and calcium homeostasis. We previously reported that the slow inactivating component of the Na+ current (late INa) is augmented in myocytes from failing hearts, and this appears to play a significant role in abnormal ventricular myocytes repolarization and function. We tested the effect of ranolazine, a novel drug being developed to treat angina, on 1) action potential duration (APD), 2) peak transient and late INa (INaT and INaL respectively), 3) early afterdepolarizations (EADs), and 4) twitch contraction (TC) including aftercontractions and contracture. Methods: Myocytes were isolated from the left ventricle of normal dogs and of dogs with chronic HF caused by multiple sequential intracoronary microembolizations. INaT and INaL were recorded using conventional whole-cell patch-clamp techniques. APs were recorded using the β-escin perforated patch-clamp configuration at frequencies of 0.25 and 0.5 Hz. TCs were recorded using an edge movement detector at stimulation frequencies ranging from 0.5 to 2.0 Hz. Results Ranolazine significantly (p < 0.05) and reversibly shortened the APD of myocytes stimulated at either 0.5 or 0.25 Hz in a concentration-dependent manner. At a stimulation frequency of 0.5 Hz, 5, 10 and 20 μM ranolazine shortened the APD90 (APD measured at 90% repolarization) from 516 ± 51 to 304 ± 22, 212 ± 34 and 160 ± 11 ms, respectively, and markedly decreased beat-to-beat variability of APD90, EADs and dispersion of APDs. Ranolazine preferentially blocked INaL relative to INaT in a state-dependent manner; with a ~ 38-fold greater potency against INaL to produce tonic block (IC50 = 6.5 μ M) than INaT (IC50 =294 μM). When we evaluated inactivated state blockade of INaL from the steady-state inactivation mid-potential shift using a theoretical model, ranolazine was found to bind more tightly to the inactivated state than the resting state of the sodium channel underlying INaL, with apparent dissociation constants Kdr=7.47μ M and Kdi=1.71μ M, respectively. TCs of myocytes stimulated at 0.5 Hz were characterized by an initial spike followed by a dome-like aftercontraction, which was observed in75% of myocytes from failing hearts and coincided with the long AP plateau and EADs. Ranolazine at 5, and 10 μM reversibly shortened duration of TCs and abolished the aftercontraction. When the rate of myocyte stimulation was increased from 1.0 to 2.0 Hz, there was a progressive increase in diastolic “tension”, i.e., contracture. Ranolazine at 5, and 10 μM reversibly prevented this frequency-dependent contracture. PMID:16686675

Parturition dysfunction in obesity: time to target the pathobiology.

PubMed

Carlson, Nicole S; Hernandez, Teri L; Hurt, K Joseph

2015-12-18

Over a third of women of childbearing age in the United States are obese, and during pregnancy they are at increased risk for delayed labor onset and slow labor progress that often results in unplanned cesarean delivery. The biology behind this dysfunctional parturition is not well understood. Studies of obesity-induced changes in parturition physiology may facilitate approaches to optimize labor in obese women. In this review, we summarize known and proposed biologic effects of obesity on labor preparation, contraction/synchronization, and endurance, drawing on both clinical observation and experimental data. We present evidence from human and animal studies of interactions between obesity and parturition signaling in all elements of the birth process, including: delayed cervical ripening, prostaglandin insensitivity, amniotic membrane strengthening, decreased myometrial oxytocin receptor expression, decreased myocyte action potential initiation and contractility, decreased myocyte gap junction formation, and impaired myocyte neutralization of reactive oxygen species. We found convincing clinical data on the effect of obesity on labor initiation and successful delivery, but few studies on the underlying pathobiology. We suggest research opportunities and therapeutic interventions based on plausible biologic mechanisms.

Effect of a hawthorn extract on contraction and energy turnover of isolated rat cardiomyocytes.

PubMed

Pöpping, S; Rose, H; Ionescu, I; Fischer, Y; Kammermeier, H

1995-11-01

The hawthorn extract LI 132 (crataegus), prepared from leaves and flowers, and standardised to 2.2% flavonoids, was investigated with respect to its effect on (1) the contraction, (2) the energy-turnover and (3) the apparent refractory period (t(ref)) of isolated cardiac myocytes from adult rats. (1) The contractile behaviour of attached myocytes was analyzed by an image processing system. (2) The energy turnover was calculated from the decrease in oxygen content in the myocyte suspension, brought about by cellular respiration. It was differentiated between energy turnover related to cell shortening and that required for ionic transport processes by application of the contraction-inhibiting agent 2,3-butanedione monoxime. (3) The apparent refractory period (t(ref)) was evaluated by pacing the myocytes with increasing stimulation rates and determining the frequency at which failure of single contractions occurred. For these purposes, the myocytes were incubated in a stimulation chamber, which is part of a computer-assisted system allowing to simultaneously evaluate the mechanics and energetics of electrically induced contraction. Within a range of 30-180 microg/ml, the hawthorn extract exhibited a positive inotropic effect on the contraction amplitude accompanied by a moderate increase of energy turnover both for mechanical and ionic processes. In comparison with other positive inotropic interventions, such as application of the beta-adrenergic agonist isoprenaline, or of the cardiac glycoside ouabain (g-strophantin), or elevation of the extracellular Ca++-concentration, the effects of the hawthorn extract were significantly more economical with respect to the energetics of the myocytes. Furthermore the extract prolonged the apparent refractory period in the presence and the absence of isoprenaline, which be indicative for an antiarrhythmic potential.

Polygons and adhesion plaques and the disassembly and assembly of myofibrils in cardiac myocytes.

PubMed

Lin, Z X; Holtzer, S; Schultheiss, T; Murray, J; Masaki, T; Fischman, D A; Holtzer, H

1989-06-01

Successive stages in the disassembly of myofibrils and the subsequent assembly of new myofibrils have been studied in cultures of dissociated chick cardiac myocytes. The myofibrils in trypsinized and dispersed myocytes are sequentially disassembled during the first 3 d of culture. They split longitudinally and then assemble into transitory polygons. Multiples of single sarcomeres, the cardiac polygons, are analogous to the transitory polygonal configurations assumed by stress fibers in spreading fibroblasts. They differ from their counterparts in fibroblasts in that they consist of muscle alpha-actinin vertices and muscle myosin heavy chain struts, rather than of the nonmuscle contractile protein isoforms of stress fiber polygons. EM sections reveal the vertices and struts in cardiac polygons to be typical Z and A bands. Most cardiac polygons are eliminated by day 5 of culture. Concurrent with the disassembly and elimination of the original myofibrils new myofibrils are rapidly assembled elsewhere in the same myocyte. Without exception both distal tips of each nascent myofibril terminate in adhesion plaques. The morphology and composition of the adhesion plaques capping each end of each myofibril are similar to those of the termini of stress fibers in fibroblasts. However, whereas the adhesion complexes involving stress fibers in fibroblasts consist of vinculin/nonmuscle alpha-actinin/beta- and gamma-actins, the analogous structures in myocytes involving myofibrils consist of vinculin/muscle alpha-actinin/alpha-actin. The addition of 1.7-2.0 microns sarcomeres to the distal tips of an elongating myofibril, irrespective of whether the myofibril consists of 1, 10, or several hundred tandem sarcomeres, occurs while the myofibril appears to remain linked to its respective adhesion plaques. The adhesion plaques in vitro are the equivalent of the in vivo intercalated discs, both in terms of their molecular composition and with respect to their functioning as initiating sites for the assembly of new sarcomeres. How 1.7-2.0 microns nascent sarcomeres can be added distally during elongation while the tips of the myofibrils remain inserted into submembranous adhesion plaques is unknown.

Polygons and adhesion plaques and the disassembly and assembly of myofibrils in cardiac myocytes

PubMed Central

1989-01-01

Successive stages in the disassembly of myofibrils and the subsequent assembly of new myofibrils have been studied in cultures of dissociated chick cardiac myocytes. The myofibrils in trypsinized and dispersed myocytes are sequentially disassembled during the first 3 d of culture. They split longitudinally and then assemble into transitory polygons. Multiples of single sarcomeres, the cardiac polygons, are analogous to the transitory polygonal configurations assumed by stress fibers in spreading fibroblasts. They differ from their counterparts in fibroblasts in that they consist of muscle alpha-actinin vertices and muscle myosin heavy chain struts, rather than of the nonmuscle contractile protein isoforms of stress fiber polygons. EM sections reveal the vertices and struts in cardiac polygons to be typical Z and A bands. Most cardiac polygons are eliminated by day 5 of culture. Concurrent with the disassembly and elimination of the original myofibrils new myofibrils are rapidly assembled elsewhere in the same myocyte. Without exception both distal tips of each nascent myofibril terminate in adhesion plaques. The morphology and composition of the adhesion plaques capping each end of each myofibril are similar to those of the termini of stress fibers in fibroblasts. However, whereas the adhesion complexes involving stress fibers in fibroblasts consist of vinculin/nonmuscle alpha-actinin/beta- and gamma-actins, the analogous structures in myocytes involving myofibrils consist of vinculin/muscle alpha-actinin/alpha-actin. The addition of 1.7-2.0 microns sarcomeres to the distal tips of an elongating myofibril, irrespective of whether the myofibril consists of 1, 10, or several hundred tandem sarcomeres, occurs while the myofibril appears to remain linked to its respective adhesion plaques. The adhesion plaques in vitro are the equivalent of the in vivo intercalated discs, both in terms of their molecular composition and with respect to their functioning as initiating sites for the assembly of new sarcomeres. How 1.7-2.0 microns nascent sarcomeres can be added distally during elongation while the tips of the myofibrils remain inserted into submembranous adhesion plaques is unknown. PMID:2472405

Small interfering RNA targeting focal adhesion kinase prevents cardiac dysfunction in endotoxemia.

PubMed

Guido, Maria C; Clemente, Carolina F; Moretti, Ana I; Barbeiro, Hermes V; Debbas, Victor; Caldini, Elia G; Franchini, Kleber G; Soriano, Francisco G

2012-01-01

Sepsis and septic shock are associated with cardiac depression. Cardiovascular instability is a major cause of death in patients with sepsis. Focal adhesion kinase (FAK) is a potential mediator of cardiomyocyte responses to oxidative and mechanical stress. Myocardial collagen deposition can affect cardiac compliance and contractility. The aim of the present study was to determine whether the silencing of FAK is protective against endotoxemia-induced alterations of cardiac structure and function. In male Wistar rats, endotoxemia was induced by intraperitoneal injection of lipopolysaccharide (10 mg/kg). Cardiac morphometry and function were studied in vivo by left ventricular catheterization and histology. Intravenous injection of small interfering RNA targeting FAK was used to silence myocardial expression of the kinase. The hearts of lipopolysaccharide-injected rats showed collagen deposition, increased matrix metalloproteinase 2 activity, and myocyte hypertrophy, as well as reduced 24-h +dP/dt and -dP/dt, together with hypotension, increased left ventricular end-diastolic pressure, and elevated levels of FAK (phosphorylated and unphosphorylated). Focal adhesion kinase silencing reduced the expression and activation of the kinase in cardiac tissue, as well as protecting against the increased collagen deposition, greater matrix metalloproteinase 2 activity, and reduced cardiac contractility that occur during endotoxemia. In conclusion, FAK is activated in endotoxemia, playing a role in cardiac remodeling and in the impairment of cardiac function. This kinase represents a potential therapeutic target for the protection of cardiac function in patients with sepsis.

Dedifferentiation, Proliferation, and Redifferentiation of Adult Mammalian Cardiomyocytes After Ischemic Injury.

PubMed

Wang, Wei Eric; Li, Liangpeng; Xia, Xuewei; Fu, Wenbin; Liao, Qiao; Lan, Cong; Yang, Dezhong; Chen, Hongmei; Yue, Rongchuan; Zeng, Cindy; Zhou, Lin; Zhou, Bin; Duan, Dayue Darrel; Chen, Xiongwen; Houser, Steven R; Zeng, Chunyu

2017-08-29

Adult mammalian hearts have a limited ability to generate new cardiomyocytes. Proliferation of existing adult cardiomyocytes (ACMs) is a potential source of new cardiomyocytes. Understanding the fundamental biology of ACM proliferation could be of great clinical significance for treating myocardial infarction (MI). We aim to understand the process and regulation of ACM proliferation and its role in new cardiomyocyte formation of post-MI mouse hearts. β-Actin-green fluorescent protein transgenic mice and fate-mapping Myh6-MerCreMer-tdTomato/lacZ mice were used to trace the fate of ACMs. In a coculture system with neonatal rat ventricular myocytes, ACM proliferation was documented with clear evidence of cytokinesis observed with time-lapse imaging. Cardiomyocyte proliferation in the adult mouse post-MI heart was detected by cell cycle markers and 5-ethynyl-2-deoxyuridine incorporation analysis. Echocardiography was used to measure cardiac function, and histology was performed to determine infarction size. In vitro, mononucleated and bi/multinucleated ACMs were able to proliferate at a similar rate (7.0%) in the coculture. Dedifferentiation proceeded ACM proliferation, which was followed by redifferentiation. Redifferentiation was essential to endow the daughter cells with cardiomyocyte contractile function. Intercellular propagation of Ca 2+ from contracting neonatal rat ventricular myocytes into ACM daughter cells was required to activate the Ca 2+ -dependent calcineurin-nuclear factor of activated T-cell signaling pathway to induce ACM redifferentiation. The properties of neonatal rat ventricular myocyte Ca 2+ transients influenced the rate of ACM redifferentiation. Hypoxia impaired the function of gap junctions by dephosphorylating its component protein connexin 43, the major mediator of intercellular Ca 2+ propagation between cardiomyocytes, thereby impairing ACM redifferentiation. In vivo, ACM proliferation was found primarily in the MI border zone. An ischemia-resistant connexin 43 mutant enhanced the redifferentiation of ACM-derived new cardiomyocytes after MI and improved cardiac function. Mature ACMs can reenter the cell cycle and form new cardiomyocytes through a 3-step process: dedifferentiation, proliferation, and redifferentiation. Intercellular Ca 2+ signal from neighboring functioning cardiomyocytes through gap junctions induces the redifferentiation process. This novel mechanism contributes to new cardiomyocyte formation in post-MI hearts in mammals. © 2017 American Heart Association, Inc.

Gene expression changes controlling distinct adaptations in the heart and skeletal muscle of a hibernating mammal

PubMed Central

Vermillion, Katie L.; Anderson, Kyle J.; Hampton, Marshall

2015-01-01

Throughout the hibernation season, the thirteen-lined ground squirrel (Ictidomys tridecemlineatus) experiences extreme fluctuations in heart rate, metabolism, oxygen consumption, and body temperature, along with prolonged fasting and immobility. These conditions necessitate different functional requirements for the heart, which maintains contractile function throughout hibernation, and the skeletal muscle, which remains largely inactive. The adaptations used to maintain these contractile organs under such variable conditions serves as a natural model to study a variety of medically relevant conditions including heart failure and disuse atrophy. To better understand how two different muscle tissues maintain function throughout the extreme fluctuations of hibernation we performed Illumina HiSeq 2000 sequencing of cDNAs to compare the transcriptome of heart and skeletal muscle across the circannual cycle. This analysis resulted in the identification of 1,076 and 1,466 differentially expressed genes in heart and skeletal muscle, respectively. In both heart and skeletal muscle we identified a distinct cold-tolerant mechanism utilizing peroxisomal metabolism to make use of elevated levels of unsaturated depot fats. The skeletal muscle transcriptome also shows an early increase in oxidative capacity necessary for the altered fuel utilization and increased oxygen demand of shivering. Expression of the fetal gene expression profile is used to maintain cardiac tissue, either through increasing myocyte size or proliferation of resident cardiomyocytes, while skeletal muscle function and mass are protected through transcriptional regulation of pathways involved in protein turnover. This study provides insight into how two functionally distinct muscles maintain function under the extreme conditions of mammalian hibernation. PMID:25572546

Impaired Ca2+ cycling of nonischemic myocytes contributes to sarcomere dysfunction early after myocardial infarction.

PubMed

Kronenbitter, Annette; Funk, Florian; Hackert, Katarzyna; Gorreßen, Simone; Glaser, Dennis; Boknik, Peter; Poschmann, Gereon; Stühler, Kai; Isić, Malgorzata; Krüger, Martina; Schmitt, Joachim P

2018-06-01

Changes in the nonischemic remote myocardium of the heart contribute to left ventricular dysfunction after ischemia and reperfusion (I/R). Understanding the underlying mechanisms early after I/R is crucial to improve the adaptation of the viable myocardium to increased mechanical demands. Here, we investigated the role of myocyte Ca 2+ handling in the remote myocardium 24 h after 60 min LAD occlusion. Cardiomyocytes isolated from the basal noninfarct-related parts of wild type mouse hearts demonstrated depressed beat-to-beat Ca 2+ handling. The amplitude of the Ca 2+ transients as well as the kinetics of Ca 2+ transport were reduced by up to 25%. These changes were associated with impaired sarcomere contraction. While expression levels of Ca 2+ regulatory proteins were unchanged in remote myocardium compared to the corresponding regions of sham-operated hearts, mobility shift analyses of phosphorylated protein showed 2.9 ± 0.4-fold more unphosphorylated phospholamban (PLN) monomers, the PLN species that inhibits the Ca 2+ ATPase SERCA2a (P ≤ 0.001). Phospho-specific antibodies revealed normal phosphorylation of PLN at T17 in remote myocardium, but markedly reduced phosphorylation at its PKA-dependent phosphorylation site, S16 (P ≤ 0.01). The underlying cause involved enhanced activity of protein phosphatases, particularly PP2A (P ≤ 0.01). In contrast, overall PKA activity was normal. The PLN interactome, as determined by co-immunoprecipitation and mass spectrometry, and the phosphorylation state of PKA targets other than PLN were also unchanged. Isoproterenol enhanced cellular Ca 2+ cycling much stronger in remote myocytes than in healthy controls and improved sarcomere function. We conclude that the reduced phosphorylation state of PLN at S16 impairs myocyte Ca 2+ cycling in the remote myocardium 24 h after I/R and contributes to contractile dysfunction. Copyright © 2018 Elsevier Ltd. All rights reserved.

Computational model based approach to analysis ventricular arrhythmias: Effects of dysfunction calcium channels

NASA Astrophysics Data System (ADS)

Gulothungan, G.; Malathi, R.

2018-04-01

Disturbed sodium (Na+) and calcium (Ca2+) handling is known to be a major predisposing factor for life-threatening cardiac arrhythmias. Cardiac contractility in ventricular tissue is prominent by Ca2+ channels like voltage dependent Ca2+ channels, sodium-calcium exchanger (Na+-Ca2+x) and sacroplasmicrecticulum (SR) Ca2+ pump and leakage channels. Experimental and clinical possibilities for studying cardiac arrhythmias in human ventricular myocardium are very limited. Therefore, the use of alternative methods such as computer simulations is of great importance. Our aim of this article is to study the impact on action potential (AP) generation and propagation in single ventricular myocyte and ventricular tissue under different dysfunction Ca2+ channels condition. In enhanced activity of Na+-Ca2+x, single myocyte produces AP duration (APD90) and APD50 is significantly smaller (266 ms and 235 ms). Its Na+-Ca2+x current at depolarization is increases 60% from its normal level and repolarization current goes more negative (nonfailing= -0.28 pA/pF and failing= -0.47 pA/pF). Similarly, same enhanced activity of Na+-Ca2+x in 10 mm region of ventricular sheet, raises the plateau potential abruptly, which ultimately affects the diastolic repolarization. Compare with normal ventricular sheet region of 10 mm, 10% of ventricular sheet resting state is reduces and ventricular sheet at time 250 ms is goes to resting state very early. In hypertrophy condition, single myocyte produces APD90 and APD50 is worthy of attention smaller (232 mS and 198 ms). Its sodium-potassium (Na+-K+) pump current is 75% reduces from its control conditions (0.13 pA/pF). Hypertrophy condition, 50% of ventricular sheet is reduces to minimum plateau potential state, that starts the repolarization process very early and reduces the APD. In a single failing SR Ca2+ channels myocyte, recovery of Ca2+ concentration level in SR reduces upto 15% from its control myocytes. At time 290 ms, 70% of ventricular sheet is in dysfunction resting potential state in the range -83 mV and ventricular sheet at time 295 ms is goes to 65% dysfunction resting state. Therefore we concluded that shorter APD, instability resting potential and affected calcium induced calcium release (CICR) due to dysfunction Ca2+ channels is potentially have a substantial effect on cardiac contractility and relaxation. Computational study on ventricular tissue AP and its underlying ionic channel currents could help to elucidate possible arrhythmogenic mechanism on a cellular level.

Extracellular cyclic ADP-ribose potentiates ACh-induced contraction in bovine tracheal smooth muscle.

PubMed

Franco, L; Bruzzone, S; Song, P; Guida, L; Zocchi, E; Walseth, T F; Crimi, E; Usai, C; De Flora, A; Brusasco, V

2001-01-01

Cyclic ADP-ribose (cADPR), a universal calcium releaser, is generated from NAD(+) by an ADP-ribosyl cyclase and is degraded to ADP-ribose by a cADPR hydrolase. In mammals, both activities are expressed as ectoenzymes by the transmembrane glycoprotein CD38. CD38 was identified in both epithelial cells and smooth myocytes isolated from bovine trachea. Intact tracheal smooth myocytes (TSMs) responded to extracellular cADPR (100 microM) with an increase in intracellular calcium concentration ([Ca(2+)](i)) both at baseline and after acetylcholine (ACh) stimulation. The nonhydrolyzable analog 3-deaza-cADPR (10 nM) elicited the same effects as cADPR, whereas the cADPR antagonist 8-NH(2)-cADPR (10 microM) inhibited both basal and ACh-stimulated [Ca(2+)](i) levels. Extracellular cADPR or 3-deaza-cADPR caused a significant increase of ACh-induced contraction in tracheal smooth muscle strips, whereas 8-NH(2)-cADPR decreased it. Tracheal mucosa strips, by releasing NAD(+), enhanced [Ca(2+)](i) in isolated TSMs, and this increase was abrogated by either NAD(+)-ase or 8-NH(2)-cADPR. These data suggest the existence of a paracrine mechanism whereby mucosa-released extracellular NAD(+) plays a hormonelike function and cADPR behaves as second messenger regulating calcium-related contractility in TSMs.

T-type Ca2+ channels regulate the exit of cardiac myocytes from the cell cycle after birth

PubMed Central

Wang, Fang; Gao, Hui; Kubo, Hajime; Fan, Xiaoxuan; Zhang, Hongyu; Berretta, Remus; Chen, Xiongwen; Sharp, Thomas; Starosta, Timothy; Makarewich, Catherine; Li, Ying; Molkentin, Jeffrey D.; Houser, Steven R.

2013-01-01

T-type Ca2+ channels (TTCCs) are expressed in the fetal heart and then disappear from ventricular myocytes after birth. The hypothesis examined in this study was the α1G TTCCs' influence in myocyte maturation and their rapid withdrawal from the cell cycle after birth. Methods Cardiac myocytes were isolated from neonatal and adult wild type (WT), α1G−/− and α1G over expressing (α1GDT) mice. Bromodeoxyuridine (BrdU) uptake, myocyte nucleation, cell cycle analysis, and T-type Ca2+ currents were measured. Results All myocytes were mono-nucleated at birth and 35% of WT myocytes expressed functional TTCCs. Very few neonatal myocytes had functional TTCCs in α1G−/− hearts. By the end of the first week after birth no WT or α1G−/− had functional TTCCs. During the first week after birth about 25% of WT myocytes were BrdU+ and became bi-nucleated. Significantly fewer α1G−/− myocytes became bi-nucleated and fewer of these myocytes were BrdU+. Neonatal α1G−/− myocytes were also smaller than WT. Adult WT and α1G−/− hearts were similar in size, but α1G−/− myocytes were smaller and a greater % were mono-nucleated. α1G over expressing hearts were smaller than WT but their myocytes were larger. Conclusions The studies performed show that loss of functional TTCCs is associated with bi-nucleation and myocyte withdrawal from the cell cycle. Loss of α1G TTCCs slowed the transition from mono- to bi-nucleation and resulted in an adult heart with a greater number of small cardiac myocytes. These results suggest that TTCCs are involved in the regulation of myocyte size and the exit of myocytes from the cell cycle during the first week after birth. PMID:23743021

Molecular and functional analyses of the contractile apparatus in lymphatic muscle

NASA Technical Reports Server (NTRS)

Muthuchamy, Mariappan; Gashev, Anatoliy; Boswell, Niven; Dawson, Nancy; Zawieja, David; Delp, Z. (Principal Investigator)

2003-01-01

Lymphatics are necessary for the generation and regulation of lymph flow. Lymphatics use phasic contractions and extrinsic compressions to generate flow; tonic contractions alter resistance. Lymphatic muscle exhibits important differences from typical vascular smooth muscle. In this study, the thoracic duct exhibited significant functional differences from mesenteric lymphatics. To understand the molecular basis for these differences, we examined the profiles of contractile proteins and their messages in mesenteric lymphatics, thoracic duct, and arterioles. Results demonstrated that mesenteric lymphatics express only SMB smooth muscle myosin heavy chain (SM-MHC), whereas thoracic duct and arterioles expressed both SMA and SMB isoforms. Both SM1 and SM2 isoforms of SM-MHC were detected in arterioles and mesenteric and thoracic lymphatics. In addition, the fetal cardiac/skeletal slow-twitch muscle-specific beta-MHC message was detected only in mesenteric lymphatics. All four actin messages, cardiac alpha-actin, vascular alpha-actin, enteric gamma-actin, and skeletal alpha-actin, were present in both mesenteric lymphatics and arterioles. However, in thoracic duct, predominantly cardiac alpha-actin and vascular alpha-actin were found. Western blot and immunohistochemical analyses corroborated the mRNA studies. However, in arterioles only vascular alpha-actin protein was detected. These data indicate that lymphatics display genotypic and phenotypic characteristics of vascular, cardiac, and visceral myocytes, which are needed to fulfill the unique roles of the lymphatic system.

Use of a fiber glass optical system to measure the contractile characteristics of a single isolated muscle cell

NASA Astrophysics Data System (ADS)

Chen, Chulung; Yin, Shizhuo; Li, Jiang; Yu, Francis T. S.; Cheung, Joseph Y.; Zhang, Xueqian; Lei, Xiaoxiao; Wu, Zhongkong

1998-05-01

Cell is the basic structural and fundamental unit of all organisms; the smallest structure capable of performing all the activities vital to life. One goal of current research interest is to learn how the muscle varies the strength of its contraction in response to electric stimuli. A wide variety of techniques have been developed to monitor the mechanical response of isolated cardiac myocytes. Some success has been reported either with the use of intact rat myocytes supported by suction micropipettes or in guinea pig myocytes adhering to glass beams. However, the usual measuring techniques exhibit destructive contact performance on live cells. They could not solve the problem, since the cell may die during or after the time-consuming attachment process at the beginning of each experiment. In contrast, a novel optical system, which consists of a microglass tube with an inner diameter the same size of a real cardiac cell, is proposed to simulate real cell's twitch process. the physical parameters of synthetic cell are well known. By comparing the dynamics of the real cell with that of the simulated cell, the twitch characteristics of the real cell can be measured.

Lethal effect of electric fields on isolated ventricular myocytes.

PubMed

de Oliveira, Pedro Xavier; Bassani, Rosana Almada; Bassani, José Wilson Magalhães

2008-11-01

Defibrillator-type shocks may cause electric and contractile dysfunction. In this study, we determined the relationship between probability of lethal injury and electric field intensity (E in isolated rat ventricular myocytes, with emphasis on field orientation and stimulus waveform. This relationship was sigmoidal with irreversible injury for E > 50 V/cm . During both threshold and lethal stimulation, cells were twofold more sensitive to the field when it was applied longitudinally (versus transversally) to the cell major axis. For a given E, the estimated maximum variation of transmembrane potential (Delta V(max)) was greater for longitudinal stimuli, which might account for the greater sensitivity to the field. Cell death, however, occurred at lower maximum Delta V(max) values for transversal shocks. This might be explained by a less steep spatial decay of transmembrane potential predicted for transversal stimulation, which would possibly result in occurrence of electroporation in a larger membrane area. For the same stimulus duration, cells were less sensitive to field-induced injury when shocks were biphasic (versus monophasic). Ours results indicate that, although significant myocyte death may occur in the E range expected during clinical defibrillation, biphasic shocks are less likely to produce irreversible cell injury.

Familial hypertrophic cardiomyopathy: functional variance among individual cardiomyocytes as a trigger of FHC-phenotype development

PubMed Central

Brenner, Bernhard; Seebohm, Benjamin; Tripathi, Snigdha; Montag, Judith; Kraft, Theresia

2014-01-01

Familial hypertrophic cardiomyopathy (FHC) is the most frequent inherited cardiac disease. It has been related to numerous mutations in many sarcomeric and even some non-sarcomeric proteins. So far, however, no common mechanism has been identified by which the many different mutations in different sarcomeric and non-sarcomeric proteins trigger development of the FHC phenotype. Here we show for different MYH7 mutations variance in force pCa-relations from normal to highly abnormal as a feature common to all mutations we studied, while direct functional effects of the different FHC-mutations, e.g., on force generation, ATPase or calcium sensitivity of the contractile system, can be quite different. The functional variation among individual M. soleus fibers of FHC-patients is accompanied by large variation in mutant vs. wildtype β-MyHC-mRNA. Preliminary results show a similar variation in mutant vs. wildtype β-MyHC-mRNA among individual cardiomyocytes. We discuss our previously proposed concept as to how different mutations in the β-MyHC and possibly other sarcomeric and non-sarcomeric proteins may initiate an FHC-phenotype by functional variation among individual cardiomyocytes that results in structural distortions within the myocardium, leading to cellular and myofibrillar disarray. In addition, distortions can activate stretch-sensitive signaling in cardiomyocytes and non-myocyte cells which is known to induce cardiac remodeling with interstitial fibrosis and hypertrophy. Such a mechanism will have major implications for therapeutic strategies to prevent FHC-development, e.g., by reducing functional imbalances among individual cardiomyocytes or by inhibition of their triggering of signaling paths initiating remodeling. Targeting increased or decreased contractile function would require selective targeting of mutant or wildtype protein to reduce functional imbalances. PMID:25346696

Extramitochondrial domain rich in carbonic anhydrase activity improves myocardial energetics.

PubMed

Schroeder, Marie A; Ali, Mohammad A; Hulikova, Alzbeta; Supuran, Claudiu T; Clarke, Kieran; Vaughan-Jones, Richard D; Tyler, Damian J; Swietach, Pawel

2013-03-05

CO2 is produced abundantly by cardiac mitochondria. Thus an efficient means for its venting is required to support metabolism. Carbonic anhydrase (CA) enzymes, expressed at various sites in ventricular myocytes, may affect mitochondrial CO2 clearance by catalyzing CO2 hydration (to H(+) and HCO3(-)), thereby changing the gradient for CO2 venting. Using fluorescent dyes to measure changes in pH arising from the intracellular hydration of extracellularly supplied CO2, overall CA activity in the cytoplasm of isolated ventricular myocytes was found to be modest (2.7-fold above spontaneous kinetics). Experiments on ventricular mitochondria demonstrated negligible intramitochondrial CA activity. CA activity was also investigated in intact hearts by (13)C magnetic resonance spectroscopy from the rate of H(13)CO3(-) production from (13)CO2 released specifically from mitochondria by pyruvate dehydrogenase-mediated metabolism of hyperpolarized [1-(13)C]pyruvate. CA activity measured upon [1-(13)C]pyruvate infusion was fourfold higher than the cytoplasm-averaged value. A fluorescent CA ligand colocalized with a mitochondrial marker, indicating that mitochondria are near a CA-rich domain. Based on immunoreactivity, this domain comprises the nominally cytoplasmic CA isoform CAII and sarcoplasmic reticulum-associated CAXIV. Inhibition of extramitochondrial CA activity acidified the matrix (as determined by fluorescence measurements in permeabilized myocytes and isolated mitochondria), impaired cardiac energetics (indexed by the phosphocreatine-to-ATP ratio measured by (31)P magnetic resonance spectroscopy of perfused hearts), and reduced contractility (as measured from the pressure developed in perfused hearts). These data provide evidence for a functional domain of high CA activity around mitochondria to support CO2 venting, particularly during elevated and fluctuating respiratory activity. Aberrant distribution of CA activity therefore may reduce the heart's energetic efficiency.

Cardio-oncology: cardiovascular complications of cancer therapy.

PubMed

Henning, Robert J; Harbison, Raymond D

2017-07-01

This paper focuses on three classes of commonly used anticancer drugs, which can cause cardiotoxicity: anthracyclines, monoclonal antibodies exemplified by trastuzumab and tyrosine kinase inhibitors. Anthracyclines can induce cardiomyocyte necrosis and fibrosis. Trastuzumab can cause cardiac stunning. The tyrosine kinase inhibitors can increase systemic arterial pressure and impair myocyte contractility. In addition, radiation therapy to the mediastinum or left chest can exacerbate the cardiotoxicity of these anticancer drugs and can also cause accelerated atherosclerosis, myocardial infarction, heart failure and arrhythmias. Left ventricular ejection fraction measurements are most commonly used to assess cardiac function in patients who receive chemo- or radiation-therapy. However, echocardiographic determinations of global longitudinal strain are more sensitive for detection of early left ventricular systolic dysfunction. Information on patient-risk stratification and monitoring is presented and guidelines for the medical treatment of cardiac dysfunction due to cancer therapies are summarized.

Exposure to low mercury concentration in vivo impairs myocardial contractile function

DOE Office of Scientific and Technical Information (OSTI.GOV)

Furieri, Lorena Barros; Fioresi, Mirian; Junior, Rogerio Faustino Ribeiro

2011-09-01

Increased cardiovascular risk after mercury exposure has been described but cardiac effects resulting from controlled chronic treatment are not yet well explored. We analyzed the effects of chronic exposure to low mercury concentrations on hemodynamic and ventricular function of isolated hearts. Wistar rats were treated with HgCl{sub 2} (1st dose 4.6 {mu}g/kg, subsequent dose 0.07 {mu}g/kg/day, im, 30 days) or vehicle. Mercury treatment did not affect blood pressure (BP) nor produced cardiac hypertrophy or changes of myocyte morphometry and collagen content. This treatment: 1) in vivo increased left ventricle end diastolic pressure (LVEDP) without changing left ventricular systolic pressure (LVSP)more » and heart rate; 2) in isolated hearts reduced LV isovolumic systolic pressure and time derivatives, and {beta}-adrenergic response; 3) increased myosin ATPase activity; 4) reduced Na{sup +}-K{sup +} ATPase (NKA) activity; 5) reduced protein expression of SERCA and phosphorylated phospholamban on serine 16 while phospholamban expression increased; as a consequence SERCA/phospholamban ratio reduced; 6) reduced sodium/calcium exchanger (NCX) protein expression and {alpha}-1 isoform of NKA, whereas {alpha}-2 isoform of NKA did not change. Chronic exposure for 30 days to low concentrations of mercury does not change BP, heart rate or LVSP but produces small but significant increase of LVEDP. However, in isolated hearts mercury treatment promoted contractility dysfunction as a result of the decreased NKA activity, reduction of NCX and SERCA and increased PLB protein expression. These findings offer further evidence that mercury chronic exposure, even at small concentrations, is an environmental risk factor affecting heart function. - Highlights: > Unchanges blood pressure, heart rate, systolic pressure. > Increases end diastolic pressure. > Promotes cardiac contractility dysfunction. > Decreases NKA activity, NCX and SERCA, increases PLB protein expression. > Small concentrations constitutes environmental cardiovascular risk factor.« less

Cardiac P2X purinergic receptors as a new pathway for increasing Na+ entry in cardiac myocytes

PubMed Central

Shen, Jian-Bing; Yang, Ronghua; Pappano, Achilles

2014-01-01

P2X4 receptors (P2X4Rs) are ligand-gated ion channels capable of conducting cations such as Na+. Endogenous cardiac P2X4R can mediate ATP-activated current in adult murine cardiomyocytes. In the present study, we tested the hypothesis that cardiac P2X receptors can induce Na+ entry and modulate Na+ handling. We further determined whether P2X receptor-induced stimulation of the Na+/Ca2+ exchanger (NCX) has a role in modulating the cardiac contractile state. Changes in Na+-K+-ATPase current (Ip) and NCX current (INCX) after agonist stimulation were measured in ventricular myocytes of P2X4 transgenic mice using whole cell patch-clamp techniques. The agonist 2-methylthio-ATP (2-meSATP) increased peak Ip from a basal level of 0.52 ± 0.02 to 0.58 ± 0.03 pA/pF. 2-meSATP also increased the Ca2+ entry mode of INCX (0.55 ± 0.09 pA/pF under control conditions vs. 0.82 ± 0.14 pA/pF with 2-meSATP) at a membrane potential of +50 mV. 2-meSATP shifted the reversal potential of INCX from −14 ± 2.3 to −25 ± 4.1 mV, causing an estimated intracellular Na+ concentration increase of 1.28 ± 0.42 mM. These experimental results were closely mimicked by mathematical simulations based on previously established models. KB-R7943 or a structurally different agent preferentially opposing the Ca2+ entry mode of NCX, YM-244769, could inhibit the 2-meSATP-induced increase in cell shortening in transgenic myocytes. Thus, the Ca2+ entry mode of INCX participates in P2X agonist-stimulated contractions. In ventricular myocytes from wild-type mice, the P2X agonist could increase INCX, and KB-R7943 was able to inhibit the contractile effect of endogenous P2X4Rs, indicating a physiological role of these receptors in wild-type cells. The data demonstrate a novel Na+ entry pathway through ligand-gated P2X4Rs in cardiomyocytes. PMID:25239801

Cardiac P2X purinergic receptors as a new pathway for increasing Na⁺ entry in cardiac myocytes.

PubMed

Shen, Jian-Bing; Yang, Ronghua; Pappano, Achilles; Liang, Bruce T

2014-11-15

P2X4 receptors (P2X4Rs) are ligand-gated ion channels capable of conducting cations such as Na(+). Endogenous cardiac P2X4R can mediate ATP-activated current in adult murine cardiomyocytes. In the present study, we tested the hypothesis that cardiac P2X receptors can induce Na(+) entry and modulate Na(+) handling. We further determined whether P2X receptor-induced stimulation of the Na(+)/Ca(2+) exchanger (NCX) has a role in modulating the cardiac contractile state. Changes in Na(+)-K(+)-ATPase current (Ip) and NCX current (INCX) after agonist stimulation were measured in ventricular myocytes of P2X4 transgenic mice using whole cell patch-clamp techniques. The agonist 2-methylthio-ATP (2-meSATP) increased peak Ip from a basal level of 0.52 ± 0.02 to 0.58 ± 0.03 pA/pF. 2-meSATP also increased the Ca(2+) entry mode of INCX (0.55 ± 0.09 pA/pF under control conditions vs. 0.82 ± 0.14 pA/pF with 2-meSATP) at a membrane potential of +50 mV. 2-meSATP shifted the reversal potential of INCX from -14 ± 2.3 to -25 ± 4.1 mV, causing an estimated intracellular Na(+) concentration increase of 1.28 ± 0.42 mM. These experimental results were closely mimicked by mathematical simulations based on previously established models. KB-R7943 or a structurally different agent preferentially opposing the Ca(2+) entry mode of NCX, YM-244769, could inhibit the 2-meSATP-induced increase in cell shortening in transgenic myocytes. Thus, the Ca(2+) entry mode of INCX participates in P2X agonist-stimulated contractions. In ventricular myocytes from wild-type mice, the P2X agonist could increase INCX, and KB-R7943 was able to inhibit the contractile effect of endogenous P2X4Rs, indicating a physiological role of these receptors in wild-type cells. The data demonstrate a novel Na(+) entry pathway through ligand-gated P2X4Rs in cardiomyocytes. Copyright © 2014 the American Physiological Society.

Androgen signaling in myocytes contributes to the maintenance of muscle mass and fiber type regulation but not to muscle strength or fatigue.

PubMed

Ophoff, Jill; Van Proeyen, Karen; Callewaert, Filip; De Gendt, Karel; De Bock, Katrien; Vanden Bosch, An; Verhoeven, Guido; Hespel, Peter; Vanderschueren, Dirk

2009-08-01

Muscle frailty is considered a major cause of disability in the elderly and chronically ill. However, the exact role of androgen receptor (AR) signaling in muscle remains unclear. Therefore, a postmitotic myocyte-specific AR knockout (mARKO) mouse model was created and investigated together with a mouse model with ubiquitous AR deletion. Muscles from mARKO mice displayed a marked reduction in AR protein (60-88%). Interestingly, body weights and lean body mass were lower in mARKO vs. control mice (-8%). The weight of the highly androgen-sensitive musculus levator ani was significantly reduced (-46%), whereas the weights of other peripheral skeletal muscles were not or only slightly reduced. mARKO mice had lower intra-abdominal fat but did not demonstrate a cortical or trabecular bone phenotype, indicating that selective ablation of the AR in myocytes affected male body composition but not skeletal homeostasis. Furthermore, muscle contractile performance in mARKO mice did not differ from their controls. Myocyte-specific AR ablation resulted in a conversion of fast toward slow fibers, without affecting muscle strength or fatigue. Similar results were obtained in ubiquitous AR deletion, showing lower body weight, whereas some but not all muscle weights were reduced. The percent slow fibers was increased, but no changes in muscle strength or fatigue could be detected. Together, our findings show that myocyte AR signaling contributes to the maintenance of muscle mass and fiber type regulation but not to muscle strength or fatigue. The levator ani weight remains the most sensitive and specific marker of AR-mediated anabolic action on muscle.

Acute heat tolerance of cardiac excitation in the brown trout (Salmo trutta fario).

PubMed

Vornanen, Matti; Haverinen, Jaakko; Egginton, Stuart

2014-01-15

The upper thermal tolerance and mechanisms of heat-induced cardiac failure in the brown trout (Salmo trutta fario) was examined. The point above which ion channel function and sinoatrial contractility in vitro, and electrocardiogram (ECG) in vivo, started to fail (break point temperature, BPT) was determined by acute temperature increases. In general, electrical excitation of the heart was most sensitive to heat in the intact animal (electrocardiogram, ECG) and least sensitive in isolated cardiac myocytes (ion currents). BPTs of Ca(2+) and K(+) currents of cardiac myocytes were much higher (>28°C) than BPT of in vivo heart rate (23.5 ± 0.6°C) (P<0.05). A striking exception among sarcolemmal ion conductances was the Na(+) current (INa), which was the most heat-sensitive molecular function, with a BPT of 20.9 ± 0.5°C. The low heat tolerance of INa was reflected as a low BPT for the rate of action potential upstroke in vitro (21.7 ± 1.2°C) and the velocity of impulse transmission in vivo (21.9 ± 2.2°C). These findings from different levels of biological organization strongly suggest that heat-dependent deterioration of Na(+) channel function disturbs normal spread of electrical excitation over the heart, leading to progressive variability of cardiac rhythmicity (missed beats, bursts of fast beating), reduction of heart rate and finally cessation of the normal heartbeat. Among the cardiac ion currents INa is 'the weakest link' and possibly a limiting factor for upper thermal tolerance of electrical excitation in the brown trout heart. Heat sensitivity of INa may result from functional requirements for very high flux rates and fast gating kinetics of the Na(+) channels, i.e. a trade-off between high catalytic activity and thermal stability.

Maternal high-altitude hypoxia and suppression of ryanodine receptor-mediated Ca2+ sparks in fetal sheep pulmonary arterial myocytes

PubMed Central

Hadley, Scott R.; Blood, Quintin; Rubalcava, Monica; Waskel, Edith; Lumbard, Britney; Le, Petersen; Longo, Lawrence D.; Buchholz, John N.

2012-01-01

Ca2+ sparks are fundamental Ca2+ signaling events arising from ryanodine receptor (RyR) activation, events that relate to contractile and dilatory events in the pulmonary vasculature. Recent studies demonstrate that long-term hypoxia (LTH) can affect pulmonary arterial reactivity in fetal, newborn, and adult animals. Because RyRs are important to pulmonary vascular reactivity and reactivity changes with ontogeny and LTH we tested the hypothesis that RyR-generated Ca2+ signals are more active before birth and that LTH suppresses these responses. We examined these hypotheses by performing confocal imaging of myocytes in living arteries and by performing wire myography studies. Pulmonary arteries (PA) were isolated from fetal, newborn, or adult sheep that lived at low altitude or from those that were acclimatized to 3,801 m for > 100 days. Confocal imaging demonstrated preservation of the distance between the sarcoplasmic reticulum, nucleus, and plasma membrane in PA myocytes. Maturation increased global Ca2+ waves and Ca2+ spark activity, with sparks becoming larger, wider, and slower. LTH preferentially depressed Ca2+ spark activity in immature pulmonary arterial myocytes, and these sparks were smaller, wider, and slower. LTH also suppressed caffeine-elicited contraction in fetal PA but augmented contraction in the newborn and adult. The influence of both ontogeny and LTH on RyR-dependent cell excitability shed new light on the therapeutic potential of these channels for the treatment of pulmonary vascular disease in newborns as well as adults. PMID:22962012

β-adrenergic effects on cardiac myofilaments and contraction in an integrated rabbit ventricular myocyte model

PubMed Central

Negroni, Jorge A.; Morotti, Stefano; Lascano, Elena C.; Gomes, Aldrin V.; Grandi, Eleonora; Puglisi, José L; Bers, Donald M.

2015-01-01

A five-state model of myofilament contraction was integrated into a well-established rabbit ventricular myocyte model of ion channels, Ca2+ transporters and kinase signaling to analyze the relative contribution of different phosphorylation targets to the overall mechanical response driven by β-adrenergic stimulation (β-AS). β-AS effect on sarcoplasmic reticulum Ca2+ handling, Ca2+, K+ and Cl− currents, and Na+/K+-ATPase properties were included based on experimental data. The inotropic effect on the myofilaments was represented as reduced myofilament Ca2+ sensitivity (XBCa) and titin stiffness, and increased cross-bridge (XB) cycling rate (XBcy). Assuming independent roles of XBCa and XBcy, the model reproduced experimental β-AS responses on action potentials and Ca2+ transient amplitude and kinetics. It also replicated the behavior of force-Ca2+, release-restretch, length-step, stiffness-frequency and force-velocity relationships, and increased force and shortening in isometric and isotonic twitch contractions. The β-AS effect was then switched off from individual targets to analyze their relative impact on contractility. Preventing β-AS effects on L-type Ca2+ channels or phospholamban limited Ca2+ transients and contractile responses in parallel, while blocking phospholemman and K+ channel (IKs) effects enhanced Ca2+ and inotropy. Removal of β-AS effects from XBCa enhanced contractile force while decreasing peak Ca2+ (due to greater Ca2+ buffering), but had less effect on shortening. Conversely, preventing β-AS effects on XBcy preserved Ca2+ transient effects, but blunted inotropy (both isometric force and especially shortening). Removal of titin effects had little impact on contraction. Finally, exclusion of β-AS from XBCa and XBcy while preserving effects on other targets resulted in preserved peak isometric force response (with slower kinetics) but nearly abolished enhanced shortening. β-AS effects on XBCa vs. XBcy have greater impact on isometric vs. isotonic contraction, respectively. PMID:25724724

Probenecid: novel use as a non-injurious positive inotrope acting via cardiac TRPV2 stimulation

PubMed Central

Koch, Sheryl E.; Gao, Xiaoqian; Haar, Lauren; Jiang, Min; Lasko, Valerie M.; Robbins, Nathan; Cai, Wenfeng; Brokamp, Cole; Varma, Priyanka; Tranter, Michael; Liu, Yong; Ren, Xiaoping; Lorenz, John N.; Wang, Hong-Sheng; Jones, W. Keith; Rubinstein, Jack

2012-01-01

Probenecid is a highly lipid soluble benzoic acid derivative originally used to increase serum antibiotic concentrations. It was later discovered to have uricosuric effects and was FDA approved for gout therapy. It has recently been found to be a potent agonist of Transient Receptor Potential Vanilloid 2 (TRPV2). We have shown that this receptor is in the cardiomyocyte and report a positive inotropic effect of the drug. Using echocardiography, Langendorff and isolated myocytes, we measured the change in contractility and, using TRPV2−/− mice, proved that the effect was mediated by TRPV2 channels in the cardiomyocytes. Analysis of the expression of Ca2+ handling and β-adrenergic signaling pathway proteins showed that the contractility was not increased through activation of the β-ADR. We propose that the response to probenecid is due to activation of TRPV2 channels secondary to SR release of Ca2+. PMID:22561103

Matrigel Mattress: A Method for the Generation of Single Contracting Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

PubMed

Feaster, Tromondae K; Cadar, Adrian G; Wang, Lili; Williams, Charles H; Chun, Young Wook; Hempel, Jonathan E; Bloodworth, Nathaniel; Merryman, W David; Lim, Chee Chew; Wu, Joseph C; Knollmann, Björn C; Hong, Charles C

2015-12-04

The lack of measurable single-cell contractility of human-induced pluripotent stem cell-derived cardiac myocytes (hiPSC-CMs) currently limits the utility of hiPSC-CMs for evaluating contractile performance for both basic research and drug discovery. To develop a culture method that rapidly generates contracting single hiPSC-CMs and allows quantification of cell shortening with standard equipment used for studying adult CMs. Single hiPSC-CMs were cultured for 5 to 7 days on a 0.4- to 0.8-mm thick mattress of undiluted Matrigel (mattress hiPSC-CMs) and compared with hiPSC-CMs maintained on a control substrate (<0.1-mm thick 1:60 diluted Matrigel, control hiPSC-CMs). Compared with control hiPSC-CMs, mattress hiPSC-CMs had more rod-shape morphology and significantly increased sarcomere length. Contractile parameters of mattress hiPSC-CMs measured with video-based edge detection were comparable with those of freshly isolated adult rabbit ventricular CMs. Morphological and contractile properties of mattress hiPSC-CMs were consistent across cryopreserved hiPSC-CMs generated independently at another institution. Unlike control hiPSC-CMs, mattress hiPSC-CMs display robust contractile responses to positive inotropic agents, such as myofilament calcium sensitizers. Mattress hiPSC-CMs exhibit molecular changes that include increased expression of the maturation marker cardiac troponin I and significantly increased action potential upstroke velocity because of a 2-fold increase in sodium current (INa). The Matrigel mattress method enables the rapid generation of robustly contracting hiPSC-CMs and enhances maturation. This new method allows quantification of contractile performance at the single-cell level, which should be valuable to disease modeling, drug discovery, and preclinical cardiotoxicity testing. © 2015 American Heart Association, Inc.

The stretch-dependent potassium channel TREK-1 and its function in murine myometrium

PubMed Central

Monaghan, Kevin; Baker, Salah A; Dwyer, Laura; Hatton, William C; Sik Park, Kyung; Sanders, Kenton M; Koh, Sang Don

2011-01-01

Smooth muscle of the uterus stays remarkably quiescent during normal pregnancy to allow sufficient time for development of the fetus. At present the mechanisms leading to uterine quiescence during pregnancy and how the suppression of activity is relieved at term are poorly understood. Myometrial excitability is governed by ion channels, and a major hypothesis regarding the regulation of contractility during pregnancy has been that expression of certain channels is regulated by hormonal influences. We have explored the expression and function of stretch-dependent K+ (SDK) channels, which are likely to be due to TREK channels, in murine myometrial tissues and myocytes using PCR, Western blots, patch clamp, intracellular microelectrode and isometric force measurements. TREK-1 is more highly expressed than TREK-2 in myometrium, and there was no detectable expression of TRAAK. Expression of TREK-1 transcripts and protein was regulated during pregnancy and delivery. SDK channels were activated in response to negative pressure applied to patches. SDK channels were insensitive to a broad-spectrum of K+ channel blockers, including tetraethylammonium and 4-aminopyridine, and insensitive to intracellular Ca2+. SDK channels were activated by stretch and arachidonic acid and inhibited by reagents that block TREK-1 channels, l-methionine and/or methioninol. Our data suggest that uterine excitability and contractility during pregnancy is regulated by the expression of SDK/TREK-1 channels. Up-regulation of these channels stabilizes membrane potential and controls contraction during pregnancy and down-regulation of these channels induces the onset of delivery. PMID:21224218

Novel insights into the development of chagasic cardiomyopathy: role of PI3Kinase/NO axis.

PubMed

Roman-Campos, Danilo; Sales-Junior, Policarpo; Duarte, Hugo L; Gomes, Enéas R; Lara, Aline; Campos, Paula; Rocha, Nazareth N; Resende, Rodrigo R; Ferreira, Anderson; Guatimosim, Sílvia; Gazzinelli, Ricardo T; Ropert, Catherine; Cruz, Jader S

2013-09-10

Chagas' disease is one of the leading causes of heart failure in Latin American countries. Despite its great social impact, there is no direct evidence in the literature explaining the development of heart failure in Chagas' disease. Therefore, the main objective of the study was to investigate the development of the Chagas' disease towards its chronic phase and correlate with modifications in the cellular electrophysiological characteristics of the infected heart. Using a murine model of Chagas' disease, we confirmed and extended previous findings of altered electrocardiogram and echocardiogram in this cardiomyopathy. The observed changes in the electrocardiogram were correlated with the prolonged action potential and reduced transient outward potassium current density. Reduced heart function was associated with remodeling of intracellular calcium handling, altered extracellular matrix content, and to a set of proteins involved in the control of cellular contractility in ventricular myocytes. Furthermore, disruption of calcium homeostasis was partially due to activation of the PI3Kinase/nitric oxide signaling pathway. Finally, we propose a causal link between the inflammatory mediators and heart remodeling during chagasic cardiomyopathy. Altogether our results demonstrate that heart failure in Chagas' disease may occur due to electrical and mechanical remodeling of cardiac myocytes, and suggest that AKT/PI3K/NO axis could be an important pharmacological target to improve the disease outcome. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

Does altered myogenic activity contribute to OAB symptoms from detrusor overactivity? ICI-RS 2013.

PubMed

Chacko, Sam; Cortes, Eduard; Drake, Marcus J; Fry, Christopher H

2014-06-01

To highlight novel experimental approaches that test if the Myogenic Hypothesis remains viable as a contributor to the aetiology of detrusor overactivity. To summarise the conclusions of a workshop held under the auspices of ICI-RS in 2013. Several theories may explain the pathology of detrusor overactivity and include a myogenic theory with fundamental changes to detrusor muscle excitation-contraction coupling. The isolated bladder displays micromotions that do not normally translate into significant changes of intravesical pressure. However, their amplitude and frequency are altered in animal models of bladder dysfunction. The origin of micromotions, if they generate significant changes of intravesical pressure and contribute to urinary tract sensations remain unanswered. Within the myocyte, changes to contractile protein phosphorylation through accessory proteins and cytoplasmic regulatory pathways occur in lower urinary tract pathologies associated with detrusor overactivity. Furthermore, myocytes isolated from overactive human bladders generate greater spontaneous activity, but a complete description of changes to ionic currents remains to be characterised. Finally, several growth factors, including mechano-growth factor, are released when bladder wall stress is increased, as with outflow obstruction. However the phenotype of the transformed detrusor myocytes remains to be measured. A number of lines of evidence suggest that the Myogenic Hypothesis remains viable as a contributor to detrusor overactivity. © 2014 Wiley Periodicals, Inc.

Tumor necrosis factor regulates NMDA receptor-mediated airway smooth muscle contractile function and airway responsiveness.

PubMed

Anaparti, Vidyanand; Pascoe, Christopher D; Jha, Aruni; Mahood, Thomas H; Ilarraza, Ramses; Unruh, Helmut; Moqbel, Redwan; Halayko, Andrew J

2016-08-01

We have shown that N-methyl-d-aspartate receptors (NMDA-Rs) are receptor-operated calcium entry channels in human airway smooth muscle (HASM) during contraction. Tumor necrosis factor (TNF) augments smooth muscle contractility by influencing pathways that regulate intracellular calcium flux and can alter NMDA-R expression and activity in cortical neurons and glial cells. We hypothesized that NMDA-R-mediated Ca(2+) and contractile responses of ASM can be altered by inflammatory mediators, including TNF. In cultured HASM cells, we assessed TNF (10 ng/ml, 48 h) effect on NMDA-R subunit abundance by quantitative PCR, confocal imaging, and immunoblotting. We observed dose- and time-dependent changes in NMDA-R composition: increased obligatory NR1 subunit expression and altered regulatory NR2 and inhibitory NR3 subunits. Measuring intracellular Ca(2+) flux in Fura-2-loaded HASM cultures, we observed that TNF exposure enhanced cytosolic Ca(2+) mobilization and changed the temporal pattern of Ca(2+) flux in individual myocytes induced by NMDA, an NMDA-R selective analog of glutamate. We measured airway responses to NMDA in murine thin-cut lung slices (TCLS) from allergen-naive animals and observed significant airway contraction. However, NMDA acted as a bronchodilator in TCLS from house dust mice-challenged mice and in allergen-naive TCLS subjected to TNF exposure. All contractile or bronchodilator responses were blocked by a selective NMDA-R antagonist, (2R)-amino-5-phosphonopentanoate, and bronchodilator responses were prevented by N(G)-nitro-l-arginine methyl ester (nitric oxide synthase inhibitor) or indomethacin (cyclooxygenase inhibitor). Collectively, we show that TNF augments NMDA-R-mediated Ca(2+) mobilization in HASM cells, whereas in multicellular TCLSs allergic inflammation and TNF exposure leads to NMDA-R-mediated bronchodilation. These findings reveal the unique contribution of ionotrophic NMDA-R to airway hyperreactivity. Copyright © 2016 the American Physiological Society.

Effect of aspirin on the contractility of aortic smooth muscle and the course of blood pressure development in male spontaneously hypertensive rats.

PubMed

Rahmani, M A; David, V; Huang, M; DeGray, G

1998-01-01

The effects of acetylsalicylic acid (ASA) on aortic smooth muscle contractility were studied in aortic rings of male SHR and WKY rats. The rats were administered two intraperitoneal injections of 10 mg/kg of ASA per week for ten weeks. Blood pressure of each rat was monitored twice weekly prior to the i.p. injections. Twenty four hours after the last injection the aortic smooth muscles were evaluated for generation of active tension in response to KCl, Phenylephrine (PE), Clonidine and Norepinephrine (NE). In another set of experiments calcium conductance was evaluated in the presence or absence of endothelium both in ASA treated and non treated animals. We report that aortic rings from ASA-treated SHR animals were more responsive to contractile agents than rings from non-treated SHR male rats. Also, the Ca2+ conductance in vitro was enhanced appreciably in SHR aortic rings denuded of their monolayer of endothelium in response to ASA treatment. No decrease in systolic blood pressure was observed in response to ASA treatment in SHR male rats. These results suggest that acetylsalicylic acid not only may modulate aortic smooth muscle contractility through the metabolites of arachidonic acid but may repair to a great extent the hypertension associated plasma membrane permeability defect of vascular myocytes.

Right Ventricular Perfusion: Physiology and Clinical Implications.

PubMed

Crystal, George J; Pagel, Paul S

2018-01-01

Regulation of blood flow to the right ventricle differs significantly from that to the left ventricle. The right ventricle develops a lower systolic pressure than the left ventricle, resulting in reduced extravascular compressive forces and myocardial oxygen demand. Right ventricular perfusion has eight major characteristics that distinguish it from left ventricular perfusion: (1) appreciable perfusion throughout the entire cardiac cycle; (2) reduced myocardial oxygen uptake, blood flow, and oxygen extraction; (3) an oxygen extraction reserve that can be recruited to at least partially offset a reduction in coronary blood flow; (4) less effective pressure-flow autoregulation; (5) the ability to downregulate its metabolic demand during coronary hypoperfusion and thereby maintain contractile function and energy stores; (6) a transmurally uniform reduction in myocardial perfusion in the presence of a hemodynamically significant epicardial coronary stenosis; (7) extensive collateral connections from the left coronary circulation; and (8) possible retrograde perfusion from the right ventricular cavity through the Thebesian veins. These differences promote the maintenance of right ventricular oxygen supply-demand balance and provide relative resistance to ischemia-induced contractile dysfunction and infarction, but they may be compromised during acute or chronic increases in right ventricle afterload resulting from pulmonary arterial hypertension. Contractile function of the thin-walled right ventricle is exquisitely sensitive to afterload. Acute increases in pulmonary arterial pressure reduce right ventricular stroke volume and, if sufficiently large and prolonged, result in right ventricular failure. Right ventricular ischemia plays a prominent role in these effects. The risk of right ventricular ischemia is also heightened during chronic elevations in right ventricular afterload because microvascular growth fails to match myocyte hypertrophy and because microvascular dysfunction is present. The right coronary circulation is more sensitive than the left to α-adrenergic-mediated constriction, which may contribute to its greater propensity for coronary vasospasm. This characteristic of the right coronary circulation may increase its vulnerability to coronary vasoconstriction and impaired right ventricular perfusion during administration of α-adrenergic receptor agonists.

Action Potential Shortening and Impairment of Cardiac Function by Ablation of Slc26a6.

PubMed

Sirish, Padmini; Ledford, Hannah A; Timofeyev, Valeriy; Thai, Phung N; Ren, Lu; Kim, Hyo Jeong; Park, Seojin; Lee, Jeong Han; Dai, Gu; Moshref, Maryam; Sihn, Choong-Ryoul; Chen, Wei Chun; Timofeyeva, Maria Valeryevna; Jian, Zhong; Shimkunas, Rafael; Izu, Leighton T; Chiamvimonvat, Nipavan; Chen-Izu, Ye; Yamoah, Ebenezer N; Zhang, Xiao-Dong

2017-10-01

Intracellular pH (pH i ) is critical to cardiac excitation and contraction; uncompensated changes in pH i impair cardiac function and trigger arrhythmia. Several ion transporters participate in cardiac pH i regulation. Our previous studies identified several isoforms of a solute carrier Slc26a6 to be highly expressed in cardiomyocytes. We show that Slc26a6 mediates electrogenic Cl - /HCO 3 - exchange activities in cardiomyocytes, suggesting the potential role of Slc26a6 in regulation of not only pH i , but also cardiac excitability. To test the mechanistic role of Slc26a6 in the heart, we took advantage of Slc26a6 knockout ( Slc26a6 -/ - ) mice using both in vivo and in vitro analyses. Consistent with our prediction of its electrogenic activities, ablation of Slc26a6 results in action potential shortening. There are reduced Ca 2+ transient and sarcoplasmic reticulum Ca 2+ load, together with decreased sarcomere shortening in Slc26a6 -/ - cardiomyocytes. These abnormalities translate into reduced fractional shortening and cardiac contractility at the in vivo level. Additionally, pH i is elevated in Slc26a6 -/ - cardiomyocytes with slower recovery kinetics from intracellular alkalization, consistent with the Cl - /HCO 3 - exchange activities of Slc26a6. Moreover, Slc26a6 -/ - mice show evidence of sinus bradycardia and fragmented QRS complex, supporting the critical role of Slc26a6 in cardiac conduction system. Our study provides mechanistic insights into Slc26a6, a unique cardiac electrogenic Cl - /HCO 3 - transporter in ventricular myocytes, linking the critical roles of Slc26a6 in regulation of pH i , excitability, and contractility. pH i is a critical regulator of other membrane and contractile proteins. Future studies are needed to investigate possible changes in these proteins in Slc26a6 -/ - mice. © 2017 American Heart Association, Inc.

Regulation of adult cardiocyte growth: effects of active and passive mechanical loading

NASA Technical Reports Server (NTRS)

Decker, M. L.; Janes, D. M.; Barclay, M. M.; Harger, L.; Decker, R. S.

1997-01-01

Fluctuations in hemodynamic load have been documented to modulate contractile protein turnover and myofibrillar structure in the heart; however, the relative importance of active and passive loading in regulating adult cardiocyte growth remains unresolved. To address this issue at the cellular level, adult feline cardiocytes were cultured either on Silastic membranes or plastic surfaces. Cardiocyte-laden membranes were stretched 10% of their rest length to enhance passive loading, whereas heart cells cultured on plastic or Silastic were field stimulated at 1 Hz to mimic active loading. Turnover of contractile proteins and structural integrity of the contractile-cytoskeletal apparatus were monitored for periods ranging from 4 to 72 h. Active and passive loading elevated contractile protein synthesis nearly equally (approximately 50%) and promoted the attachment of remodeled myofibrils to vinculin-positive focal contacts and/or costameres during the first 24 h of loading. Thereafter, rates of contractile protein synthesis returned to control values in passively stretched heart cells but remained elevated in field-stimulated cultures. The fractional rate of growth was increased significantly (approximately 8%/day) in electrically paced cells, whereas in passively stretched cardiocytes the growth rate rose only modestly (approximately 2%/day). Changes in the rate of myocyte growth appeared more closely correlated with the development of focal contacts and myofibril remodeling than with changes in myofibrillar protein turnover per se. 2,3-Butanedione monoxime, nifedipine, and, to a lesser extent, ryanodine blocked field-stimulated contractile protein synthesis and myofibrillar remodeling but had no impact on protein turnover or myofibril reassembly in passively loaded cardiocytes. The results of these experiments imply that both active and passive loading stimulate contractile protein turnover and myofibril remodeling, but the generation of active tension accelerates cardiocyte growth to a greater extent than passive loading. Furthermore, pharmacological interventions suggest that unique pathways may mediate these cellular events in actively and passively loaded adult cardiocytes.

Regulation of adult cardiocyte growth: effects of active and passive mechanical loading.

PubMed

Decker, M L; Janes, D M; Barclay, M M; Harger, L; Decker, R S

1997-06-01

Fluctuations in hemodynamic load have been documented to modulate contractile protein turnover and myofibrillar structure in the heart; however, the relative importance of active and passive loading in regulating adult cardiocyte growth remains unresolved. To address this issue at the cellular level, adult feline cardiocytes were cultured either on Silastic membranes or plastic surfaces. Cardiocyte-laden membranes were stretched 10% of their rest length to enhance passive loading, whereas heart cells cultured on plastic or Silastic were field stimulated at 1 Hz to mimic active loading. Turnover of contractile proteins and structural integrity of the contractile-cytoskeletal apparatus were monitored for periods ranging from 4 to 72 h. Active and passive loading elevated contractile protein synthesis nearly equally (approximately 50%) and promoted the attachment of remodeled myofibrils to vinculin-positive focal contacts and/or costameres during the first 24 h of loading. Thereafter, rates of contractile protein synthesis returned to control values in passively stretched heart cells but remained elevated in field-stimulated cultures. The fractional rate of growth was increased significantly (approximately 8%/day) in electrically paced cells, whereas in passively stretched cardiocytes the growth rate rose only modestly (approximately 2%/day). Changes in the rate of myocyte growth appeared more closely correlated with the development of focal contacts and myofibril remodeling than with changes in myofibrillar protein turnover per se. 2,3-Butanedione monoxime, nifedipine, and, to a lesser extent, ryanodine blocked field-stimulated contractile protein synthesis and myofibrillar remodeling but had no impact on protein turnover or myofibril reassembly in passively loaded cardiocytes. The results of these experiments imply that both active and passive loading stimulate contractile protein turnover and myofibril remodeling, but the generation of active tension accelerates cardiocyte growth to a greater extent than passive loading. Furthermore, pharmacological interventions suggest that unique pathways may mediate these cellular events in actively and passively loaded adult cardiocytes.

The Ku Protein Complex Interacts with YY1, Is Up-Regulated in Human Heart Failure, and Represses α Myosin Heavy-Chain Gene Expression

PubMed Central

Sucharov, Carmen C.; Helmke, Steve M.; Langer, Stephen J.; Perryman, M. Benjamin; Bristow, Michael; Leinwand, Leslie

2004-01-01

Human heart failure is accompanied by repression of genes such as α myosin heavy chain (αMyHC) and SERCA2A and the induction of fetal genes such as βMyHC and atrial natriuretic factor. It seems likely that changes in MyHC isoforms contribute to the poor contractility seen in heart failure, because small changes in isoform composition can have a major effect on the contractility of cardiac myocytes and the heart. Our laboratory has recently shown that YY1 protein levels are increased in human heart failure and that YY1 represses the activity of the human αMyHC promoter. We have now identified a region of the αMyHC promoter that binds a factor whose expression is increased sixfold in failing human hearts. Through peptide mass spectrometry, we identified this binding activity to be a heterodimer of Ku70 and Ku80. Expression of Ku represses the human αMyHC promoter in neonatal rat ventricular myocytes. Moreover, overexpression of Ku70/80 decreases αMyHC mRNA expression and increases skeletal α-actin. Interestingly, YY1 interacts with Ku70 and Ku80 in HeLa cells. Together, YY1, Ku70, and Ku80 repress the αMyHC promoter to an extent that is greater than that with YY1 or Ku70/80 alone. Our results suggest that Ku is an important factor in the repression of the human αMyHC promoter during heart failure. PMID:15367688

Hypertrophic Stimulation Increases β-actin Dynamics in Adult Feline Cardiomyocytes

PubMed Central

Balasubramanian, Sundaravadivel; Mani, Santhosh K.; Kasiganesan, Harinath; Baicu, Catalin C.; Kuppuswamy, Dhandapani

2010-01-01

The myocardium responds to hemodynamic stress through cellular growth and organ hypertrophy. The impact of cytoskeletal elements on this process, however, is not fully understood. While α-actin in cardiomyocytes governs muscle contraction in combination with the myosin motor, the exact role of β-actin has not been established. We hypothesized that in adult cardiomyocytes, as in non-myocytes, β-actin can facilitate cytoskeletal rearrangement within cytoskeletal structures such as Z-discs. Using a feline right ventricular pressure overload (RVPO) model, we measured the level and distribution of β-actin in normal and pressure overloaded myocardium. Resulting data demonstrated enriched levels of β-actin and enhanced translocation to the Triton-insoluble cytoskeletal and membrane skeletal complexes. In addition, RVPO in vivo and in vitro hypertrophic stimulation with endothelin (ET) or insulin in isolated adult cardiomyocytes enhanced the content of polymerized fraction (F-actin) of β-actin. To determine the localization and dynamics of β-actin, we adenovirally expressed GFP-tagged β-actin in isolated adult cardiomyocytes. The ectopically expressed β-actin-GFP localized to the Z-discs, costameres, and cell termini. Fluorescence recovery after photobleaching (FRAP) measurements of β-actin dynamics revealed that β-actin at the Z-discs is constantly being exchanged with β-actin from cytoplasmic pools and that this exchange is faster upon hypertrophic stimulation with ET or insulin. In addition, in electrically stimulated isolated adult cardiomyocytes, while β-actin overexpression improved cardiomyocyte contractility, immunoneutralization of β-actin resulted in a reduced contractility suggesting that β-actin could be important for the contractile function of adult cardiomyocytes. These studies demonstrate the presence and dynamics of β-actin in the adult cardiomyocyte and reinforce its usefulness in measuring cardiac cytoskeletal rearrangement during hypertrophic stimulation. PMID:20635003

Hypertrophic stimulation increases beta-actin dynamics in adult feline cardiomyocytes.

PubMed

Balasubramanian, Sundaravadivel; Mani, Santhosh K; Kasiganesan, Harinath; Baicu, Catalin C; Kuppuswamy, Dhandapani

2010-07-12

The myocardium responds to hemodynamic stress through cellular growth and organ hypertrophy. The impact of cytoskeletal elements on this process, however, is not fully understood. While alpha-actin in cardiomyocytes governs muscle contraction in combination with the myosin motor, the exact role of beta-actin has not been established. We hypothesized that in adult cardiomyocytes, as in non-myocytes, beta-actin can facilitate cytoskeletal rearrangement within cytoskeletal structures such as Z-discs. Using a feline right ventricular pressure overload (RVPO) model, we measured the level and distribution of beta-actin in normal and pressure overloaded myocardium. Resulting data demonstrated enriched levels of beta-actin and enhanced translocation to the Triton-insoluble cytoskeletal and membrane skeletal complexes. In addition, RVPO in vivo and in vitro hypertrophic stimulation with endothelin (ET) or insulin in isolated adult cardiomyocytes enhanced the content of polymerized fraction (F-actin) of beta-actin. To determine the localization and dynamics of beta-actin, we adenovirally expressed GFP-tagged beta-actin in isolated adult cardiomyocytes. The ectopically expressed beta-actin-GFP localized to the Z-discs, costameres, and cell termini. Fluorescence recovery after photobleaching (FRAP) measurements of beta-actin dynamics revealed that beta-actin at the Z-discs is constantly being exchanged with beta-actin from cytoplasmic pools and that this exchange is faster upon hypertrophic stimulation with ET or insulin. In addition, in electrically stimulated isolated adult cardiomyocytes, while beta-actin overexpression improved cardiomyocyte contractility, immunoneutralization of beta-actin resulted in a reduced contractility suggesting that beta-actin could be important for the contractile function of adult cardiomyocytes. These studies demonstrate the presence and dynamics of beta-actin in the adult cardiomyocyte and reinforce its usefulness in measuring cardiac cytoskeletal rearrangement during hypertrophic stimulation.

Temporal changes in expression of connexin 43 after load-induced hypertrophy in vitro.

PubMed

Bupha-Intr, Tepmanas; Haizlip, Kaylan M; Janssen, Paul M L

2009-03-01

Upon remodeling of the ventricle after a provoking stimulus, such as hypertension, connections between adjacent myocytes may need to be "reformatted" to preserve a synchronization of excitation of the remodeling heart. In the mammalian heart, the protein connexin forms the gap junctions that allow electrical and chemical signaling communication between neighboring cells. We aim to elucidate whether mechanical load, in isolation, potentially changes the expression of connexin 43 (Cx43), the major isoform of the connexin family in the ventricle, and its phosphorylation. Cx43 expression levels and contractile function of multicellular rabbit cardiac preparations were assessed in a newly developed in vitro system that allows for the study of the transition of healthy multicellular rabbit myocardium to hypertrophied myocardium. We found that in mechanically loaded cardiac trabeculae, Cx43 levels remained stable for about 12 h and then rapidly declined. Phosphorylation at Ser368 declined much faster, being almost absent after 2 h of high-load conditions. No-load conditions did not affect Cx43 levels, nor did phosphorylation at Ser368. The downregulation of Cx43 under mechanical load did not correspond with the contractile changes that were observed. Furthermore, blocking paracrine activity of the muscle could only partially prevent the downregulation of Cx43. Additionally, no effect of mechanical loading on the expression of N-cadherin and zonula occludens-1 was observed, indicating a specificity of the connexin response. High mechanical load induced a rapid loss of Cx43 phosphorylation, followed by a decrease in Cx43 protein levels. Paracrine factors are partly responsible for the underlying mechanism of action, whereas no direct correlation to contractile ability was observed.

Temporal changes in expression of connexin 43 after load-induced hypertrophy in vitro

PubMed Central

Bupha-Intr, Tepmanas; Haizlip, Kaylan M.; Janssen, Paul M. L.

2009-01-01

Upon remodeling of the ventricle after a provoking stimulus, such as hypertension, connections between adjacent myocytes may need to be “reformatted” to preserve a synchronization of excitation of the remodeling heart. In the mammalian heart, the protein connexin forms the gap junctions that allow electrical and chemical signaling communication between neighboring cells. We aim to elucidate whether mechanical load, in isolation, potentially changes the expression of connexin 43 (Cx43), the major isoform of the connexin family in the ventricle, and its phosphorylation. Cx43 expression levels and contractile function of multicellular rabbit cardiac preparations were assessed in a newly developed in vitro system that allows for the study of the transition of healthy multicellular rabbit myocardium to hypertrophied myocardium. We found that in mechanically loaded cardiac trabeculae, Cx43 levels remained stable for about 12 h and then rapidly declined. Phosphorylation at Ser368 declined much faster, being almost absent after 2 h of high-load conditions. No-load conditions did not affect Cx43 levels, nor did phosphorylation at Ser368. The downregulation of Cx43 under mechanical load did not correspond with the contractile changes that were observed. Furthermore, blocking paracrine activity of the muscle could only partially prevent the downregulation of Cx43. Additionally, no effect of mechanical loading on the expression of N-cadherin and zonula occludens-1 was observed, indicating a specificity of the connexin response. High mechanical load induced a rapid loss of Cx43 phosphorylation, followed by a decrease in Cx43 protein levels. Paracrine factors are partly responsible for the underlying mechanism of action, whereas no direct correlation to contractile ability was observed. PMID:19136602

[Image processing applying in analysis of motion features of cultured cardiac myocyte in rat].

PubMed

Teng, Qizhi; He, Xiaohai; Luo, Daisheng; Wang, Zhengrong; Zhou, Beiyi; Yuan, Zhirun; Tao, Dachang

2007-02-01

Study of mechanism of medicine actions, by quantitative analysis of cultured cardiac myocyte, is one of the cutting edge researches in myocyte dynamics and molecular biology. The characteristics of cardiac myocyte auto-beating without external stimulation make the research sense. Research of the morphology and cardiac myocyte motion using image analysis can reveal the fundamental mechanism of medical actions, increase the accuracy of medicine filtering, and design the optimal formula of medicine for best medical treatments. A system of hardware and software has been built with complete sets of functions including living cardiac myocyte image acquisition, image processing, motion image analysis, and image recognition. In this paper, theories and approaches are introduced for analysis of living cardiac myocyte motion images and implementing quantitative analysis of cardiac myocyte features. A motion estimation algorithm is used for motion vector detection of particular points and amplitude and frequency detection of a cardiac myocyte. Beatings of cardiac myocytes are sometimes very small. In such case, it is difficult to detect the motion vectors from the particular points in a time sequence of images. For this reason, an image correlation theory is employed to detect the beating frequencies. Active contour algorithm in terms of energy function is proposed to approximate the boundary and detect the changes of edge of myocyte.

Effects of acetylcholine and noradrenalin on action potentials of isolated rabbit sinoatrial and atrial myocytes.

PubMed

Verkerk, Arie O; Geuzebroek, Guillaume S C; Veldkamp, Marieke W; Wilders, Ronald

2012-01-01

The autonomic nervous system controls heart rate and contractility through sympathetic and parasympathetic inputs to the cardiac tissue, with acetylcholine (ACh) and noradrenalin (NA) as the chemical transmitters. In recent years, it has become clear that specific Regulators of G protein Signaling proteins (RGS proteins) suppress muscarinic sensitivity and parasympathetic tone, identifying RGS proteins as intriguing potential therapeutic targets. In the present study, we have identified the effects of 1 μM ACh and 1 μM NA on the intrinsic action potentials of sinoatrial (SA) nodal and atrial myocytes. Single cells were enzymatically isolated from the SA node or from the left atrium of rabbit hearts. Action potentials were recorded using the amphotericin-perforated patch-clamp technique in the absence and presence of ACh, NA, or a combination of both. In SA nodal myocytes, ACh increased cycle length and decreased diastolic depolarization rate, whereas NA decreased cycle length and increased diastolic depolarization rate. Both ACh and NA increased maximum upstroke velocity. Furthermore, ACh hyperpolarized the maximum diastolic potential. In atrial myocytes stimulated at 2 Hz, both ACh and NA hyperpolarized the maximum diastolic potential, increased the action potential amplitude, and increased the maximum upstroke velocity. Action potential duration at 50 and 90% repolarization was decreased by ACh, but increased by NA. The effects of both ACh and NA on action potential duration showed a dose dependence in the range of 1-1000 nM, while a clear-cut frequency dependence in the range of 1-4 Hz was absent. Intermediate results were obtained in the combined presence of ACh and NA in both SA nodal and atrial myocytes. Our data uncover the extent to which SA nodal and atrial action potentials are intrinsically dependent on ACh, NA, or a combination of both and may thus guide further experiments with RGS proteins.

Effects of Acetylcholine and Noradrenalin on Action Potentials of Isolated Rabbit Sinoatrial and Atrial Myocytes

PubMed Central

Verkerk, Arie O.; Geuzebroek, Guillaume S. C.; Veldkamp, Marieke W.; Wilders, Ronald

2012-01-01

The autonomic nervous system controls heart rate and contractility through sympathetic and parasympathetic inputs to the cardiac tissue, with acetylcholine (ACh) and noradrenalin (NA) as the chemical transmitters. In recent years, it has become clear that specific Regulators of G protein Signaling proteins (RGS proteins) suppress muscarinic sensitivity and parasympathetic tone, identifying RGS proteins as intriguing potential therapeutic targets. In the present study, we have identified the effects of 1 μM ACh and 1 μM NA on the intrinsic action potentials of sinoatrial (SA) nodal and atrial myocytes. Single cells were enzymatically isolated from the SA node or from the left atrium of rabbit hearts. Action potentials were recorded using the amphotericin-perforated patch-clamp technique in the absence and presence of ACh, NA, or a combination of both. In SA nodal myocytes, ACh increased cycle length and decreased diastolic depolarization rate, whereas NA decreased cycle length and increased diastolic depolarization rate. Both ACh and NA increased maximum upstroke velocity. Furthermore, ACh hyperpolarized the maximum diastolic potential. In atrial myocytes stimulated at 2 Hz, both ACh and NA hyperpolarized the maximum diastolic potential, increased the action potential amplitude, and increased the maximum upstroke velocity. Action potential duration at 50 and 90% repolarization was decreased by ACh, but increased by NA. The effects of both ACh and NA on action potential duration showed a dose dependence in the range of 1–1000 nM, while a clear-cut frequency dependence in the range of 1–4 Hz was absent. Intermediate results were obtained in the combined presence of ACh and NA in both SA nodal and atrial myocytes. Our data uncover the extent to which SA nodal and atrial action potentials are intrinsically dependent on ACh, NA, or a combination of both and may thus guide further experiments with RGS proteins. PMID:22754533

An Elevation in Physical Coupling of Type 1 IP3 Receptors to TRPC3 Channels Constricts Mesenteric Arteries in Genetic Hypertension

PubMed Central

Adebiyi, Adebowale; Thomas-Gatewood, Candice M.; Leo, M. Dennis; Kidd, Michael W.; Neeb, Zachary P.; Jaggar, Jonathan H.

2013-01-01

Hypertension is associated with an elevation in agonist-induced vasoconstriction, but mechanisms involved require further investigation. Many vasoconstrictors bind to phospholipase C-coupled receptors, leading to an elevation in inositol 1,4,5-trisphosphate (IP3) that activates sarcoplasmic reticulum (SR) IP3 receptors (IP3Rs). In cerebral artery myocytes, IP3Rs release SR Ca2+ and can physically couple to canonical transient receptor potential 3 (TRPC3) channels in a caveolin-1-containing macromolecular complex, leading to cation current (ICat) activation that stimulates vasoconstriction. Here, we investigated mechanisms by which IP3Rs control vascular contractility in systemic arteries and IP3R involvement in elevated agonist-induced vasoconstriction during hypertension. Total and plasma membrane-localized TRPC3 protein was ~2.7- and 2-fold higher in mesenteric arteries of hypertensive spontaneously hypertensive rats (SHR) than in Wistar-Kyoto (WKY) rat controls, respectively. In contrast, IP3R1, TRPC1, TRPC6, and caveolin-1 expression was similar. TRPC3 expression was also similar in arteries of pre-hypertensive SHR and WKY rats. Control, IP3- and endothelin-1 (ET-1)-induced FRET between IP3R1 and TRPC3 was higher in hypertensive SHR than WKY myocytes. IP3-induced ICat was ~3-fold larger in SHR myocytes. Pyr3, a selective TRPC3 channel blocker, and CIRBP-TAT, an IP3R-TRP physical coupling inhibitor, reduced IP3-induced ICat and ET-1-induced vasoconstriction more in SHR than WKY myocytes and arteries. Thapsigargin, a SR Ca2+-ATPase blocker, did not alter ET-1-stimulated vasoconstriction in SHR or WKY arteries. These data indicate that ET-1 stimulates physical coupling of IP3R1 to TRPC3 channels in mesenteric artery myocytes, leading to vasoconstriction. Furthermore, an elevation in IP3R1 to TRPC3 channel molecular coupling augments ET-1-induced vasoconstriction during hypertension. PMID:23045459

Chromium (D-phenylalanine)3 improves obesity-induced cardiac contractile defect in ob/ob mice.

PubMed

Dong, Feng; Yang, Xiaoping; Sreejayan, Nair; Ren, Jun

2007-11-01

Low-molecular weight chromium compounds, such as chromium picolinate [Cr(pic)(3)], improve insulin sensitivity, although toxicity is a concern. We synthesized a novel chromium complex, chromium (d-phenylalanine)(3) [Cr(d-phe)(3)], in an attempt to improve insulin sensitivity with reduced toxicity. The aim of this study was to compare the two chromium compounds on cardiac contractile function in ob/ob obese mice. C57BL lean and ob/ob obese mice were randomly divided into three groups: H(2)O, Cr(d-phe)(3), or Cr(pic)(3) (45 mug/kg per day orally for 6 months). The glucose tolerance test displayed improved glucose clearance by Cr(d-phe)(3) but not Cr(pic)(3). Myocytes from ob/ob mice exhibited depressed peak shortening (PS) and maximal velocity of shortening/relengthening (+/-dL/dt), prolonged time-to-PS and time-to-90% relengthening (TR90), reduced electrically stimulated rise in intracellular Ca(2+) (Deltafura-2 fluorescence intensity), and slowed intracellular Ca(2+) decay. Although a 3-month Cr(d-phe)(3) treatment for a separate group of ob/ob and lean 2-month-old mice only rectified reduced +/-dL/dt in ob/ob mice, all mechanical and intracellular Ca(2+) abnormalities were significantly attenuated or ablated by 6 months of Cr(d-phe)(3) but not Cr(pic)(3) treatment (except TR90). Sarco(endo)plasmic reticulum Ca(2+) ATPase activity and Na(+)-Ca(2+) exchanger expression were depressed in ob/ob mice, which were reversed by both Cr(d-phe)(3) and Cr(pic)(3), with a more pronounced effect from Cr(d-phe)(3). Cr(d-phe)(3) corrected reduced insulin-stimulated glucose uptake and improved basal phosphorylation of Akt and insulin receptor, as well as insulin-stimulated phosphorylation of Akt and insulin receptor in ob/ob myocytes. Heart homogenates from ob/ob mice had enhanced oxidative stress and protein carbonyl formation compared with the lean group, which were attenuated by both Cr(d-phe)(3) and Cr(pic)(3). Our data suggest that the new Cr(d-phe)(3) compound possesses better cardio-protective and insulin-sensitizing properties against obesity.

Cardiomyocyte-enriched protein CIP protects against pathophysiological stresses and regulates cardiac homeostasis.

PubMed

Huang, Zhan-Peng; Kataoka, Masaharu; Chen, Jinghai; Wu, Gengze; Ding, Jian; Nie, Mao; Lin, Zhiqiang; Liu, Jianming; Hu, Xiaoyun; Ma, Lixin; Zhou, Bin; Wakimoto, Hiroko; Zeng, Chunyu; Kyselovic, Jan; Deng, Zhong-Liang; Seidman, Christine E; Seidman, J G; Pu, William T; Wang, Da-Zhi

2015-11-02

Cardiomyopathy is a common human disorder that is characterized by contractile dysfunction and cardiac remodeling. Genetic mutations and altered expression of genes encoding many signaling molecules and contractile proteins are associated with cardiomyopathy; however, how cardiomyocytes sense pathophysiological stresses in order to then modulate cardiac remodeling remains poorly understood. Here, we have described a regulator in the heart that harmonizes the progression of cardiac hypertrophy and dilation. We determined that expression of the myocyte-enriched protein cardiac ISL1-interacting protein (CIP, also known as MLIP) is reduced in patients with dilated cardiomyopathy. As CIP is highly conserved between human and mouse, we evaluated the effects of CIP deficiency on cardiac remodeling in mice. Deletion of the CIP-encoding gene accelerated progress from hypertrophy to heart failure in several cardiomyopathy models. Conversely, transgenic and AAV-mediated CIP overexpression prevented pathologic remodeling and preserved cardiac function. CIP deficiency combined with lamin A/C deletion resulted in severe dilated cardiomyopathy and cardiac dysfunction in the absence of stress. Transcriptome analyses of CIP-deficient hearts revealed that the p53- and FOXO1-mediated gene networks related to homeostasis are disturbed upon pressure overload stress. Moreover, FOXO1 overexpression suppressed stress-induced cardiomyocyte hypertrophy in CIP-deficient cardiomyocytes. Our studies identify CIP as a key regulator of cardiomyopathy that has potential as a therapeutic target to attenuate heart failure progression.

Cardiomyocyte-enriched protein CIP protects against pathophysiological stresses and regulates cardiac homeostasis

PubMed Central

Huang, Zhan-Peng; Kataoka, Masaharu; Chen, Jinghai; Wu, Gengze; Ding, Jian; Nie, Mao; Lin, Zhiqiang; Liu, Jianming; Hu, Xiaoyun; Ma, Lixin; Zhou, Bin; Wakimoto, Hiroko; Zeng, Chunyu; Kyselovic, Jan; Deng, Zhong-Liang; Seidman, Christine E.; Seidman, J.G.; Pu, William T.; Wang, Da-Zhi

2015-01-01

Cardiomyopathy is a common human disorder that is characterized by contractile dysfunction and cardiac remodeling. Genetic mutations and altered expression of genes encoding many signaling molecules and contractile proteins are associated with cardiomyopathy; however, how cardiomyocytes sense pathophysiological stresses in order to then modulate cardiac remodeling remains poorly understood. Here, we have described a regulator in the heart that harmonizes the progression of cardiac hypertrophy and dilation. We determined that expression of the myocyte-enriched protein cardiac ISL1-interacting protein (CIP, also known as MLIP) is reduced in patients with dilated cardiomyopathy. As CIP is highly conserved between human and mouse, we evaluated the effects of CIP deficiency on cardiac remodeling in mice. Deletion of the CIP-encoding gene accelerated progress from hypertrophy to heart failure in several cardiomyopathy models. Conversely, transgenic and AAV-mediated CIP overexpression prevented pathologic remodeling and preserved cardiac function. CIP deficiency combined with lamin A/C deletion resulted in severe dilated cardiomyopathy and cardiac dysfunction in the absence of stress. Transcriptome analyses of CIP-deficient hearts revealed that the p53- and FOXO1-mediated gene networks related to homeostasis are disturbed upon pressure overload stress. Moreover, FOXO1 overexpression suppressed stress-induced cardiomyocyte hypertrophy in CIP-deficient cardiomyocytes. Our studies identify CIP as a key regulator of cardiomyopathy that has potential as a therapeutic target to attenuate heart failure progression. PMID:26436652

Increase in neurokinin-1 receptor-mediated colonic motor response in a rat model of irritable bowel syndrome.

PubMed

La, Jun-Ho; Kim, Tae-Wan; Sung, Tae-Sik; Kim, Hyn-Ju; Kim, Jeom-Yong; Yang, Il-Suk

2005-01-14

Irritable bowel syndrome (IBS) is a functional bowel disorder. Its major symptom is bowel dysmotility, yet the mechanism of the symptom is poorly understood. Since the neurokinin-1 receptor (NK1R)-mediated signaling in the gut is important in the control of normal bowel motor function, we aimed to investigate whether the NK1R-mediated bowel motor function was altered in IBS, using a rat IBS model that was previously reported to show colonic dysmotility in response to restraint stress. IBS symptoms were produced in male Sprague-Dawley rats by inducing colitis with acetic acid. Rats were left to recover from colitis for 6 d, and used for experiments 7 d post-induction of colitis. Motor activities of distal colon were recorded in vitro. The contractile sensitivity of isolated colon to a NK1R agonist (Sar9,Met(O2)11)-substance P (1-30 nmol/L) was higher in IBS rats than that in normal rats. After the enteric neurotransmission was blocked by tetrodotoxin (TTX, 1 micromol/L), the contractile sensitivity to the NK1R agonist was increased in normal colon but not in IBS rat colon. The NK1R agonist-induced contraction was not different between the two groups when the agonist was challenged to the TTX-treated colon or the isolated colonic myocytes. A nitric oxide synthase inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME, 100 micromol/L) augmented the NK1R agonist-induced contraction only in normal rat colon. These results suggest that the NK1R-meidated colonic motor response is increased in IBS rats, due to the decrease in the nitrergic inhibitory neural component.

Mechanical signaling coordinates the embryonic heart

NASA Astrophysics Data System (ADS)

Chiou, Kevin; Rocks, Jason; Prosser, Benjamin; Discher, Dennis; Liu, Andrea

The heart is an active material which relies on robust signaling mechanisms between cells in order to produce well-timed, coordinated beats. Heart tissue is composed primarily of active heart muscle cells (cardiomyocytes) embedded in a passive extracellular matrix. During a heartbeat, cardiomyocyte contractions are coordinated across the heart to form a wavefront that propagates through the tissue to pump blood. In the adult heart, this contractile wave is coordinated via intercellular electrical signaling.Here we present theoretical and experimental evidence for mechanical coordination of embryonic heartbeats. We model cardiomyocytes as mechanically excitable Eshelby inclusions embedded in an overdamped elastic-fluid biphasic medium. For physiological parameters, this model replicates recent experimental measurements of the contractile wavefront which are not captured by electrical signaling models. We additionally challenge our model by pharmacologically blocking gap junctions, inhibiting electrical signaling between myocytes. We find that while adult hearts stop beating almost immediately after gap junctions are blocked, embryonic hearts continue beating even at significantly higher concentrations, providing strong support for a mechanical signaling mechanism.

Probenecid: novel use as a non-injurious positive inotrope acting via cardiac TRPV2 stimulation.

PubMed

Koch, Sheryl E; Gao, Xiaoqian; Haar, Lauren; Jiang, Min; Lasko, Valerie M; Robbins, Nathan; Cai, Wenfeng; Brokamp, Cole; Varma, Priyanka; Tranter, Michael; Liu, Yong; Ren, Xiaoping; Lorenz, John N; Wang, Hong-Sheng; Jones, W Keith; Rubinstein, Jack

2012-07-01

Probenecid is a highly lipid soluble benzoic acid derivative originally used to increase serum antibiotic concentrations. It was later discovered to have uricosuric effects and was FDA approved for gout therapy. It has recently been found to be a potent agonist of transient receptor potential vanilloid 2 (TRPV2). We have shown that this receptor is in the cardiomyocyte and report a positive inotropic effect of the drug. Using echocardiography, Langendorff and isolated myocytes, we measured the change in contractility and, using TRPV2(-/-) mice, proved that the effect was mediated by TRPV2 channels in the cardiomyocytes. Analysis of the expression of Ca(2+) handling and β-adrenergic signaling pathway proteins showed that the contractility was not increased through activation of the β-ADR. We propose that the response to probenecid is due to activation of TRPV2 channels secondary to SR release of Ca(2+). Copyright © 2012 Elsevier Ltd. All rights reserved.

Synthesis and Chemical and Biological Comparison of Nitroxyl and Nitric Oxide Releasing Diazeniumdiolate-based Aspirin Derivatives

PubMed Central

Basudhar, Debashree; Bharadwaj, Gaurav; Cheng, Robert Y.; Jain, Sarthak; Shi, Sa; Heinecke, Julie L.; Holland, Ryan J.; Ridnour, Lisa A.; Caceres, Viviane M.; Spadari-Bratfisch, Regina C.; Paolocci, Nazareno; Velázquez-Martínez, Carlos A.; Wink, David A.; Miranda, Katrina M.

2013-01-01

Structural modifications of non-steroidal anti-inflammatory drugs (NSAIDs) have successfully reduced the side effect of gastrointestinal ulceration without affecting anti-inflammatory activity, but may increase risk of myocardial infarction with chronic use. That nitroxyl (HNO) reduces platelet aggregation, preconditions against myocardial infarction and enhances contractility led us to synthesize a diazeniumdiolate-based HNO releasing aspirin and to compare it to an NO-releasing analogue. Here, the decomposition mechanisms are described for these compounds. In addition to protection against stomach ulceration, these prodrugs also exhibited significantly enhanced cytotoxcity compared to either aspirin or the parent diazeniumdiolate toward non-small cell lung carcinoma cells (A549) but were not appreciably toxic toward endothelial cells (HUVECs). The HNO-NSAID prodrug inhibited cylcooxgenase-2 and glyceraldehyde 3-phosphate dehydrogenase activity and triggered significant sarcomere shortening compared to control on murine ventricular myocytes. Together, these anti-inflammatory, anti-neoplasic and contractile properties suggest the potential of HNO-NSAIDs in the treatment of inflammation, cancer or heart failure. PMID:24102516

Some growth factors stimulate cultured adult rabbit ventricular myocyte hypertrophy in the absence of mechanical loading

NASA Technical Reports Server (NTRS)

Decker, R. S.; Cook, M. G.; Behnke-Barclay, M.; Decker, M. L.

1995-01-01

Cultured adult rabbit cardiac myocytes treated with recombinant growth factors display enhanced rates of protein accumulation (ie, growth) in response to insulin and insulin-like growth factors (IGFs), but epidermal growth factor, acidic or basic fibroblast growth factor, and platelet-derived growth factor failed to increase contractile protein synthesis or growth of the heart cells. Insulin and IGF-1 increased growth rates by stimulating anabolic while simultaneously inhibiting catabolic pathways, whereas IGF-2 elevated growth modestly by apparently inhibiting lysosomal proteolysis. Neutralizing antibodies directed against either IGF-1 or IGF-2 or IGF binding protein 3 blocked protein accumulation. A monoclonal antibody directed against the IGF-1 receptor also inhibited changes in protein turnover provoked by recombinant human IGF-1 but not IGF-2. Of the other growth factors tested, only transforming growth factor-beta 1 increased the fractional rate of myosin heavy chain (MHC) synthesis, with beta-MHC synthesis being elevated and alpha-MHC synthesis being suppressed. However, the other growth factors were able to modestly stimulate the rate of DNA synthesis in this preparation. Bromodeoxyuridine labeling revealed that these growth factors increased DNA synthesis in myocytes and nonmyocytes alike, but the heart cells displayed neither karyokinesis or cytokinesis. In contrast, cocultures of cardiac myocytes and nonmyocytes and nonmyocyte-conditioned culture medium failed to enhance the rate of cardiac MHC synthesis or its accumulation, implying that quiescent heart cells do not respond to "conditioning" by cardiac nonmyocytes. These findings demonstrated that insulin and the IGFs promote passively loaded cultured adult rabbit heart cells to hypertrophy but suggest that other growth factors tested may be limited in this regard.

Urocortin2 prolongs action potential duration and modulates potassium currents in guinea pig myocytes and HEK293 cells.

PubMed

Yang, Li-Zhen; Zhu, Yi-Chun

2015-07-05

We previously reported that activation of corticotropin releasing factor receptor type 2 by urocortin2 up-regulates both L-type Ca(2+) channels and intracellular Ca(2+) concentration in ventricular myocytes and plays an important role in cardiac contractility and arrhythmogenesis. This study goal was to further test the hypothesis that urocortin2 may modulate action potentials as well as rapidly and slowly activating delayed rectifier potassium currents. With whole cell patch-clamp techniques, action potentials and slowly activating delayed rectifier potassium currents were recorded in isolated guinea pig ventricular myocytes, respectively. And rapidly activating delayed rectifier potassium currents were tested in hERG-HEK293 cells. Urocortin2 produced a time- and concentration-dependent prolongation of action potential duration. The EC50 values of action potential duration and action potential duration at 90% of repolarization were 14.73 and 24.3nM respectively. The prolongation of action potential duration of urocortin2 was almost completely or partly abolished by H-89 (protein kinase A inhibitor) or KB-R7943 (Na(+)/Ca(2+) exchange inhibitor) pretreatment respectively. And urocortin2 caused reduction of rapidly activating delayed rectifier potassium currents in hERG-HEK293 cells. In addition, urocortin2 slowed the rate of slowly activating delayed rectifier potassium channel activation, and rightward shifted the threshold of slowly activating delayed rectifier potassium currents to more positive potentials. Urocortin2 prolonged action potential duration via activation of protein kinase A and Na(+)/ Ca(2+) exchange in isolated guinea pig ventricular myocytes in a time- and concentration- dependent manner. In hERG-HEK293 cells, urocortin2 reduced rapidly activating delayed rectifier potassium current density which may contribute to action potential duration prolongation. Copyright © 2015 Elsevier B.V. All rights reserved.

β-Adrenergic Regulation of Cardiac Progenitor Cell Death Versus Survival and Proliferation

PubMed Central

Khan, Mohsin; Mohsin, Sadia; Avitabile, Daniele; Siddiqi, Sailay; Nguyen, Jonathan; Wallach, Kathleen; Quijada, Pearl; McGregor, Michael; Gude, Natalie; Alvarez, Roberto; Tilley, Douglas G.; Koch, Walter J.; Sussman, Mark A.

2013-01-01

Rationale Short-term β-adrenergic stimulation promotes contractility in response to stress but is ultimately detrimental in the failing heart because of accrual of cardiomyocyte death. Endogenous cardiac progenitor cell (CPC) activation may partially offset cardiomyocyte losses, but consequences of long-term β-adrenergic drive on CPC survival and proliferation are unknown. Objective We sought to determine the relationship between β-adrenergic activity and regulation of CPC function. Methods and Results Mouse and human CPCs express only β2 adrenergic receptor (β2-AR) in conjunction with stem cell marker c-kit. Activation of β2-AR signaling promotes proliferation associated with increased AKT, extracellular signal-regulated kinase 1/2, and endothelial NO synthase phosphorylation, upregulation of cyclin D1, and decreased levels of G protein–coupled receptor kinase 2. Conversely, silencing of β2-AR expression or treatment with β2-antagonist ICI 118, 551 impairs CPC proliferation and survival. β1-AR expression in CPC is induced by differentiation stimuli, sensitizing CPC to isoproterenol-induced cell death that is abrogated by metoprolol. Efficacy of β1-AR blockade by metoprolol to increase CPC survival and proliferation was confirmed in vivo by adoptive transfer of CPC into failing mouse myocardium. Conclusions β-adrenergic stimulation promotes expansion and survival of CPCs through β2-AR, but acquisition of β1-AR on commitment to the myocyte lineage results in loss of CPCs and early myocyte precursors. PMID:23243208

The Permeability Transition Pore Controls Cardiac Mitochondrial Maturation and Myocyte Differentiation

PubMed Central

Hom, Jennifer R.; Quintanilla, Rodrigo A.; Hoffman, David L.; Karen L., de Mesy Bentley; Molkentin, Jeffery D.; Sheu, Shey-Shing; Porter, George A.

2011-01-01

SUMMARY Although mature myocytes rely on mitochondria as the primary source of energy, the role of mitochondria in the developing heart is not well known. Here, we find closure of the mitochondrial permeability transition pore (mPTP) drives maturation of mitochondrial structure and function and myocyte differentiation. Cardiomyocytes at embryonic day (E) 9.5, when compared to E13.5, displayed fragmented mitochondria with few cristae, a less polarized mitochondrial membrane potential, higher reactive oxygen species (ROS) levels, and an open mPTP. Pharmacologic and genetic closing of the mPTP yielded maturation of mitochondrial structure and function, lowered ROS, and increased myocyte differentiation (measured by counting Z-bands). Furthermore, myocyte differentiation was inhibited and enhanced with oxidant and antioxidant treatment, respectively, suggesting that redox signaling pathways lie downstream of mitochondria to regulate cardiac myocyte differentiation. PMID:21920313

Andrographis paniculata extract protect against isoproterenol-induced myocardial injury by mitigating cardiac dysfunction and oxidative injury in rats.

PubMed

Ojha, Shreesh; Bharti, Saurabh; Golechha, Mahaveer; Sharma, Ashok K; Rani, Neha; Kumari, Santosh; Arya, Dharamvir Singh

2012-01-01

Present study evaluated the cardioprotective effect of Andrographis paniculata (100, 200 or 400 mg/kg) against isoproterenol (85 mg/kg, b.w.)-induced cardiotoxicity referred as myocardial infarction in rats. Isoproterenol significantly (p < 0.05) decreased mean arterial pressure, heart rate, contractility and relaxation and increased left ventricular end diastolic pressure. Isoproterenol also significantly (p < 0.05) decreased antioxidants, superoxide dismutase, catalase, glutathione peroxidase, glutathione and increased leakage of cardiac injury markers; creatine phosphokinase-MB isoenzyme, lactate dehydrogenase concomitant to increased lipid peroxidation and histopathological perturbations. However, pretreatment with A. paniculata favorably restored hemodynamic parameters and left ventricular function and significantly (p < 0.05) prevented the depletion of endogenous antioxidants and myocyte marker enzymes as well as inhibited lipid peroxidation. Significant (p < 0.05) reversal of almost all the hemodynamic, biochemical and histopathological parameters by A. paniculata pretreatment in isoproterenol-induced cardiotoxicity depicted the cardioprotective effect of A. paniculata. Results showed that A. paniculata protected heart against cardiotoxic effects of isoproterenol by boosting endogenous antioxidant network, restoring ventricular function and maintaining structural integrity of heart.

Histomorphologic changes of esophageal mucosa in experimental third degree stricture.

PubMed

Shaprynskyi, Volodymyr O; Shaprinskiy, Yevgeniy V; Karyi, Yaroslav V; Lysenko, Serhii A

Nowadays the level of early and late complications after the operations for esophageal corrosive strictures such as esophago-organ anastomotic leak, development of infections, pneumonia, pleural empyema, mediastinitis, peritonitis, postoperative corrosive stricture development etc. remains rather high. Besides, postoperative mortality rate is high as well - 3.5-30 %. For that reason, an experimental model of esophageal stricture was suggested and ultrastructural mucosal changes in the stricture itself were studied to elaborate the unified pathogenic approach in treatment of esophageal stricture and improvement of its results. The aim of our work was to study the dynamics of ultrastructural changes both in normal esophageal walls and in third degree esophageal stricture Materials and Methods: The experiment was carried out on white male rats weighting 250-300 grams, to whom the third degree esophageal stricture model was created. After layer-by-layer incision of anterior abdominal wall abdominal portion of the esophagus was completely ligated (10 rats). In the control group (6 rats) anterior abdominal wall was opened with its subsequent layered closure. The animals were withdrawn from the experiment on the third day by ketamine overdose, and the samples were taken for ultrastructural study. Electron microscopic study of submicroscopic organization of basal, prickle, superficial epithelial cells in stratified non-squamous epithelium, smooth myocytes of muscle plate and contractile elements in esophageal muscular layer was carried out. Nuclear membrane, membranes of mitochondria, endoplasmic reticulum and cytoplasmic Golgi complex were found to be subjected to focal lysis. The third degree esophageal stricture caused destructive lesions in ultrastructural architectonics of stratified non-squamous epithelium cells, smooth myocytes of muscle plate and contractile elements in esophageal muscular layer of rats. Thus, catabolic processes leading to organelle disintegration develop in esophageal cells of rats with third degree stricture.

Creation of a genetic calcium channel blocker by targeted gem gene transfer in the heart.

PubMed

Murata, Mitsushige; Cingolani, Eugenio; McDonald, Amy D; Donahue, J Kevin; Marbán, Eduardo

2004-08-20

Calcium channel blockers are among the most commonly used therapeutic drugs. Nevertheless, the utility of calcium channel blockers for heart disease is limited because of the potent vasodilatory effect that causes hypotension, and other side effects attributable to blockade of noncardiac channels. Therefore, focal calcium channel blockade by gene transfer is highly desirable. With a view to creating a focally applicable genetic calcium channel blocker, we overexpressed the ras-related small G-protein Gem in the heart by somatic gene transfer. Adenovirus-mediated delivery of Gem markedly decreased L-type calcium current density in ventricular myocytes, resulting in the abbreviation of action potential duration. Furthermore, transduction of Gem resulted in a significant shortening of the electrocardiographic QTc interval and reduction of left ventricular systolic function. Focal delivery of Gem to the atrioventricular (AV) node significantly slowed AV nodal conduction (prolongation of PR and AH intervals), which was effective in the reduction of heart rate during atrial fibrillation. Thus, these results indicate that gene transfer of Gem functions as a genetic calcium channel blocker, the local application of which can effectively modulate cardiac electrical and contractile function.

Phagocyte-Myocyte Interactions and Consequences during Hypoxic Wound Healing

PubMed Central

Zhang, Shuang; Dehn, Shirley; DeBerge, Matthew; Rhee, KJ; Hudson, Barry; Thorp, Edward

2014-01-01

Myocardial infarction (MI), secondary to atherosclerotic plaque rupture and occlusive thrombi, triggers acute margination of inflammatory neutrophils and monocyte phagocyte subsets to the damaged heart, the latter of which may give rise briefly to differentiated macrophage-like or dendritic-like cells. Within the injured myocardium, a primary function of these phagocytic cells is to remove damaged extracellular matrix, necrotic and apoptotic cardiac cells, as well as immune cells that turn over. Recognition of dying cellular targets by phagocytes triggers intracellular signaling, particularly in macrophages, wherein cytokines and lipid mediators are generated to promote inflammation resolution, fibrotic scarring, angiogenesis, and compensatory organ remodeling. These actions cooperate in an effort to preserve myocardial contractility and prevent heart failure. Immune cell function is modulated by local tissue factors that include secreted protease activity, oxidative stress during clinical reperfusion, and hypoxia. Importantly, experimental evidence suggests that monocyte function and phagocytosis efficiency is compromised in the setting of MI risk factors, including hyperlipidemia and ageing, however underlying mechanisms remain unclear. Herein we review seminal phagocyte and cardiac molecular factors that lead to, and culminate in, the recognition and removal of dying injured myocardium, the effects of hypoxia, and their relationship to cardiac infarct size and heart healing. PMID:24862542

Amphiphile-induced heart muscle-cell (myocyte) injury: effects of intracellular fatty acid overload.

PubMed

Janero, D R; Burghardt, C; Feldman, D

1988-10-01

Lipid amphiphile toxicity may be an important contributor to myocardial injury, especially during ischemia/reperfusion. In order to investigate directly the potential biochemical and metabolic effects of amphiphile overload on the functioning heart muscle cell (myocyte), a novel model of nonesterified fatty acid (NEFA)-induced myocyte damage has been defined. The model uses intact, beating neonatal rat myocytes in primary monolayer culture as a study object and 5-(tetradecyloxy)-2-furoic acid (TOFA) as a nonmetabolizable fatty acid. Myocytes incubated with TOFA accumulated it as NEFA, and the consequent NEFA amphiphile overload elicited a variety of cellular defects (including decreased beating rate, depletion of high-energy stores and glycogen pools, and breakdown of myocyte membrane phospholipid) and culminated in cell death. The amphiphile-induced cellular pathology could be reversed by removing TOFA from the culture medium, which resulted in intracellular TOFA "wash-out." Although the development and severity of amphiphile-induced myocyte injury could be correlated with both the intracellular TOFA/NEFA content (i.e., the level of TOFA to which the cells were exposed) and the duration of this exposure, removal of amphiphile overload did not inevitably lead to myocyte recovery. TOFA had adverse effects on myocyte mitochondrial function in situ (decoupling of oxidative phosphorylation, impairing respiratory control) and on myocyte oxidative catabolism (transiently increasing fatty acid beta oxidation, citric acid cycle flux, and glucose oxidation). The amphiphile-induced bioenergetic abnormalities appeared to constitute a state of "metabolic anoxia" underlying the progression of myocyte injury to cell death. This anoxic state could be ameliorated to some extent, but not prevented, by carbohydrate catabolism.

S-nitrosothiols and the S-nitrosoproteome of the cardiovascular system.

PubMed

Maron, Bradley A; Tang, Shiow-Shih; Loscalzo, Joseph

2013-01-20

Since their discovery in the early 1990's, S-nitrosylated proteins have been increasingly recognized as important determinants of many biochemical processes. Specifically, S-nitrosothiols in the cardiovascular system exert many actions, including promoting vasodilation, inhibiting platelet aggregation, and regulating Ca(2+) channel function that influences myocyte contractility and electrophysiologic stability. Contemporary developments in liquid chromatography-mass spectrometry methods, the development of biotin- and His-tag switch assays, and the availability of cyanide dye-labeling for S-nitrosothiol detection in vitro have increased significantly the identification of a number of cardiovascular protein targets of S-nitrosylation in vivo. Recent analyses using modern S-nitrosothiol detection techniques have revealed the mechanistic significance of S-nitrosylation to the pathophysiology of numerous cardiovascular diseases, including essential hypertension, pulmonary hypertension, ischemic heart disease, stroke, and congestive heart failure, among others. Despite enhanced insight into S-nitrosothiol biochemistry, translating these advances into beneficial pharmacotherapies for patients with cardiovascular diseases remains a primary as-yet unmet goal for investigators within the field.

Cardiomyocytes from phorbol myristate acetate-activated mesenchymal stem cells restore electromechanical function in infarcted rat hearts

PubMed Central

Song, Heesang; Hwang, Hye Jin; Chang, Woochul; Song, Byeong-Wook; Cha, Min-Ji; Lim, Soyeon; Choi, Eun Ju; Ham, Onju; Lee, Chang Youn; Park, Jun-Hee; Lee, Se-Yeon; Choi, Eunmi; Lee, Chungkeun; Lee, Myoungho; Lee, Moon-Hyoung; Kim, Sung-Hou; Jang, Yangsoo; Hwang, Ki-Chul

2011-01-01

Despite the safety and feasibility of mesenchymal stem cell (MSC) therapy, an optimal cell type has not yet emerged in terms of electromechanical integration in infarcted myocardium. We found that poor to moderate survival benefits of MSC-implanted rats were caused by incomplete electromechanical integration induced by tissue heterogeneity between myocytes and engrafted MSCs in the infarcted myocardium. Here, we report the development of cardiogenic cells from rat MSCs activated by phorbol myristate acetate, a PKC activator, that exhibited high expressions of cardiac-specific markers and Ca2+ homeostasis-related proteins and showed adrenergic receptor signaling by norepinephrine. Histological analysis showed high connexin 43 coupling, few inflammatory cells, and low fibrotic markers in myocardium implanted with these phorbol myristate acetate-activated MSCs. Infarct hearts implanted with these cells exhibited restoration of conduction velocity through decreased tissue heterogeneity and improved myocardial contractility. These findings have major implications for the development of better cell types for electromechanical integration of cell-based treatment for infarcted myocardium. PMID:21173226

Redox modification of ryanodine receptors by mitochondria-derived reactive oxygen species contributes to aberrant Ca2+ handling in ageing rabbit hearts

PubMed Central

Cooper, Leroy L; Li, Weiyan; Lu, Yichun; Centracchio, Jason; Terentyeva, Radmila; Koren, Gideon; Terentyev, Dmitry

2013-01-01

Ageing is associated with a blunted response to sympathetic stimulation and an increased risk of arrhythmia and sudden cardiac death. Aberrant calcium (Ca2+) handling is an important contributor to the electrical and contractile dysfunction associated with ageing. Yet, the specific molecular mechanisms underlying abnormal Ca2+ handling in ageing heart remain poorly understood. In this study, we used ventricular myocytes isolated from young (5–9 months) and old (4–6 years) rabbit hearts to test the hypothesis that changes in Ca2+ homeostasis are caused by post-translational modification of ryanodine receptors (RyRs) by mitochondria-derived reactive oxygen species (ROS) generated in the ageing heart. Changes in parameters of Ca2+ handling were determined by measuring cytosolic and intra-sarcoplasmic reticulum (SR) Ca2+ dynamics in intact and permeabilized ventricular myocytes using confocal microscopy. We also measured age-related changes in ROS production and mitochondria membrane potential using a ROS-sensitive dye and a mitochondrial voltage-sensitive fluorescent indicator, respectively. In permeablized myocytes, ageing did not change SERCA activity and spark frequency but decreased spark amplitude and SR Ca2+ load suggesting increased RyR activity. Treatment with the antioxidant dithiothreitol reduced RyR-mediated SR Ca2+ leak in permeabilized myocytes from old rabbit hearts to the level comparable to young. Moreover, myocytes from old rabbits had more depolarized mitochondria membrane potential and increased rate of ROS production. Under β-adrenergic stimulation, Ca2+ transient amplitude, SR Ca2+ load, and latency of pro-arrhythmic spontaneous Ca2+ waves (SCWs) were decreased while RyR-mediated SR Ca2+ leak was increased in cardiomyocytes from old rabbits. Additionally, with β-adrenergic stimulation, scavenging of mitochondrial ROS in myocytes from old rabbit hearts restored redox status of RyRs, which reduced SR Ca2+ leak, ablated most SCWs, and increased latency to levels comparable to young. These data indicate that an age-associated increase of ROS production by mitochondria leads to the thiol-oxidation of RyRs, which underlies the hyperactivity of RyRs and thereby shortened refractoriness of Ca2+ release in cardiomyocytes from the ageing heart. This mechanism probably plays an important role in the increased incidence of arrhythmia and sudden death in the ageing population. PMID:24042501

Altered in vivo left ventricular torsion and principal strains in hypothyroid rats

PubMed Central

Chen, Yong; Somji, Aleefia; Yu, Xin

2010-01-01

The twisting and untwisting motions of the left ventricle (LV) lead to efficient ejection of blood during systole and filling of the ventricle during diastole. Global LV mechanical performance is dependent on the contractile properties of cardiac myocytes; however, it is not known how changes in contractile protein expression affect the pattern and timing of LV rotation. At the myofilament level, contractile performance is largely dependent on the isoforms of myosin heavy chain (MHC) that are expressed. Therefore, in this study, we used MRI to examine the in vivo mechanical consequences of altered MHC isoform expression by comparing the contractile properties of hypothyroid rats, which expressed only the slow β-MHC isoform, and euthyroid rats, which predominantly expressed the fast α-MHC isoform. Unloaded shortening velocity (Vo) and apparent rate constants of force development (ktr) were measured in the skinned ventricular myocardium isolated from euthyroid and hypothyroid hearts. Increased expression of β-MHC reduced LV torsion and fiber strain and delayed the development of peak torsion and strain during systole. Depressed in vivo mechanical performance in hypothyroid rats was related to slowed cross-bridge performance, as indicated by significantly slower Vo and ktr, compared with euthyroid rats. Dobutamine infusion in hypothyroid hearts produced smaller increases in torsion and strain and aberrant transmural torsion patterns, suggesting that the myocardial response to β-adrenergic stress is compromised. Thus, increased expression of β-MHC alters the pattern and decreases the magnitude of LV rotation, contributing to reduced mechanical performance during systole, especially in conditions of increased workload. PMID:20729398

Decrease in coronary vascular volume in systole augments cardiac contraction.

PubMed

Willemsen, M J; Duncker, D J; Krams, R; Dijkman, M A; Lamberts, R R; Sipkema, P; Westerhof, N

2001-08-01

Coronary arterial inflow is impeded and venous outflow is increased as a result of the decrease in coronary vascular volume due to cardiac contraction. We evaluated whether cardiac contraction is influenced by interfering with the changes of the coronary vascular volume over the heart cycle. Length-tension relationships were determined in Tyrode-perfused rat papillary muscle and when coronary vascular volume changes were partly inhibited by filling it with congealed gelatin or perfusing it with a high viscosity dextran buffer. Also, myocyte thickening during contraction was reduced by placing a silicon tube around the muscle. Increasing perfusion pressure from 8 to 80 cmH2O, increased developed tension by approximately 40%. When compared with the low perfusion state, developed tension of the gelatin-filled vasculature was reduced to 43 +/- 6% at the muscle length where the muscle generates the largest developed tension (n = 5, means +/- SE). Dextran reduced developed tension to 73 +/- 6% (n = 6). The silicon tube, in low perfusion state, reduced the developed tension to 83 +/- 7% (n = 4) of control. Time-control and oxygen-lowering experiments show that the findings are based on mechanical effects. Thus interventions to prevent myocyte thickening reduce developed tension. We hypothesize that when myocyte thickening is prevented, intracellular pressure increases and counteracts the force produced by the contractile apparatus. We conclude that emptying of the coronary vasculature serves a physiological purpose by facilitating cardiomyocyte thickening thereby augmenting force development.

The Effect of Substrate Stiffness on Cardiomyocyte Action Potentials.

PubMed

Boothe, Sean D; Myers, Jackson D; Pok, Seokwon; Sun, Junping; Xi, Yutao; Nieto, Raymond M; Cheng, Jie; Jacot, Jeffrey G

2016-12-01

The stiffness of myocardial tissue changes significantly at birth and during neonatal development, concurrent with significant changes in contractile and electrical maturation of cardiomyocytes. Previous studies by our group have shown that cardiomyocytes generate maximum contractile force when cultured on a substrate with a stiffness approximating native cardiac tissue. However, effects of substrate stiffness on the electrophysiology and ion currents in cardiomyocytes have not been fully characterized. In this study, neonatal rat ventricular myocytes were cultured on the surface of flat polyacrylamide hydrogels with elastic moduli ranging from 1 to 25 kPa. Using whole-cell patch clamping, action potentials and L-type calcium currents were recorded. Cardiomyocytes cultured on hydrogels with a 9 kPa elastic modulus, similar to that of native myocardium, had the longest action potential duration. Additionally, the voltage at maximum calcium flux significantly decreased in cardiomyocytes on hydrogels with an elastic modulus higher than 9 kPa, and the mean inactivation voltage decreased with increasing stiffness. Interestingly, the expression of the L-type calcium channel subunit α gene and channel localization did not change with stiffness. Substrate stiffness significantly affects action potential length and calcium flux in cultured neonatal rat cardiomyocytes in a manner that may be unrelated to calcium channel expression. These results may explain functional differences in cardiomyocytes resulting from changes in the elastic modulus of the extracellular matrix, as observed during embryonic development, in ischemic regions of the heart after myocardial infarction, and during dilated cardiomyopathy.

Myomaker mediates fusion of fast myocytes in zebrafish embryos

DOE Office of Scientific and Technical Information (OSTI.GOV)

Landemaine, Aurélie; Rescan, Pierre-Yves; Gabillard, Jean-Charles, E-mail: Jean-charles.gabillard@rennes.inra.fr

2014-09-05

Highlights: • Myomaker is transiently expressed in fast myocytes during embryonic myogenesis. • Myomaker is essential for fast myocyte fusion in zebrafish. • The function of myomaker is conserved among Teleostomi. - Abstract: Myomaker (also called Tmem8c), a new membrane activator of myocyte fusion was recently discovered in mice. Using whole mount in situ hybridization on zebrafish embryos at different stages of embryonic development, we show that myomaker is transiently expressed in fast myocytes forming the bulk of zebrafish myotome. Zebrafish embryos injected with morpholino targeted against myomaker were alive after yolk resorption and appeared morphologically normal, but they weremore » unable to swim, even under effect of a tactile stimulation. Confocal observations showed a marked phenotype characterized by the persistence of mononucleated muscle cells in the fast myotome at developmental stages where these cells normally fuse to form multinucleated myotubes. This indicates that myomaker is essential for myocyte fusion in zebrafish. Thus, there is an evolutionary conservation of myomaker expression and function among Teleostomi.« less

Inhibition of glycogen synthase kinase 3beta during heart failure is protective.

PubMed

Hirotani, Shinichi; Zhai, Peiyong; Tomita, Hideharu; Galeotti, Jonathan; Marquez, Juan Pablo; Gao, Shumin; Hong, Chull; Yatani, Atsuko; Avila, Jesús; Sadoshima, Junichi

2007-11-26

Glycogen synthase kinase (GSK)-3, a negative regulator of cardiac hypertrophy, is inactivated in failing hearts. To examine the histopathological and functional consequence of the persistent inhibition of GSK-3beta in the heart in vivo, we generated transgenic mice with cardiac-specific overexpression of dominant negative GSK-3beta (Tg-GSK-3beta-DN) and tetracycline-regulatable wild-type GSK-3beta. GSK-3beta-DN significantly reduced the kinase activity of endogenous GSK-3beta, inhibited phosphorylation of eukaryotic translation initiation factor 2B epsilon, and induced accumulation of beta-catenin and myeloid cell leukemia-1, confirming that GSK-3beta-DN acts as a dominant negative in vivo. Tg-GSK-3beta-DN exhibited concentric hypertrophy at baseline, accompanied by upregulation of the alpha-myosin heavy chain gene and increases in cardiac function, as evidenced by a significantly greater Emax after dobutamine infusion and percentage of contraction in isolated cardiac myocytes, indicating that inhibition of GSK-3beta induces well-compensated hypertrophy. Although transverse aortic constriction induced a similar increase in hypertrophy in both Tg-GSK-3beta-DN and nontransgenic mice, Tg-GSK-3beta-DN exhibited better left ventricular function and less fibrosis and apoptosis than nontransgenic mice. Induction of the GSK-3beta transgene in tetracycline-regulatable wild-type GSK-3beta mice induced left ventricular dysfunction and premature death, accompanied by increases in apoptosis and fibrosis. Overexpression of GSK-3beta-DN in cardiac myocytes inhibited tumor necrosis factor-alpha-induced apoptosis, and the antiapoptotic effect of GSK-3beta-DN was abrogated in the absence of myeloid cell leukemia-1. These results suggest that persistent inhibition of GSK-3beta induces compensatory hypertrophy, inhibits apoptosis and fibrosis, and increases cardiac contractility and that the antiapoptotic effect of GSK-3beta inhibition is mediated by myeloid cell leukemia-1. Thus, downregulation of GSK-3beta during heart failure could be compensatory.

Rapid frequency‐dependent changes in free mitochondrial calcium concentration in rat cardiac myocytes

PubMed Central

Wüst, Rob C. I.; Helmes, Michiel; Martin, Jody L.; van der Wardt, Thomas J. T.; Musters, René J. P.; van der Velden, Jolanda

2017-01-01

Key points Calcium ions regulate mitochondrial ATP production and contractile activity and thus play a pivotal role in matching energy supply and demand in cardiac muscle.The magnitude and kinetics of the changes in free mitochondrial calcium concentration in cardiac myocytes are largely unknown.Rapid stimulation frequency‐dependent increases but relatively slow decreases in free mitochondrial calcium concentration were observed in rat cardiac myocytes. This asymmetry caused a rise in the mitochondrial calcium concentration with stimulation frequency.These results provide insight into the mechanisms of mitochondrial calcium uptake and release that are important in healthy and diseased myocardium. Abstract Calcium ions regulate mitochondrial ATP production and contractile activity and thus play a pivotal role in matching energy supply and demand in cardiac muscle. Little is known about the magnitude and kinetics of the changes in free mitochondrial calcium concentration in cardiomyocytes. Using adenoviral infection, a ratiometric mitochondrially targeted Förster resonance energy transfer (FRET)‐based calcium indicator (4mtD3cpv, MitoCam) was expressed in cultured adult rat cardiomyocytes and the free mitochondrial calcium concentration ([Ca2+]m) was measured at different stimulation frequencies (0.1–4 Hz) and external calcium concentrations (1.8–3.6 mm) at 37°C. Cytosolic calcium concentrations were assessed under the same experimental conditions in separate experiments using Fura‐4AM. The increases in [Ca2+]m during electrical stimulation at 0.1 Hz were rapid (rise time = 49 ± 2 ms), while the decreases in [Ca2+]m occurred more slowly (decay half time = 1.17 ± 0.07 s). Model calculations confirmed that this asymmetry caused the rise in [Ca2+]m during diastole observed at elevated stimulation frequencies. Inhibition of the mitochondrial sodium–calcium exchanger (mNCE) resulted in a rise in [Ca2+]m at baseline and, paradoxically, in an acceleration of Ca2+ release. In conclusion: rapid increases in [Ca2+]m allow for fast adjustment of mitochondrial ATP production to increases in myocardial demand on a beat‐to‐beat basis and mitochondrial calcium release depends on mNCE activity and mitochondrial calcium buffering. PMID:28028811

An elevation in physical coupling of type 1 inositol 1,4,5-trisphosphate (IP3) receptors to transient receptor potential 3 (TRPC3) channels constricts mesenteric arteries in genetic hypertension.

PubMed

Adebiyi, Adebowale; Thomas-Gatewood, Candice M; Leo, M Dennis; Kidd, Michael W; Neeb, Zachary P; Jaggar, Jonathan H

2012-11-01

Hypertension is associated with an elevation in agonist-induced vasoconstriction, but mechanisms involved require further investigation. Many vasoconstrictors bind to phospholipase C-coupled receptors, leading to an elevation in inositol 1,4,5-trisphosphate (IP(3)) that activates sarcoplasmic reticulum IP(3) receptors. In cerebral artery myocytes, IP(3) receptors release sarcoplasmic reticulum Ca(2+) and can physically couple to canonical transient receptor potential 3 (TRPC3) channels in a caveolin-1-containing macromolecular complex, leading to cation current activation that stimulates vasoconstriction. Here, we investigated mechanisms by which IP(3) receptors control vascular contractility in systemic arteries and IP(3)R involvement in elevated agonist-induced vasoconstriction during hypertension. Total and plasma membrane-localized TRPC3 protein was ≈2.7- and 2-fold higher in mesenteric arteries of spontaneously hypertensive rats (SHRs) than in Wistar-Kyoto (WKY) rat controls, respectively. In contrast, IP(3)R1, TRPC1, TRPC6, and caveolin-1 expression was similar. TRPC3 expression was also similar in arteries of pre-SHRs and WKY rats. Control, IP(3)-induced and endothelin-1 (ET-1)-induced fluorescence resonance energy transfer between IP3R1 and TRPC3 was higher in SHR than WKY myocytes. IP3-induced cation current was ≈3-fold larger in SHR myocytes. Pyr3, a selective TRPC3 channel blocker, and calmodulin and IP(3) receptor binding domain peptide, an IP(3)R-TRP physical coupling inhibitor, reduced IP(3)-induced cation current and ET-1-induced vasoconstriction more in SHR than WKY myocytes and arteries. Thapsigargin, a sarcoplasmic reticulum Ca(2+)-ATPase blocker, did not alter ET-1-stimulated vasoconstriction in SHR or WKY arteries. These data indicate that ET-1 stimulates physical coupling of IP(3)R1 to TRPC3 channels in mesenteric artery myocytes, leading to vasoconstriction. Furthermore, an elevation in IP(3)R1 to TRPC3 channel molecular coupling augments ET-1-induced vasoconstriction during hypertension.

Functional subcellular distribution of β1- and β2-adrenergic receptors in rat ventricular cardiac myocytes

PubMed Central

Cros, Caroline; Brette, Fabien

2013-01-01

β-adrenergic stimulation is a key regulator of cardiac function. The localization of major cardiac adrenergic receptors (β1 and β2) has been investigated using biochemical and biophysical approaches and has led to contradictory results. This study investigates the functional subcellular localization of β1- and β2-adrenergic receptors in rat ventricular myocytes using a physiological approach. Ventricular myocytes were isolated from the hearts of rat and detubulated using formamide. Physiological cardiac function was measured as Ca2+ transient using Fura-2-AM and cell shortening. Selective activation of β1- and β2-adrenergic receptors was induced with isoproterenol (0.1 μmol/L) and ICI-118,551 (0.1 μmol/L); and with salbutamol (10 μmol/L) and atenolol (1 μmol/L), respectively. β1- and β2-adrenergic stimulations induced a significant increase in Ca2+ transient amplitude and cell shortening in intact rat ventricular myocytes (i.e., surface sarcolemma and t-tubules) and in detubulated cells (depleted from t-tubules, surface sarcolemma only). Both β1- and β2-adrenergic receptors stimulation caused a greater effect on Ca2+ transient and cell shortening in detubulated myocytes than in control myocytes. Quantitative analysis indicates that β1-adrenergic stimulation is ∼3 times more effective at surface sarcolemma compared to t-tubules, whereas β2- adrenergic stimulation occurs almost exclusively at surface sarcolemma (∼100 times more effective). These physiological data demonstrate that in rat ventricular myocytes, β1-adrenergic receptors are functionally present at surface sarcolemma and t-tubules, while β2-adrenergic receptors stimulation occurs only at surface sarcolemma of cardiac cells. PMID:24303124

α4-Integrin Mediates Neutrophil-Induced Free Radical Injury to Cardiac Myocytes

PubMed Central

Poon, Betty Y.; Ward, Christopher A.; Cooper, Conan B.; Giles, Wayne R.; Burns, Alan R.; Kubes, Paul

2001-01-01

Previous work has demonstrated that circulating neutrophils (polymorphonuclear leukocytes [PMNs]) adhere to cardiac myocytes via β2-integrins and cause cellular injury via the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase enzyme system. Since PMNs induced to leave the vasculature (emigrated PMNs) express the α4-integrin, we asked whether (a) these PMNs also induce myocyte injury via NADPH oxidase; (b) β2-integrins (CD18) still signal oxidant production, or if this process is now coupled to the α4-integrin; and (c) dysfunction is superoxide dependent within the myocyte or at the myocyte–PMN interface. Emigrated PMNs exposed to cardiac myocytes quickly induced significant changes in myocyte function. Myocyte shortening was decreased by 30–50% and rates of contraction and relaxation were reduced by 30% within the first 10 min. Both α4-integrin antibody (Ab)-treated PMNs and NADPH oxidase–deficient PMNs were unable to reduce myocyte shortening. An increased level of oxidative stress was detected in myocytes within 5 min of PMN adhesion. Addition of an anti–α4-integrin Ab, but not an anti-CD18 Ab, prevented oxidant production, suggesting that in emigrated PMNs the NADPH oxidase system is uncoupled from CD18 and can be activated via the α4-integrin. Addition of exogenous superoxide dismutase (SOD) inhibited all parameters of dysfunction measured, whereas overexpression of intracellular SOD within the myocytes did not inhibit the oxidative stress or the myocyte dysfunction caused by the emigrated PMNs. These findings demonstrate that profound molecular changes occur within PMNs as they emigrate, such that CD18 and associated intracellular signaling pathways leading to oxidant production are uncoupled and newly expressed α4-integrin functions as the ligand that signals oxidant production. The results also provide pathological relevance as the emigrated PMNs have the capacity to injure cardiac myocytes through the α4-integrin–coupled NADPH oxidase pathway that can be inhibited by extracellular, but not intracellular SOD. PMID:11238444

Sympathetic neurons are a powerful driver of myocyte function in cardiovascular disease.

PubMed

Larsen, Hege E; Lefkimmiatis, Konstantinos; Paterson, David J

2016-12-14

Many therapeutic interventions in disease states of heightened cardiac sympathetic activity are targeted to the myocytes. However, emerging clinical data highlights a dominant role in disease progression by the neurons themselves. Here we describe a novel experimental model of the peripheral neuro-cardiac axis to study the neuron's ability to drive a myocyte cAMP phenotype. We employed a co-culture of neonatal ventricular myocytes and sympathetic stellate neurons from normal (WKY) and pro-hypertensive (SHR) rats that are sympathetically hyper-responsive and measured nicotine evoked cAMP responses in the myocytes using a fourth generation FRET cAMP sensor. We demonstrated the dominant role of neurons in driving the myocyte ß-adrenergic phenotype, where SHR cultures elicited heightened myocyte cAMP responses during neural activation. Moreover, cross-culturing healthy neurons onto diseased myocytes rescued the diseased cAMP response of the myocyte. Conversely, healthy myocytes developed a diseased cAMP response if diseased neurons were introduced. Our results provide evidence for a dominant role played by the neuron in driving the adrenergic phenotype seen in cardiovascular disease. We also highlight the potential of using healthy neurons to turn down the gain of neurotransmission, akin to a smart pre-synaptic ß-blocker.

From overload to failure: what happens inside the myocyte.

PubMed

Harding, S E; Davia, K; Davies, C H; del Monte, F; Money-Kyrle, A R; Poole-Wilson, P A

1998-08-01

To determine whether there is a defect in the surviving muscle cells of the failing human heart, studies have been performed on individual myocytes isolated from normal and failing human myocardium. Myocytes from the failing ventricle contract and relax more slowly, and have a reduced contraction amplitude at physiological (but not low) stimulation frequencies. Slow relaxation is seen irrespective of the aetiology of the heart disease studied, and is more pronounced in myocytes from hypertrophied ventricles. Myocytes from hypertrophied ventricles are larger than normal, but the relaxation deficit is independent of cell size. Beta-adrenoceptor desensitization is evident in myocytes and it varies according to the severity of disease and with the age of the patient. Action potentials are longer in myocytes from failing human heart, probably because of an alteration in K+ current density. Many of the functional changes identified in failing human myocardium are seen at the level of the single cardiac myocyte, which implies that pharmacological or genetic manipulation of surviving cells is a logical therapeutic strategy.

Phagocyte-myocyte interactions and consequences during hypoxic wound healing.

PubMed

Zhang, Shuang; Dehn, Shirley; DeBerge, Matthew; Rhee, Ki-Jong; Hudson, Barry; Thorp, Edward B

2014-01-01

Myocardial infarction (MI), secondary to atherosclerotic plaque rupture and occlusive thrombi, triggers acute margination of inflammatory neutrophils and monocyte phagocyte subsets to the damaged heart, the latter of which may give rise briefly to differentiated macrophage-like or dendritic-like cells. Within the injured myocardium, a primary function of these phagocytic cells is to remove damaged extracellular matrix, necrotic and apoptotic cardiac cells, as well as immune cells that turn over. Recognition of dying cellular targets by phagocytes triggers intracellular signaling, particularly in macrophages, wherein cytokines and lipid mediators are generated to promote inflammation resolution, fibrotic scarring, angiogenesis, and compensatory organ remodeling. These actions cooperate in an effort to preserve myocardial contractility and prevent heart failure. Immune cell function is modulated by local tissue factors that include secreted protease activity, oxidative stress during clinical reperfusion, and hypoxia. Importantly, experimental evidence suggests that monocyte function and phagocytosis efficiency is compromised in the setting of MI risk factors, including hyperlipidemia and ageing, however underlying mechanisms remain unclear. Herein we review seminal phagocyte and cardiac molecular factors that lead to, and culminate in, the recognition and removal of dying injured myocardium, the effects of hypoxia, and their relationship to cardiac infarct size and heart healing. Copyright © 2014 Elsevier Inc. All rights reserved.

Rate-dependent electrical, contractile and restitution properties of isolated left ventricular myocytes in guinea-pig hypertrophy.

PubMed

Davey, P; Bryant, S; Hart, G

2001-01-01

Left ventricular hypertrophy predisposes to sudden cardiac death (SCD) and studies of human SCD suggest that the antecedent heart rate (HR) is usually < 100 beats min(-1). This is surprising in view of the known association between adrenergic receptor stimulation and SCD which by itself would suggest that it is more likely to occur from high rather than low HR. We therefore hypothesized that there may be electrical or mechanical abnormalities present in myocytes isolated from animals with left ventricular hypertrophy that predispose to SCD at low stimulation frequencies but which may not be present at high HR. Mild left ventricular hypertrophy was induced in guinea-pigs by infra-renal aortic banding. Electrical and mechanical properties of isolated myocytes were studied at different stimulation frequencies between 0.1 and 3 Hz. Action potential duration (APD) is prolonged in hypertrophy at stimulation frequencies < 1 Hz but not at faster rates. Contraction size, time-to-peak contraction (TTPC) and half-relaxation time are greatly enhanced in hypertrophy at all frequencies between 0.1 and 3 Hz. Electrical (50.3 +/- 5.2 ms in hypertrophy and 78.4 +/- 12.1 ms in control, P < 0.03) and mechanical (205 +/- 16 ms for hypertrophy and 266 +/- 24 ms for control cells, P < 0.03) restitution time constants are quicker in hypertrophy. The finding of APD prolongation at low but not at high frequencies is consistent with the finding that SCD arises from low and not high HR. This data supports the role of abnormal repolarization in SCD.

Structural and Functional Maturation of Cardiomyocytes Derived from Human Pluripotent Stem Cells

PubMed Central

Lundy, Scott D.; Zhu, Wei-Zhong

2013-01-01

Despite preclinical studies demonstrating the functional benefit of transplanting human pluripotent stem cell-derived cardiomyocytes (PSC-CMs) into damaged myocardium, the ability of these immature cells to adopt a more adult-like cardiomyocyte (CM) phenotype remains uncertain. To address this issue, we tested the hypothesis that prolonged in vitro culture of human embryonic stem cell (hESC)- and human induced pluripotent stem cell (hiPSC)-derived CMs would result in the maturation of their structural and contractile properties to a more adult-like phenotype. Compared to their early-stage counterparts (PSC-CMs after 20–40 days of in vitro differentiation and culture), late-stage hESC-CMs and hiPSC-CMs (80–120 days) showed dramatic differences in morphology, including increased cell size and anisotropy, greater myofibril density and alignment, sarcomeres visible by bright-field microscopy, and a 10-fold increase in the fraction of multinucleated CMs. Ultrastructural analysis confirmed improvements in the myofibrillar density, alignment, and morphology. We measured the contractile performance of late-stage hESC-CMs and hiPSC-CMs and noted a doubling in shortening magnitude with slowed contraction kinetics compared to the early-stage cells. We then examined changes in the calcium-handling properties of these matured CMs and found an increase in calcium release and reuptake rates with no change in the maximum amplitude. Finally, we performed electrophysiological assessments in hESC-CMs and found that late-stage myocytes have hyperpolarized maximum diastolic potentials, increased action potential amplitudes, and faster upstroke velocities. To correlate these functional changes with gene expression, we performed qPCR and found a robust induction of the key cardiac structural markers, including β-myosin heavy chain and connexin-43, in late-stage hESC-CMs and hiPSC-CMs. These findings suggest that PSC-CMs are capable of slowly maturing to more closely resemble the phenotype of adult CMs and may eventually possess the potential to regenerate the lost myocardium with robust de novo force-producing tissue. PMID:23461462

Heart-Derived Stem Cells in Miniature Swine with Coronary Microembolization: Novel Ischemic Cardiomyopathy Model to Assess the Efficacy of Cell-Based Therapy

PubMed Central

Young, Rebeccah F.; Leiker, Merced M.; Suzuki, Takayuki

2016-01-01

A major problem in translating stem cell therapeutics is the difficulty of producing stable, long-term severe left ventricular (LV) dysfunction in a large animal model. For that purpose, extensive infarction was created in sinclair miniswine by injecting microspheres (1.5 × 106 microspheres, 45 μm diameter) in LAD. At 2 months after embolization, animals (n = 11) were randomized to receive allogeneic cardiosphere-derived cells derived from atrium (CDCs: 20 × 106, n = 5) or saline (untreated, n = 6). Four weeks after therapy myocardial function, myocyte proliferation (Ki67), mitosis (phosphor-Histone H3; pHH3), apoptosis, infarct size (TTC), myocyte nuclear density, and cell size were evaluated. CDCs injected into infarcted and remodeled remote myocardium (global infusion) increased regional function and global function contrasting no change in untreated animals. CDCs reduced infarct volume and stimulated Ki67 and pHH3 positive myocytes in infarct and remote regions. As a result, myocyte number (nuclear density) increased and myocyte cell diameter decreased in both infarct and remote regions. Coronary microembolization produces stable long-term ischemic cardiomyopathy. Global infusion of CDCs stimulates myocyte regeneration and improves left ventricular ejection fraction. Thus, global infusion of CDCs could become a new therapy to reverse LV dysfunction in patients with asymptomatic heart failure. PMID:27738436

Bacopa monnieri extract increases rat coronary flow and protects against myocardial ischemia/reperfusion injury.

PubMed

Srimachai, Sirintorn; Devaux, Sylvie; Demougeot, Celine; Kumphune, Sarawut; Ullrich, Nina D; Niggli, Ernst; Ingkaninan, Kornkanok; Kamkaew, Natakorn; Scholfield, C Norman; Tapechum, Sompol; Chootip, Krongkarn

2017-02-20

This study explored Bacopa monnieri, a medicinal Ayurvedic herb, as a cardioprotectant against ischemia/reperfusion injury using cardiac function and coronary flow as end-points. In normal isolated rat hearts, coronary flow, left ventricular developed pressure, heart rate, and functional recovery were measured using the Langendorff preparation. Hearts were perfused with either (i) Krebs-Henseleit (normal) solution, (control), or with 30, 100 μg/ml B. monnieri ethanolic extract (30 min), or (ii) with normal solution or extract for 10 min preceding no-perfusion ischemia (30 min) followed by reperfusion (30 min) with normal solution. Infarct volumes were measured by triphenyltetrazolium staining. L-type Ca 2+ -currents (I Ca, L ) were measured by whole-cell patching in HL-1 cells, a mouse atrial cardiomyocyte cell line. Cytotoxicity of B. monnieri was assessed in rat isolated ventricular myocytes by trypan blue exclusion. In normally perfused hearts, B. monnieri increased coronary flow by 63 ± 13% (30 μg/ml) and 216 ± 21% (100 μg/ml), compared to control (5 ± 3%) (n = 8-10, p < 0.001). B. monnieri treatment preceding ischemia/reperfusion improved left ventricular developed pressure by 84 ± 10% (30 μg/ml), 82 ± 10% (100 μg/ml) and 52 ± 6% (control) compared to pre- ischemia/reperfusion. Similarly, functional recovery showed a sustained increase. Moreover, B. monnieri (100 μg/ml) reduced the percentage of infarct size from 51 ± 2% (control) to 25 ± 2% (n = 6-8, p < 0.0001). B. monnieri (100 μg/ml) reduced I Ca, L by 63 ± 4% in HL-1 cells. Ventricular myocyte survival decreased at higher concentrations (50-1000 μg/ml) B. monnieri. B. monnieri improves myocardial function following ischemia/reperfusion injury through recovery of coronary blood flow, contractile force and decrease in infarct size. Thus this may lead to a novel cardioprotectant strategy.

Numerical analysis of the effect of T-tubule location on calcium transient in ventricular myocytes.

PubMed

George, Uduak Z; Wang, Jun; Yu, Zeyun

2014-01-01

Intracellular calcium (Ca2+) signaling in cardiac myocytes is vital for proper functioning of the heart. Understanding the intracellular Ca2+ dynamics would give an insight into the functions of normal and diseased hearts. In the current study, spatiotemporal Ca2+ dynamics is investigated in ventricular myocytes by considering Ca2+ release and re-uptake via sarcolemma and transverse tubules (T-tubules), Ca2+ diffusion and buffering in the cytosol, and the blockade of Ca2+ activities associated with the sarcoplasmic reticulum. This study is carried out using a three dimensional (3D) geometric model of a branch of T-tubule extracted from the electron microscopy (EM) images of a partial ventricular myocyte. Mathematical modeling is done by using a system of partial differential equations involving Ca2+, buffers, and membrane channels. Numerical simulation results suggest that a lack of T-tubule structure at the vicinity of the cell surface could increase the peak time of Ca2+ concentration in myocytes. The results also show that T-tubules and mobile buffers play an important role in the regulation of Ca2+ transient in ventricular myocytes.

S-Nitrosothiols and the S-Nitrosoproteome of the Cardiovascular System

PubMed Central

Maron, Bradley A.; Tang, Shiow-Shih

2013-01-01

Abstract Significance: Since their discovery in the early 1990's, S-nitrosylated proteins have been increasingly recognized as important determinants of many biochemical processes. Specifically, S-nitrosothiols in the cardiovascular system exert many actions, including promoting vasodilation, inhibiting platelet aggregation, and regulating Ca2+ channel function that influences myocyte contractility and electrophysiologic stability. Recent Advances: Contemporary developments in liquid chromatography–mass spectrometry methods, the development of biotin- and His-tag switch assays, and the availability of cyanide dye-labeling for S-nitrosothiol detection in vitro have increased significantly the identification of a number of cardiovascular protein targets of S-nitrosylation in vivo. Critical Issues: Recent analyses using modern S-nitrosothiol detection techniques have revealed the mechanistic significance of S-nitrosylation to the pathophysiology of numerous cardiovascular diseases, including essential hypertension, pulmonary hypertension, ischemic heart disease, stroke, and congestive heart failure, among others. Future Directions: Despite enhanced insight into S-nitrosothiol biochemistry, translating these advances into beneficial pharmacotherapies for patients with cardiovascular diseases remains a primary as-yet unmet goal for investigators within the field. Antioxid. Redox Signal. 18, 270–287. PMID:22770551

Ranolazine improves cardiac diastolic dysfunction through modulation of myofilament calcium sensitivity

PubMed Central

Lovelock, Joshua D.; Monasky, Michelle M.; Jeong, Euy-Myoung; Lardin, Harvey A.; Liu, Hong; Patel, Bindiya G.; Taglieri, Domenico M.; Gu, Lianzhi; Kumar, Praveen; Pokhrel, Narayan; Zeng, Dewan; Belardinelli, Luiz; Sorescu, Dan; Solaro, R. John; Dudley, Samuel C.

2012-01-01

Rationale Previously, we demonstrated that a deoxycorticosterone acetate (DOCA)-salt hypertensive mouse model produces cardiac oxidative stress and diastolic dysfunction with preserved systolic function. Oxidative stress has been shown to increase late inward sodium current (INa), reducing the net cytosolic Ca2+ efflux. Objective Oxidative stress in the DOCA-salt model may increase late INa resulting in diastolic dysfunction amenable to treatment with ranolazine. Methods and Results Echocardiography detected evidence of diastolic dysfunction in hypertensive mice that improved after treatment with ranolazine (E/E′, sham 31.9 ± 2.8, sham+ranolazine 30.2 ± 1.9, DOCA-salt 41.8 ± 2.6, and DOCA-salt+ranolazine 31.9 ± 2.6, p = 0.018). The end diastolic pressure volume relationship slope was elevated in DOCA-salt mice, improving to sham levels with treatment (sham 0.16 ± 0.01 vs. sham+ranolazine 0.18 ± 0.01 vs. DOCA-salt 0.23 ± 0.2 vs. DOCA-salt+ranolazine 0.17 ± 0.01 mm Hg/L, p < 0.005). DOCA-salt myocytes demonstrated impaired relaxation, τ, improving with ranolazine (DOCA-salt 0.18 ± 0.02, DOCA-salt + ranolazine 0.13 ± 0.01, Sham 0.11 ± 0.01, Sham + ranolazine 0.09 ± 0.02 s, p = 0.0004). Neither late INa nor the Ca2+ transients were different from sham myocytes. Detergent extracted fiber bundles from DOCA-salt hearts demonstrated increased myofilament response to Ca2+ with glutathionylation of myosin binding protein C. Treatment with ranolazine ameliorated the Ca2+ response and cross-bridge kinetics. Conclusions Therefore, diastolic dysfunction could be reversed by ranolazine, likely resulting from a direct effect on myofilaments, indicating that cardiac oxidative stress may mediate diastolic dysfunction through altering the contractile apparatus. PMID:22343711

Ranolazine improves cardiac diastolic dysfunction through modulation of myofilament calcium sensitivity.

PubMed

Lovelock, Joshua D; Monasky, Michelle M; Jeong, Euy-Myoung; Lardin, Harvey A; Liu, Hong; Patel, Bindiya G; Taglieri, Domenico M; Gu, Lianzhi; Kumar, Praveen; Pokhrel, Narayan; Zeng, Dewan; Belardinelli, Luiz; Sorescu, Dan; Solaro, R John; Dudley, Samuel C

2012-03-16

Previously, we demonstrated that a deoxycorticosterone acetate (DOCA)-salt hypertensive mouse model produces cardiac oxidative stress and diastolic dysfunction with preserved systolic function. Oxidative stress has been shown to increase late inward sodium current (I(Na)), reducing the net cytosolic Ca(2+) efflux. Oxidative stress in the DOCA-salt model may increase late I(Na), resulting in diastolic dysfunction amenable to treatment with ranolazine. Echocardiography detected evidence of diastolic dysfunction in hypertensive mice that improved after treatment with ranolazine (E/E':sham, 31.9 ± 2.8, sham+ranolazine, 30.2 ± 1.9, DOCA-salt, 41.8 ± 2.6, and DOCA-salt+ranolazine, 31.9 ± 2.6; P=0.018). The end-diastolic pressure-volume relationship slope was elevated in DOCA-salt mice, improving to sham levels with treatment (sham, 0.16 ± 0.01 versus sham+ranolazine, 0.18 ± 0.01 versus DOCA-salt, 0.23 ± 0.2 versus DOCA-salt+ranolazine, 0.17 ± 0.0 1 mm Hg/L; P<0.005). DOCA-salt myocytes demonstrated impaired relaxation, τ, improving with ranolazine (DOCA-salt, 0.18 ± 0.02, DOCA-salt+ranolazine, 0.13 ± 0.01, sham, 0.11 ± 0.01, sham+ranolazine, 0.09 ± 0.02 seconds; P=0.0004). Neither late I(Na) nor the Ca(2+) transients were different from sham myocytes. Detergent extracted fiber bundles from DOCA-salt hearts demonstrated increased myofilament response to Ca(2+) with glutathionylation of myosin binding protein C. Treatment with ranolazine ameliorated the Ca(2+) response and cross-bridge kinetics. Diastolic dysfunction could be reversed by ranolazine, probably resulting from a direct effect on myofilaments, indicating that cardiac oxidative stress may mediate diastolic dysfunction through altering the contractile apparatus.

Left ventricular dysfunction in ischemic heart disease: fundamental importance of the fibrous matrix.

PubMed

Swan, H J

1994-05-01

The contractile function of the myocardium is coordinated by a fibrous matrix of exquisite organization and complexity. In the normal heart, and apparently in physiological hypertrophy, this matrix is submicroscopic. In pathological states changes are frequent, and usually progressive. Thickening of the many elements of the fine structure is due to an increased synthesis of Type I collagen, This change, which affects the myocardium in a global manner, can be observed by light microscopy using special techniques. Perivascular fibrosis, with an increase in vascular smooth muscle, is accompanied by development of fibrous septa, with a decrease in diastolic compliance. These structural changes are believed to be due to increased activation of the renin-angiotensin-aldosterone system, and to be independent of the processes of myocyte hypertrophy. Reparative or replacement fibrosis is a separate process by means of which small and large areas of necrosis heal, with the development of coarse collagen structures, which lack a specific organizational pattern. Regarding ischemic heart disease, an increase in tissue collagenase is found in experimental myocardial "stunning" and in the very early phase of acute infarction. Absence of elements of the fibrous matrix allow for myocyte slippage, and--if the affected area is large--cardiac dilatation. If, subsequently, the necrosis becomes transmural, there is further disturbance of collagen due to both mechanical strain and continued autolysis, During healing collagen synthesis increases greatly to allow for reparative scarring in the available tissue matrix. In cases of infarction with moderate or severe initial dilatation, pathological hypertrophy of the spared myocardium is progressive, accounting for late heart failure and poor survival.(ABSTRACT TRUNCATED AT 250 WORDS)

Cardiac myosin binding protein C regulates postnatal myocyte cytokinesis

PubMed Central

Jiang, Jianming; Burgon, Patrick G.; Wakimoto, Hiroko; Onoue, Kenji; Gorham, Joshua M.; O’Meara, Caitlin C.; Fomovsky, Gregory; McConnell, Bradley K.; Lee, Richard T.; Seidman, J. G.; Seidman, Christine E.

2015-01-01

Homozygous cardiac myosin binding protein C-deficient (Mybpct/t) mice develop dramatic cardiac dilation shortly after birth; heart size increases almost twofold. We have investigated the mechanism of cardiac enlargement in these hearts. Throughout embryogenesis myocytes undergo cell division while maintaining the capacity to pump blood by rapidly disassembling and reforming myofibrillar components of the sarcomere throughout cell cycle progression. Shortly after birth, myocyte cell division ceases. Cardiac MYBPC is a thick filament protein that regulates sarcomere organization and rigidity. We demonstrate that many Mybpct/t myocytes undergo an additional round of cell division within 10 d postbirth compared with their wild-type counterparts, leading to increased numbers of mononuclear myocytes. Short-hairpin RNA knockdown of Mybpc3 mRNA in wild-type mice similarly extended the postnatal window of myocyte proliferation. However, adult Mybpct/t myocytes are unable to fully regenerate the myocardium after injury. MYBPC has unexpected inhibitory functions during postnatal myocyte cytokinesis and cell cycle progression. We suggest that human patients with homozygous MYBPC3-null mutations develop dilated cardiomyopathy, coupled with myocyte hyperplasia (increased cell number), as observed in Mybpct/t mice. Human patients, with heterozygous truncating MYBPC3 mutations, like mice with similar mutations, have hypertrophic cardiomyopathy. However, the mechanism leading to hypertrophic cardiomyopathy in heterozygous MYBPC3+/− individuals is myocyte hypertrophy (increased cell size), whereas the mechanism leading to cardiac dilation in homozygous Mybpc3−/− mice is primarily myocyte hyperplasia. PMID:26153423

Cardiac myosin missense mutations cause dilated cardiomyopathy in mouse models and depress molecular motor function.

PubMed

Schmitt, Joachim P; Debold, Edward P; Ahmad, Ferhaan; Armstrong, Amy; Frederico, Andrea; Conner, David A; Mende, Ulrike; Lohse, Martin J; Warshaw, David; Seidman, Christine E; Seidman, J G

2006-09-26

Dilated cardiomyopathy (DCM) leads to heart failure, a leading cause of death in industrialized nations. Approximately 30% of DCM cases are genetic in origin, with some resulting from point mutations in cardiac myosin, the molecular motor of the heart. The effects of these mutations on myosin's molecular mechanics have not been determined. We have engineered two murine models characterizing the physiological, cellular, and molecular effects of DCM-causing missense mutations (S532P and F764L) in the alpha-cardiac myosin heavy chain and compared them with WT mice. Mutant mice developed morphological and functional characteristics of DCM consistent with the human phenotypes. Contractile function of isolated myocytes was depressed and preceded left ventricular dilation and reduced fractional shortening. In an in vitro motility assay, both mutant cardiac myosins exhibited a reduced ability to translocate actin (V(actin)) but had similar force-generating capacities. Actin-activated ATPase activities were also reduced. Single-molecule laser trap experiments revealed that the lower V(actin) in the S532P mutant was due to a reduced ability of the motor to generate a step displacement and an alteration of the kinetics of its chemomechanical cycle. These results suggest that the depressed molecular function in cardiac myosin may initiate the events that cause the heart to remodel and become pathologically dilated.

Loss of Adult Cardiac Myocyte GSK-3 Leads to Mitotic Catastrophe Resulting in Fatal Dilated Cardiomyopathy

PubMed Central

Zhou, Jibin; Ahmad, Firdos; Parikh, Shan; Hoffman, Nichole E.; Rajan, Sudarsan; Verma, Vipin K.; Song, Jianliang; Yuan, Ancai; Shanmughapriya, Santhanam; Guo, Yuanjun; Gao, Erhe; Koch, Walter; Woodgett, James R.; Muniswamy, Madesh; Kishore, Raj; Lal, Hind; Force, Thomas

2016-01-01

Rationale Cardiac myocyte-specific deletion of either Glycogen Synthase Kinase (GSK)3A or GSK3B leads to cardiac protection following myocardial infarction, suggesting that deletion of both isoforms may provide synergistic protection. This is an important consideration due to the fact that all GSK-3–targeted drugs including the drugs already in clinical trial target both isoforms of GSK-3 and none are isoform specific. Objective To identify the consequences of combined deletion of cardiac myocyte GSK3A and GSK3B in heart function. Methods and Results We generated tamoxifen-inducible cardiac myocyte-specific mice lacking both GSK-3 isoforms (double knockout, DKO). We unexpectedly found that cardiac myocyte GSK-3 is essential for cardiac homeostasis and overall survival. Serial echocardiographic analysis reveals that within 2 weeks of tamoxifen treatment, DKO hearts leads to excessive dilatative remodeling and ventricular dysfunction. Further experimentation with isolated adult cardiac myocytes and fibroblasts from DKO implicated cardiac myocytes intrinsic factors responsible for observed phenotype. Mechanistically, loss of GSK-3 in adult cardiac myocytes resulted in induction of mitotic catastrophe, a previously unreported event in cardiac myocytes. DKO cardiac myocytes showed cell cycle progression resulting in increased DNA content and multi-nucleation. However, increased cell cycle activity was rivaled by marked activation of DNA damage, cell cycle checkpoint activation, and mitotic catastrophe induced apoptotic cell death. Importantly, mitotic catastrophe was also confirmed in isolated adult cardiac myocytes. Conclusion Together, our findings suggest that cardiac myocyte GSK-3 is required to maintain normal cardiac homeostasis and its loss is incompatible with life due to cell cycle dysregulation that ultimately results in a severe fatal dilated cardiomyopathy. PMID:26976650

Loss of Adult Cardiac Myocyte GSK-3 Leads to Mitotic Catastrophe Resulting in Fatal Dilated Cardiomyopathy.

PubMed

Zhou, Jibin; Ahmad, Firdos; Parikh, Shan; Hoffman, Nichole E; Rajan, Sudarsan; Verma, Vipin K; Song, Jianliang; Yuan, Ancai; Shanmughapriya, Santhanam; Guo, Yuanjun; Gao, Erhe; Koch, Walter; Woodgett, James R; Madesh, Muniswamy; Kishore, Raj; Lal, Hind; Force, Thomas

2016-04-15

Cardiac myocyte-specific deletion of either glycogen synthase kinase (GSK)-3α and GSK-3β leads to cardiac protection after myocardial infarction, suggesting that deletion of both isoforms may provide synergistic protection. This is an important consideration because of the fact that all GSK-3-targeted drugs, including the drugs already in clinical trial target both isoforms of GSK-3, and none are isoform specific. To identify the consequences of combined deletion of cardiac myocyte GSK-3α and GSK-3β in heart function. We generated tamoxifen-inducible cardiac myocyte-specific mice lacking both GSK-3 isoforms (double knockout). We unexpectedly found that cardiac myocyte GSK-3 is essential for cardiac homeostasis and overall survival. Serial echocardiographic analysis reveals that within 2 weeks of tamoxifen treatment, double-knockout hearts leads to excessive dilatative remodeling and ventricular dysfunction. Further experimentation with isolated adult cardiac myocytes and fibroblasts from double-knockout implicated cardiac myocytes intrinsic factors responsible for observed phenotype. Mechanistically, loss of GSK-3 in adult cardiac myocytes resulted in induction of mitotic catastrophe, a previously unreported event in cardiac myocytes. Double-knockout cardiac myocytes showed cell cycle progression resulting in increased DNA content and multinucleation. However, increased cell cycle activity was rivaled by marked activation of DNA damage, cell cycle checkpoint activation, and mitotic catastrophe-induced apoptotic cell death. Importantly, mitotic catastrophe was also confirmed in isolated adult cardiac myocytes. Together, our findings suggest that cardiac myocyte GSK-3 is required to maintain normal cardiac homeostasis, and its loss is incompatible with life because of cell cycle dysregulation that ultimately results in a severe fatal dilated cardiomyopathy. © 2016 American Heart Association, Inc.

Melatonin mediates vasodilation through both direct and indirect activation of BKCa channels.

PubMed

Zhao, T; Zhang, H; Jin, C; Qiu, F; Wu, Y; Shi, L

2017-10-01

Melatonin, synthesized primarily by the pineal gland, is a neuroendocrine hormone with high membrane permeability. The vascular effects of melatonin, including vasoconstriction and vasodilation, have been demonstrated in numerous studies. However, the mechanisms underlying these effects are not fully understood. Large-conductance Ca 2+ -activated K + (BK Ca ) channels are expressed broadly on smooth muscle cells and play an important role in vascular tone regulation. This study explored the mechanisms of myocyte BK Ca channels and endothelial factors underlying the action of melatonin on the mesenteric arteries (MAs). Vascular contractility and patch-clamp studies were performed on myocytes of MAs from Wistar rats. Melatonin induced significant vasodilation on MAs. In the presence of N ω -nitro-l-arginine methyl ester (l-NAME), a potent endothelial oxide synthase (eNOS) inhibitor, melatonin elicited concentration-dependent relaxation, with lowered pIC 50 The effect of melatonin was significantly attenuated in the presence of BK Ca channel blocker iberiotoxin or MT1/MT2 receptor antagonist luzindole in both (+) l-NAME and (-) l-NAME groups. In the (+) l-NAME group, iberiotoxin caused a parallel rightward shift of the melatonin concentration-relaxation curve, with pIC 50 lower than that of luzindole. Both inside-out and cell-attached patch-clamp recordings showed that melatonin significantly increased the open probability, mean open time and voltage sensitivity of BK Ca channels. In a cell-attached patch-clamp configuration, the melatonin-induced enhancement of BK Ca channel activity was significantly suppressed by luzindole. These findings indicate that in addition to the activation of eNOS, melatonin-induced vasorelaxation of MAs is partially attributable to its direct (passing through the cell membrane) and indirect (via MT1/MT2 receptors) activation of the BK Ca channels on mesenteric arterial myocytes. © 2017 Society for Endocrinology.

Mitochondrial function in engineered cardiac tissues is regulated by extracellular matrix elasticity and tissue alignment.

PubMed

Lyra-Leite, Davi M; Andres, Allen M; Petersen, Andrew P; Ariyasinghe, Nethika R; Cho, Nathan; Lee, Jezell A; Gottlieb, Roberta A; McCain, Megan L

2017-10-01

Mitochondria in cardiac myocytes are critical for generating ATP to meet the high metabolic demands associated with sarcomere shortening. Distinct remodeling of mitochondrial structure and function occur in cardiac myocytes in both developmental and pathological settings. However, the factors that underlie these changes are poorly understood. Because remodeling of tissue architecture and extracellular matrix (ECM) elasticity are also hallmarks of ventricular development and disease, we hypothesize that these environmental factors regulate mitochondrial function in cardiac myocytes. To test this, we developed a new procedure to transfer tunable polydimethylsiloxane disks microcontact-printed with fibronectin into cell culture microplates. We cultured Sprague-Dawley neonatal rat ventricular myocytes within the wells, which consistently formed tissues following the printed fibronectin, and measured oxygen consumption rate using a Seahorse extracellular flux analyzer. Our data indicate that parameters associated with baseline metabolism are predominantly regulated by ECM elasticity, whereas the ability of tissues to adapt to metabolic stress is regulated by both ECM elasticity and tissue alignment. Furthermore, bioenergetic health index, which reflects both the positive and negative aspects of oxygen consumption, was highest in aligned tissues on the most rigid substrate, suggesting that overall mitochondrial function is regulated by both ECM elasticity and tissue alignment. Our results demonstrate that mitochondrial function is regulated by both ECM elasticity and myofibril architecture in cardiac myocytes. This provides novel insight into how extracellular cues impact mitochondrial function in the context of cardiac development and disease. NEW & NOTEWORTHY A new methodology has been developed to measure O 2 consumption rates in engineered cardiac tissues with independent control over tissue alignment and matrix elasticity. This led to the findings that matrix elasticity regulates basal mitochondrial function, whereas both matrix elasticity and tissue alignment regulate mitochondrial stress responses. Copyright © 2017 the American Physiological Society.

HDAC Inhibition Improves the Sarcoendoplasmic Reticulum Ca2+-ATPase Activity in Cardiac Myocytes.

PubMed

Meraviglia, Viviana; Bocchi, Leonardo; Sacchetto, Roberta; Florio, Maria Cristina; Motta, Benedetta M; Corti, Corrado; Weichenberger, Christian X; Savi, Monia; D'Elia, Yuri; Rosato-Siri, Marcelo D; Suffredini, Silvia; Piubelli, Chiara; Pompilio, Giulio; Pramstaller, Peter P; Domingues, Francisco S; Stilli, Donatella; Rossini, Alessandra

2018-01-31

SERCA2a is the Ca 2+ ATPase playing the major contribution in cardiomyocyte (CM) calcium removal. Its activity can be regulated by both modulatory proteins and several post-translational modifications. The aim of the present work was to investigate whether the function of SERCA2 can be modulated by treating CMs with the histone deacetylase (HDAC) inhibitor suberanilohydroxamic acid (SAHA). The incubation with SAHA (2.5 µM, 90 min) of CMs isolated from rat adult hearts resulted in an increase of SERCA2 acetylation level and improved ATPase activity. This was associated with a significant improvement of calcium transient recovery time and cell contractility. Previous reports have identified K464 as an acetylation site in human SERCA2. Mutants were generated where K464 was substituted with glutamine (Q) or arginine (R), mimicking constitutive acetylation or deacetylation, respectively. The K464Q mutation ameliorated ATPase activity and calcium transient recovery time, thus indicating that constitutive K464 acetylation has a positive impact on human SERCA2a (hSERCA2a) function. In conclusion, SAHA induced deacetylation inhibition had a positive impact on CM calcium handling, that, at least in part, was due to improved SERCA2 activity. This observation can provide the basis for the development of novel pharmacological approaches to ameliorate SERCA2 efficiency.

Low molecular weight fucoidan alleviates cardiac dysfunction in diabetic Goto-Kakizaki rats by reducing oxidative stress and cardiomyocyte apoptosis.

PubMed

Yu, Xinfeng; Zhang, Quanbin; Cui, Wentong; Zeng, Zheng; Yang, Wenzhe; Zhang, Chao; Zhao, Hongwei; Gao, Weidong; Wang, Xiaomin; Luo, Dali

2014-01-01

Diabetic cardiomyopathy (DCM) is characterized by cardiac dysfunction and cardiomyocyte apoptosis. Oxidative stress is suggested to be the major contributor to the development of DCM. This study was intended to evaluate the protective effect of low molecular weight fucoidan (LMWF) against cardiac dysfunction in diabetic rats. Type 2 diabetic goto-kakizaki rats were untreated or treated with LMWF (50 and 100 mg/kg/day) for three months. The establishment of DCM model and the effects of LMWF on cardiac function were evaluated by echocardiography and isolated heart perfusion. Ventricle staining with H-E or Sirius Red was performed to investigate the structural changes in myocardium. Functional evaluation demonstrated that LMWF has a beneficial effect on DCM by enhancing myocardial contractility and mitigating cardiac fibrosis. Additionally, LMWF exerted significant inhibitory effects on the reactive oxygen species production and myocyte apoptosis in diabetic hearts. The depressed activity of superoxide dismutase in diabetic heart was also improved by intervention with LMWF. Moreover, LMWF robustly inhibited the enhanced expression of protein kinase C β, an important contributor to oxidative stress, in diabetic heart and high glucose-treated cardiomyocytes. In conclusion, LMWF possesses a protective effect against DCM through ameliorations of PKCβ-mediated oxidative stress and subsequent cardiomyocyte apoptosis in diabetes.

Cardiac Myocyte Cell Cycle Control in Development, Disease and Regeneration

PubMed Central

Ahuja, Preeti; Sdek, Patima; Maclellan, W. Robb

2009-01-01

Cardiac myocytes rapidly proliferate during fetal life but exit the cell cycle soon after birth in mammals. Although the extent to which adult cardiac myocytes are capable of cell cycle reentry is controversial and species-specific differences may exist, it appears that for the vast majority of adult cardiac myocytes the predominant form of growth postnatally is an increase in cell size (hypertrophy) not number. Unfortunately, this limits the ability of the heart to restore function after any significant injury. Interst in novel regenerative therapies has led to the accumulation of much information on the mechanisms that regulate the rapid proliferation of cardiac myocytes in utero, their cell cycle exit in the perinatal period and the permanent arrest (terminal differentiation) in adult myocytes. The recent identification of cardiac progenitor cells capable of giving rise to cardiac myocyte-like cells has challenged the dogma that the heart is a terminally differentiated organ and opened new prospects for cardiac regeneration. In this review, we summarize the current understanding of cardiomyocyte cell cycle control in normal development and disease. In addition, we also discuss the potential usefulness of cardiomyocyte self-renewal as well as feasibility of therapeutic manipulation of the cardiac myocyte cell cycle for cardiac regeneration. PMID:17429040

Sildenafil ameliorates left ventricular T-tubule remodeling in a pressure overload-induced murine heart failure model

PubMed Central

Huang, Chun-kai; Chen, Bi-yi; Guo, Ang; Chen, Rong; Zhu, Yan-qi; Kutschke, William; Hong, Jiang; Song, Long-sheng

2016-01-01

Aim: Sildenafil, a phosphodiesterase 5 (PDE5) inhibitor, has been shown to exert beneficial effects in heart failure. The purpose of this study was to test whether sildenafil suppressed transverse-tubule (T-tubule) remodeling in left ventricular (LV) failure and thereby providing the therapeutic benefits. Methods: A pressure overload-induced murine heart failure model was established in mice by thoracic aortic banding (TAB). One day after TAB, the mice received sildenafil (100 mg·kg−1·d−1, sc) or saline for 5 weeks. At the end of treatment, echocardiography was used to examine LV function. Then the intact hearts were dissected out and placed in Langendorff-perfusion chamber for in situ confocal imaging of T-tubule ultrastructure from epicardial myocytes. Results: TAB surgery resulted in heart failure accompanied by remarkable T-tubule remodeling. Sildenafil treatment significantly attenuated TAB-induced cardiac hypertrophy and congestive heart failure, improved LV contractile function, and preserved T-tubule integrity in LV cardiomyocytes. But sildenafil treatment did not significantly affect the chamber dilation. The integrity of LV T-tubule structure was correlated with cardiac hypertrophy (R2=0.74, P<0.01) and global LV function (R2=0.47, P<0.01). Conclusion: Sildenafil effectively ameliorates LV T-tubule remodeling in TAB mice, revealing a novel mechanism underlying the therapeutic benefits of sildenafil in heart failure. PMID:26972492

Novel insights on the relationship between T-tubular defects and contractile dysfunction in a mouse model of hypertrophic cardiomyopathy.

PubMed

Crocini, C; Ferrantini, C; Scardigli, M; Coppini, R; Mazzoni, L; Lazzeri, E; Pioner, J M; Scellini, B; Guo, A; Song, L S; Yan, P; Loew, L M; Tardiff, J; Tesi, C; Vanzi, F; Cerbai, E; Pavone, F S; Sacconi, L; Poggesi, C

2016-02-01

Abnormalities of cardiomyocyte Ca(2+) homeostasis and excitation-contraction (E-C) coupling are early events in the pathogenesis of hypertrophic cardiomyopathy (HCM) and concomitant determinants of the diastolic dysfunction and arrhythmias typical of the disease. T-tubule remodelling has been reported to occur in HCM but little is known about its role in the E-C coupling alterations of HCM. Here, the role of T-tubule remodelling in the electro-mechanical dysfunction associated to HCM is investigated in the Δ160E cTnT mouse model that expresses a clinically-relevant HCM mutation. Contractile function of intact ventricular trabeculae is assessed in Δ160E mice and wild-type siblings. As compared with wild-type, Δ160E trabeculae show prolonged kinetics of force development and relaxation, blunted force-frequency response with reduced active tension at high stimulation frequency, and increased occurrence of spontaneous contractions. Consistently, prolonged Ca(2+) transient in terms of rise and duration are also observed in Δ160E trabeculae and isolated cardiomyocytes. Confocal imaging in cells isolated from Δ160E mice reveals significant, though modest, remodelling of T-tubular architecture. A two-photon random access microscope is employed to dissect the spatio-temporal relationship between T-tubular electrical activity and local Ca(2+) release in isolated cardiomyocytes. In Δ160E cardiomyocytes, a significant number of T-tubules (>20%) fails to propagate action potentials, with consequent delay of local Ca(2+) release. At variance with wild-type, we also observe significantly increased variability of local Ca(2+) transient rise as well as higher Ca(2+)-spark frequency. Although T-tubule structural remodelling in Δ160E myocytes is modest, T-tubule functional defects determine non-homogeneous Ca(2+) release and delayed myofilament activation that significantly contribute to mechanical dysfunction. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

Impact of vitamin D3 on cardiovascular responses to glucocorticoid excess.

PubMed

Ahmed, Mona A

2013-06-01

Although the cardiovascular system is not a classical target for 1,25-dihydroxyvitamin D3, both cardiac myocytes and vascular smooth muscle cells respond to this hormone. The present study aimed to elucidate the effect of active vitamin D3 on cardiovascular functions in rats exposed to glucocorticoid excess. Adult male Wistar rats were allocated into three groups: control group, dexamethasone (Dex)-treated group receiving Dex (200 μg/kg) subcutaneously for 12 days, and vitamin D3-Dex-treated group receiving 1,25-(OH)2D3 (100 ng/kg) and Dex (200 μg/kg) subcutaneously for 12 days. Rats were subjected to measurement of systolic (SBP), diastolic (DBP), and mean arterial (MAP) blood pressures and heart rate. Rate pressure product (RPP) was calculated. Rats' isolated hearts were perfused in Langendorff preparation and studied for basal activities (heart rate, peaked developed tension, time to peak tension, half relaxation time, and myocardial flow rate) and their responses to isoproterenol infusion. Blood samples were collected for determination of plasma level of nitrite, nitric oxide surrogate. Dex-treated group showed significant increase in SBP, DBP, MAP, and RPP, as well as cardiac hypertrophy and enhancement of basal cardiac performance evidenced by increased heart rate, rapid and increased contractility, and accelerated lusitropy, together with impaired contractile and myocardial flow rate responsiveness to beta-adrenergic activation and depressed inotropic and coronary vascular reserves. Such alterations were accompanied by low plasma nitrite. These changes were markedly improved by vitamin D3 treatment. In conclusion, vitamin D3 is an efficacious modulator of the deleterious cardiovascular responses induced by glucocorticoid excess, probably via accentuation of nitric oxide.

THG113.31, a specific PGF2alpha receptor antagonist, induces human myometrial relaxation and BKCa channel activation

PubMed Central

Doheny, Helen C; O'Reilly, Michael J; Sexton, Donal J; Morrison, John J

2007-01-01

Background PGF2alpha exerts a significant contractile effect on myometrium and is central to human labour. THG113.31, a specific non-competitive PGF2alpha receptor (FP) antagonist, exerts an inhibitory effect on myometrial contractility. The BKCa channel is ubiquitously encountered in human uterine tissue and plays a significant role in modulating myometrial cell membrane potential and excitability. The objective of this study was to investigate potential BKCa channel involvement in the response of human myometrium to THG113.31. Methods Single and whole-cell electrophysiological BKCa channel recordings from freshly dispersed myocytes, were investigated in the presence and absence of THG113.31. Functional studies investigated the effects of THG113.31 on isolated spontaneous myometrial contractions, in the presence and absence of the BKCa channel blocker, iberiotoxin. Results Single channel recordings identified the BKCa channel as a target of THG113.31. THG113.31 significantly increased the open state probability of these channels [control 0.023+/-0.006; 10 microM THG113.31 0.087+/-0.012 (P = 0.009); and 50 microM THG113.31 0.1356+/-0.018 (P = 0.001)]. In addition, THG113.31 increased whole-cell BKCa currents over a range of membrane potentials, and this effect was reversed by 100 nanoM IbTX. Isometric tension studies demonstrated that THG113.31 exerted a significant concentration-dependent relaxant effect on human myometrial tissue and pre-incubation of strips with IbTX abolished this effect on spontaneously occurring contractions. Conclusion These data suggests that activation of the BKCa channel may contribute, at least partially, to the uterorelaxant effect of THG113.31. PMID:17367527

Magnetic Resonance Assessment of Hypertrophic and Pseudo-Hypertrophic Changes in Lower Leg Muscles of Boys with Duchenne Muscular Dystrophy and Their Relationship to Functional Measurements.

PubMed

Vohra, Ravneet S; Lott, Donovan; Mathur, Sunita; Senesac, Claudia; Deol, Jasjit; Germain, Sean; Bendixen, Roxanna; Forbes, Sean C; Sweeney, H Lee; Walter, Glenn A; Vandenborne, Krista

2015-01-01

The primary objectives of this study were to evaluate contractile and non-contractile content of lower leg muscles of boys with Duchenne muscular dystrophy (DMD) and determine the relationships between non-contractile content and functional abilities. Lower leg muscles of thirty-two boys with DMD and sixteen age matched unaffected controls were imaged. Non-contractile content, contractile cross sectional area and non-contractile cross sectional area of lower leg muscles (tibialis anterior, extensor digitorum longus, peroneal, medial gastrocnemius and soleus) were assessed by magnetic resonance imaging (MRI). Muscle strength, timed functional tests and the Brooke lower extremity score were also assessed. Non-contractile content of lower leg muscles (peroneal, medial gastrocnemius, and soleus) was significantly greater than control group (p<0.05). Non-contractile content of lower leg muscles correlated with Brooke score (rs = 0.64-0.84) and 30 feet walk (rs = 0.66-0.80). Dorsiflexor (DF) and plantarflexor (PF) specific torque was significantly different between the groups. Overall, non-contractile content of the lower leg muscles was greater in DMD than controls. Furthermore, there was an age dependent increase in contractile content in the medial gastrocnemius of boys with DMD. The findings of this study suggest that T1 weighted MR images can be used to monitor disease progression and provide a quantitative estimate of contractile and non-contractile content of tissue in children with DMD.

Magnetic Resonance Assessment of Hypertrophic and Pseudo-Hypertrophic Changes in Lower Leg Muscles of Boys with Duchenne Muscular Dystrophy and Their Relationship to Functional Measurements

PubMed Central

Vohra, Ravneet S.; Lott, Donovan; Mathur, Sunita; Senesac, Claudia; Deol, Jasjit; Germain, Sean; Bendixen, Roxanna; Forbes, Sean C.; Sweeney, H. Lee; Walter, Glenn A.; Vandenborne, Krista

2015-01-01

Introduction The primary objectives of this study were to evaluate contractile and non-contractile content of lower leg muscles of boys with Duchenne muscular dystrophy (DMD) and determine the relationships between non-contractile content and functional abilities. Methods Lower leg muscles of thirty-two boys with DMD and sixteen age matched unaffected controls were imaged. Non-contractile content, contractile cross sectional area and non-contractile cross sectional area of lower leg muscles (tibialis anterior, extensor digitorum longus, peroneal, medial gastrocnemius and soleus) were assessed by magnetic resonance imaging (MRI). Muscle strength, timed functional tests and the Brooke lower extremity score were also assessed. Results Non-contractile content of lower leg muscles (peroneal, medial gastrocnemius, and soleus) was significantly greater than control group (p<0.05). Non-contractile content of lower leg muscles correlated with Brooke score (rs = 0.64-0.84) and 30 feet walk (rs = 0.66-0.80). Dorsiflexor (DF) and plantarflexor (PF) specific torque was significantly different between the groups. Discussion Overall, non-contractile content of the lower leg muscles was greater in DMD than controls. Furthermore, there was an age dependent increase in contractile content in the medial gastrocnemius of boys with DMD. The findings of this study suggest that T1 weighted MR images can be used to monitor disease progression and provide a quantitative estimate of contractile and non-contractile content of tissue in children with DMD. PMID:26103164

Co-regulation of the atrial natriuretic factor and cardiac myosin light chain-2 genes during alpha-adrenergic stimulation of neonatal rat ventricular cells. Identification of cis sequences within an embryonic and a constitutive contractile protein gene which mediate inducible expression.

PubMed

Knowlton, K U; Baracchini, E; Ross, R S; Harris, A N; Henderson, S A; Evans, S M; Glembotski, C C; Chien, K R

1991-04-25

To study the mechanisms which mediate the transcriptional activation of cardiac genes during alpha adrenergic stimulation, the present study examined the regulated expression of three cardiac genes, a ventricular embryonic gene (atrial natriuretic factor, ANF), a constitutively expressed contractile protein gene (cardiac MLC-2), and a cardiac sodium channel gene. alpha 1-Adrenergic stimulation activates the expression and release of ANF from neonatal ventricular cells. As assessed by RNase protection analyses, treatment with alpha-adrenergic agonists increases the steady-state levels of ANF mRNA by greater than 15-fold. However, a rat cardiac sodium channel gene mRNA is not induced, indicating that alpha-adrenergic stimulation does not lead to an increase in the expression of all cardiac genes. Studies employing a series of rat ANF luciferase and rat MLC-2 luciferase fusion genes identify 315- and 92-base pair cis regulatory sequences within an embryonic gene (ANF) and a constitutively expressed contractile protein gene (MLC-2), respectively, which mediate alpha-adrenergic-inducible gene expression. Transfection of various ANF luciferase reporters into neonatal rat ventricular cells demonstrated that upstream sequences which mediate tissue-specific expression (-3003 to -638) can be segregated from those responsible for inducibility. The lack of inducibility of a cardiac Na+ channel gene, and the segregation of ANF gene sequences which mediate cardiac specific from those which mediate inducible expression, provides further insight into the relationship between muscle-specific and inducible expression during cardiac myocyte hypertrophy. Based on these results, a testable model is proposed for the induction of embryonic cardiac genes and constitutively expressed contractile protein genes and the noninducibility of a subset of cardiac genes during alpha-adrenergic stimulation of neonatal rat ventricular cells.

Catecholamines and myocardial contractile function during hypodynamia and with an altered thyroid hormone balance

NASA Technical Reports Server (NTRS)

Pruss, G. M.; Kuznetsov, V. I.; Zhilinskaya, A. A.

1980-01-01

The dynamics of catecholamine content and myocardial contractile function during hypodynamia were studied in 109 white rats whose motor activity was severely restricted for up to 30 days. During the first five days myocardial catecholamine content, contractile function, and physical load tolerance decreased. Small doses of thyroidin counteracted this tendency. After 15 days, noradrenalin content and other indices approached normal levels and, after 30 days, were the same as control levels, although cardiac functional reserve was decreased. Thyroidin administration after 15 days had no noticeable effect. A detailed table shows changes in 17 indices of myocardial contractile function during hypodynamia.

Cardiac myocyte-protective effect of microRNA-22 during ischemia and reperfusion through disrupting the caveolin-3/eNOS signaling

PubMed Central

Chen, Zhenfei; Qi, Yinliang; Gao, Chao

2015-01-01

MicroRNA-22 (miR-22) was previously reported to elicit cardiac myocyte hypertrophy and had an anti-apoptotic effect on neurons. However, its effects on cardiac myocyte apoptosis and cardiac function during ischemia and reperfusion (I/R) are not clear. In the present study, we demonstrate that pre-administration of miR-22 mimic reduced I/R-induced cardiac dysfunction significantly in a rat model. We found that miR-22 overexpression inhibited cardiac myocyte apoptosis, and reduced cardiac remodeling during I/R. Significant cardiac myocyte apoptosis was also observed in a cardiac myocyte model after hypoxia/reoxygenation (H/R), a representative process of I/R. Further experiments showed that eNOS activity and the following NO production were significantly decreased during I/R and H/R, while such decrease was inhibited by overexpression of miR-22. Mechanistically, overexpression of miR-22 had little effect on the total protein level of eNOS, but restored the level of p-eNOS (Ser1177) which was down-regulated during H/R. Further RT-PCR results demonstrated that Caveolin 3 (Cav3), an upstream negative regulator of eNOS, was upregulated during H/R, resulting in a decrease of p-eNOS. However, such upregulation of Cav3 transcript level was inhibited directly by miR-22 during H/R, leading to a restored p-eNOS level and followed NO production in cardiac myocytes. Together, the present study revealed that miR-22 down-regulated Cav3, leading to restored eNOS activity and NO production, which further inhibited cardiac myocyte apoptosis and promoted cardiac function after I/R. Of clinical interest, the present study may highlight miR-22 as a potential therapeutic agent for reducing I/R induced cardiac injury. PMID:26191152

Genetic engineering and therapy for inherited and acquired cardiomyopathies.

PubMed

Day, Sharlene; Davis, Jennifer; Westfall, Margaret; Metzger, Joseph

2006-10-01

The cardiac myofilaments consist of a highly ordered assembly of proteins that collectively generate force in a calcium-dependent manner. Defects in myofilament function and its regulation have been implicated in various forms of acquired and inherited human heart disease. For example, during cardiac ischemia, cardiac myocyte contractile performance is dramatically downregulated due in part to a reduced sensitivity of the myofilaments to calcium under acidic pH conditions. Over the last several years, the thin filament regulatory protein, troponin I, has been identified as an important mediator of this response. Mutations in troponin I and other sarcomere genes are also linked to several distinct inherited cardiomyopathic phenotypes, including hypertrophic, dilated, and restrictive cardiomyopathies. With the cardiac sarcomere emerging as a central player for such a diverse array of human heart diseases, genetic-based strategies that target the myofilament will likely have broad therapeutic potential. The development of safe vector systems for efficient gene delivery will be a critical hurdle to overcome before these types of therapies can be successfully applied. Nonetheless, studies focusing on the principles of acute genetic engineering of the sarcomere hold value as they lay the essential foundation on which to build potential gene-based therapies for heart disease.

Store-Operated Ca2+ Entry (SOCE) Contributes to Normal Skeletal Muscle Contractility in young but not in aged skeletal muscle

PubMed Central

Brotto, Leticia S.; Bougoin, Sylvain; Nosek, Thomas M.; Reid, Michael; Hardin, Brian; Pan, Zui; Ma, Jianjie; Parness, Jerome

2011-01-01

Muscle atrophy alone is insufficient to explain the significant decline in contractile force of skeletal muscle during normal aging. One contributing factor to decreased contractile force in aging skeletal muscle could be compromised excitation-contraction (E-C) coupling, without sufficient available Ca2+ to allow for repetitive muscle contractility, skeletal muscles naturally become weaker. Using biophysical approaches, we previously showed that store-operated Ca2+ entry (SOCE) is compromised in aged skeletal muscle but not in young ones. While important, a missing component from previous studies is whether or not SOCE function correlates with contractile function during aging. Here we test the contribution of extracellular Ca2+ to contractile function of skeletal muscle during aging. First, we demonstrate graded coupling between SR Ca2+ release channel-mediated Ca2+ release and activation of SOCE. Inhibition of SOCE produced significant reduction of contractile force in young skeletal muscle, particularly at high frequency stimulation, and such effects were completely absent in aged skeletal muscle. Our data indicate that SOCE contributes to the normal physiological contractile response of young healthy skeletal muscle and that defective extracellular Ca2+ entry through SOCE contributes to the reduced contractile force characteristic of aged skeletal muscle. PMID:21666285

Store-operated Ca(2+) entry (SOCE) contributes to normal skeletal muscle contractility in young but not in aged skeletal muscle.

PubMed

Thornton, Angela M; Zhao, Xiaoli; Weisleder, Noah; Brotto, Leticia S; Bougoin, Sylvain; Nosek, Thomas M; Reid, Michael; Hardin, Brian; Pan, Zui; Ma, Jianjie; Parness, Jerome; Brotto, Marco

2011-06-01

Muscle atrophy alone is insufficient to explain the significant decline in contractile force of skeletal muscle during normal aging. One contributing factor to decreased contractile force in aging skeletal muscle could be compromised excitation-contraction (E-C) coupling, without sufficient available Ca(2+) to allow for repetitive muscle contractility, skeletal muscles naturally become weaker. Using biophysical approaches, we previously showed that store-operated Ca(2+) entry (SOCE) is compromised in aged skeletal muscle but not in young ones. While important, a missing component from previous studies is whether or not SOCE function correlates with contractile function during aging. Here we test the contribution of extracellular Ca(2+) to contractile function of skeletal muscle during aging. First, we demonstrate graded coupling between SR Ca(2+) release channel-mediated Ca(2+) release and activation of SOCE. Inhibition of SOCE produced significant reduction of contractile force in young skeletal muscle, particularly at high frequency stimulation, and such effects were completely absent in aged skeletal muscle. Our data indicate that SOCE contributes to the normal physiological contractile response of young healthy skeletal muscle and that defective extracellular Ca(2+) entry through SOCE contributes to the reduced contractile force characteristic of aged skeletal muscle.

Heterologous desensitization of cardiac β-adrenergic signal via hormone-induced βAR/arrestin/PDE4 complexes

PubMed Central

Shi, Qian; Li, Minghui; Mika, Delphine; Fu, Qin; Kim, Sungjin; Phan, Jason; Shen, Ao; Vandecasteele, Gregoire; Xiang, Yang K.

2017-01-01

Aims Cardiac β-adrenergic receptor (βAR) signalling is susceptible to heterologous desensitization by different neurohormonal stimuli in clinical conditions associated with heart failure. We aim to examine the underlying mechanism of cross talk between βARs and a set of G-protein coupled receptors (GPCRs) activated by hormones/agonists. Methods and results Rat ventricular cardiomyocytes were used to determine heterologous phosphorylation of βARs under a series of GPCR agonists. Activation of Gs-coupled dopamine receptor, adenosine receptor, relaxin receptor and prostaglandin E2 receptor, and Gq-coupled α1 adrenergic receptor and angiotensin II type 1 receptor promotes phosphorylation of β1AR and β2AR at putative protein kinase A (PKA) phosphorylation sites; but activation of Gi-coupled α2 adrenergic receptor and activation of protease-activated receptor does not. The GPCR agonists that promote β2AR phosphorylation effectively inhibit βAR agonist isoproterenol-induced PKA phosphorylation of phospholamban and contractile function in ventricular cardiomyocytes. Heterologous GPCR stimuli have minimal to small effect on isoproterenol-induced β2AR activation and G-protein coupling for cyclic adenosine monophosphate (cAMP) production. However, these GPCR stimuli significantly promote phosphorylation of phosphodiesterase 4D (PDE4D), and recruit PDE4D to the phosphorylated β2AR in a β-arrestin 2 dependent manner without promoting β2AR endocytosis. The increased binding between β2AR and PDE4D effectively hydrolyzes cAMP signal generated by subsequent stimulation with isoproterenol. Mutation of PKA phosphorylation sites in β2AR, inhibition of PDE4, or genetic ablation of PDE4D or β-arrestin 2 abolishes this heterologous inhibitory effect. Ablation of β-arrestin 2 or PDE4D gene also rescues β-adrenergic stimuli-induced myocyte contractile function. Conclusions These data reveal essential roles of β-arrestin 2 and PDE4D in a common mechanism for heterologous desensitization of cardiac βARs under hormonal stimulation, which is associated with impaired cardiac function during the development of pathophysiological conditions. PMID:28339772

Heterologous desensitization of cardiac β-adrenergic signal via hormone-induced βAR/arrestin/PDE4 complexes.

PubMed

Shi, Qian; Li, Minghui; Mika, Delphine; Fu, Qin; Kim, Sungjin; Phan, Jason; Shen, Ao; Vandecasteele, Gregoire; Xiang, Yang K

2017-05-01

Cardiac β-adrenergic receptor (βAR) signalling is susceptible to heterologous desensitization by different neurohormonal stimuli in clinical conditions associated with heart failure. We aim to examine the underlying mechanism of cross talk between βARs and a set of G-protein coupled receptors (GPCRs) activated by hormones/agonists. Rat ventricular cardiomyocytes were used to determine heterologous phosphorylation of βARs under a series of GPCR agonists. Activation of Gs-coupled dopamine receptor, adenosine receptor, relaxin receptor and prostaglandin E2 receptor, and Gq-coupled α1 adrenergic receptor and angiotensin II type 1 receptor promotes phosphorylation of β1AR and β2AR at putative protein kinase A (PKA) phosphorylation sites; but activation of Gi-coupled α2 adrenergic receptor and activation of protease-activated receptor does not. The GPCR agonists that promote β2AR phosphorylation effectively inhibit βAR agonist isoproterenol-induced PKA phosphorylation of phospholamban and contractile function in ventricular cardiomyocytes. Heterologous GPCR stimuli have minimal to small effect on isoproterenol-induced β2AR activation and G-protein coupling for cyclic adenosine monophosphate (cAMP) production. However, these GPCR stimuli significantly promote phosphorylation of phosphodiesterase 4D (PDE4D), and recruit PDE4D to the phosphorylated β2AR in a β-arrestin 2 dependent manner without promoting β2AR endocytosis. The increased binding between β2AR and PDE4D effectively hydrolyzes cAMP signal generated by subsequent stimulation with isoproterenol. Mutation of PKA phosphorylation sites in β2AR, inhibition of PDE4, or genetic ablation of PDE4D or β-arrestin 2 abolishes this heterologous inhibitory effect. Ablation of β-arrestin 2 or PDE4D gene also rescues β-adrenergic stimuli-induced myocyte contractile function. These data reveal essential roles of β-arrestin 2 and PDE4D in a common mechanism for heterologous desensitization of cardiac βARs under hormonal stimulation, which is associated with impaired cardiac function during the development of pathophysiological conditions. Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2017. For permissions please email: journals.permissions@oup.com.

Regional effects of streptozotocin-induced diabetes on shortening and calcium transport in epicardial and endocardial myocytes from rat left ventricle.

PubMed

Smail, Manal M A; Qureshi, Muhammad A; Shmygol, Anatoliy; Oz, Murat; Singh, Jaipaul; Sydorenko, Vadym; Arabi, Alya; Howarth, Frank C; Al Kury, Lina

2016-11-01

In the heart, the left ventricle pumps blood at higher pressure than the right ventricle. Within the left ventricle, the electromechanical properties of ventricular cardiac myocytes vary transmurally and this may be related to the gradients of stress and strain experienced in vivo across the ventricular wall. Diabetes is also associated with alterations in hemodynamic function. The aim of this study was to investigate shortening and Ca 2+ transport in epicardial (EPI) and endocardial (ENDO) left ventricular myocytes in the streptozotocin (STZ)-induced diabetic rat. Shortening, intracellular Ca 2+ and L-type Ca 2+ current (I Ca,L ) were measured by video detection, fura-2 microfluorimetry, and whole-cell patch clamp techniques, respectively. Time to peak (TPK) shortening was prolonged to similar extents in ENDO and EPI myocytes from STZ-treated rats compared to ENDO and EPI myocytes from controls. Time to half (THALF) relaxation of shortening was prolonged in ENDO myocytes from STZ-treated rats compared to ENDO controls. TPK Ca 2+ transient was prolonged in ENDO myocytes from STZ-treated rats compared to ENDO controls. THALF decay of the Ca 2+ transient was prolonged in ENDO myocytes from STZ-treated rats compared to ENDO controls. Sarcoplasmic reticulum (SR) fractional release of Ca 2+ was reduced in EPI myocytes from STZ-treated rats compared to EPI controls. I C a,L activation, inactivation, and recovery from inactivation were not significantly altered in EPI and ENDO myocytes from STZ-treated rats or controls. Regional differences in Ca 2+ transport may partly underlie differences in ventricular myocyte shortening across the wall of the healthy and the STZ-treated rat left ventricle. © 2016 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.

Relationship between improvement in left ventricular dyssynchrony and contractile function and clinical outcome with cardiac resynchronization therapy: the MADIT-CRT trial.

PubMed

Pouleur, Anne-Catherine; Knappe, Dorit; Shah, Amil M; Uno, Hajime; Bourgoun, Mikhail; Foster, Elyse; McNitt, Scott; Hall, W Jackson; Zareba, Wojciech; Goldenberg, Ilan; Moss, Arthur J; Pfeffer, Marc A; Solomon, Scott D

2011-07-01

To assess long-term effects of cardiac resynchronization therapy (CRT) on left ventricular (LV) dyssynchrony and contractile function, by two-dimensional speckle-tracking echocardiography, compared with implantable cardioverter defibrillator (ICD) only in MADIT-CRT. We studied 761 patients in New York Heart Association I/II, ejection fraction ≤30%, and QRS ≥130 ms [n = 434, CRT-defibrillator (CRT-D), n = 327, ICD] with echocardiographic studies available at baseline and 12 months. Dyssynchrony was determined as the standard deviation of time to peak transverse strain between 12 segments of apical four- and two-chamber views, and contractile function as global longitudinal strain (GLS) by averaging longitudinal strain over these 12 segments. We compared changes in LV dyssynchrony and contractile function between treatment groups and assessed relationships between these changes over the first year and subsequent outcomes (median post 1-year follow-up = 14.9 months). Mean changes in LV dyssynchrony and contractile function measured by GLS in the overall population were, respectively, -29 ± 83 ms and -1 ± 2.9%. However, both LV dyssynchrony (CRT-D: -47 ± 83 ms vs. ICD: -6 ± 76 ms, P < 0.001) and contractile function (CRT-D: -1.4 ± 3.1% vs. ICD: -0.4 ± 2.5%, P < 0.001) improved to a greater extent in the CRT-D group compared with the ICD-only group. A greater improvement in dyssynchrony and contractile function at 1 year was associated with lower rates of the subsequent primary outcome of death or heart failure, adjusting for baseline dyssynchrony and contractile function, treatment arm, ischaemic status, and change in LV end-systolic volume. Each 20 ms decrease in LV dyssynchrony was associated with a 7% reduction in the primary outcome (P = 0.047); each 1% improvement in GLS over the 12-month period was associated with a 24% reduction in the primary outcome (P < 0.001). Cardiac resynchronization therapy resulted in a significant improvement in both LV dyssynchrony and contractile function measured by GLS compared with ICD only and these improvements were associated with better subsequent outcomes.

Three-dimensional engineered heart tissue from neonatal rat cardiac myocytes.

PubMed

Zimmermann, W H; Fink, C; Kralisch, D; Remmers, U; Weil, J; Eschenhagen, T

2000-04-05

A technique is presented that allows neonatal rat cardiac myocytes to form spontaneously and coherently beating 3-dimensional engineered heart tissue (EHT) in vitro, either as a plane biconcaval matrix anchored at both sides on Velcro-coated silicone tubes or as a ring. Contractile activity was monitored in standard organ baths or continuously in a CO(2) incubator for up to 18 days (=26 days after casting). Long-term measurements showed an increase in force between days 8 and 18 after casting and stable forces thereafter. At day 10, the twitch amplitude (TA) of electrically paced EHTs (average length x width x thickness, 11 x 6 x 0.4 mm) was 0.51 mN at length of maximal force development (L(max)) and a maximally effective calcium concentration. EHTs showed typical features of neonatal rat heart: a positive force-length and a negative force-frequency relation, high sensitivity to calcium (EC(50) 0.24 mM), modest positive inotropic (increase in TA by 46%) and pronounced positive lusitropic effect of isoprenaline (decrease in twitch duration by 21%). Both effects of isoprenaline were sensitive to the muscarinic receptor agonist carbachol in a pertussis toxin-sensitive manner. Adenovirus-mediated gene transfer of beta-galactosidase into EHTs reached 100% efficiency. In summary, EHTs retain many of the physiological characteristics of rat cardiac tissue and allow efficient gene transfer with subsequent force measurement. Copyright 2000 John Wiley & Sons, Inc.

Orientation and length of mammalian skeletal myocytes in response to a unidirectional stretch

NASA Technical Reports Server (NTRS)

Collinsworth, A. M.; Torgan, C. E.; Nagda, S. N.; Rajalingam, R. J.; Kraus, W. E.; Truskey, G. A.

2000-01-01

Effects of mechanical forces exerted on mammalian skeletal muscle cells during development were studied using an in vitro model to unidirectionally stretch cultured C2C12 cells grown on silastic membrane. Previous models to date have not studied these responses of the mammalian system specifically. The silastic membrane upon which these cells were grown exhibited linear strain behavior over the range of 3.6-14.6% strain, with a Poisson's ratio of approximately 0.5. To mimic murine in utero long bone growth, cell substrates were stretched at an average strain rate of 2.36%/day for 4 days or 1.77%/day for 6 days with an overall membrane strain of 9.5% and 10.6%, respectively. Both control and stretched fibers stained positively for the contractile protein, alpha-actinin, demonstrating muscle fiber development. An effect of stretch on orientation and length of myofibers was observed. At both strain rates, stretched fibers aligned at a smaller angle relative to the direction of stretch and were significantly longer compared to randomly oriented control fibers. There was no effect of duration of stretch on orientation or length, suggesting the cellular responses are independent of strain rate for the range tested. These results demonstrate that, under conditions simulating mammalian long bone growth, cultured myocytes respond to mechanical forces by lengthening and orienting along the direction of stretch.

β-Adrenergic induced SR Ca2+ leak is mediated by an Epac-NOS pathway.

PubMed

Pereira, Laëtitia; Bare, Dan J; Galice, Samuel; Shannon, Thomas R; Bers, Donald M

2017-07-01

Cardiac β-adrenergic receptors (β-AR) and Ca 2+ -Calmodulin dependent protein kinase (CaMKII) regulate both physiological and pathophysiological Ca 2+ signaling. Elevated diastolic Ca 2+ leak from the sarcoplasmic reticulum (SR) contributes to contractile dysfunction in heart failure and to arrhythmogenesis. β-AR activation is known to increase SR Ca 2+ leak via CaMKII-dependent phosphorylation of the ryanodine receptor. Two independent and reportedly parallel pathways have been implicated in this β-AR-CaMKII cascade, one involving exchange protein directly activated by cAMP (Epac2) and another involving nitric oxide synthase 1 (NOS1). Here we tested whether Epac and NOS function in a single series pathway to increase β-AR induced and CaMKII-dependent SR Ca 2+ leak. Leak was measured as both Ca 2+ spark frequency and tetracaine-induced shifts in SR Ca 2+ , in mouse and rabbit ventricular myocytes. Direct Epac activation by 8-CPT (8-(4-chlorophenylthio)-2'-O-methyl-cAMP) mimicked β-AR-induced SR Ca 2+ leak, and both were blocked by NOS inhibition. The same was true for myocyte CaMKII activation (assessed via a FRET-based reporter) and ryanodine receptor phosphorylation. Inhibitor and phosphorylation studies also implicated phosphoinositide 3-kinase (PI3K) and protein kinase B (Akt) downstream of Epac and above NOS activation in this pathway. We conclude that these two independently characterized parallel pathways function mainly via a single series arrangement (β-AR-cAMP-Epac-PI3K-Akt-NOS1-CaMKII) to mediate increased SR Ca 2+ leak. Thus, for β-AR activation the cAMP-PKA branch effects inotropy and lusitropy (by effects on Ca 2+ current and SR Ca 2+ -ATPase), this cAMP-Epac-NOS pathway increases pathological diastolic SR Ca 2+ leak. This pathway distinction may allow novel SR Ca 2+ leak therapeutic targeting in treatment of arrhythmias in heart failure that spare the inotropic and lusitropic effects of the PKA branch. Copyright © 2017 Elsevier Ltd. All rights reserved.

Temporal Adaptive Changes in Contractility and Fatigability of Diaphragm Muscles from Streptozotocin-Diabetic Rats

PubMed Central

Brotto, Marco; Brotto, Leticia; Jin, J.-P.; Nosek, Thomas M.; Romani, Andrea

2010-01-01

Diabetes is characterized by ventilatory depression due to decreased diaphragm (DPH) function. This study investigated the changes in contractile properties of rat DPH muscles over a time interval encompassing from 4 days to 14 weeks after the onset of streptozotocin-induced diabetes, with and without insulin treatment for 2 weeks. Maximum tetanic force in intact DPH muscle strips and recovery from fatiguing stimulation were measured. An early (4-day) depression in contractile function in diabetic DPH was followed by gradual improvement in muscle function and fatigue recovery (8 weeks). DPH contractile function deteriorated again at 14 weeks, a process that was completely reversed by insulin treatment. Maximal contractile force and calcium sensitivity assessed in Triton-skinned DPH fibers showed a similar bimodal pattern and the same beneficial effect of insulin treatment. While an extensive analysis of the isoforms of the contractile and regulatory proteins was not conducted, Western blot analysis of tropomyosin suggests that the changes in diabetic DPH response depended, at least in part, on a switch in fiber type. PMID:20467472

Temporal adaptive changes in contractility and fatigability of diaphragm muscles from streptozotocin-diabetic rats.

PubMed

Brotto, Marco; Brotto, Leticia; Jin, J-P; Nosek, Thomas M; Romani, Andrea

2010-01-01

Diabetes is characterized by ventilatory depression due to decreased diaphragm (DPH) function. This study investigated the changes in contractile properties of rat DPH muscles over a time interval encompassing from 4 days to 14 weeks after the onset of streptozotocin-induced diabetes, with and without insulin treatment for 2 weeks. Maximum tetanic force in intact DPH muscle strips and recovery from fatiguing stimulation were measured. An early (4-day) depression in contractile function in diabetic DPH was followed by gradual improvement in muscle function and fatigue recovery (8 weeks). DPH contractile function deteriorated again at 14 weeks, a process that was completely reversed by insulin treatment. Maximal contractile force and calcium sensitivity assessed in Triton-skinned DPH fibers showed a similar bimodal pattern and the same beneficial effect of insulin treatment. While an extensive analysis of the isoforms of the contractile and regulatory proteins was not conducted, Western blot analysis of tropomyosin suggests that the changes in diabetic DPH response depended, at least in part, on a switch in fiber type.

Role of Non-Myocyte Gap Junctions and Connexin Hemichannels in Cardiovascular Health and Disease: Novel Therapeutic Targets?

PubMed

Johnson, Robert D; Camelliti, Patrizia

2018-03-15

The heart is a complex organ composed of multiple cell types, including cardiomyocytes and different non-myocyte populations, all working closely together to determine the hearts properties and maintain normal cardiac function. Connexins are abundantly expressed proteins that form plasma membrane hemichannels and gap junctions between cells. Gap junctions are intracellular channels that allow for communication between cells, and in the heart they play a crucial role in cardiac conduction by coupling adjacent cardiomyocytes. Connexins are expressed in both cardiomyocytes and non-myocytes, including cardiac fibroblasts, endothelial cells, and macrophages. Non-myocytes are the largest population of cells in the heart, and therefore it is important to consider what roles connexins, hemichannels, and gap junctions play in these cell types. The aim of this review is to provide insight into connexin-based signalling in non-myocytes during health and disease, and highlight how targeting these proteins could lead to the development of novel therapies. We conclude that connexins in non-myocytes contribute to arrhythmias and adverse ventricular remodelling following myocardial infarction, and are associated with the initiation and development of atherosclerosis. Therefore, therapeutic interventions targeting these connexins represent an exciting new research avenue with great potential.

HDAC Inhibition Improves the Sarcoendoplasmic Reticulum Ca2+-ATPase Activity in Cardiac Myocytes

PubMed Central

Meraviglia, Viviana; Sacchetto, Roberta; Motta, Benedetta M.; Corti, Corrado; D’Elia, Yuri; Rosato-Siri, Marcelo D.; Suffredini, Silvia; Pompilio, Giulio; Pramstaller, Peter P.; Stilli, Donatella

2018-01-01

SERCA2a is the Ca2+ ATPase playing the major contribution in cardiomyocyte (CM) calcium removal. Its activity can be regulated by both modulatory proteins and several post-translational modifications. The aim of the present work was to investigate whether the function of SERCA2 can be modulated by treating CMs with the histone deacetylase (HDAC) inhibitor suberanilohydroxamic acid (SAHA). The incubation with SAHA (2.5 µM, 90 min) of CMs isolated from rat adult hearts resulted in an increase of SERCA2 acetylation level and improved ATPase activity. This was associated with a significant improvement of calcium transient recovery time and cell contractility. Previous reports have identified K464 as an acetylation site in human SERCA2. Mutants were generated where K464 was substituted with glutamine (Q) or arginine (R), mimicking constitutive acetylation or deacetylation, respectively. The K464Q mutation ameliorated ATPase activity and calcium transient recovery time, thus indicating that constitutive K464 acetylation has a positive impact on human SERCA2a (hSERCA2a) function. In conclusion, SAHA induced deacetylation inhibition had a positive impact on CM calcium handling, that, at least in part, was due to improved SERCA2 activity. This observation can provide the basis for the development of novel pharmacological approaches to ameliorate SERCA2 efficiency. PMID:29385061

Mechanical signaling and the cellular response to extracellular matrix in angiogenesis and cardiovascular physiology

NASA Technical Reports Server (NTRS)

Ingber, Donald E.

2002-01-01

Great advances have been made in the identification of the soluble angiogenic factors, insoluble extracellular matrix (ECM) molecules, and receptor signaling pathways that mediate control of angiogenesis--the growth of blood capillaries. This review focuses on work that explores how endothelial cells integrate these chemical signals with mechanical cues from their local tissue microenvironment so as to produce functional capillary networks that exhibit specialized form as well as function. These studies have revealed that ECM governs whether an endothelial cell will switch between growth, differentiation, motility, or apoptosis programs in response to a soluble stimulus based on its ability to mechanically resist cell tractional forces and thereby produce cell and cytoskeletal distortion. Transmembrane integrin receptors play a key role in this mechanochemical transduction process because they both organize a cytoskeletal signaling complex within the focal adhesion and preferentially focus mechanical forces on this site. Molecular filaments within the internal cytoskeleton--microfilaments, microtubules, and intermediate filaments--also contribute to the cell's structural and functional response to mechanical stress through their role as discrete support elements within a tensegrity-stabilized cytoskeletal array. Importantly, a similar form of mechanical control also has been shown to be involved in the regulation of contractility in vascular smooth muscle cells and cardiac myocytes. Thus, the mechanism by which cells perform mechanochemical transduction and the implications of these findings for morphogenetic control are discussed in the wider context of vascular development and cardiovascular physiology.

Increase in parasympathetic tone by pyridostigmine prevents ventricular dysfunction during the onset of heart failure.

PubMed

Lataro, Renata M; Silva, Carlos A A; Fazan, Rubens; Rossi, Marcos A; Prado, Cibele M; Godinho, Rosely O; Salgado, Helio C

2013-10-15

Heart failure (HF) is characterized by elevated sympathetic activity and reduced parasympathetic control of the heart. Experimental evidence suggests that the increase in parasympathetic function can be a therapeutic alternative to slow HF evolution. The parasympathetic neurotransmission can be improved by acetylcholinesterase inhibition. We investigated the long-term (4 wk) effects of the acetylcholinesterase inhibitor pyridostigmine on sympathovagal balance, cardiac remodeling, and cardiac function in the onset of HF following myocardial infarction. Myocardial infarction was elicited in adult male Wistar rats. After 4 wk of pyridostigmine administration, per os, methylatropine and propranolol were used to evaluate the cardiac sympathovagal balance. The tachycardic response caused by methylatropine was considered to be the vagal tone, whereas the bradycardic response caused by propranolol was considered to be the sympathetic tone. In conscious HF rats, pyridostigmine reduced the basal heart rate, increased vagal, and reduced sympathetic control of heart rate. Pyridostigmine reduced the myocyte diameter and collagen density of the surviving left ventricle. Pyridostigmine also increased vascular endothelial growth factor protein in the left ventricle, suggesting myocardial angiogenesis. Cardiac function was assessed by means of the pressure-volume conductance catheter system. HF rats treated with pyridostigmine exhibited a higher stroke volume, ejection fraction, cardiac output, and contractility of the left ventricle. It was demonstrated that the long-term administration of pyridostigmine started right after coronary artery ligation augmented cardiac vagal and reduced sympathetic tone, attenuating cardiac remodeling and left ventricular dysfunction during the progression of HF in rats.

In permanent atrial fibrillation, PDE3 reduces force responses to 5‐HT, but PDE3 and PDE4 do not cause the blunting of atrial arrhythmias

PubMed Central

Schwarz, Simon; Ravens, Ursula; Knaut, Michael

2016-01-01

Abstract Background and Purpose 5‐HT increases force and L‐type Ca2 + current (ICa,L) and causes arrhythmias through 5‐HT4 receptors in human atrium. In permanent atrial fibrillation (peAF), atrial force responses to 5‐HT are blunted, arrhythmias abolished but ICa,L responses only moderately attenuated. We investigated whether, in peAF, this could be due to an increased function of PDE3 and/or PDE4, using the inhibitors cilostamide (300 nM) and rolipram (1 μM) respectively. Experimental Approach Contractile force, arrhythmic contractions and ICa,L were assessed in right atrial trabeculae and myocytes, obtained from patients with sinus rhythm (SR), paroxysmal atrial fibrillation (pAF) and peAF. Key Results Maximum force responses to 5‐HT were reduced to 15% in peAF, but not in pAF. Cilostamide, but not rolipram, increased both the blunted force responses to 5‐HT in peAF and the inotropic potency of 5‐HT fourfold to sevenfold in trabeculae of patients with SR, pAF and peAF. Lusitropic responses to 5‐HT were not decreased in peAF. Responses of ICa,L to 5‐HT did not differ and were unaffected by cilostamide or rolipram in myocytes from patients with SR or peAF. Concurrent cilostamide and rolipram increased 5‐HT's propensity to elicit arrhythmias in trabeculae from patients with SR, but not with peAF. Conclusions and Implications PDE3, but not PDE4, reduced inotropic responses to 5‐HT in peAF, independently of lusitropy and ICa,L, but PDE3 activity was the same as that in patients with SR and pAF. Atrial remodelling in peAF abolished the facilitation of 5‐HT to induce arrhythmias by inhibition of PDE3 plus PDE4. PMID:27238373

Pressure-overload-induced angiotensin-mediated early remodeling in mouse heart

PubMed Central

Kim, Jeremy H.; Jiang, Ya-Ping; Cohen, Ira S.; Lin, Richard Z.; Mathias, Richard T.

2017-01-01

Our previous work on angiotensin II-mediated electrical-remodeling in canine left ventricle, in connection with a long history of other studies, suggested the hypothesis: increases in mechanical load induce autocrine secretion of angiotensin II (A2), which coherently regulates a coterie of membrane ion transporters in a manner that increases contractility. However, the relation between load and A2 secretion was correlative. We subsequently showed a similar or identical system was present in murine heart. To investigate whether the relation between mechanical load and A2-mediated electrical remodeling was causal, we employed transverse aortic constriction in mice to subject the left ventricle to pressure overload for short-term (1 to 2 days) or long-term (1 to 2 weeks) periods. Heart-to-body weight ratios and cell capacitance measurements were used to determine hypertrophy. Whole-cell patch clamp recordings of the predominant repolarization currents Ito,fast and IK,slow were used to assess electrical remodeling. Hearts or myocytes subjected to long-term load displayed significant hypertrophy, which was not evident in short-term load. However, short-term load induced significant reductions in Ito,fast and IK,slow. Incubation of these myocytes with the angiotensin II type 1 receptor inhibitor saralasin for 2 hours restored Ito,fast and IK,slow to control levels. The number of Ito.fast or IK,slow channels did not change with A2 or long-term load, however the hypertrophic increase in membrane area reduced the current densities for both channels. For Ito,fast but not IK,slow there was an additional reduction that was reversed by inhibition of angiotensin receptors. These results suggest increased load activates an endogenous renin angiotensin system that initially reduces Ito,fast and IK,slow prior to the onset of hypertrophic growth. However, there are functional interactions between electrical and anatomical remodeling. First, hypertrophy tends to reduce all current densities. Second, the hypertrophic program can modify signaling between the angiotensin receptor and target current. PMID:28464037

Ion channel mechanisms of rat tail artery contraction-relaxation by menthol involving, respectively, TRPM8 activation and L-type Ca2+ channel inhibition

PubMed Central

Melanaphy, Donal; Kustov, Maxim V.; Watson, Conall A.; Borysova, Lyudmyla; Burdyga, Theodor V.; Zholos, Alexander V.

2016-01-01

Transient receptor potential melastatin 8 (TRPM8) is the principal cold and menthol receptor channel. Characterized primarily for its cold-sensing role in sensory neurons, it is expressed and functional in several nonneuronal tissues, including vasculature. We previously demonstrated that menthol causes variable mechanical responses (vasoconstriction, vasodilatation, or biphasic reactions) in isolated arteries, depending on vascular tone. Here we aimed to dissect the specific ion channel mechanisms and corresponding Ca2+ signaling pathways underlying such complex responses to menthol and other TRPM8 ligands in rat tail artery myocytes using patch-clamp electrophysiology, confocal Ca2+ imaging, and ratiometric Ca2+ recording. Menthol (300 μM, a concentration typically used to induce TRPM8 currents) strongly inhibited L-type Ca2+ channel current (L-ICa) in isolated myocytes, especially its sustained component, most relevant for depolarization-induced vasoconstriction. In contraction studies, with nifedipine present (10 μM) to abolish L-ICa contribution to phenylephrine (PE)-induced vasoconstrictions of vascular rings, a marked increase in tone was observed with menthol, similar to resting (i.e., without α-adrenoceptor stimulation by PE) conditions, when L-type channels were mostly deactivated. Menthol-induced increases in PE-induced vasoconstrictions could be inhibited both by the TRPM8 antagonist AMTB (thus confirming the specific role of TRPM8) and by cyclopiazonic acid treatment to deplete Ca2+ stores, pointing to a major contribution of Ca2+ release from the sarcoplasmic reticulum in these contractile responses. Immunocytochemical analysis has indeed revealed colocalization of TRPM8 and InsP3 receptors. Moreover, menthol Ca2+ responses, which were somewhat reduced under Ca2+-free conditions, were strongly reduced by cyclopiazonic acid treatment to deplete Ca2+ store, whereas caffeine-induced Ca2+ responses were blunted in the presence of menthol. Finally, two other common TRPM8 agonists, WS-12 and icilin, also inhibited L-ICa. With respect to L-ICa inhibition, WS-12 is the most selective agonist. It augmented PE-induced contractions, whereas any secondary phase of vasorelaxation (as with menthol) was completely lacking. Thus TRPM8 channels are functionally active in rat tail artery myocytes and play a distinct direct stimulatory role in control of vascular tone. However, indirect effects of TRPM8 agonists, which are unrelated to TRPM8, are mediated by inhibition of L-type Ca2+ channels and largely obscure TRPM8-mediated vasoconstriction. These findings will promote our understanding of the vascular TRPM8 role, especially the well-known hypotensive effect of menthol, and may also have certain translational implications (e.g., in cardiovascular surgery, organ storage, transplantation, and Raynaud's phenomenon). PMID:27765744

Heterokaryon analysis of muscle differentiation: regulation of the postmitotic state.

PubMed

Clegg, C H; Hauschka, S D

1987-08-01

MM14 mouse myoblasts withdraw irreversibly from the cell cycle and become postmitotic within a few hours of being deprived of fibroblast growth factor (Clegg, C. H., T. A. Linkhart, B. B. Olwin, and S. D. Hauschka, 1987, J. Cell Biol., 105:949-956). To examine the mechanisms that may regulate this developmental state of skeletal muscle, we tested the mitogen responsiveness of various cell types after their polyethylene glycol-mediated fusion with post-mitotic myocytes. Heterokaryons containing myocytes and quiescent nonmyogenic cells such as 3T3, L cell, and a differentiation-defective myoblast line (DD-1) responded to mitogen-rich medium by initiating DNA synthesis. Myonuclei replicated DNA and reexpressed thymidine kinase. In contrast, (myocyte x G1 myoblast) heterokaryons failed to replicate DNA in mitogen-rich medium and became postmitotic. This included cells with a nuclear ratio of three myoblasts to one myocyte. Proliferation dominance in (myocyte x 3T3 cell) and (myocyte x DD-1) heterokaryons was conditionally regulated by the timing of mitogen treatment; such cells became postmitotic when mitogen exposure was delayed for as little as 6 h after cell fusion. In addition, (myocyte x DD-1) heterokaryons expressed a muscle-specific trait and lost epidermal growth factor receptors when they became postmitotic. These results demonstrate that DNA synthesis is not irreversibly blocked in skeletal muscle; myonuclei readily express proliferation-related functions when provided with a mitogenic signal. Rather, myocyte-specific repression of DNA synthesis in heterokaryons argues that the postmitotic state of skeletal muscle is regulated by diffusible factors that inhibit processes of cellular mitogenesis.

Negative feedback exerted by cAMP-dependent protein kinase and cAMP phosphodiesterase on subsarcolemmal cAMP signals in intact cardiac myocytes: an in vivo study using adenovirus-mediated expression of CNG channels.

PubMed

Rochais, Francesca; Vandecasteele, Grégoire; Lefebvre, Florence; Lugnier, Claire; Lum, Hazel; Mazet, Jean-Luc; Cooper, Dermot M F; Fischmeister, Rodolphe

2004-12-10

Intracardiac cAMP levels are modulated by hormones and neuromediators with specific effects on contractility and metabolism. To understand how the same second messenger conveys different information, mutants of the rat olfactory cyclic nucleotide-gated (CNG) channel alpha-subunit CNGA2, encoded into adenoviruses, were used to monitor cAMP in adult rat ventricular myocytes. CNGA2 was not found in native myocytes but was strongly expressed in infected cells. In whole cell patch-clamp experiments, the forskolin analogue L-858051 (L-85) elicited a non-selective, Mg2+ -sensitive current observed only in infected cells, which was thus identified as the CNG current (ICNG). The beta-adrenergic agonist isoprenaline (ISO) also activated ICNG, although the maximal efficiency was approximately 5 times lower than with L-85. However, ISO and L-85 exerted a similar maximal increase of the L-type Ca2+ current. The use of a CNGA2 mutant with a higher sensitivity for cAMP indicated that this difference is caused by the activation of a localized fraction of CNG channels by ISO. cAMP-dependent protein kinase (PKA) blockade with H89 or PKI, or phosphodiesterase (PDE) inhibition with IBMX, dramatically potentiated ISO- and L-85-stimulated ICNG. A similar potentiation of beta-adrenergic stimulation occurred when PDE4 was blocked, whereas PDE3 inhibition had a smaller effect (by 2-fold). ISO and L-85 increased total PDE3 and PDE4 activities in cardiomyocytes, although this effect was insensitive to H89. However, in the presence of IBMX, H89 had no effect on ISO stimulation of ICNG. This study demonstrates that subsarcolemmal cAMP levels are dynamically regulated by a negative feedback involving PKA stimulation of subsarcolemmal cAMP-PDE.

Effects of glutamine treatment on myocardial damage and cardiac function in rats after severe burn injury.

PubMed

Yan, Hong; Zhang, Yong; Lv, Shang-jun; Wang, Lin; Liang, Guang-ping; Wan, Qian-xue; Peng, Xi

2012-01-01

Treatment with glutamine has been shown to reduce myocardial damage associated with ischemia/reperfusion injury. However, the cardioprotective effect of glutamine specifically after burn injury remains unclear. The present study explores the ability of glutamine to protect against myocardial damage in rats that have been severely burned. Seventy-two Wistar rats were randomly divided into three groups: normal controls (C), burned controls (B) and a glutamine-treated group (G). Groups B and G were subjected to full thickness burns comprising 30% of total body surface area. Group G was administered 1.5 g/ (kg•d) glutamine and group B was given the same dose of alanine via intragastric administration for 3 days. Levels of serum creatine kinase (CK), lactate dehydrogenase (LDH), aspartate transaminase (AST) and blood lactic acid were measured, as well as myocardial ATP and glutathione (GSH) contents. Cardiac function indices and histopathological changes were analyzed at 12, 24, 48 and 72 post-burn hours. In both burned groups, levels of serum CK, LDH, AST and blood lactic acid increased significantly, while myocardial ATP and GSH contents decreased. Compared with group B, CK, LDH, and AST levels were lower and blood lactic acid, myocardial ATP and GSH levels were higher in group G. Moreover, cardiac contractile function inhibition and myocardial histopathological damage were significantly reduced in group G compared to B. Taken together, these results show that glutamine supplementation protects myocardial structure and function after burn injury by improving energy metabolism and by promoted the synthesis of ATP and GSH in cardiac myocytes.

Effects of glutamine treatment on myocardial damage and cardiac function in rats after severe burn injury

PubMed Central

Yan, Hong; Zhang, Yong; Lv, Shang-jun; Wang, Lin; Liang, Guang-ping; Wan, Qian-xue; Peng, Xi

2012-01-01

Treatment with glutamine has been shown to reduce myocardial damage associated with ischemia/reperfusion injury. However, the cardioprotective effect of glutamine specifically after burn injury remains unclear. The present study explores the ability of glutamine to protect against myocardial damage in rats that have been severely burned. Seventy-two Wistar rats were randomly divided into three groups: normal controls (C), burned controls (B) and a glutamine-treated group (G). Groups B and G were subjected to full thickness burns comprising 30% of total body surface area. Group G was administered 1.5 g/ (kg•d) glutamine and group B was given the same dose of alanine via intragastric administration for 3 days. Levels of serum creatine kinase (CK), lactate dehydrogenase (LDH), aspartate transaminase (AST) and blood lactic acid were measured, as well as myocardial ATP and glutathione (GSH) contents. Cardiac function indices and histopathological changes were analyzed at 12, 24, 48 and 72 post-burn hours. In both burned groups, levels of serum CK, LDH, AST and blood lactic acid increased significantly, while myocardial ATP and GSH contents decreased. Compared with group B, CK, LDH, and AST levels were lower and blood lactic acid, myocardial ATP and GSH levels were higher in group G. Moreover, cardiac contractile function inhibition and myocardial histopathological damage were significantly reduced in group G compared to B. Taken together, these results show that glutamine supplementation protects myocardial structure and function after burn injury by improving energy metabolism and by promotedthe synthesis of ATP and GSH in cardiac myocytes. PMID:22977661

Combinatorial Polymer Matrices Enhance In Vitro Maturation of Human Induced Pluripotent Cell Cell-Derived Cardiomyocytes

PubMed Central

Chun, Young Wook; Balikov, Daniel A.; Feaster, Tromondae K.; Williams, Charles H.; Sheng, Calvin C.; Lee, Jung-Bok; Boire, Timothy C.; Neely, M. Diana; Bellan, Leon M.; Ess, Kevin C.; Bowman, Aaron B.; Sung, Hak-Joon; Hong, Charles C.

2015-01-01

Cardiomyocytes derived from human induced pluripotent stem cells (iPSC-CMs) hold great promise for modeling human heart diseases. However, iPSC-CMs studied to date resemble immature embryonic myocytes and therefore do not adequately recapitulate native adult cardiomyocyte phenotypes. Since extracellular matrix plays an essential role in heart development and maturation in vivo, we sought to develop a synthetic culture matrix that could enhance functional maturation of iPSC-CMs in vitro. In this study, we employed a library of combinatorial polymers comprising of three functional subunits - poly-ε-caprolacton (PCL), polyethylene glycol (PEG), and carboxylated PCL (cPCL) - as synthetic substrates for culturing human iPSC-CMs. Of these, iPSC-CMs cultured on 4%PEG-96%PCL (each % indicates the corresponding molar ratio) exhibit the greatest contractility and mitochondrial function. These functional enhancements are associated with increased expression of cardiac myosin light chain-2v, cardiac troponin I and integrin alpha-7. Importantly, iPSC-CMs cultured on 4%PEG-95%PCL demonstrate troponin I (TnI) isoform switch from the fetal slow skeletal TnI (ssTnI) to the postnatal cardiac TnI (cTnI), the first report of such transition in vitro. Finally, culturing iPSC-CMs on 4%PEG-96%PCL also significantly increased expression of genes encoding intermediate filaments known to transduce integrin-mediated mechanical signals to the myofilaments. In summary, our study demonstrates that synthetic culture matrices engineered from combinatorial polymers can be utilized to promote in vitro maturation of human iPSC-CMs through the engagement of critical matrix-integrin interactions. PMID:26204225

[Ca2+]i Elevation and Oxidative Stress Induce KCNQ1 Protein Translocation from the Cytosol to the Cell Surface and Increase Slow Delayed Rectifier (IKs) in Cardiac Myocytes*

PubMed Central

Wang, Yuhong; Zankov, Dimitar P.; Jiang, Min; Zhang, Mei; Henderson, Scott C.; Tseng, Gea-Ny

2013-01-01

Our goals are to simultaneously determine the three-dimensional distribution patterns of KCNQ1 and KCNE1 in cardiac myocytes and to study the mechanism and functional implications for variations in KCNQ1/KCNE1 colocalization in myocytes. We monitored the distribution patterns of KCNQ1, KCNE1, and markers for subcellular compartments/organelles using immunofluorescence/confocal microscopy and confirmed the findings in ventricular myocytes by directly observing fluorescently tagged KCNQ1-GFP and KCNE1-dsRed expressed in these cells. We also monitored the effects of stress on KCNQ1-GFP and endoplasmic reticulum (ER) remodeling during live cell imaging. The data showed that 1) KCNE1 maintained a stable cell surface localization, whereas KCNQ1 exhibited variations in the cytosolic compartment (striations versus vesicles) and the degree of presence on the cell surface; 2) the degree of cell surface KCNQ1/KCNE1 colocalization was positively correlated with slow delayed rectifier (IKs) current density; 3) KCNQ1 and calnexin (an ER marker) shared a cytosolic compartment; and 4) in response to stress ([Ca2+]i elevation, oxidative overload, or AT1R stimulation), KCNQ1 exited the cytosolic compartment and trafficked to the cell periphery in vesicles. This was accompanied by partial ER fragmentation. We conclude that the cellular milieu regulates KCNQ1 distribution in cardiac myocytes and that stressful conditions can increase IKs by inducing KCNQ1 movement to the cell surface. This represents a hitherto unrecognized mechanism by which IKs fulfills its function as a repolarization reserve in ventricular myocytes. PMID:24142691

Altered transient outward current in human atrial myocytes of patients with reduced left ventricular function.

PubMed

Schreieck, J; Wang, Y; Overbeck, M; Schömig, A; Schmitt, C

2000-02-01

Electrophysiologic remodeling is involved in the self-perpetuation of atrial fibrillation. To define whether differences in atrial electrophysiology already are present in patients with increased susceptibility for atrial fibrillation, we compared patients in sinus rhythm with and without heart failure. Atrial specimens were obtained from patients with reduced left ventricular ejection fraction (LVEF; n = 10) and normal LVEF (n = 16) who were undergoing aortocoronary bypass surgery and from donor hearts (n = 4). Enzymatically isolated atrial myocytes were investigated by whole cell, patch clamp techniques. Total outward current was significantly larger in myocytes of hearts with low LVEF than normal LVEF (19.4 +/- 1.3 vs 15.1 +/- 1.2 pA/pF at pulses to +60 mV, respectively). Analysis of inactivation time courses of different outward current components revealed that the observed current difference is due to the transient calcium-independent outward current I(to1) which is twice as large in the low LVEF group than in the normal LVEF group (9.4 +/- 0.9 vs 4.7 +/- 0.4 pA/pF at pulses to +60 mV, respectively). I(to1) recovery from inactivation was significantly more rapid in myocytes of hearts with low LVEF, and action potential plateau in these cells was significantly shorter. The results of I(to1) and action potential measurements in atrial myocytes of donor hearts were very similar to the results of patients with preserved heart function. I(to1) in human atrial myocytes of patients with reduced LVEF has an increased density and altered kinetics in sinus rhythm. These differences in outward current may explain the reduced plateau phase of action potentials.

Antioxidant Phytochemicals of Opuntia ficus-indica (L.) Mill. Cladodes with Potential Anti-spasmodic Activity.

PubMed

Lanuzza, Francesco; Occhiuto, Francesco; Monforte, Maria Teresa; Tripodo, Maria Marcella; D'Angelo, Valeria; Galati, Enza Maria

2017-10-01

Opuntia ficus-indica (OFI) (L.) Mill. (Cactaceae), a plant widespread in dry regions of the world, shows interesting biological activities (cicatrizant, antiulcer, anti-inflammatory, and hypolipidemic) and is widely used in traditional medicine. Phytochemical analysis and antispasmodic effect of wild OFI cladodes were carried out. Polyphenols and Vitamin E occurrence, in antioxidant pool of OFI cladodes, were quantified by high-performance liquid chromatography. The antispasmodic effect of OFI cladodes was assessed in isolated rabbit smooth muscle tissues. The experiments were carried out with preparations of rabbit jejunum and uterus with the spontaneous contractile activity, to evaluate the effect of cumulative concentrations of the extract on basal tone, amplitude, and frequency of contractions. Catechin, quercetin, kaempferol, isorhamnetin and chlorogenic, ferulic, and p-coumaric acid were identified. α-, β-, and γ-tocopherols have been highlighted and α-tocopherol is the major component. OFI cladodes contain significant amount of polyphenols and tocopherols that are effective radical scavengers and inhibited ethanol 1,1-diphenyl-2-picrylhydrazyl formation by 50%. OFI cladodes caused a light inhibition of amplitude and frequency of spontaneous contractions and a marked decrease in muscle basal tone of rabbit jejunum preparations. On spontaneously contracting uterus preparations, the addition of increasing concentrations of cladode extract caused uterine muscle relaxation. The contraction of smooth muscle preparations depends on an increase in cytoplasmic free calcium ion concentration, which activates the contractile elements. The flavonoids may suppress the contractility of smooth myocytes, by an inhibition of availability of Ca 2+ for muscle contraction. Opuntia ficus-indica (OFI) cladodes contain significant amount of polyphenols and tocopherols that are effective radical scavengers and inhibited ethanol 1,1-diphenyl-2-picrylhydrazyl formation by 50%Polyphenols and Vitamin E complex occurrence in OFI cladodes were characterized by high-performance liquid chromatographyOFI cladodes exhibited significative antispasmodic activity. The antispasmodic effect was assessed in isolated rabbit smooth muscle tissues. The experiments were carried out with preparations of rabbit jejunum and uterus with the spontaneous contractile activity, to evaluate the effect of cumulative concentrations of the extract on basal tone, amplitude, and frequency of contractions. Abbreviations used: OFI: Opuntia ficus-indica , DPPH: Ethanol 1,1-diphenyl-2-picrylhydrazyl.

The role of spatial organization of Ca2+ release sites in the generation of arrhythmogenic diastolic Ca2+ release in myocytes from failing hearts

PubMed Central

Ho, Hsiang-Ting; Bonilla, Ingrid M.; Terentyeva, Radmila; Schober, Karsten E.; Terentyev, Dmitry; Carnes, Cynthia A.

2018-01-01

In heart failure (HF), dysregulated cardiac ryanodine receptors (RyR2) contribute to the generation of diastolic Ca2+ waves (DCWs), thereby predisposing adrenergically stressed failing hearts to life-threatening arrhythmias. However, the specific cellular, subcellular, and molecular defects that account for cardiac arrhythmia in HF remain to be elucidated. Patch-clamp techniques and confocal Ca2+ imaging were applied to study spatially defined Ca2+ handling in ventricular myocytes isolated from normal (control) and failing canine hearts. Based on their activation time upon electrical stimulation, Ca2+ release sites were categorized as coupled, located in close proximity to the sarcolemmal Ca2+ channels, and uncoupled, the Ca2+ channel-free non-junctional Ca2+ release units. In control myocytes, stimulation of β-adrenergic receptors with isoproterenol (Iso) resulted in a preferential increase in Ca2+ spark rate at uncoupled sites. This site-specific effect of Iso was eliminated by the phosphatase inhibitor okadaic acid, which caused similar facilitation of Ca2+ sparks at coupled and uncoupled sites. Iso-challenged HF myocytes exhibited increased predisposition to DCWs compared to control myocytes. In addition, the overall frequency of Ca2+ sparks was increased in HF cells due to preferential stimulation of coupled sites. Furthermore, coupled sites exhibited accelerated recovery from functional refractoriness in HF myocytes compared to control myocytes. Spatially resolved subcellular Ca2+ mapping revealed that DCWs predominantly originated from coupled sites. Inhibition of CaMK∏ suppressed DCWs and prevented preferential stimulation of coupled sites in Iso-challenged HF myocytes. These results suggest that CaMK∏-(and phosphatase)-dependent dysregulation of junctional Ca2+ release sites contributes to Ca2+-dependent arrhythmogenesis in HF. PMID:28612155

Rapid fusion between mesenchymal stem cells and cardiomyocytes yields electrically active, non-contractile hybrid cells.

PubMed

Shadrin, Ilya Y; Yoon, Woohyun; Li, Liqing; Shepherd, Neal; Bursac, Nenad

2015-07-10

Cardiac cell therapies involving bone marrow-derived human mesenchymal stem cells (hMSCs) have shown promising results, although their mechanisms of action are still poorly understood. Here, we investigated direct interactions between hMSCs and cardiomyocytes in vitro. Using a genetic Ca(2+) indicator gCaMP3 to efficiently label hMSCs in co-cultures with neonatal rat ventricular myocytes (NRVMs), we determined that 25-40% of hMSCs (from 4 independent donors) acquired periodic Ca(2+) transients and cardiac markers through spontaneous fusion with NRVMs. Sharp electrode and voltage-clamp recordings in fused cells showed action potential properties and Ca(2+) current amplitudes in between those of non-fused hMSCs and NRVMs. Time-lapse video-microscopy revealed the first direct evidence of active fusion between hMSCs and NRVMs within several hours of co-culture. Application of blebbistatin, nifedipine or verapamil caused complete and reversible inhibition of fusion, suggesting potential roles for actomyosin bridging and Ca(2+) channels in the fusion process. Immunostaining for Cx43, Ki67, and sarcomeric α-actinin showed that fused cells remain strongly coupled to surrounding NRVMs, but downregulate sarcomeric structures over time, acquiring a non-proliferative and non-contractile phenotype. Overall, these results describe the phenotype and mechanisms of hybrid cell formation via fusion of hMSCs and cardiomyocytes with potential implications for cardiac cell therapy.

[Gallbladder contractility in children with functional abdominal pain or irritable bowel syndrome].

PubMed

Iwańczak, Franciszek; Siedlecka-Dawidko, Jolanta; Iwanczak, Barbara

2013-07-01

III Rome Criteria of functional gastrointestinal disorders in children, distinguished the disturbances with abdominal pain, to which irritable bowel syndrome, functional abdominal pains, functional dyspepsia and abdominal migraine were included. THE AIM OF THE STUDY was sonographic assessment of the gallbladder and its contractility in functional abdominal pain and irritable bowel syndrome in children. The study comprised 96 children aged 6 to 18 years, 59 girls and 37 boys. Depending on diagnosis, the children were divided into three groups. 38 children with functional abdominal pain constituted the first group, 26 children with irritable bowel syndrome were included to the second group, the third group consisted of 32 healthy children (control group). Diagnosis of functional abdominal pain and irritable bowel syndrome was made based on the III Rome Criteria. In irritable bowel syndrome both forms with diarrhea (13) and with constipation (13) were observed. Anatomy and contractility of the gallbladder were assessed by ultrasound examination. The presence of septum, wall thickness, thick bile, vesicle volume in fasting state and 30th and 60th minute after test meal were taken into consideration. Test meal comprised about 15% of caloric requirement of moderate metabolism. Children with bile stones and organic diseases were excluded from the study. Thickened vesicle wall and thick bile were present more frequently in children with irritable bowel syndrome and functional abdominal pain than in control group (p < 0.02). Fasting vesicle volume was significantly greater in children with functional abdominal pain than in irritable bowel syndrome and control group (p = 0.003, p = 0.05). Vesicle contractility after test meal was greatest in children with functional abdominal pain. Evaluation of diminished (smaller than 30%) and enlarged (greater then 80%) gallbladder contractility at 30th and 60th minute after test meal demonstrated disturbances of contractility in children with irritable bowel syndrome and functional abdominal pain. In children with functional abdominal pain and irritable bowel syndrome disturbances of gallbladder anatomy, fasting volume and contractility after test meal were demonstrated. The observed disturbances require further studies for explanation of their role in functional gastrointestinal disturbances with abdominal pain in children.

Combined atorvastatin and coenzyme Q10 improve the left ventricular function in isoproterenol-induced heart failure in rat.

PubMed

Garjani, Alireza; Andalib, Sina; Biabani, Sajjad; Soraya, Hamid; Doustar, Yousef; Garjani, Afagh; Maleki-Dizaji, Nasrin

2011-09-01

The effect of atorvastatin on cardiac remodeling, function, and homodynamic parameters in isoproterenol-induced heart failure was evaluated in the present study. A subcutaneous injection of isoproterenol (5mg/kg/day) for 10 days was used for the induction of heart failure. Isoproterenol administration produced intensive myocardial necrosis and fibrosis with a significant decrease in the arterial pressure indices, heart rate, contractility (LVdP/dt(max)) and relaxation (LVdP/dt(min)), but an increase in the left ventricular end-diastolic pressure. Rats were randomly assigned to control, treatment with only atorvastatin, and treatment with atorvastatin plus coenzyme Q10. Histopathological analysis showed a marked attenuation of myocyte necrosis and interstitial fibrosis in all atorvastatin treated groups (P<0.001). A low dose of atorvastatin (5mg/kg/day) significantly improved the left ventricular systolic pressure, contractility and relaxation (P<0.01). On the contrary, a high dose of atorvastatin (20mg/kg/day) worsened the isoproterenol-induced left ventricular dysfunction by a further reduction of LVdP/dt(max) from +2780 ± 94 to +1588 ± 248 (mmHg/s; P<0.01) and LVdP/dt(min) from -2007 ± 190 to -2939 ± 291 (mmHg/s; P<0.05). Co-administration of coenzyme Q10 with atorvastatin reversed the hemodynamic depression and the left ventricular dysfunction to a high level (P<0.001). There was a lower level of LVEDPs in the atorvastatin+coenzyme Q10 treated groups (3 ± 1 and 4 ± 1.4 versus 8 ± 3.5 and 14 ± 3.6 mmHg, respectively), thereby suggesting improvement in the myocardial stiffness by the combined coenzyme Q10 and atorvastatin treatment. The atorvastatin therapy attenuated myocardial necrosis and fibrosis in isoproterenol-induced heart failure. However, a high dose of the drug considerably worsened the left ventricular dysfunction and hemodynamic depression, which was reversed by coenzyme Q10 co-administration. Copyright © 2011 Elsevier B.V. All rights reserved.

Repeated high-intensity exercise modulates Ca(2+) sensitivity of human skeletal muscle fibers.

PubMed

Gejl, K D; Hvid, L G; Willis, S J; Andersson, E; Holmberg, H-C; Jensen, R; Frandsen, U; Hansen, J; Plomgaard, P; Ørtenblad, N

2016-05-01

The effects of short-term high-intensity exercise on single fiber contractile function in humans are unknown. Therefore, the purposes of this study were: (a) to access the acute effects of repeated high-intensity exercise on human single muscle fiber contractile function; and (b) to examine whether contractile function was affected by alterations in the redox balance. Eleven elite cross-country skiers performed four maximal bouts of 1300 m treadmill skiing with 45 min recovery. Contractile function of chemically skinned single fibers from triceps brachii was examined before the first and following the fourth sprint with respect to Ca(2+) sensitivity and maximal Ca(2+) -activated force. To investigate the oxidative effects of exercise on single fiber contractile function, a subset of fibers was incubated with dithiothreitol (DTT) before analysis. Ca(2+) sensitivity was enhanced by exercise in both MHC I (17%, P < 0.05) and MHC II (15%, P < 0.05) fibers. This potentiation was not present after incubation of fibers with DTT. Specific force of both MHC I and MHC II fibers was unaffected by exercise. In conclusion, repeated high-intensity exercise increased Ca(2+) sensitivity in both MHC I and MHC II fibers. This effect was not observed in a reducing environment indicative of an exercise-induced oxidation of the human contractile apparatus. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Bone marrow cells adopt the cardiomyogenic fate in vivo

PubMed Central

Rota, Marcello; Kajstura, Jan; Hosoda, Toru; Bearzi, Claudia; Vitale, Serena; Esposito, Grazia; Iaffaldano, Grazia; Padin-Iruegas, M. Elena; Gonzalez, Arantxa; Rizzi, Roberto; Small, Narissa; Muraski, John; Alvarez, Roberto; Chen, Xiongwen; Urbanek, Konrad; Bolli, Roberto; Houser, Steven R.; Leri, Annarosa; Sussman, Mark A.; Anversa, Piero

2007-01-01

The possibility that adult bone marrow cells (BMCs) retain a remarkable degree of developmental plasticity and acquire the cardiomyocyte lineage after infarction has been challenged, and the notion of BMC transdifferentiation has been questioned. The center of the controversy is the lack of unequivocal evidence in favor of myocardial regeneration by the injection of BMCs in the infarcted heart. Because of the interest in cell-based therapy for heart failure, several approaches including gene reporter assay, genetic tagging, cell genotyping, PCR-based detection of donor genes, and direct immunofluorescence with quantum dots were used to prove or disprove BMC transdifferentiation. Our results indicate that BMCs engraft, survive, and grow within the spared myocardium after infarction by forming junctional complexes with resident myocytes. BMCs and myocytes express at their interface connexin 43 and N-cadherin, and this interaction may be critical for BMCs to adopt the cardiomyogenic fate. With time, a large number of myocytes and coronary vessels are generated. Myocytes show a diploid DNA content and carry, at most, two sex chromosomes. Old and new myocytes show synchronicity in calcium transients, providing strong evidence in favor of the functional coupling of these two cell populations. Thus, BMCs transdifferentiate and acquire the cardiomyogenic and vascular phenotypes restoring the infarcted heart. Together, our studies reveal that locally delivered BMCs generate de novo myocardium composed of integrated cardiomyocytes and coronary vessels. This process occurs independently of cell fusion and ameliorates structurally and functionally the outcome of the heart after infarction. PMID:17965233

Myocardin: A novel player in atherosclerosis.

PubMed

Xia, Xiao-Dan; Zhou, Zhen; Yu, Xiao-Hua; Zheng, Xi-Long; Tang, Chao-Ke

2017-02-01

Myocardin (MYOCD) the most important coactivator of serum response factor (SRF), plays a critical role specifically in the development of cardiac myocytes and vascular smooth muscle cells (VSMCs). Binding of Myocardin to the SRF on the CArG box-containing target genes can transcriptionally activate a variety of downstream muscle-specific genes, such as Sm22α, Acta2, Myh11, and several other signaling pathways. Myocardin expression represents a contractile and differentiated SMC phenotype. Loss of Myocardin, however, represents a synthetic and dedifferentiated phenotype, a hallmark in atherosclerosis. Growing evidence shows that Myocardin is involved in lipid metabolism and vascular inflammation, the primary pathogenesis of atherosclerosis. Moreover, Myocardin expression level is altered in atherosclerotic patients and animal models, suggesting more extensive and important roles for Myocardin in atherosclerosis. In the current review, we summarized recent progress on the regulation and signaling of Myocardin, and highlighted its impacts on atherosclerotic disease. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Tissue Cells Feel and Respond to the Stiffness of Their Substrate

NASA Astrophysics Data System (ADS)

Discher, Dennis E.; Janmey, Paul; Wang, Yu-li

2005-11-01

Normal tissue cells are generally not viable when suspended in a fluid and are therefore said to be anchorage dependent. Such cells must adhere to a solid, but a solid can be as rigid as glass or softer than a baby's skin. The behavior of some cells on soft materials is characteristic of important phenotypes; for example, cell growth on soft agar gels is used to identify cancer cells. However, an understanding of how tissue cells-including fibroblasts, myocytes, neurons, and other cell types-sense matrix stiffness is just emerging with quantitative studies of cells adhering to gels (or to other cells) with which elasticity can be tuned to approximate that of tissues. Key roles in molecular pathways are played by adhesion complexes and the actin-myosin cytoskeleton, whose contractile forces are transmitted through transcellular structures. The feedback of local matrix stiffness on cell state likely has important implications for development, differentiation, disease, and regeneration.

Cardiac biomarkers as sensitive tools to evaluate the impact of xenobiotics on amphibians: the effects of anionic surfactant linear alkylbenzene sulfonate (LAS).

PubMed

Jones-Costa, Monica; Franco-Belussi, Lilian; Vidal, Felipe Augusto Pinto; Gongora, Nathália Penteado; Castanho, Luciano Mendes; Dos Santos Carvalho, Cleoni; Silva-Zacarin, Elaine Cristina Mathias; Abdalla, Fabio Camargo; Duarte, Iolanda Cristina Silveira; Oliveira, Classius De; de Oliveira, Cristiane Ronchi; Salla, Raquel Fernanda

2018-04-30

Amphibian populations have been experiencing a drastic decline worldwide. Aquatic contaminants are among the main factors responsible for this decline, especially in the aquatic environment. The linear alkylbenzene sulfonate (LAS) is of particular concern, since it represents 84% of the anionic surfactants' trade. In Brazil, the maximal LAS concentration allowed in fresh waters is 0.5mgL -1 , but its potential harmful effects in amphibians remain unknown. Therefore, this study aimed to analyze the effects of a sublethal concentration of LAS (0.5mgL -1 ) for 96h on sensitive cardiac biomarkers of bullfrog tadpoles, Lithobates catesbeianus (Shaw, 1802). For this, we measured the activity level (AL - % of animals), in situ heart rate (f H - bpm), relative ventricular mass (RVM - % of body mass), in vitro myocardial contractility and cardiac histology of the ventricles. Tadpoles' AL and f H decreased in LAS group. In contrast, the RVM increased, as a result of a hypertrophy of the myocardium, which was corroborated by the enlargement of the nuclear measures and the increase of myocytes' diameters. These cellular effects resulted in an elevation of the in vitro contractile force of ventricle strips. Acceleration in the contraction (TPT - ms) also occurred, although no alterations in the time to relaxation (THR -ms) were observed. Therefore, it can be concluded that even when exposed to an environmentally safe concentration, this surfactant promotes several alterations in the cardiac function of bullfrog tadpoles that can impair their development, making them more susceptible to predators and less competitive in terms of reproduction success. Thus, LAS concentrations that are considered safe by Brazilian by regulatory agencies must be revised in order to minimize a drastic impact over amphibian populations. This study demonstrates the relevance of employing cardiac biomarkers at different levels (e.g., morphological, physiological and cellular) to evaluate effects of xenobiotics in tadpoles. Copyright © 2018 Elsevier Inc. All rights reserved.

Targeted Gene Silencing of Tumor Necrosis Factor Attenuates the Negative Inotropic Effects of Lipopolysaccharide in Isolated Contracting Cardiac Myocytes

PubMed Central

Ramabadran, R. S.; Chancey, Amanda; Vallejo, Jesus G.; Barger, Philip M.; Sivasubramanian, Natarajan; Mann, Douglas L.

2008-01-01

Bacterial endotoxin (lipopolysaccharide) depresses cardiovascular function; however, the mediators and signaling pathways that are responsible for the negative inotropic effects of lipopolysaccharide are not fully known. We used RNA interference to determine the relative role of tumor necrosis factor with respect to mediating the negative inotropic effects of lipopolysaccharide in isolated cardiac myocytes. Cardiac myocyte cultures were treated with lipopolysaccharide in the presence or absence of small interfering RNAs (siRNA) for tumor necrosis factor. We examined the effects of tumor necrosis factor siRNA on lipopolysaccharide-induced tumor necrosis factor messenger RNA (mRNA) and protein biosynthesis, as well as the negative inotropic effects of lipopolysaccharide in isolated contracting cardiac myocytes. Treatment of adult cardiac myocyte cultures with tumor necrosis factor siRNA significantly attenuated lipopolysaccharide-induced tumor necrosis factor mRNA and protein biosynthesis, whereas transfection with a double-stranded RNA that does not target mammalian mRNA had no effect. Pretreatment with tumor necrosis factor siRNA significantly attenuated, but did not abrogate, the lipopolysaccharide-induced decrease in sarcomere shortening in isolated contracting cardiac myocytes. In contrast, tumor necrosis factor siRNA had a comparatively smaller effect on improving sarcomere shortening once the negative inotropic effects of lipopolysaccharide were fully established. These results suggest that tumor necrosis factor plays an important upstream role in lipopolysaccharide-induced negative inotropic effects in isolated contracting cardiac myocytes and that other molecular mechanisms are responsible for the decrease in sarcomere shortening after sustained lipopolysaccharide signaling. PMID:18427645

Characterization of L-type calcium channel activity in atrioventricular nodal myocytes from rats with streptozotocin-induced Diabetes mellitus

PubMed Central

Yuill, Kathryn H; Al Kury, Lina T; Howarth, Frank Christopher

2015-01-01

Cardiovascular complications are common in patients with Diabetes mellitus (DM). In addition to changes in cardiac muscle inotropy, electrical abnormalities are also commonly observed in these patients. We have previously shown that spontaneous cellular electrical activity is altered in atrioventricular nodal (AVN) myocytes, isolated from the streptozotocin (STZ) rat model of type-1 DM. In this study, utilizing the same model, we have characterized the changes in L-type calcium channel activity in single AVN myocytes. Ionic currents were recorded from AVN myocytes isolated from the hearts of control rats and from those with STZ-induced diabetes. Patch-clamp recordings were used to assess the changes in cellular electrical activity in individual myocytes. Type-1 DM significantly altered the cellular characteristics of L-type calcium current. A reduction in peak ICaL density was observed, with no corresponding changes in the activation parameters of the current. L-type calcium channel current also exhibited faster time-dependent inactivation in AVN myocytes from diabetic rats. A negative shift in the voltage dependence of inactivation was also evident, and a slowing of restitution parameters. These findings demonstrate that experimentally induced type-1 DM significantly alters AVN L-type calcium channel cellular electrophysiology. These changes in ion channel activity may contribute to the abnormalities in cardiac electrical function that are associated with high mortality levels in patients with DM. PMID:26603460

Myocyte-Derived Hsp90 Modulates Collagen Upregulation via Biphasic Activation of STAT-3 in Fibroblasts during Cardiac Hypertrophy

PubMed Central

Datta, Ritwik; Bansal, Trisha; Rana, Santanu; Datta, Kaberi; Datta Chaudhuri, Ratul; Chawla-Sarkar, Mamta

2016-01-01

ABSTRACT Signal transducer and activator of transcription 3 (STAT-3)-mediated signaling in relation to upregulated collagen expression in fibroblasts during cardiac hypertrophy is well defined. Our recent findings have identified heat shock protein 90 (Hsp90) to be a critical modulator of fibrotic signaling in cardiac fibroblasts in this disease milieu. The present study was therefore intended to analyze the role of Hsp90 in the STAT-3-mediated collagen upregulation process. Our data revealed a significant difference between in vivo and in vitro results, pointing to a possible involvement of myocyte-fibroblast cross talk in this process. Cardiomyocyte-targeted knockdown of Hsp90 in rats (Rattus norvegicus) in which the renal artery was ligated showed downregulated collagen synthesis. Furthermore, the results obtained with cardiac fibroblasts conditioned with Hsp90-inhibited hypertrophied myocyte supernatant pointed toward cardiomyocytes' role in the regulation of collagen expression in fibroblasts during hypertrophy. Our study also revealed a novel signaling mechanism where myocyte-derived Hsp90 orchestrates not only p65-mediated interleukin-6 (IL-6) synthesis but also its release in exosomal vesicles. Such myocyte-derived exosomes and myocyte-secreted IL-6 are responsible in unison for the biphasic activation of STAT-3 signaling in cardiac fibroblasts that culminates in excess collagen synthesis, leading to severely compromised cardiac function during cardiac hypertrophy. PMID:28031326

Myocyte-Derived Hsp90 Modulates Collagen Upregulation via Biphasic Activation of STAT-3 in Fibroblasts during Cardiac Hypertrophy.

PubMed

Datta, Ritwik; Bansal, Trisha; Rana, Santanu; Datta, Kaberi; Datta Chaudhuri, Ratul; Chawla-Sarkar, Mamta; Sarkar, Sagartirtha

2017-03-15

Signal transducer and activator of transcription 3 (STAT-3)-mediated signaling in relation to upregulated collagen expression in fibroblasts during cardiac hypertrophy is well defined. Our recent findings have identified heat shock protein 90 (Hsp90) to be a critical modulator of fibrotic signaling in cardiac fibroblasts in this disease milieu. The present study was therefore intended to analyze the role of Hsp90 in the STAT-3-mediated collagen upregulation process. Our data revealed a significant difference between in vivo and in vitro results, pointing to a possible involvement of myocyte-fibroblast cross talk in this process. Cardiomyocyte-targeted knockdown of Hsp90 in rats ( Rattus norvegicus ) in which the renal artery was ligated showed downregulated collagen synthesis. Furthermore, the results obtained with cardiac fibroblasts conditioned with Hsp90-inhibited hypertrophied myocyte supernatant pointed toward cardiomyocytes' role in the regulation of collagen expression in fibroblasts during hypertrophy. Our study also revealed a novel signaling mechanism where myocyte-derived Hsp90 orchestrates not only p65-mediated interleukin-6 (IL-6) synthesis but also its release in exosomal vesicles. Such myocyte-derived exosomes and myocyte-secreted IL-6 are responsible in unison for the biphasic activation of STAT-3 signaling in cardiac fibroblasts that culminates in excess collagen synthesis, leading to severely compromised cardiac function during cardiac hypertrophy. Copyright © 2017 American Society for Microbiology.

Mammalian target of rapamycin (mTOR) signaling is required for a late-stage fusion process during skeletal myotube maturation.

PubMed

Park, In-Hyun; Chen, Jie

2005-09-09

Skeletal myogenesis is a well orchestrated cascade of events regulated by multiple signaling pathways, one of which is recently characterized by its sensitivity to the bacterial macrolide rapamycin. Previously we reported that the mammalian target of rapamycin (mTOR) regulates the initiation of the differentiation program in mouse C2C12 myoblasts by controlling the expression of insulin-like growth factor-II in a kinase-independent manner. Here we provide experimental evidence suggesting that a different mode of mTOR signaling regulates skeletal myogenesis at a later stage. In the absence of endogenous mTOR function in C2C12 cells treated with rapamycin, a kinase-inactive mTOR fully supports myogenin expression, but causes a delay in contractile protein expression. Myoblasts fuse to form nascent myotubes in the absence of kinase-active mTOR, whereas the formation of mature myotubes by further fusion requires the catalytic activity of mTOR. Therefore, the two stages of myocyte fusion are molecularly separable at the level of mTOR signaling. In addition, our data suggest that a factor secreted into the culture medium is responsible for mediating the function of mTOR in regulating the late-stage fusion leading to mature myotubes. Furthermore, taking advantage of the unique features of cells stably expressing a mutant mTOR, we have performed cDNA microarray analysis to compare global gene expression profiles between mature and nascent myotubes, the results of which have implicated classes of genes and revealed candidate regulators in myotube maturation or functions of mature myotubes.

Cardiac Remodeling: Endothelial Cells Have More to Say Than Just NO

PubMed Central

Segers, Vincent F. M.; Brutsaert, Dirk L.; De Keulenaer, Gilles W.

2018-01-01

The heart is a highly structured organ consisting of different cell types, including myocytes, endothelial cells, fibroblasts, stem cells, and inflammatory cells. This pluricellularity provides the opportunity of intercellular communication within the organ, with subsequent optimization of its function. Intercellular cross-talk is indispensable during cardiac development, but also plays a substantial modulatory role in the normal and failing heart of adults. More specifically, factors secreted by cardiac microvascular endothelial cells modulate cardiac performance and either positively or negatively affect cardiac remodeling. The role of endothelium-derived small molecules and peptides—for instance NO or endothelin-1—has been extensively studied and is relatively well defined. However, endothelial cells also secrete numerous larger proteins. Information on the role of these proteins in the heart is scattered throughout the literature. In this review, we will link specific proteins that modulate cardiac contractility or cardiac remodeling to their expression by cardiac microvascular endothelial cells. The following proteins will be discussed: IL-6, periostin, tenascin-C, thrombospondin, follistatin-like 1, frizzled-related protein 3, IGF-1, CTGF, dickkopf-3, BMP-2 and−4, apelin, IL-1β, placental growth factor, LIF, WISP-1, midkine, and adrenomedullin. In the future, it is likely that some of these proteins can serve as markers of cardiac remodeling and that the concept of endothelial function and dysfunction might have to be redefined as we learn more about other factors secreted by ECs besides NO. PMID:29695980

Metabolic coupling of glutathione between mouse and quail cardiac myocytes and its protective role against oxidative stress.

PubMed

Nakamura, T Y; Yamamoto, I; Kanno, Y; Shiba, Y; Goshima, K

1994-05-01

Cultured quail myocytes were much more resistant to H2O2 toxicity than cultured mouse myocytes. The intracellular concentration of glutathione ([GSH]i) and the activity of gamma-glutamylcysteine synthetase (gamma-GCS) in quail heart cells were about five and three times higher, respectively, than in mouse heart cells, although catalase and glutathione peroxidase (GSHpx) activity was similar in both. Preloading of gamma-glutamylcysteine monoethyl ester (gamma-GCE), a membrane-permeating GSH precursor, increased the H2O2 resistance of cultured mouse myocytes. These observations suggest that the high [GSH]i and the high activity of gamma-GCS in quail myocytes are responsible for their high resistance to H2O2. Both H2O2 sensitivity and [GSH]i of mosaic sheets composed of equal amounts of mouse and quail myocytes approximated those of sheets composed entirely of quail myocytes. From these observations, it is hypothesized that GSH was transferred from quail myocytes to mouse myocytes, probably through gap junctions between them, and that quail myocytes resynthesized GSH by a feedback mechanism, thus maintaining their intracellular GSH levels. When the fluorescent dye lucifer yellow was injected into a beating quail myocyte in a mosaic sheet, it spread to neighboring mouse myocytes but not to neighboring L cells (a cell line derived from mouse connective tissue). These observations indicate that existence of gap junctions in the region of cell contact between mouse and quail myocytes but not between quail myocytes and L cells. When quail myocytes preloaded with [3H]gamma-GCE were cocultured with mouse myocytes and L cells, the radioactivity was transmitted to neighboring mouse myocytes but not L cells. These observations show that GSH and/or its precursors can be transmitted from quail myocytes to mouse myocytes through gap junctions and that this can protect mouse myocytes from H2O2 toxicity. Mouse myocyte sheets composed of 10(4) cells or more showed higher resistance to H2O2 toxicity than single isolated mouse myocytes. Metabolic coupling of GSH between myocytes may contribute at least in part to this high resistance of the cell sheets.

Kinetics of Mechanical Stretch-Induced Nitric Oxide Production in Rat Ventricular Cardiac Myocytes.

PubMed

Shim, A L; Mitrokhin, V M; Gorbacheva, L R; Savinkova, I G; Pustovit, K B; Mladenov, M I; Kamkin, A G

2017-09-01

Discrete mechanical stretch of isolated spontaneously contracting cardiac myocytes was employed to examine the kinetics of NO production in these cells. NO oscillations were detected with fluorescent dye 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate. The mechanisms underlying stretch-induced changes in NO concentration remain unclear and further studies are needed to evaluate the role of NO oscillation in the regulation of cardiomyocyte function.

Improvement of left ventricular remodeling after myocardial infarction with eight weeks L-thyroxine treatment in rats.

PubMed

Chen, Yue-Feng; Weltman, Nathan Y; Li, Xiang; Youmans, Steven; Krause, David; Gerdes, Anthony Martin

2013-02-14

Left ventricular (LV) remodeling following large transmural myocardial infarction (MI) remains a pivotal clinical issue despite the advance of medical treatment over the past few decades. Identification of new medications to improve the remodeling process and prevent progression to heart failure after MI is critical. Thyroid hormones (THs) have been shown to improve LV function and remodeling in animals post-MI and in the human setting. However, changes in underlying cellular remodeling resulting from TH treatment are not clear. MI was produced in adult female Sprague-Dawley rats by ligation of the left descending coronary artery. L-thyroxine (T4) pellet (3.3 mg, 60 days sustained release) was used to treat MI rats for 8 weeks. Isolated myocyte shape, arterioles, and collagen deposition in the non-infarcted area were measured at terminal study. T4 treatment improved LV ±dp/dt, normalized TAU, and increased myocyte cross-sectional area without further increasing myocyte length in MI rats. T4 treatment increased the total LV tissue area by 34%, increased the non-infarcted tissue area by 41%, and increased the thickness of non-infarcted area by 36% in MI rats. However, myocyte volume accounted for only ~1/3 of the increase in myocyte mass in the non-infarct area, indicating the presence of more myocytes with treatment. T4 treatment tended to increase the total length of smaller arterioles (5 to 15 μm) proportional to LV weight increase and also decreased collagen deposition in the LV non-infarcted area. A tendency for increased metalloproteinase-2 (MMP-2) expression and tissue inhibitor of metalloproteinases (TIMPs) -1 to -4 expression was also observed in T4 treated MI rats. These results suggest that long-term T4 treatment after MI has beneficial effects on myocyte, arteriolar, and collagen matrix remodeling in the non-infarcted area. Most importantly, results suggest improved survival of myocytes in the peri-infarct area.

Smooth muscle architecture within cell-dense vascular tissues influences functional contractility.

PubMed

Win, Zaw; Vrla, Geoffrey D; Steucke, Kerianne E; Sevcik, Emily N; Hald, Eric S; Alford, Patrick W

2014-12-01

The role of vascular smooth muscle architecture in the function of healthy and dysfunctional vessels is poorly understood. We aimed at determining the relationship between vascular smooth muscle architecture and contractile output using engineered vascular tissues. We utilized microcontact printing and a microfluidic cell seeding technique to provide three different initial seeding conditions, with the aim of influencing the cellular architecture within the tissue. Cells seeded in each condition formed confluent and aligned tissues but within the tissues, the cellular architecture varied. Tissues with a more elongated cellular architecture had significantly elevated basal stress and produced more contractile stress in response to endothelin-1 stimulation. We also found a correlation between the contractile phenotype marker expression and the cellular architecture, contrary to our previous findings in non-confluent tissues. Taken with previous results, these data suggest that within cell-dense vascular tissues, smooth muscle contractility is strongly influenced by cell and tissue architectures.

Optimum periodicity of repeated contractile actions applied in mass transport

NASA Astrophysics Data System (ADS)

Ahn, Sungsook; Lee, Sang Joon

2015-01-01

Dynamically repeated periodic patterns are abundant in natural and artificial systems, such as tides, heart beats, stock prices, and the like. The characteristic repeatability and periodicity are expected to be optimized in effective system-specific functions. In this study, such optimum periodicity is experimentally evaluated in terms of effective mass transport using one-valve and multi-valve systems working in contractile fluid flows. A set of nanoscale gating functions is utilized, operating in nanocomposite networks through which permeates selectively pass under characteristic contractile actions. Optimized contractile periodicity exists for effective energy impartment to flow in a one-valve system. In the sequential contractile actions for a multi-valve system, synchronization with the fluid flow is critical for effective mass transport. This study provides fundamental understanding on the various repeated periodic patterns and dynamic repeatability occurring in nature and mechanical systems, which are useful for broad applications.

Effects of regular exercise training on skeletal muscle contractile function

NASA Technical Reports Server (NTRS)

Fitts, Robert H.

2003-01-01

Skeletal muscle function is critical to movement and one's ability to perform daily tasks, such as eating and walking. One objective of this article is to review the contractile properties of fast and slow skeletal muscle and single fibers, with particular emphasis on the cellular events that control or rate limit the important mechanical properties. Another important goal of this article is to present the current understanding of how the contractile properties of limb skeletal muscle adapt to programs of regular exercise.

Effect of Piper betle on cardiac function, marker enzymes, and oxidative stress in isoproterenol-induced cardiotoxicity in rats.

PubMed

Arya, Dharamvir Singh; Arora, Sachin; Malik, Salma; Nepal, Saroj; Kumari, Santosh; Ojha, Shreesh

2010-11-01

The present study was designed to investigate the cardioprotective potential of Piper betle (P. betle) against isoproterenol (ISP)-induced myocardial infarction in rats. Rats were randomly divided into eight groups viz. control, ISP, P. betle (75, 150, and 300 mg/kg) and P. betle (75, 150, and 300 mg/kg) + ISP treated group. P. betle leaf extract (75, 150, or 300 mg/kg) or saline was orally administered for 30 days. ISP (85 mg/kg, s.c.) was administered at an interval of 24 h on the 28(th) and 29(th) day and on day 30 the functional and biochemical parameters were measured. ISP administration showed a significant decrease in systolic, diastolic, mean arterial pressure (SAP, DAP, MAP), heart rate (HR), contractility (+LVdP/dt), and relaxation (-LVdP/dt) and increased left ventricular end-diastolic pressure (LVEDP). ISP also caused significant decrease in myocardial antioxidants; superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), reduced glutathione (GSH), and myocyte injury marker enzymes; creatine phosphokinase-MB (CK-MB) isoenzyme and lactate dehydrogenase (LDH) along with enhanced lipid peroxidation; thiobarbituric acid reacting species (TBARS) in heart. Pre-treatment with P. betle favorably modulated hemodynamic (SAP, DAP, and MAP) and ventricular function parameters (-LVdP/dt and LVEDP). P. betle pre-treatment also restored SOD, CAT, GSH, and GPx, reduced the leakage of CK-MB isoenzyme and LDH along with decreased lipid peroxidation in the heart. Taken together, the biochemical and functional parameters indicate that P. betle 150 and 300 mg/kg has a significant cardioprotective effect against ISP-induced myocardial infarction. Results of the present study suggest the cardioprotective potential of P. betle.

MEN16132, a novel potent and selective nonpeptide antagonist for the human bradykinin B2 receptor. In vitro pharmacology and molecular characterization.

PubMed

Cucchi, Paola; Meini, Stefania; Bressan, Alessandro; Catalani, Claudio; Bellucci, Francesca; Santicioli, Paolo; Lecci, Alessandro; Faiella, Angela; Rotondaro, Luigi; Giuliani, Sandro; Giolitti, Alessandro; Quartara, Laura; Maggi, Carlo Alberto

2005-12-28

The pharmacological characterization of the novel nonpeptide antagonist for the B2 receptor, namely MEN16132 (4-(S)-Amino-5-(4-{4-[2,4-dichloro-3-(2,4-dimethyl-8-quinolyloxymethyl)phenylsulfonamido]-tetrahydro-2H-4-pyranylcarbonyl}piperazino)-5-oxopentyl](trimethyl)ammonium chloride hydrochloride) is presented. The affinity of MEN16132 for the bradykinin B2 receptor has been investigated by means of competition studies at [3H]bradykinin binding to membranes prepared from Chinese Hamster Ovary (CHO) cells expressing the human bradykinin B2 receptor (pKi 10.5), human lung fibroblasts (pKi 10.5), guinea pig airways (pKi 10.0), guinea pig ileum longitudinal smooth muscle (pKi 10.2), or guinea pig cultured colonic myocytes (pKi 10.3). In all assays MEN16132 was as potent as the peptide antagonist Icatibant, and from 3- to 100-fold more potent than the reference nonpeptide antagonists FR173657 or LF16-0687. The selectivity for the bradykinin B2 receptor was checked at the human bradykinin B1 receptor (pKi<5), and at a panel of 26 different receptors and channels. The antagonist potency was measured in functional assays, i.e., in blocking the bradykinin induced inositolphosphates (IP) accumulation at the human (CHO: pKB 10.3) and guinea pig (colonic myocytes: pKB 10.3) B2 receptor, or in antagonizing the bradykinin induced contractile responses in human (detrusor smooth muscle: pKB 9.9) and guinea pig (ileum longitudinal smooth muscle: pKB 10.1) tissues. In both functional assay types MEN16132 exerted a different antagonist pattern, i.e., surmountable at the human and insurmountable at the guinea pig bradykinin B2 receptors. Moreover, the receptor determinants important for the high affinity interaction of MEN16132 with the human bradykinin B2 receptor were investigated by means of radioligand binding studies performed at 24 point-mutated receptors. The results obtained revealed that residues in transmembrane segment 2 (W86A), 3 (I110A), 6 (W256A), and 7 (Y295A, Y295F but not much Y295W), were crucial for the high affinity of MEN16132. In conclusion, MEN16132 is a new, potent, and selective nonpeptide bradykinin B2 receptor antagonist.

Role of microtubules in the contractile dysfunction of hypertrophied myocardium

NASA Technical Reports Server (NTRS)

Zile, M. R.; Koide, M.; Sato, H.; Ishiguro, Y.; Conrad, C. H.; Buckley, J. M.; Morgan, J. P.; Cooper, G. 4th

1999-01-01

OBJECTIVES: We sought to determine whether the ameliorative effects of microtubule depolymerization on cellular contractile dysfunction in pressure overload cardiac hypertrophy apply at the tissue level. BACKGROUND: A selective and persistent increase in microtubule density causes decreased contractile function of cardiocytes from cats with hypertrophy produced by chronic right ventricular (RV) pressure overloading. Microtubule depolymerization by colchicine normalizes contractility in these isolated cardiocytes. However, whether these changes in cellular function might contribute to changes in function at the more highly integrated and complex cardiac tissue level was unknown. METHODS: Accordingly, RV papillary muscles were isolated from 25 cats with RV pressure overload hypertrophy induced by pulmonary artery banding (PAB) for 4 weeks and 25 control cats. Contractile state was measured using physiologically sequenced contractions before and 90 min after treatment with 10(-5) mol/liter colchicine. RESULTS: The PAB significantly increased RV systolic pressure and the RV weight/body weight ratio in PAB; it significantly decreased developed tension from 59+/-3 mN/mm2 in control to 25+/-4 mN/mm2 in PAB, shortening extent from 0.21+/-0.01 muscle lengths (ML) in control to 0.12+/-0.01 ML in PAB, and shortening rate from 1.12+/-0.07 ML/s in control to 0.55+/-0.03 ML/s in PAB. Indirect immunofluorescence confocal microscopy showed that PAB muscles had a selective increase in microtubule density and that colchicine caused complete microtubule depolymerization in both control and PAB papillary muscles. Microtubule depolymerization normalized myocardial contractility in papillary muscles of PAB cats but did not alter contractility in control muscles. CONCLUSIONS: Excess microtubule density, therefore, is equally important to both cellular and to myocardial contractile dysfunction caused by chronic, severe pressure-overload cardiac hypertrophy.

Does vector-free gravity simulate microgravity? Functional and morphologic attributes of clinorotated nerve and muscle grown in cell culture

NASA Technical Reports Server (NTRS)

Gruener, Raphael; Hoeger, Glenn

1988-01-01

Cocultured Xenopus neurons and myocytes were subjected to nonvectorial gravity by clinostat rotation to determine the effects of microgravity on cell development and communications. Observed effects included increases in the myocyte and its nuclear area, fragmentation of nucleoli, the appearance of neuritic aneurysms, decreased growth in the presence of trophic factors, and decreased yolk utilization. These effects were most notable at 1-10 rpm and depended on the onset and duration of rotation. It is found that, in microgravity, cell differentiation is altered by interference with cytoskeleton-related mechanisms. It is suggested that the alteration of the distribution of acetylcholine receptor aggregates on myocytes which occurs might indicate that microgravity affects brain development.

Altered myocardial force generation in end-stage human heart failure.

PubMed

Papp, Zoltán; van der Velden, Jolanda; Borbély, Attila; Édes, István; Stienen, Ger J M

2014-12-01

This study aimed to elucidate the molecular background of increased Ca 2+ sensitivity of force production in cardiomyocytes of end-stage human heart failure. Ca 2+ -activated isometric force and the cross-bridge specific rate of force redevelopment (k tr ) were determined in Triton-skinned myocytes from end-stage failing and non-failing donor hearts. Measurements (control: pH 7.2, 0 mM inorganic phosphate (P i )) were performed under test conditions that probed either the Ca 2+ -regulatory function of the thin filaments (pH 6.5), the kinetics of the actin-myosin cross-bridge cycle (10 mM P i ), or both (pH 6.5, 10 mM P i ). The control maximal Ca 2+ -activated force (F o ) and k trmax did not differ between failing and non-failing myocytes. At submaximal [Ca 2+ ], however, both force and k tr were higher in failing than in donor myocytes. The difference in the Ca 2+ sensitivities of force production was preserved when the thin filament regulatory function was perturbed by acidosis (pH 6.5) but was abolished by cross-bridge modulation (i.e. by P i ) both at pH 7.2 and at pH 6.5. P i induced a larger reduction in force but a smaller increase in k tr in the failing myocytes than in the non-failing myocytes at submaximal [Ca 2+ ]. The enhanced P i sensitivity of the actin-myosin interaction suggests that the P i release step of the actin-myosin cross-bridge cycle is modified during end-stage human heart failure. This might be of functional importance when P i accumulates (e.g. during cardiac ischaemia). Moreover, this alteration can influence cardiac energetics and the clinical efficacy of sarcomere targeted agents in human heart failure. © 2015 The Authors. ESC Heart Failure published by John Wiley & Sons Ltd on behalf of the European Society of Cardiology.

Maturation status of sarcomere structure and function in human iPSC-derived cardiac myocytes.

PubMed

Bedada, Fikru B; Wheelwright, Matthew; Metzger, Joseph M

2016-07-01

Human heart failure due to myocardial infarction is a major health concern. The paucity of organs for transplantation limits curative approaches for the diseased and failing adult heart. Human induced pluripotent stem cell-derived cardiac myocytes (hiPSC-CMs) have the potential to provide a long-term, viable, regenerative-medicine alternative. Significant progress has been made with regard to efficient cardiac myocyte generation from hiPSCs. However, directing hiPSC-CMs to acquire the physiological structure, gene expression profile and function akin to mature cardiac tissue remains a major obstacle. Thus, hiPSC-CMs have several hurdles to overcome before they find their way into translational medicine. In this review, we address the progress that has been made, the void in knowledge and the challenges that remain. This article is part of a Special Issue entitled: Cardiomyocyte Biology: Integration of Developmental and Environmental Cues in the Heart edited by Marcus Schaub and Hughes Abriel. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

Stage-specific effects of Notch activation during skeletal myogenesis

PubMed Central

Bi, Pengpeng; Yue, Feng; Sato, Yusuke; Wirbisky, Sara; Liu, Weiyi; Shan, Tizhong; Wen, Yefei; Zhou, Daoguo; Freeman, Jennifer; Kuang, Shihuan

2016-01-01

Skeletal myogenesis involves sequential activation, proliferation, self-renewal/differentiation and fusion of myogenic stem cells (satellite cells). Notch signaling is known to be essential for the maintenance of satellite cells, but its function in late-stage myogenesis, i.e. post-differentiation myocytes and post-fusion myotubes, is unknown. Using stage-specific Cre alleles, we uncovered distinct roles of Notch1 in mononucleated myocytes and multinucleated myotubes. Specifically, constitutive Notch1 activation dedifferentiates myocytes into Pax7 quiescent satellite cells, leading to severe defects in muscle growth and regeneration, and postnatal lethality. By contrast, myotube-specific Notch1 activation improves the regeneration and exercise performance of aged and dystrophic muscles. Mechanistically, Notch1 activation in myotubes upregulates the expression of Notch ligands, which modulate Notch signaling in the adjacent satellite cells to enhance their regenerative capacity. These results highlight context-dependent effects of Notch activation during myogenesis, and demonstrate that Notch1 activity improves myotube’s function as a stem cell niche. DOI: http://dx.doi.org/10.7554/eLife.17355.001 PMID:27644105

[Effects of vitamin K3 on the contractile activity of the colonic smooth muscles of guinea pig through the calcium activated potassium channel].

PubMed

Li, Jun; Luo, He-sheng; He, Xiao-gu

2006-07-25

To study the mechanism of relaxation of gastrointestinal smooth muscles by vitamin K(3). Stripes of proximal colon were collected from guinea pigs. Suspension of single cells was created from these stripes. TD-112S transducer was used to measure the contraction of the stripes stimulated by vitamin K(3) of the concentrations of 40, 100, 400, and 800 micromol/L respectively. The Ca(2+)-activated K(+) current [IK(Ca)] of the cytomembrane of the colon smooth muscle was recorded with an EPC 10 amplifier under conventional whole cell patterns. The contraction frequencies of the muscle stripes stimulated by vitamin K(3) of the concentrations of 40, 100, 400, and 800 micromol/L were 79% +/- 4%, 58% +/- 5%, 33% +/- 4%, and 12% +/- 3% respectively of that of the control group (all P < 0.01), and the contraction strength was reduced to 77% +/- 10%, 54% +/- 7%, 30% +/- 6%, and 11% +/- 4% respectively (all P < 0.01). The IK((Ca)) of the cytomembrane of the colon smooth muscle at the voltage of +60 mV was increased to 120% +/- 18%, 149% +/- 12%, 197% +/- 19%, and 223% +/- 14% respectively (all P < 0.01). Vitamin K(3) inhibits the contractile activity of the colonic muscle stripes and increases the IK(Ca) of single myocytes concentration-dependently. The mechanism is activation of the Ca(2+)-activated K(+) channel, thus promoting the potassium efflux.

Estrogen and testosterone in concert with EFNB3 regulate vascular smooth muscle cell contractility and blood pressure.

PubMed

Wang, Yujia; Wu, Zenghui; Thorin, Eric; Tremblay, Johanne; Lavoie, Julie L; Luo, Hongyu; Peng, Junzheng; Qi, Shijie; Wu, Tao; Chen, Fei; Shen, Jianzhong; Hu, Shenjiang; Wu, Jiangping

2016-04-01

EPH kinases and their ligands, ephrins (EFNs), have vital and diverse biological functions, although their function in blood pressure (BP) control has not been studied in detail. In the present study, we report that Efnb3 gene knockout (KO) led to increased BP in female but not male mice. Vascular smooth muscle cells (VSMCs) were target cells for EFNB3 function in BP regulation. The deletion of EFNB3 augmented contractility of VSMCs from female but not male KO mice, compared with their wild-type (WT) counterparts. Estrogen augmented VSMC contractility while testosterone reduced it in the absence of EFNB3, although these sex hormones had no effect on the contractility of VSMCs from WT mice. The effect of estrogen on KO VSMC contractility was via a nongenomic pathway involving GPER, while that of testosterone was likely via a genomic pathway, according to VSMC contractility assays and GPER knockdown assays. The sex hormone-dependent contraction phenotypes in KO VSMCs were reflected in BP in vivo. Ovariectomy rendered female KO mice normotensive. At the molecular level, EFNB3 KO in VSMCs resulted in reduced myosin light chain kinase phosphorylation, an event enhancing sensitivity to Ca(2+)flux in VSMCs. Our investigation has revealed previously unknown EFNB3 functions in BP regulation and show that EFNB3 might be a hypertension risk gene in certain individuals. Copyright © 2016 the American Physiological Society.

Operative contractility: a functional concept of the inotropic state.

PubMed

Curiel, Roberto; Perez-Gonzalez, Juan; Torres, Edwar; Landaeta, Ruben; Cerrolaza, Miguel

2005-10-01

1. Initial unsuccessful attempts to evaluate ventricular function in terms of the 'heart as a pump' led to focusing on the 'heart as a muscle' and to the concept of myocardial contractility. However, no clinically ideal index exists to assess the contractile state. The aim of the present study was to develop a mathematical model to assess cardiac contractility. 2. A tri-axial system was conceived for preload (PL), afterload (AL) and contractility, where stroke volume (SV) was represented as the volume of the tetrahedron. Based on this model, 'operative' contractility ('OperCon') was calculated from the readily measured values of PL, AL and SV. The model was tested retrospectively under a variety of different experimental and clinical conditions, in 71 studies in humans and 29 studies in dogs. A prospective echocardiographic study was performed in 143 consecutive subjects to evaluate the ability of the model to assess contractility when SV and PL were measured volumetrically (mL) or dimensionally (cm). 3. With inotropic interventions, OperCon changes were comparable to those of ejection fraction (EF), velocity of shortening (Vcf) and dP/dt-max. Only with positive inotropic interventions did elastance (Ees) show significantly larger changes. With load manipulations, OperCon showed significantly smaller changes than EF and Ees and comparable changes to Vcf and dP/dt-max. Values of OperCon were similar when AL was represented by systolic blood pressure or wall stress and when volumetric or dimensional values were used. 4. Operative contractility is a reliable, simple and versatile method to assess cardiac contractility.

Overexpression of connexin 43 using a retroviral vector improves electrical coupling of skeletal myoblasts with cardiac myocytes in vitro

PubMed Central

Tolmachov, Oleg; Ma, Yu-Ling; Themis, Michael; Patel, Pravina; Spohr, Hilmar; MacLeod, Kenneth T; Ullrich, Nina D; Kienast, Yvonne; Coutelle, Charles; Peters, Nicholas S

2006-01-01

Background Organ transplantation is presently often the only available option to repair a damaged heart. As heart donors are scarce, engineering of cardiac grafts from autologous skeletal myoblasts is a promising novel therapeutic strategy. The functionality of skeletal muscle cells in the heart milieu is, however, limited because of their inability to integrate electrically and mechanically into the myocardium. Therefore, in pursuit of improved cardiac integration of skeletal muscle grafts we sought to modify primary skeletal myoblasts by overexpression of the main gap-junctional protein connexin 43 and to study electrical coupling of connexin 43 overexpressing myoblasts to cardiac myocytes in vitro. Methods To create an efficient means for overexpression of connexin 43 in skeletal myoblasts we constructed a bicistronic retroviral vector MLV-CX43-EGFP expressing the human connexin 43 cDNA and the marker EGFP gene. This vector was employed to transduce primary rat skeletal myoblasts in optimised conditions involving a concomitant use of the retrovirus immobilising protein RetroNectin® and the polycation transduction enhancer Transfectam®. The EGFP-positive transduced cells were then enriched by flow cytometry. Results More than four-fold overexpression of connexin 43 in the transduced skeletal myoblasts, compared with non-transduced cells, was shown by Western blotting. Functionality of the overexpressed connexin 43 was demonstrated by microinjection of a fluorescent dye showing enhanced gap-junctional intercellular transfer in connexin 43 transduced myoblasts compared with transfer in non-transduced myoblasts. Rat cardiac myocytes were cultured in multielectrode array culture dishes together with connexin 43/EGFP transduced skeletal myoblasts, control non-transduced skeletal myoblasts or alone. Extracellular field action potential activation rates in the co-cultures of connexin 43 transduced skeletal myoblasts with cardiac myocytes were significantly higher than in the co-cultures of non-transduced skeletal myoblasts with cardiac myocytes and similar to the rates in pure cultures of cardiac myocytes. Conclusion The observed elevated field action potential activation rate in the co-cultures of cardiac myocytes with connexin 43 transduced skeletal myoblasts indicates enhanced cell-to-cell electrical coupling due to overexpression of connexin 43 in skeletal myoblasts. This study suggests that retroviral connexin 43 transduction can be employed to augment engineering of the electrocompetent cardiac grafts from patients' own skeletal myoblasts. PMID:16756651

Overexpression of connexin 43 using a retroviral vector improves electrical coupling of skeletal myoblasts with cardiac myocytes in vitro.

PubMed

Tolmachov, Oleg; Ma, Yu-Ling; Themis, Michael; Patel, Pravina; Spohr, Hilmar; Macleod, Kenneth T; Ullrich, Nina D; Kienast, Yvonne; Coutelle, Charles; Peters, Nicholas S

2006-06-06

Organ transplantation is presently often the only available option to repair a damaged heart. As heart donors are scarce, engineering of cardiac grafts from autologous skeletal myoblasts is a promising novel therapeutic strategy. The functionality of skeletal muscle cells in the heart milieu is, however, limited because of their inability to integrate electrically and mechanically into the myocardium. Therefore, in pursuit of improved cardiac integration of skeletal muscle grafts we sought to modify primary skeletal myoblasts by overexpression of the main gap-junctional protein connexin 43 and to study electrical coupling of connexin 43 overexpressing myoblasts to cardiac myocytes in vitro. To create an efficient means for overexpression of connexin 43 in skeletal myoblasts we constructed a bicistronic retroviral vector MLV-CX43-EGFP expressing the human connexin 43 cDNA and the marker EGFP gene. This vector was employed to transduce primary rat skeletal myoblasts in optimised conditions involving a concomitant use of the retrovirus immobilising protein RetroNectin and the polycation transduction enhancer Transfectam. The EGFP-positive transduced cells were then enriched by flow cytometry. More than four-fold overexpression of connexin 43 in the transduced skeletal myoblasts, compared with non-transduced cells, was shown by Western blotting. Functionality of the overexpressed connexin 43 was demonstrated by microinjection of a fluorescent dye showing enhanced gap-junctional intercellular transfer in connexin 43 transduced myoblasts compared with transfer in non-transduced myoblasts. Rat cardiac myocytes were cultured in multielectrode array culture dishes together with connexin 43/EGFP transduced skeletal myoblasts, control non-transduced skeletal myoblasts or alone. Extracellular field action potential activation rates in the co-cultures of connexin 43 transduced skeletal myoblasts with cardiac myocytes were significantly higher than in the co-cultures of non-transduced skeletal myoblasts with cardiac myocytes and similar to the rates in pure cultures of cardiac myocytes. The observed elevated field action potential activation rate in the co-cultures of cardiac myocytes with connexin 43 transduced skeletal myoblasts indicates enhanced cell-to-cell electrical coupling due to overexpression of connexin 43 in skeletal myoblasts. This study suggests that retroviral connexin 43 transduction can be employed to augment engineering of the electrocompetent cardiac grafts from patients' own skeletal myoblasts.

Ca2+ handling remodeling and STIM1L/Orai1/TRPC1/TRPC4 upregulation in monocrotaline-induced right ventricular hypertrophy.

PubMed

Jessica, Sabourin; Angèle, Boet; Catherine, Rucker-Martin; Mélanie, Lambert; Ana-Maria, Gomez; Jean-Pierre, Benitah; Frédéric, Perros; Marc, Humbert; Fabrice, Antigny

2018-05-01

Right ventricular (RV) function is the most important prognostic factor for pulmonary arterial hypertension (PAH) patients. The progressive increase of pulmonary vascular resistance induces RV hypertrophy (RVH) and at term RV failure (RVF). However, the molecular mechanisms of RVH and RVF remain understudied. In this study, we gained insights into cytosolic Ca 2+ signaling remodeling in ventricular cardiomyocytes during the pathogenesis of severe pulmonary hypertension (PH) induced in rats by monocrotaline (MCT) exposure, and we further identified molecular candidates responsible for this Ca 2+ remodeling. After PH induction, hypertrophied RV myocytes presented longer action potential duration, higher and faster [Ca 2+ ] i transients and increased sarcoplasmic reticulum (SR) Ca 2+ content, whereas no changes in these parameters were detected in left ventricular (LV) myocytes. These modifications were associated with increased P-Ser 16 -phospholamban pentamer expression without altering SERCA2a (Sarco/Endoplasmic Reticulum Ca 2+ -ATPase) pump abundance. Moreover, after PH induction, Ca 2+ sparks frequency were higher in hypertrophied RV cells, while total RyR2 (Ryanodine Receptor) expression and phosphorylation were unaffected. Together with cellular hypertrophy, the T-tubules network was disorganized. Hypertrophied RV cardiomyocytes from MCT-exposed rats showed decreased expression of classical STIM1 (Stromal Interaction molecule) associated with increased expression of muscle-specific STIM1 Long isoform, glycosylated-Orai1 channel form, and TRPC1 and TRPC4 channels, which was correlated with an enhanced Ca 2+ -release-activated Ca 2+ (CRAC)-like current. Pharmacological inhibition of TRPCs/Orai1 channels in hypertrophied RV cardiomyocytes normalized [Ca 2+ ] i transients amplitude, the SR Ca 2+ content and cell contractility to control levels. Finally, we showed that most of these changes did not appear in LV cardiomyocytes. These new findings demonstrate RV-specific cellular Ca 2+ cycling remodeling in PH rats with maladaptive RVH and that the STIM1L/Orai1/TRPC1/C4-dependent Ca 2+ current participates in this Ca 2+ remodeling in RVH secondary to PH. Copyright © 2018 Elsevier Ltd. All rights reserved.

Antioxidant Phytochemicals of Opuntia ficus-indica (L.) Mill. Cladodes with Potential Anti-spasmodic Activity

PubMed Central

Lanuzza, Francesco; Occhiuto, Francesco; Monforte, Maria Teresa; Tripodo, Maria Marcella; D’Angelo, Valeria; Galati, Enza Maria

2017-01-01

Background: Opuntia ficus-indica (OFI) (L.) Mill. (Cactaceae), a plant widespread in dry regions of the world, shows interesting biological activities (cicatrizant, antiulcer, anti-inflammatory, and hypolipidemic) and is widely used in traditional medicine. Objectives: Phytochemical analysis and antispasmodic effect of wild OFI cladodes were carried out. Material and Methods: Polyphenols and Vitamin E occurrence, in antioxidant pool of OFI cladodes, were quantified by high-performance liquid chromatography. The antispasmodic effect of OFI cladodes was assessed in isolated rabbit smooth muscle tissues. The experiments were carried out with preparations of rabbit jejunum and uterus with the spontaneous contractile activity, to evaluate the effect of cumulative concentrations of the extract on basal tone, amplitude, and frequency of contractions. Results: Catechin, quercetin, kaempferol, isorhamnetin and chlorogenic, ferulic, and p-coumaric acid were identified. α-, β-, and γ-tocopherols have been highlighted and α-tocopherol is the major component. OFI cladodes contain significant amount of polyphenols and tocopherols that are effective radical scavengers and inhibited ethanol 1,1-diphenyl-2-picrylhydrazyl formation by 50%. OFI cladodes caused a light inhibition of amplitude and frequency of spontaneous contractions and a marked decrease in muscle basal tone of rabbit jejunum preparations. On spontaneously contracting uterus preparations, the addition of increasing concentrations of cladode extract caused uterine muscle relaxation. Conclusion: The contraction of smooth muscle preparations depends on an increase in cytoplasmic free calcium ion concentration, which activates the contractile elements. The flavonoids may suppress the contractility of smooth myocytes, by an inhibition of availability of Ca2+ for muscle contraction. SUMMARY Opuntia ficus-indica (OFI) cladodes contain significant amount of polyphenols and tocopherols that are effective radical scavengers and inhibited ethanol 1,1-diphenyl-2-picrylhydrazyl formation by 50%Polyphenols and Vitamin E complex occurrence in OFI cladodes were characterized by high-performance liquid chromatographyOFI cladodes exhibited significative antispasmodic activity. The antispasmodic effect was assessed in isolated rabbit smooth muscle tissues. The experiments were carried out with preparations of rabbit jejunum and uterus with the spontaneous contractile activity, to evaluate the effect of cumulative concentrations of the extract on basal tone, amplitude, and frequency of contractions. Abbreviations used: OFI: Opuntia ficus-indica, DPPH: Ethanol 1,1-diphenyl-2-picrylhydrazyl. PMID:29142394

Difference in propagation of Ca2+ release in atrial and ventricular myocytes.

PubMed

Tanaami, Takeo; Ishida, Hideyuki; Seguchi, Hidetaka; Hirota, Yuki; Kadono, Toshie; Genka, Chokoh; Nakazawa, Hiroe; Barry, William H

2005-04-01

Intracellular [Ca2+] ([Ca2+]i) was imaged in atrial and ventricular rat myocytes by means of a high-speed Nipkow confocal microscope. Atrial myocytes with an absent t-tubule system on 8-di- ANEPPS staining showed an initial rise in Ca2+ at the periphery of the cell, which propagated to the interior of the cell. Ventricular myocytes showed a uniform rise in [Ca2+]i after electrical stimulation, consistent with a prominent t-tubular network. In atrial myocytes, there was a much shorter time between the peak of the [Ca2+]i transient and the peak contraction as compared to ventricular myocytes. A regional release of Ca2+ induced by an exposure of one end of the myocyte to caffeine with a rapid solution switcher resulted in a uniform propagation of Ca2+ down the length of the cell in atrial myocytes, but we found no propagation in ventricular myocytes. A staining with rhodamine 123 indicated a much greater density of mitochondria in ventricular myocytes than in atrial myocytes. Thus the atrial myocytes display a lack of "local control" of Ca2+ release, with propagation after the Ca2+ release at the periphery induced by stimulation or at one end of the cell induced by exposure to caffeine. Ventricular myocytes showed the presence of local control, as indicated by an absence of the propagation of a local caffeine-induced Ca2+ transient. We suggest that this finding, as well as a reduced delay between the peak of the [Ca2+]i transient and the peak shortening in atrial myocytes, could be due in part to reduced Ca2+ buffering provided by mitochondria in atrial myocytes as opposed to ventricular myocytes.

Changes in contractile activation characteristics of rat fast and slow skeletal muscle fibres during regeneration.

PubMed

Gregorevic, Paul; Plant, David R; Stupka, Nicole; Lynch, Gordon S

2004-07-15

Damaged skeletal muscle fibres are replaced with new contractile units via muscle regeneration. Regenerating muscle fibres synthesize functionally distinct isoforms of contractile and regulatory proteins but little is known of their functional properties during the regeneration process. An advantage of utilizing single muscle fibre preparations is that assessment of their function is based on the overall characteristics of the contractile apparatus and regulatory system and as such, these preparations are sensitive in revealing not only coarse, but also subtle functional differences between muscle fibres. We examined the Ca(2+)- and Sr(2+)-activated contractile characteristics of permeabilized fibres from rat fast-twitch (extensor digitorum longus) and slow-twitch (soleus) muscles at 7, 14 and 21 days following myotoxic injury, to test the hypothesis that fibres from regenerating fast and slow muscles have different functional characteristics to fibres from uninjured muscles. Regenerating muscle fibres had approximately 10% of the maximal force producing capacity (P(o)) of control (uninjured) fibres, and an altered sensitivity to Ca(2+) and Sr(2+) at 7 days post-injury. Increased force production and a shift in Ca(2+) sensitivity consistent with fibre maturation were observed during regeneration such that P(o) was restored to 36-45% of that in control fibres by 21 days, and sensitivity to Ca(2+) and Sr(2+) was similar to that of control (uninjured) fibres. The findings support the hypothesis that regenerating muscle fibres have different contractile activation characteristics compared with mature fibres, and that they adopt properties of mature fast- or slow-twitch muscle fibres in a progressive manner as the regeneration process is completed.

High-Resolution Mapping of Chromatin Conformation in Cardiac Myocytes Reveals Structural Remodeling of the Epigenome in Heart Failure

PubMed Central

Rosa-Garrido, Manuel; Chapski, Douglas J.; Schmitt, Anthony D.; Kimball, Todd H.; Karbassi, Elaheh; Monte, Emma; Balderas, Enrique; Pellegrini, Matteo; Shih, Tsai-Ting; Soehalim, Elizabeth; Liem, David; Ping, Peipei; Galjart, Niels J.; Ren, Shuxun; Wang, Yibin; Ren, Bing

2017-01-01

Background: Cardiovascular disease is associated with epigenomic changes in the heart; however, the endogenous structure of cardiac myocyte chromatin has never been determined. Methods: To investigate the mechanisms of epigenomic function in the heart, genome-wide chromatin conformation capture (Hi-C) and DNA sequencing were performed in adult cardiac myocytes following development of pressure overload–induced hypertrophy. Mice with cardiac-specific deletion of CTCF (a ubiquitous chromatin structural protein) were generated to explore the role of this protein in chromatin structure and cardiac phenotype. Transcriptome analyses by RNA-seq were conducted as a functional readout of the epigenomic structural changes. Results: Depletion of CTCF was sufficient to induce heart failure in mice, and human patients with heart failure receiving mechanical unloading via left ventricular assist devices show increased CTCF abundance. Chromatin structural analyses revealed interactions within the cardiac myocyte genome at 5-kb resolution, enabling examination of intra- and interchromosomal events, and providing a resource for future cardiac epigenomic investigations. Pressure overload or CTCF depletion selectively altered boundary strength between topologically associating domains and A/B compartmentalization, measurements of genome accessibility. Heart failure involved decreased stability of chromatin interactions around disease-causing genes. In addition, pressure overload or CTCF depletion remodeled long-range interactions of cardiac enhancers, resulting in a significant decrease in local chromatin interactions around these functional elements. Conclusions: These findings provide a high-resolution chromatin architecture resource for cardiac epigenomic investigations and demonstrate that global structural remodeling of chromatin underpins heart failure. The newly identified principles of endogenous chromatin structure have key implications for epigenetic therapy. PMID:28802249

High-Resolution Mapping of Chromatin Conformation in Cardiac Myocytes Reveals Structural Remodeling of the Epigenome in Heart Failure.

PubMed

Rosa-Garrido, Manuel; Chapski, Douglas J; Schmitt, Anthony D; Kimball, Todd H; Karbassi, Elaheh; Monte, Emma; Balderas, Enrique; Pellegrini, Matteo; Shih, Tsai-Ting; Soehalim, Elizabeth; Liem, David; Ping, Peipei; Galjart, Niels J; Ren, Shuxun; Wang, Yibin; Ren, Bing; Vondriska, Thomas M

2017-10-24

Cardiovascular disease is associated with epigenomic changes in the heart; however, the endogenous structure of cardiac myocyte chromatin has never been determined. To investigate the mechanisms of epigenomic function in the heart, genome-wide chromatin conformation capture (Hi-C) and DNA sequencing were performed in adult cardiac myocytes following development of pressure overload-induced hypertrophy. Mice with cardiac-specific deletion of CTCF (a ubiquitous chromatin structural protein) were generated to explore the role of this protein in chromatin structure and cardiac phenotype. Transcriptome analyses by RNA-seq were conducted as a functional readout of the epigenomic structural changes. Depletion of CTCF was sufficient to induce heart failure in mice, and human patients with heart failure receiving mechanical unloading via left ventricular assist devices show increased CTCF abundance. Chromatin structural analyses revealed interactions within the cardiac myocyte genome at 5-kb resolution, enabling examination of intra- and interchromosomal events, and providing a resource for future cardiac epigenomic investigations. Pressure overload or CTCF depletion selectively altered boundary strength between topologically associating domains and A/B compartmentalization, measurements of genome accessibility. Heart failure involved decreased stability of chromatin interactions around disease-causing genes. In addition, pressure overload or CTCF depletion remodeled long-range interactions of cardiac enhancers, resulting in a significant decrease in local chromatin interactions around these functional elements. These findings provide a high-resolution chromatin architecture resource for cardiac epigenomic investigations and demonstrate that global structural remodeling of chromatin underpins heart failure. The newly identified principles of endogenous chromatin structure have key implications for epigenetic therapy. © 2017 The Authors.

Milrinone attenuates thromboxane receptor-mediated hyperresponsiveness in hypoxic pulmonary arterial myocytes

PubMed Central

Santhosh, KT; Elkhateeb, O; Nolette, N; Outbih, O; Halayko, AJ; Dakshinamurti, S

2011-01-01

BACKGROUND AND PURPOSE Neonatal pulmonary hypertension (PPHN) is characterized by pulmonary vasoconstriction, due in part to dysregulation of the thromboxane prostanoid (TP) receptor. Hypoxia induces TP receptor–mediated hyperresponsiveness, whereas serine phosphorylation mediates desensitization of TP receptors. We hypothesized that prostacyclin (IP) receptor activity induces TP receptor phosphorylation and decreases ligand affinity; that TP receptor sensitization in hypoxic myocytes is due to IP receptor inactivation; and that this would be reversible by the cAMP-specific phosphodiesterase inhibitor milrinone. EXPERIMENTAL APPROACH We examined functional regulation of TP receptors by serine phosphorylation and effects of IP receptor stimulation and protein kinase A (PKA) activity on TP receptor sensitivity in myocytes from neonatal porcine resistance pulmonary arteries after 72 h hypoxia in vitro. Ca2+ response curves to U46619 (TP receptor agonist) were determined in hypoxic and normoxic myocytes incubated with or without iloprost (IP receptor agonist), forskolin (adenylyl cyclase activator), H8 (PKA inhibitor) or milrinone. TP and IP receptor saturation binding kinetics were measured in presence of iloprost or 8-bromo-cAMP. KEY RESULTS Ligand affinity for TP receptors was normalized in vitro by IP receptor signalling intermediates. However, IP receptor affinity was compromised in hypoxic myocytes, decreasing cAMP production. Milrinone normalized TP receptor sensitivity in hypoxic myocytes by restoring PKA-mediated regulatory TP receptor phosphorylation. CONCLUSIONS AND IMPLICATIONS TP receptor sensitivity and EC50 for TP receptor agonists was regulated by PKA, as TP receptor serine phosphorylation by PKA down-regulated Ca2+ mobilization. Hypoxia decreased IP receptor activity and cAMP generation, inducing TP receptor hyperresponsiveness, which was reversed by milrinone. PMID:21385177

Microtubule depolymerization normalizes in vivo myocardial contractile function in dogs with pressure-overload left ventricular hypertrophy

NASA Technical Reports Server (NTRS)

Koide, M.; Hamawaki, M.; Narishige, T.; Sato, H.; Nemoto, S.; DeFreyte, G.; Zile, M. R.; Cooper G, I. V.; Carabello, B. A.

2000-01-01

BACKGROUND: Because initially compensatory myocardial hypertrophy in response to pressure overloading may eventually decompensate to myocardial failure, mechanisms responsible for this transition have long been sought. One such mechanism established in vitro is densification of the cellular microtubule network, which imposes a viscous load that inhibits cardiocyte contraction. METHODS AND RESULTS: In the present study, we extended this in vitro finding to the in vivo level and tested the hypothesis that this cytoskeletal abnormality is important in the in vivo contractile dysfunction that occurs in experimental aortic stenosis in the adult dog. In 8 dogs in which gradual stenosis of the ascending aorta had caused severe left ventricular (LV) pressure overloading (gradient, 152+/-16 mm Hg) with contractile dysfunction, LV function was measured at baseline and 1 hour after the intravenous administration of colchicine. Cardiocytes obtained by biopsy before and after in vivo colchicine administration were examined in tandem. Microtubule depolymerization restored LV contractile function both in vivo and in vitro. CONCLUSIONS: These and additional corroborative data show that increased cardiocyte microtubule network density is an important mechanism for the ventricular contractile dysfunction that develops in large mammals with adult-onset pressure-overload-induced cardiac hypertrophy.

Putting the past behind us: Social stress-induced urinary retention can be overcome.

PubMed

Weiss, Dana A; Butler, Stephan J; Fesi, Joanna; Long, Christopher J; Valentino, Rita J; Canning, Douglas A; Zderic, Stephen A

2015-08-01

To study the pathophysiology of dysfunctional voiding, we have previously developed a model of stress-induced voiding dysfunction. We have shown that cyclosporine A (CsA), an inhibitor of the Ca(2+)-calmodulin complex, can prevent social stress-induced urinary retention. However, treatment with cyclosporine has not had an effect on the increase in the stress peptide corticotrophin-releasing factor (CRF) in Barrington's nucleus, which is involved in the micturition pathway. We now investigate whether cyclosporine administered after stress can reverse the abnormal voiding phenotype, and whether it has effects on the bladder wall itself, or on the stress response within Barrington's nucleus. Six-week old Swiss-Webster mice were exposed to aggressor males for 1 h a day, followed by 23 h of barrier separation. In a long-term trial, 1 month of stress was followed by single-cage housing for 6 months. In a separate CsA reversal trial, mice either received CsA in drinking water or had plain drinking water during 1 month of single-cage housing during recovery. Bladder contractile function was examined on a Guth myograph. Nuclear translocation of myocyte enhancing factor (MEF)-2 and NFAT (nuclear factor of activated T cells) in the bladder was assessed using electrophoretic mobility shift assays (EMSAs). The expression of CRF was determined in Barrington's nucleus using in situ hybridization. Voiding dysfunction persisted for up to 6 months after stress exposure while mice recovered in single-cage housing. In the CsA reversal trial, voiding patterns improved when they received CsA in water during single-cage housing following stress, whereas those that underwent single-cage housing alone had persistent abnormal voiding (Fig. A). There was no difference between CRF levels in Barrington's nucleus between reversal groups (p = 0.42) (Fig. B), possibly indicating a direct effect on the bladder rather than a persistent stress effect. There were no differences in the contractility of bladder wall muscle. CsA decreased the nuclear translocation of MEF-2 and NFAT induced by stress (Fig. C,D). CsA reverses stress-induced urinary retention, but does not change the stress-induced CRF increase in Barrington's nucleus. Furthermore, bladder smooth muscle contractility is unchanged by CsA; however, there are changes in the levels of the downstream transcription factors MEF-2 and NFAT. We suspect that additional CsA responsive neural changes play a pivotal role in the abnormal voiding phenotype following social stress. Copyright © 2015 Journal of Pediatric Urology Company. Published by Elsevier Ltd. All rights reserved.

Pathophysiological roles of peroxynitrite in circulatory shock

PubMed Central

Szabó, Csaba; Módis, Katalin

2014-01-01

Summary Peroxynitrite is a reactive oxidant produced from nitric oxide (NO) and superoxide, which reacts with proteins, lipids and DNA and promotes cytotoxic and pro-inflammatory responses. Here we overview the role of peroxynitrite in various forms of circulatory shock. Immunohistochemical and biochemical evidence demonstrate the production of peroxynitrite in various experimental models of endotoxic and hemorrhagic shock, both in rodents and in large animals. In addition, biological markers of peroxynitrite have been identified in human tissues after circulatory shock. Peroxynitrite can initiate toxic oxidative reactions in vitro and in vivo. Initiation of lipid peroxidation, direct inhibition of mitochondrial respiratory chain enzymes, inactivation of glyceraldehyde-3-phosphate dehydrogenase, inhibition of membrane Na+/K+ ATP-ase activity, inactivation of membrane sodium channels, and other oxidative protein modifications contribute to the cytotoxic effect of peroxynitrite. In addition, peroxynitrite is a potent trigger of DNA strand breakage, with subsequent activation of the nuclear enzyme poly (ADP-ribose) polymerase (PARP), which promotes cellular energetic collapse and cellular necrosis. Additional actions of peroxynitrite that contribute to the pathogenesis of shock include inactivation of catecholamines and catecholamine receptors (leading to vascular failure), endothelial and epithelial injury (leading to endothelial and epithelial hyper-permeability and barrier dysfunction) as well as myocyte injury (contributing to loss of cardiac contractile function). Neutralization of peroxynitrite with potent peroxynitrite decomposition catalysts provides cytoprotective and beneficial effects in rodent and large animal models of circulatory shock. PMID:20523270

Left atrial strain predicts hemodynamic parameters in cardiovascular patients.

PubMed

Hewing, Bernd; Theres, Lena; Spethmann, Sebastian; Stangl, Karl; Dreger, Henryk; Knebel, Fabian

2017-08-01

We aimed to evaluate the predictive value of left atrial (LA) reservoir, conduit, and contractile function parameters as assessed by speckle tracking echocardiography (STE) for invasively measured hemodynamic parameters in a patient cohort with myocardial and valvular diseases. Sixty-nine patients undergoing invasive hemodynamic assessment were enrolled into the study. Invasive hemodynamic parameters were obtained by left and right heart catheterization. Transthoracic echocardiography assessment of LA reservoir, conduit, and contractile function was performed by STE. Forty-nine patients had sinus rhythm (SR) and 20 patients had permanent atrial fibrillation (AF). AF patients had significantly reduced LA reservoir function compared to SR patients. In patients with SR, LA reservoir, conduit, and contractile function inversely correlated with pulmonary capillary wedge pressure (PCWP), left ventricular end-diastolic pressure, and mean pulmonary artery pressure (PAP), and showed a moderate association with cardiac index. In AF patients, there were no significant correlations between LA reservoir function and invasively obtained hemodynamic parameters. In SR patients, LA contractile function with a cutoff value of 16.0% had the highest diagnostic accuracy (area under the curve, AUC: 0.895) to predict PCWP ≥18 mm Hg compared to the weaker diagnostic accuracy of average E/E' ratio with an AUC of 0.786 at a cutoff value of 14.3. In multivariate analysis, LA contractile function remained significantly associated with PCWP ≥18 mm Hg. In a cohort of patients with a broad spectrum of cardiovascular diseases LA strain shows a valuable prediction of hemodynamic parameters, specifically LV filling pressures, in the presence of SR. © 2017, Wiley Periodicals, Inc.

Expression of mitochondrial regulatory genes parallels respiratory capacity and contractile function in a rat model of hypoxia-induced right ventricular hypertrophy

USDA-ARS?s Scientific Manuscript database

Chronic hypobaric hypoxia (CHH) increases load on the right ventricle (RV) resulting in RV hypertrophy. We hypothesized that CHH elicits distinct responses, i.e., the hypertrophied RV, unlike the left ventricle (LV), displaying enhanced mitochondrial respiratory and contractile function. Wistar rats...

Novel Perspectives in Redox Biology and Pathophysiology of Failing Myocytes: Modulation of the Intramyocardial Redox Milieu for Therapeutic Interventions—A Review Article from the Working Group of Cardiac Cell Biology, Italian Society of Cardiology

PubMed Central

Arcaro, Alessia; Pirozzi, Flora; Angelini, Annalisa; Chimenti, Cristina; Crotti, Lia; Giordano, Carla; Mancardi, Daniele; Torella, Daniele; Tocchetti, Carlo G.

2016-01-01

The prevalence of heart failure (HF) is still increasing worldwide, with enormous human, social, and economic costs, in spite of huge efforts in understanding pathogenetic mechanisms and in developing effective therapies that have transformed this syndrome into a chronic disease. Myocardial redox imbalance is a hallmark of this syndrome, since excessive reactive oxygen and nitrogen species can behave as signaling molecules in the pathogenesis of hypertrophy and heart failure, leading to dysregulation of cellular calcium handling, of the contractile machinery, of myocardial energetics and metabolism, and of extracellular matrix deposition. Recently, following new interesting advances in understanding myocardial ROS and RNS signaling pathways, new promising therapeutical approaches with antioxidant properties are being developed, keeping in mind that scavenging ROS and RNS tout court is detrimental as well, since these molecules also play a role in physiological myocardial homeostasis. PMID:26881035

REACTIVE OXYGEN SPECIES: IMPACT ON SKELETAL MUSCLE

PubMed Central

Powers, Scott K.; Ji, Li Li; Kavazis, Andreas N.; Jackson, Malcolm J.

2014-01-01

It is well established that contracting muscles produce both reactive oxygen and nitrogen species. Although the sources of oxidant production during exercise continue to be debated, growing evidence suggests that mitochondria are not the dominant source. Regardless of the sources of oxidants in contracting muscles, intense and prolonged exercise can result in oxidative damage to both proteins and lipids in the contracting myocytes. Further, oxidants regulate numerous cell signaling pathways and modulate the expression of many genes. This oxidant-mediated change in gene expression involves changes at transcriptional, mRNA stability, and signal transduction levels. Furthermore, numerous products associated with oxidant-modulated genes have been identified and include antioxidant enzymes, stress proteins, and mitochondrial electron transport proteins. Interestingly, low and physiological levels of reactive oxygen species are required for normal force production in skeletal muscle, but high levels of reactive oxygen species result in contractile dysfunction and fatigue. Ongoing research continues to explore the redox-sensitive targets in muscle that are responsible for both redox-regulation of muscle adaptation and oxidant-mediated muscle fatigue. PMID:23737208

Autophagy as a mechanism for myolysis of cardiomyocytes in mitral regurgitation.

PubMed

Chen, Mien-Cheng; Chang, Jen-Ping; Wang, Ya-Hui; Liu, Wen-Hao; Ho, Wan-Chun; Chang, Hsueh-Wen

2011-03-01

Myolysis of atrial cardiomyocytes occurs in patients with severe mitral and tricuspid regurgitation. This morphological remodelling may involve autophagy. This study comprised 20 patients (10 with long-standing persistent atrial fibrillation and 10 with sinus rhythm) with severe mitral and tricuspid regurgitation. Atrial appendageal tissues were obtained during surgery. The appearance of autophagosomes (LC3B) in myocytes can reflect autophagy induction. Complement 9 is used as a reliable marker of oncosis. In the fibrillating right atria, 68·4 ± 18·9% of total myocytes showed moderate-to-severe myolysis, while 64·2 ± 15·8% of total myocytes comprised these cells in right atrial myocardium with sinus rhythm. Immunohistochemical study revealed LC3B-positive myocytes in 8·0% of myocytes without myolysis, 11·9% of myocytes with mild myolysis and 49·4% of myocytes with moderate-to-severe myolysis in right atrial myocardium with sinus rhythm (P < 0·0001). Similarly, in the fibrillating right atria, LC3B-positive myocytes were observed in 5·9% of myocytes without myolysis, 12·2% of myocytes with mild myolysis and 50·7% of myocytes with moderate-to-severe myolysis (P < 0·0001). Moreover, in the fibrillating left atria, LC3B-positive myocytes were observed in 4·9% of myocytes without myolysis, 12·6% of myocytes with mild myolysis and 52·0% of myocytes with moderate-to-severe myolysis (P < 0·0001). None of the atrial myocytes displayed intracellular deposition of complement 9. Induction of autophagy, but not oncosis, occurs in most cases of atrial cardiomyocytes with severe mitral and tricuspid regurgitation, even those without atrial fibrillation, and is closely associated with the development of myolysis in this disease. © 2010 The Authors. European Journal of Clinical Investigation © 2010 Stichting European Society for Clinical Investigation Journal Foundation.

β-Myosin Heavy Chain Is Induced by Pressure Overload in a Minor Sub-Population of Smaller Mouse Cardiac Myocytes

PubMed Central

López, Javier E.; Myagmar, Bat-Erdene; Swigart, Philip M.; Montgomery, Megan D.; Haynam, Stephen; Bigos, Marty; Rodrigo, Manoj C.; Simpson, Paul C.

2011-01-01

Rationale Induction of the fetal hypertrophic marker gene beta-myosin heavy chain (β-MyHC) is a signature feature of pressure overload hypertrophy in rodents. β-MyHC is assumed present in all or most enlarged myocytes. Objective To quantify the number and size of myocytes expressing endogenous β-MyHC using a flow cytometry approach. Methods and Results Myocytes were isolated from the LV of male C57Bl/6J mice after transverse aortic constriction (TAC), and the fraction of cells expressing endogenous β-MyHC was quantified by flow cytometry on 10,000–20,000 myocytes, using a validated β-MyHC antibody. Side scatter by flow cytometry in the same cells was validated as an index of myocyte size. β-MyHC-positive myocytes were 3±1% of myocytes in control hearts (n=12), increasing to 25±10% at 3d-6w after TAC (n=24, p<0.01). β-MyHC-positive myocytes did not enlarge with TAC, and were smaller at all times than myocytes without β-MyHC (~70% as large, p<0.001). β-MyHC-positive myocytes arose by addition of β-MyHC to α-MyHC, and had more total MyHC after TAC than did the hypertrophied myocytes that had α-MyHC only. Myocytes positive for β-MyHC were found in discrete regions of the LV, in 3 patterns, peri-vascular, in areas with fibrosis, and in apparently normal myocardium. Conclusion β-MyHC protein is induced by pressure overload in a minor sub-population of smaller cardiac myocytes. The hypertrophied myocytes after TAC have α-MyHC only. These data challenge the current paradigm of the fetal hypertrophic gene program, and identify a new sub-population of smaller working ventricular myocytes with more myosin. PMID:21778428

Recovery time course in contractile function of fast and slow skeletal muscle after hindlimb immobilization

NASA Technical Reports Server (NTRS)

Witzmann, F. A.; Kim, D. H.; Fitts, R. H.

1982-01-01

The present study was undertaken to characterize the time course and extent of recovery in the isometric and isotonic contractile properties of fast and slow skeletal muscle following 6 wk of hindlimb immobilization. Female Sprague-Dawley rats were randomly assigned to an immobilized group or a control group. The results of the study show that fast and slow skeletal muscles possess the ability to completely recover normal contractile function following 6 wk of hindlimb immobilization. The rate of recovery is dependent on the fiber type composition of the affected muscle.

Protection of adult rat cardiac myocytes from ischemic cell death: role of caveolar microdomains and delta-opioid receptors.

PubMed

Patel, Hemal H; Head, Brian P; Petersen, Heidi N; Niesman, Ingrid R; Huang, Diane; Gross, Garrett J; Insel, Paul A; Roth, David M

2006-07-01

The role of caveolae, membrane microenvironments enriched in signaling molecules, in myocardial ischemia is poorly defined. In the current study, we used cardiac myocytes prepared from adult rats to test the hypothesis that opioid receptors (OR), which are capable of producing cardiac protection in vivo, promote cardiac protection in cardiac myocytes in a caveolae-dependent manner. We determined protein expression and localization of delta-OR (DOR) using coimmunohistochemistry, caveolar fractionation, and immunoprecipitations. DOR colocalized in fractions with caveolin-3 (Cav-3), a structural component of caveolae in muscle cells, and could be immunoprecipitated by a Cav-3 antibody. Immunohistochemistry confirmed plasma membrane colocalization of DOR with Cav-3. Cardiac myocytes were subjected to simulated ischemia (2 h) or an ischemic preconditioning (IPC) protocol (10 min ischemia, 30 min recovery, 2 h ischemia) in the presence and absence of methyl-beta-cyclodextrin (MbetaCD, 2 mM), which binds cholesterol and disrupts caveolae. We also assessed the cardiac protective effects of SNC-121 (SNC), a selective DOR agonist, on cardiac myocytes with or without MbetaCD and MbetaCD preloaded with cholesterol. Ischemia, simulated by mineral oil layering to inhibit gas exchange, promoted cardiac myocyte cell death (trypan blue staining), a response blunted by SNC (37 +/- 3 vs. 59 +/- 3% dead cells in the presence and absence of 1 muM SNC, respectively, P < 0.01) or by use of the IPC protocol (35 +/- 4 vs. 62 +/- 3% dead cells, P < 0.01). MbetaCD treatment, which disrupted caveolae (as detected by electron microscopy), fully attenuated the protective effects of IPC or SNC, resulting in cell death comparable to that of the ischemic group. By contrast, SNC-induced protection was not abrogated in cells incubated with cholesterol-saturated MbetaCD, which maintained caveolae structure and function. These findings suggest a key role for caveolae, perhaps through enrichment of signaling molecules, in contributing to protection of cardiac myocytes from ischemic damage.

The Electrophysiological Effects of Qiliqiangxin on Cardiac Ventricular Myocytes of Rats

PubMed Central

Wei, Yidong; Liu, Xiaoyu; Wei, Haidong; Hou, Lei; Che, Wenliang; The, Erlinda; Li, Gang; Jhummon, Muktanand Vikash; Wei, Wanlin

2013-01-01

Qiliqiangxin, a Chinese herb, represents the affection in Ca channel function of cardiac myocytes. It is unknown whether Qiliqiangxin has an effect on Na current and K current because the pharmacological actions of this herb's compound are very complex. We investigated the rational usage of Qiliqiangxin on cardiac ventricular myocytes of rats. Ventricular myocytes were exposed acutely to 1, 10, and 50 mg/L Qiliqiangxin, and whole cell patch-clamp technique was used to study the acute effects of Qiliqiangxin on Sodium current (I Na), outward currents delayed rectifier outward K+ current (I K), slowly activating delayed rectifier outward K+ current (I Ks), transient outward K+ current (I to), and inward rectifier K+ current (I K1). Qiliqiangxin can decrease I Na by 28.53% ± 5.98%, and its IC50 was 9.2 mg/L. 10 and 50 mg/L Qiliqiangxin decreased by 37.2% ± 6.4% and 55.9% ± 5.5% summit current density of I to. 10 and 50 mg/L Qiliqiangxin decreased I Ks by 15.51% ± 4.03% and 21.6% ± 5.6%. Qiliqiangxin represented a multifaceted pharmacological profile. The effects of Qiliqiangxin on Na and K currents of ventricular myocytes were more profitable in antiarrhythmic therapy in the clinic. We concluded that the relative efficacy of Qiliqiangxin was another choice for the existing antiarrhythmic therapy. PMID:24250713

Effects of severe caloric restriction from birth on the hearts of adult rats.

PubMed

Melo, Dirceu Sousa; Riul, Tania Regina; Esteves, Elizabeth Adriana; Moraes, Patrícia Lanza; Ferreira, Fernanda Oliveira; Gavioli, Mariana; Alves, Márcia Netto Magalhães; Almeida, Pedro William Machado; Guatimosim, Silvia; Ferreira, Anderson José; Dias Peixoto, Marco Fabricio

2013-08-01

There has been increasing evidence suggesting that a severe caloric restriction (SCR) (above 40%) has beneficial effects on the hearts of rats. However, most of the reports have focused on the effects of SCR that started in adulthood. We investigated the consequences of SCR on the hearts of rats subjected to SCR since birth (CR50). From birth to the age of 3 months, CR50 rats were fed 50% of the food that the ad libitum group (AL) was fed. Thereafter, a maximal aerobic test was performed to indirectly evaluate global cardiovascular function. Indices of contractility (+dT/dt) and relaxation (-dT/dt) were analyzed in isolated heart preparation, and cardiomyocyte diameter, number, density, and myocardium collagen content were obtained through histologic analysis. Ventricular myocytes were isolated, using standard methods to evaluate phosphorylated AKT levels, and Ca(2+) handling was evaluated with a combination of Western blot analysis, intracellular Ca(2+) imaging, and confocal microscopy. CR50 rats exhibited increased aerobic performance and cardiac function, as shown by the increase in ±dT/dt. Despite the smaller cardiomyocyte diameter, CR50 rats had an increased heart-body weight ratio, increased cardiomyocyte density and number, and similar levels of myocardium collagen content, compared with AL rats. AKT was hyperphosphorylated in cardiomyocytes from CR50 rats, and there were no significant differences in Ca(2+) transient and SERCA2 levels in cardiomyocytes between CR50 and AL rats. Collectively, these observations reveal the beneficial effects of a 50% caloric restriction on the hearts of adult rats restricted since birth, which might involve cardiomyocyte AKT signaling.

Abnormalities of capillary microarchitecture in a rat model of coronary ischemic congestive heart failure

PubMed Central

Chen, Jiqiu; Yaniz-Galende, Elisa; Kagan, Heather J.; Liang, Lifan; Hekmaty, Saboor; Giannarelli, Chiara

2015-01-01

The aim of the present study is to explore the role of capillary disorder in coronary ischemic congestive heart failure (CHF). CHF was induced in rats by aortic banding plus ischemia-reperfusion followed by aortic debanding. Coronary arteries were perfused with plastic polymer containing fluorescent dye. Multiple fluorescent images of casted heart sections and scanning electric microscope of coronary vessels were obtained to characterize changes in the heart. Cardiac function was assessed by echocardiography and in vivo hemodynamics. Stenosis was found in all levels of the coronary arteries in CHF. Coronary vasculature volume and capillary density in remote myocardium were significantly increased in CHF compared with control. This occurred largely in microvessels with a diameter of ≤3 μm. Capillaries in CHF had a tortuous structure, while normal capillaries were linear. Capillaries in CHF had inconsistent diameters, with assortments of narrowed and bulged segments. Their surfaces appeared rough, potentially indicating endothelial dysfunction in CHF. Segments of main capillaries between bifurcations were significantly shorter in length in CHF than in control. Transiently increasing preload by injecting 50 μl of 30% NaCl demonstrated that the CHF heart had lower functional reserve; this may be associated with congestion in coronary microcirculation. Ischemic coronary vascular disorder is not limited to the main coronary arteries, as it occurs in arterioles and capillaries. Capillary disorder in CHF included stenosis, deformed structure, proliferation, and roughened surfaces. This disorder in the coronary artery architecture may contribute to the reduction in myocyte contractility in the setting of heart failure. PMID:25659485

LRRC10 is required to maintain cardiac function in response to pressure overload.

PubMed

Brody, Matthew J; Feng, Li; Grimes, Adrian C; Hacker, Timothy A; Olson, Timothy M; Kamp, Timothy J; Balijepalli, Ravi C; Lee, Youngsook

2016-01-15

We previously reported that the cardiomyocyte-specific leucine-rich repeat containing protein (LRRC)10 has critical functions in the mammalian heart. In the present study, we tested the role of LRRC10 in the response of the heart to biomechanical stress by performing transverse aortic constriction on Lrrc10-null (Lrrc10(-/-)) mice. Mild pressure overload induced severe cardiac dysfunction and ventricular dilation in Lrrc10(-/-) mice compared with control mice. In addition to dilation and cardiomyopathy, Lrrc10(-/-) mice showed a pronounced increase in heart weight with pressure overload stimulation and a more dramatic loss of cardiac ventricular performance, collectively suggesting that the absence of LRRC10 renders the heart more disease prone with greater hypertrophy and structural remodeling, although rates of cardiac fibrosis and myocyte dropout were not different from control mice. Lrrc10(-/-) cardiomyocytes also exhibited reduced contractility in response to β-adrenergic stimulation, consistent with loss of cardiac ventricular performance after pressure overload. We have previously shown that LRRC10 interacts with actin in the heart. Here, we show that His(150) of LRRC10 was required for an interaction with actin, and this interaction was reduced after pressure overload, suggesting an integral role for LRRC10 in the response of the heart to mechanical stress. Importantly, these experiments demonstrated that LRRC10 is required to maintain cardiac performance in response to pressure overload and suggest that dysregulated expression or mutation of LRRC10 may greatly sensitize human patients to more severe cardiac disease in conditions such as chronic hypertension or aortic stenosis. Copyright © 2016 the American Physiological Society.

Porcine uterus cryopreservation: an analysis of contractile function using different uterotonics.

PubMed

Schölch, Daniel; Schölch, Sebastian; Strahl, Olga; Hoffmann, Inge; Beckmann, Matthias W; Dittrich, Ralf

2012-10-01

Cryopreservation of whole organs has become increasingly successful in recent years, and establishing reliable methods for confirming the success of specific cryopreservation procedures has therefore become extremely important. On the assumption that methods such as histological evaluation do not provide definitive evidence of long-term cryopreservation and that clear signs of conserved function in an organ are good evidence of its viability, contractile function was analysed in porcine uteri (n=60), either after long-term (group A) or short-term (group B) cryopreservation and post-thaw treatment with three different uterotonics. A slow freezing protocol was used to preserve the organs. Fifteen fresh uteri were analysed similarly for contractile function, which was evaluated by measuring intrauterine pressure after administration of oxytocin, prostaglandin E(1) (PGE(1)), and carbachol. After cryopreservation, all but three uteri (95%) showed rhythmic contractions similar to those in fresh uteri except for differences in the heights of contraction peaks, with lower contractions in PGE(1) subgroup B (P<0.05). With the exception of three nonresponsive uteri in group A, there were no differences in contractility between uteri after long-term cryopreservation and fresh uteri. The results of this study thus contribute to the debate on whether slow freezing or vitrification techniques are best for whole-organ cryopreservation. In summary, (1) preservation of muscular function in porcine uteri is feasible with a slow freezing protocol; (2) measurement of contractile function following administration of uterotonics is a useful method of confirming functionality; and (3) long-term cryopreservation does not significantly impair post-thaw contractibility in comparison with fresh uteri. Copyright © 2012 Elsevier Inc. All rights reserved.

Milrinone attenuates thromboxane receptor-mediated hyperresponsiveness in hypoxic pulmonary arterial myocytes.

PubMed

Santhosh, K T; Elkhateeb, O; Nolette, N; Outbih, O; Halayko, A J; Dakshinamurti, S

2011-07-01

Neonatal pulmonary hypertension (PPHN) is characterized by pulmonary vasoconstriction, due in part to dysregulation of the thromboxane prostanoid (TP) receptor. Hypoxia induces TP receptor-mediated hyperresponsiveness, whereas serine phosphorylation mediates desensitization of TP receptors. We hypothesized that prostacyclin (IP) receptor activity induces TP receptor phosphorylation and decreases ligand affinity; that TP receptor sensitization in hypoxic myocytes is due to IP receptor inactivation; and that this would be reversible by the cAMP-specific phosphodiesterase inhibitor milrinone. We examined functional regulation of TP receptors by serine phosphorylation and effects of IP receptor stimulation and protein kinase A (PKA) activity on TP receptor sensitivity in myocytes from neonatal porcine resistance pulmonary arteries after 72 h hypoxia in vitro. Ca(2+) response curves to U46619 (TP receptor agonist) were determined in hypoxic and normoxic myocytes incubated with or without iloprost (IP receptor agonist), forskolin (adenylyl cyclase activator), H8 (PKA inhibitor) or milrinone. TP and IP receptor saturation binding kinetics were measured in presence of iloprost or 8-bromo-cAMP. Ligand affinity for TP receptors was normalized in vitro by IP receptor signalling intermediates. However, IP receptor affinity was compromised in hypoxic myocytes, decreasing cAMP production. Milrinone normalized TP receptor sensitivity in hypoxic myocytes by restoring PKA-mediated regulatory TP receptor phosphorylation. TP receptor sensitivity and EC(50) for TP receptor agonists was regulated by PKA, as TP receptor serine phosphorylation by PKA down-regulated Ca(2+) mobilization. Hypoxia decreased IP receptor activity and cAMP generation, inducing TP receptor hyperresponsiveness, which was reversed by milrinone. © 2011 The Authors. British Journal of Pharmacology © 2011 The British Pharmacological Society.

Chronic hemodynamic overload of the atria is an important factor for gap junction remodeling in human and rat hearts.

PubMed

Rucker-Martin, Catherine; Milliez, Paul; Tan, Sisareuth; Decrouy, Xavier; Recouvreur, Michel; Vranckx, Roger; Delcayre, Claude; Renaud, Jean-François; Dunia, Irene; Segretain, Dominique; Hatem, Stéphane N

2006-10-01

The expression and distribution of connexins is abnormal in a number of cardiac diseases, including atrial fibrillation, and is believed to favor conduction slowing and arrhythmia. Here, we studied the role of atrial structural remodeling in the disorganization of gap junctions and whether redistributed connexins can form new functional junction channels. Expression of connexin-43 (Cx43) was characterized by immunoblotting and immunohistochemistry in human right atrial specimens and in rat atria after myocardial infarction (MI). Gap junctions were studied by electron and 3-D microscopy, and myocyte-myocyte coupling was determined by Lucifer yellow dye transfer. In both chronically hemodynamically overloaded human atria in sinus rhythm and in dilated atria from MI-rats, Cx43 were dephosphorylated and redistributed from the intercalated disc to the lateral cell membranes as observed during atrial fibrillation. In MI-rats, the gap junctions at the intercalated disc were smaller (20% decrease) and contained very little Cx43 (0 or 1 gold particle vs. 42 to 98 in sham-operated rats). In the lateral membranes of myocytes, numerous connexon aggregates comprising non-phosphorylated Cx43 were observed. These connexon aggregates were in no case assembled into gap junction plaque-like structures. However, N-cadherin was well organized in the intercalated disc. There was very little myocyte-myocyte coupling in MI-rat atria and no myocyte-fibroblast coupling. Regression of the atrial remodeling was associated with the normalization of Cx43 localization. Structural alteration of the atrial myocardium is an important factor in the disorganization of connexins and gap junction. Moreover, redistributed Cx43 do not form junction channels.

Spiral-Wave Dynamics in a Mathematical Model of Human Ventricular Tissue with Myocytes and Fibroblasts

PubMed Central

Nayak, Alok Ranjan; Shajahan, T. K.; Panfilov, A. V.; Pandit, Rahul

2013-01-01

Cardiac fibroblasts, when coupled functionally with myocytes, can modulate the electrophysiological properties of cardiac tissue. We present systematic numerical studies of such modulation of electrophysiological properties in mathematical models for (a) single myocyte-fibroblast (MF) units and (b) two-dimensional (2D) arrays of such units; our models build on earlier ones and allow for zero-, one-, and two-sided MF couplings. Our studies of MF units elucidate the dependence of the action-potential (AP) morphology on parameters such as , the fibroblast resting-membrane potential, the fibroblast conductance , and the MF gap-junctional coupling . Furthermore, we find that our MF composite can show autorhythmic and oscillatory behaviors in addition to an excitable response. Our 2D studies use (a) both homogeneous and inhomogeneous distributions of fibroblasts, (b) various ranges for parameters such as , and , and (c) intercellular couplings that can be zero-sided, one-sided, and two-sided connections of fibroblasts with myocytes. We show, in particular, that the plane-wave conduction velocity decreases as a function of , for zero-sided and one-sided couplings; however, for two-sided coupling, decreases initially and then increases as a function of , and, eventually, we observe that conduction failure occurs for low values of . In our homogeneous studies, we find that the rotation speed and stability of a spiral wave can be controlled either by controlling or . Our studies with fibroblast inhomogeneities show that a spiral wave can get anchored to a local fibroblast inhomogeneity. We also study the efficacy of a low-amplitude control scheme, which has been suggested for the control of spiral-wave turbulence in mathematical models for cardiac tissue, in our MF model both with and without heterogeneities. PMID:24023798

Characterization and inhibition of beta-adrenergic receptor kinase in intact myocytes.

PubMed

Laugwitz, K L; Kronsbein, K; Schmitt, M; Hoffmann, K; Seyfarth, M; Schömig, A; Ungerer, M

1997-08-01

beta-Adrenergic receptor kinase (beta ARK) phosphorylates and thereby inactivates agonist-occupied beta-adrenergic receptors (beta AR). beta ARK is thought to play an important role in the regulation of cardiac function. Therefore, we studied beta ARK activation and its inhibition in intact smooth muscle cells and in cardiomyoblasts. beta AR agonist-stimulated translocation of beta ARK was monitored by immunofluorescence labelling with specific antibodies and confocal laser scanning microscopy in DDT-MF 2 hamster smooth muscle cells and in H9c2 rat cardiomyoblasts. In unstimulated cells. beta ARK was mainly located in the cytosol. After beta AR agonist stimulation, the beta ARK signal was partially translocated to the membranes. Liposomal gene transfer of the COOH-terminus of beta ARK ('beta ARKmini') as a beta ARK inhibitor led to functional expression of this protein in both cell lines with high efficiency. Western blots with beta ARK antibodies showed a gene concentration-dependent immunoreactivity of the 'beta ARKmini' protein. 'beta ARKmini'-transfected myocytes demonstrated reduced membrane targeting of the beta ARK immuno-fluorescence signal. Additionally, the effect of 'beta ARKmini' on beta AR-induced desensitization of myocytic cAMP accumulation was investigated. In control cells, desensitization with isoproterenol led to a subsequent reduction of beta AR-induced cAMP accumulation. In 'beta ARKmini'-transfected myocytes, this beta AR-induced desensitization was significantly diminished, whereas normal beta AR-induced cAMP accumulation was unaffected. A gene concentration of 2 micrograms 'beta ARKmini' DNA/100,000 cardiomyoblasts, and of 0.7 microgram 'beta ARKmini' DNA/100,000 DDT-MF2 smooth muscle cells led to approximately 5.9- and approximately 5.6-fold overexpressions of 'beta ARKmini' vs. native beta ARK, respectively. These gene doses proved sufficient to attenuate beta-adrenergic desensitization significantly. (1) beta ARK translocation was evidenced in DDT-MF2 smooth muscle cells and in cardiomyoblasts by confocal laser scanning microscopy. (2) Feasibility of 'beta ARKmini' gene transfer to myocytes was demonstrated, and necessary gene doses for beta ARK inhibition were titered. (3) Overexpression of 'beta ARKmini' functionally interacted with endogenous beta-adrenergic signal transduction, leading to sustained cAMP accumulation after prolonged beta-adrenergic stimulation.

c-Kit-positive cardiac stem cells nested in hypoxic niches are activated by stem cell factor reversing the aging myopathy.

PubMed

Sanada, Fumihiro; Kim, Junghyun; Czarna, Anna; Chan, Noel Yan-Ki; Signore, Sergio; Ogórek, Barbara; Isobe, Kazuya; Wybieralska, Ewa; Borghetti, Giulia; Pesapane, Ada; Sorrentino, Andrea; Mangano, Emily; Cappetta, Donato; Mangiaracina, Chiara; Ricciardi, Mario; Cimini, Maria; Ifedigbo, Emeka; Perrella, Mark A; Goichberg, Polina; Choi, Augustine M; Kajstura, Jan; Hosoda, Toru; Rota, Marcello; Anversa, Piero; Leri, Annarosa

2014-01-03

Hypoxia favors stem cell quiescence, whereas normoxia is required for stem cell activation, but whether cardiac stem cell (CSC) function is regulated by the hypoxic/normoxic state of the cell is currently unknown. A balance between hypoxic and normoxic CSCs may be present in the young heart, although this homeostatic control may be disrupted with aging. Defects in tissue oxygenation occur in the old myocardium, and this phenomenon may expand the pool of hypoxic CSCs, which are no longer involved in myocyte renewal. Here, we show that the senescent heart is characterized by an increased number of quiescent CSCs with intact telomeres that cannot re-enter the cell cycle and form a differentiated progeny. Conversely, myocyte replacement is controlled only by frequently dividing CSCs with shortened telomeres; these CSCs generate a myocyte population that is chronologically young but phenotypically old. Telomere dysfunction dictates their actual age and mechanical behavior. However, the residual subset of quiescent young CSCs can be stimulated in situ by stem cell factor reversing the aging myopathy. Our findings support the notion that strategies targeting CSC activation and growth interfere with the manifestations of myocardial aging in an animal model. Although caution has to be exercised in the translation of animal studies to human beings, our data strongly suggest that a pool of functionally competent CSCs persists in the senescent heart and that this stem cell compartment can promote myocyte regeneration effectively, partly correcting the aging myopathy.

Bone marrow support of the heart in pressure overload is lost with aging.

PubMed

Sopko, Nikolai A; Turturice, Benjamin A; Becker, Mitchell E; Brown, Chase R; Dong, Feng; Popović, Zoran B; Penn, Marc S

2010-12-21

Exogenous stem cell delivery is under investigation to prevent and treat cardiac dysfunction. It is less studied as to the extent endogenous bone marrow derived stem cells contribute to cardiac homeostais in response to stress and the affects of aging on this stress response. To determine the role of bone marrow (BM) derived stem cells on cardiac homeostasis in response to pressure overload (PO) and how this response is altered by aging. Young (8 weeks) and old (>40 weeks) C57/b6 mice underwent homo- and heterochronic BM transplantation prior to transverse aortic constriction (TAC). We found that older BM is associated with decreased cardiac function following TAC. This decreased function is associated with decrease in BM cell engraftment, increased myocyte apoptosis, decreased myocyte hypertrophy, increased myocardial fibrosis and decreased cardiac function. Additionally, there is a decrease in activation of resident cells within the heart in response to PO in old mice. Interestingly, these effects are not due to alterations in vascular density or inflammation in response to PO or differences in ex vivo stem cell migration between young and old mice. BM derived stem cells are activated in response to cardiac PO, and the recruitment of BM derived cells are involved in cardiac myocyte hypertrophy and maintenance of function in response to PO which is lost with aging.

Contribution of SRF, Elk-1, and myocardin to airway smooth muscle remodeling in heaves, an asthma-like disease of horses.

PubMed

Chevigny, Mylène; Guérin-Montpetit, Karine; Vargas, Amandine; Lefebvre-Lavoie, Josiane; Lavoie, Jean-Pierre

2015-07-01

Myocyte hyperplasia and hypertrophy contribute to the increased mass of airway smooth muscle (ASM) in asthma. Serum-response factor (SRF) is a transcription factor that regulates myocyte differentiation in vitro in vascular and intestinal smooth muscles. When SRF is associated with phosphorylated (p)Elk-1, it promotes ASM proliferation while binding to myocardin (MYOCD) leading to the expression of contractile elements in these tissues. The objective of this study was therefore to characterize the expression of SRF, pElk-1, and MYOCD in ASM cells from central and peripheral airways in heaves, a spontaneously occurring asthma-like disease of horses, and in controls. Six horses with heaves and five aged-matched controls kept in the same environment were studied. Nuclear protein expression of SRF, pElk-1, and MYOCD was evaluated in peripheral airways and endobronchial biopsies obtained during disease remission and after 1 and 30 days of naturally occurring antigenic exposure using immunohistochemistry and immunofluorescence techniques. Nuclear expression of SRF (P = 0.03, remission vs. 30 days) and MYOCD (P = 0.05, controls vs. heaves at 30 days) increased in the peripheral airways of horses with heaves during disease exacerbation, while MYOCD (P = 0.04, remission vs. 30 days) decreased in the central airways of control horses. No changes were observed in the expression of pElk-1 protein in either tissue. In conclusion, SRF and its cofactor MYOCD likely contribute to the hypertrophy of peripheral ASM observed in equine asthmatic airways, while the remodeling of the central airways is more static or involves different transcription factors. Copyright © 2015 the American Physiological Society.

P2X receptors in the cardiovascular system and their potential as therapeutic targets in disease.

PubMed

Ralevic, Vera

2015-01-01

This review considers the expression and roles of P2X receptors in the cardiovascular system in health and disease and their potential as therapeutic targets. P2X receptors are ligand gated ion channels which are activated by the endogenous ligand ATP. They are formed from the assembly of three P2X subunit proteins from the complement of seven (P2X1-7), which can associate to form homomeric or heteromeric P2X receptors. The P2X1 receptor is widely expressed in the cardiovascular system, being located in the heart, in the smooth muscle of the majority of blood vessels and in platelets. P2X1 receptors expressed in blood vessels can be activated by ATP coreleased with noradrenaline as a sympathetic neurotransmitter, leading to smooth muscle depolarisation and contraction. There is evidence that the purinergic component of sympathetic neurotransmission is increased in hypertension, identifying P2X1 receptors as a possible therapeutic target in this disorder. P2X3 and P2X2/3 receptors are expressed on cardiac sympathetic neurones and may, through positive feedback of neuronal ATP at this prejunctional site, amplify sympathetic neurotransmission. Activation of P2X receptors expressed in the heart increases cardiac myocyte contractility, and an important role of the P2X4 receptor in this has been identified. Deletion of P2X4 receptors in the heart depresses contractile performance in models of heart failure, while overexpression of P2X4 receptors has been shown to be cardioprotective, thus P2X4 receptors may be therapeutic targets in the treatment of heart disease. P2X receptors have been identified on endothelial cells. Although immunoreactivity for all P2X1-7 receptor proteins has been shown on the endothelium, relatively little is known about their function, with the exception of the endothelial P2X4 receptor, which has been shown to mediate endothelium-dependent vasodilatation to ATP released during shear stress. The potential of P2X receptors as therapeutic targets in the treatment of cardiovascular disease is discussed.

The Functional Lumen Imaging Probe Detects Esophageal Contractility Not Observed With Manometry in Patients With Achalasia.

PubMed

Carlson, Dustin A; Lin, Zhiyue; Kahrilas, Peter J; Sternbach, Joel; Donnan, Erica N; Friesen, Laurel; Listernick, Zoe; Mogni, Benjamin; Pandolfino, John E

2015-12-01

The functional lumen imaging probe (FLIP) could improve the characterization of achalasia subtypes by detecting nonocclusive esophageal contractions not observed with standard manometry. We aimed to evaluate esophageal contractions during volumetric distention in patients with achalasia using FLIP topography. Fifty-one treatment-naive patients with achalasia, defined and subclassified by high-resolution esophageal pressure topography, and 10 asymptomatic individuals (controls) were evaluated with the FLIP during endoscopy. During stepwise distension, simultaneous intrabag pressures and 16 channels of cross-sectional areas were measured; data were exported to software that generated FLIP topography plots. Esophageal contractility was identified by noting periods of reduced luminal diameter. Esophageal contractions were characterized further by propagation direction, repetitiveness, and based on whether they were occluding or nonoccluding. Esophageal contractility was detected in all 10 controls: 8 of 10 had repetitive antegrade contractions and 9 of 10 had occluding contractions. Contractility was detected in 27% (4 of 15) of patients with type I achalasia and in 65% (18 of 26, including 9 with occluding contractions) of patients with type II achalasia. Contractility was detected in all 10 patients with type III achalasia; 8 of these patients had a pattern of contractility that was not observed in controls (repetitive retrograde contractions). Esophageal contractility not observed with manometry can be detected in patients with achalasia using FLIP topography. The presence and patterns of contractility detected with FLIP topography may represent variations in pathophysiology, such as mechanisms of panesophageal pressurization in patients with type II achalasia. These findings could have implications for additional subclassification to supplement prediction of the achalasia disease course. Copyright © 2015 AGA Institute. Published by Elsevier Inc. All rights reserved.

The Functional Lumen Imaging Probe Detects Esophageal Contractility not Observed with Manometry in Patients with Achalasia

PubMed Central

Carlson, Dustin A.; Lin, Zhiyue; Kahrilas, Peter J.; Sternbach, Joel; Donnan, Erica N.; Friesen, Laurel; Listernick, Zoe; Mogni, Benjamin; Pandolfino, John E.

2015-01-01

Background & Aims The functional lumen imaging probe (FLIP) could improve characterization of achalasia subtypes by detecting non-occlusive esophageal contractions not observed with standard manometry. We aimed to evaluate for esophageal contractions during volumetric distention in patients with achalasia using FLIP topography. Methods Fifty one treatment-naïve patients with achalasia, defined and sub-classified by high-resolution esophageal pressure topography, and 10 asymptomatic individuals (controls) were evaluated with the FLIP during endoscopy. During stepwise distension, simultaneous intra-bag pressures and 16 channels of cross-sectional areas were measured; data were exported to software that generated FLIP topography plots. Esophageal contractility was identified by noting periods of reduced luminal diameter. Esophageal contractions were further characterized by propagation direction, repetitiveness, and based on whether they were occluding or non-occluding. Results Esophageal contractility was detected in all 10 controls: 8/10 had repetitive, antegrade, contractions and 9/10 had occluding contractions. Contractility was detected in 27% (4/15) of patients with type I achalasia and 65% (18/26, including 9 with occluding contractions) of patients with type II achalasia. Contractility was detected in all 10 patients with type III achalasia; 8 of these patients had a pattern of contractility not observed in controls (repetitive, retrograde contractions). Conclusions Esophageal contractility not observed with manometry can be detected in patients with achalasia using FLIP topography. The presence and patterns of contractility detected with FLIP topography may represent variations in pathophysiology, such as mechanisms of pan-esophageal pressurization in patients with type II achalasia. These findings could have implications for additional sub-classification to supplement prediction of the achalasia disease course. PMID:26278501

Genetic Lineage Tracing of Non-Myocyte Population by Dual Recombinases.

PubMed

Li, Yan; He, Lingjuan; Huang, Xiuzhen; Issa Bhaloo, Shirin; Zhao, Huan; Zhang, Shaohua; Pu, Wenjuan; Tian, Xueying; Li, Yi; Liu, Qiaozhen; Yu, Wei; Zhang, Libo; Liu, Xiuxiu; Liu, Kuo; Tang, Juan; Zhang, Hui; Cai, Dongqing; Adams, Ralf H; Xu, Qingbo; Lui, Kathy O; Zhou, Bin

2018-04-26

Background -Whether the adult mammalian heart harbors cardiac stem cells (CSCs) for regeneration of cardiomyocytes is an important yet contentious topic in the field of cardiovascular regeneration. The putative myocyte stem cell populations recognized without specific cell markers such as the cardiosphere-derived cells or with markers such as Sca1 + , Bmi1 + , Isl1 + or Abcg2 + CSCs have been reported. Moreover, it remains unclear whether putative CSCs with unknown or unidentified markers exist and give rise to de novo cardiomyocytes in the adult heart. Methods -To address this question without relying on a particular stem cell marker, we developed a new genetic lineage tracing system to label all non-myocyte populations that contain putative CSCs. Using dual lineage tracing system, we assessed if non-myocytes generated any new myocytes during embryonic development, adult homeostasis and after myocardial infarction. Skeletal muscle was also examined after injury for internal control of new myocytes generation from non-myocytes. Results -By this stem cell marker-free and dual recombinases-mediated cell tracking approach, our fate mapping data show that new myocytes arise from non-myocytes in the embryonic heart, but not in the adult heart during homeostasis or after myocardial infarction. As positive control, our lineage tracing system detected new myocytes derived from non-myocytes in the skeletal muscle after injury. Conclusions -This study provides in vivo genetic evidence for non-myocyte to myocyte conversion in embryonic but not adult heart, arguing again the myogenic potential of putative stem cell populations for cardiac regeneration in the adult stage. This study also provides a new genetic strategy to identify endogenous stem cells, if any, in other organ systems for tissue repair and regeneration.

Do Binucleate Cardiomyocytes Have A Role in Myocardial Repair? Insights Using Isolated Rodent Myocytes and Cell Culture

PubMed Central

Stephen, Michael J; Poindexter, Brian J; Moolman, Johan A; Sheikh-Hamad, David; Bick, Roger J

2009-01-01

Neonatal and adult cardiomyocytes were isolated from rat hearts. Some of the adult myocytes were cultured to allow for cell dedifferentiation, a phenomenon thought to mimic cell changes that occur in stressed myocardium, with myocytes regressing to a fetal pattern of metabolism and stellate neonatal shape. Using fluorescence deconvolution microscopy, cells were probed with fluorescent markers and scanned for a number of proteins associated with ion control, calcium movements and cardiac function. Image analysis of deconvoluted image stacks and sequential real-time image recordings of calcium transients of cells were made. All three myocyte groups were predominantly comprised of binucleate cells. Clustering of proteins to a single nucleus was a common observation, suggesting that one nucleus is active in protein synthesis pathways, while the other nucleus assumes a ‘dormant’ or different role and that cardiomyocytes might be mitotically active even in late development, or specific protein syntheses could be targeted and regulated for reintroduction into the cell cycle. Such possibilities would extend cardiac disease associated stem cell research and therapy options, while producing valuable insights into developmental and death pathways of binucleate cardiomyocytes (word count 183). PMID:19430572

Cardiotoxic and Cardioprotective Features of Chronic β-adrenergic Signaling

PubMed Central

Zhang, Xiaoying; Szeto, Christopher; Gao, Erhe; Tang, Mingxin; Jin, Jianguo; Fu, Qin; Makarewich, Catherine; Ai, Xiaojie; Li, Ying; Tang, Allen; Wang, Jenny; Gao, Hui; Wang, Fang; Ge, Xinyi Joy; Kunapuli, Satya P.; Zhou, Lin; Zeng, Chunyu; Xiang, Kevin Yang; Chen, Xiongwen

2012-01-01

Rationale In the failing heart, persistent β-adrenergic receptor (βAR) activation is thought to induce myocyte death by protein kinase A (PKA)-dependent and PKA-independent activation of calcium/calmodulin-dependent kinase II (CaMKII). β-Adrenergic signaling pathways are also capable of activating cardioprotective mechanisms. Objective This study used a novel PKA inhibitor peptide (PKI) to inhibit PKA activity to test the hypothesis that βAR signaling causes cell death through PKA-dependent pathways and cardioprotection through PKA-independent pathways. Methods and Results In PKI transgenic mice, chronic isoproterenol (ISO) failed to induce cardiac hypertrophy, fibrosis, myocyte apoptosis and depressed cardiac function. In cultured adult feline ventricular myocytes (AFVMs), PKA inhibition protected myocytes from death induced by β1-AR agonists by preventing cytosolic and SR Ca2+ overload and CaMKII activation. PKA inhibition revealed a cardioprotective role of β-adrenergic signaling via cAMP/EPAC /Rap1/Rac/ERK pathway. Selective PKA inhibition causes protection in the heart after myocardial infarction (MI) that was superior to β-blocker therapy. Conclusion These results suggest that selective block of PKA could be a novel heart failure therapy. PMID:23104882

Combined use of UV-labile calcium chelators and calcium-sensitive dyes in a microscope with two light sources influencing different regions in a group of coordinated contracting cardiac myocytes

NASA Astrophysics Data System (ADS)

Pilarczyk, Goetz; Greulich, Karl-Otto

1997-12-01

The coordination of excitation in a biological system of cells such as cardiac myocytes in heart tissue has crucial influence on the function of the entire organ. This coordinated behavior can be visualized in a small group of embryonic cardiac myocytes derived from the hearts of unborn chicken. Loaded with a calcium sensitive dye the excitation can be imaged via the occurring transient rise in cytosolic calcium concentration. It can be shown that in regions with physiological or morphological restrictions the transient rise in cytosolic calcium occurs with a temporal delay compared to the ordinary array of coupled myocytes. The height of the transient rise of cytosolic calcium is related to the ability of the individual cell to participate in the coordinated contraction. The free cytosolic calcium concentration is decreased with the UV-labile calcium, chelator diazo-2. Our setup allows to decrease the free cytosolic calcium in a single cell of the contracting array of cells. This allows us to introduce mismatches in selected regions of the coordinated contraction and to visualize the effects simultaneously.

Contractile function is unaltered in diaphragm from mice lacking calcium release channel isoform 3

NASA Technical Reports Server (NTRS)

Clancy, J. S.; Takeshima, H.; Hamilton, S. L.; Reid, M. B.

1999-01-01

Skeletal muscle expresses at least two isoforms of the calcium release channel in the sarcoplasmic reticulum (RyR1 and RyR3). Whereas the function of RyR1 is well defined, the physiological significance of RyR3 is unclear. Some authors have suggested that RyR3 participates in excitation-contraction coupling and that RyR3 may specifically confer resistance to fatigue. To test this hypothesis, we measured contractile function of diaphragm strips from adult RyR3-deficient mice (exon 2-targeted mutation) and their heterozygous and wild-type littermates. In unfatigued diaphragm, there were no differences in isometric contractile properties (twitch characteristics, force-frequency relationships, maximal force) among the three groups. Our fatigue protocol (30 Hz, 0.25 duty cycle, 37 degrees C) depressed force to 25% of the initial force; however, lack of RyR3 did not accelerate the decline in force production. The force-frequency relationship was shifted to higher frequencies and was depressed in fatigued diaphragm; lack of RyR3 did not exaggerate these changes. We therefore provide evidence that RyR3 deficiency does not alter contractile function of adult muscle before, during, or after fatigue.

Functional, structural, and chemical changes in myosin associated with hydrogen peroxide treatment of skeletal muscle fibers.

PubMed

Prochniewicz, Ewa; Lowe, Dawn A; Spakowicz, Daniel J; Higgins, LeeAnn; O'Conor, Kate; Thompson, LaDora V; Ferrington, Deborah A; Thomas, David D

2008-02-01

To understand the molecular mechanism of oxidation-induced inhibition of muscle contractility, we have studied the effects of hydrogen peroxide on permeabilized rabbit psoas muscle fibers, focusing on changes in myosin purified from these fibers. Oxidation by 5 mM peroxide decreased fiber contractility (isometric force and shortening velocity) without significant changes in the enzymatic activity of myofibrils and isolated myosin. The inhibitory effects were reversed by treating fibers with dithiothreitol. Oxidation by 50 mM peroxide had a more pronounced and irreversible inhibitory effect on fiber contractility and also affected enzymatic activity of myofibrils, myosin, and actomyosin. Peroxide treatment also affected regulation of contractility, resulting in fiber activation in the absence of calcium. Electron paramagnetic resonance of spin-labeled myosin in muscle fibers showed that oxidation increased the fraction of myosin heads in the strong-binding structural state under relaxing conditions (low calcium) but had no effect under activating conditions (high calcium). This change in the distribution of structural states of myosin provides a plausible explanation for the observed changes in both contractile and regulatory functions. Mass spectroscopy analysis showed that 50 mM but not 5 mM peroxide induced oxidative modifications in both isoforms of the essential light chains and in the heavy chain of myosin subfragment 1 by targeting multiple methionine residues. We conclude that 1) inhibition of muscle fiber contractility via oxidation of myosin occurs at high but not low concentrations of peroxide and 2) the inhibitory effects of oxidation suggest a critical and previously unknown role of methionines in myosin function.

Cardiac myocyte exosomes: stability, HSP60, and proteomics.

PubMed

Malik, Z A; Kott, K S; Poe, A J; Kuo, T; Chen, L; Ferrara, K W; Knowlton, A A

2013-04-01

Exosomes, which are 50- to 100-nm-diameter lipid vesicles, have been implicated in intercellular communication, including transmitting malignancy, and as a way for viral particles to evade detection while spreading to new cells. Previously, we demonstrated that adult cardiac myocytes release heat shock protein (HSP)60 in exosomes. Extracellular HSP60, when not in exosomes, causes cardiac myocyte apoptosis via the activation of Toll-like receptor 4. Thus, release of HSP60 from exosomes would be damaging to the surrounding cardiac myocytes. We hypothesized that 1) pathological changes in the environment, such as fever, change in pH, or ethanol consumption, would increase exosome permeability; 2) different exosome inducers would result in different exosomal protein content; 3) ethanol at "physiological" concentrations would cause exosome release; and 4) ROS production is an underlying mechanism of increased exosome production. We found the following: first, exosomes retained their protein cargo under different physiological/pathological conditions, based on Western blot analyses. Second, mass spectrometry demonstrated that the protein content of cardiac exosomes differed significantly from other types of exosomes in the literature and contained cytosolic, sarcomeric, and mitochondrial proteins. Third, ethanol did not affect exosome stability but greatly increased the production of exosomes by cardiac myocytes. Fourth, ethanol- and hypoxia/reoxygenation-derived exosomes had different protein content. Finally, ROS inhibition reduced exosome production but did not completely inhibit it. In conclusion, exosomal protein content is influenced by the cell source and stimulus for exosome formation. ROS stimulate exosome production. The functions of exosomes remain to be fully elucidated.

Differential effects of pertussis toxin on insulin-stimulated phosphatidylcholine hydrolysis and glycerolipid synthesis de novo. Studies in BC3H-1 myocytes and rat adipocytes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hoffman, J.M.; Standaert, M.L.; Nair, G.P.

1991-04-02

Insulin-induced increases in diacylglycerol (DAG) have been suggested to result from stimulation of de novo phosphatidic acid (PA) synthesis and phosphatidylcholine (PC) hydrolysis. Presently, the authors found that insulin decreased PC levels of BC3H-1 myocytes and rat adipocytes by approximately 10-25% within 30 s. These decreases were rapidly reversed in both cell types, apparently because of increased PC synthesis de novo. In BC3H-1 myocytes, pertussis toxin inhibited PC resynthesis and insulin effects on the pathway of de novo PA-DAG-PC synthesis, as evidenced by changes in ({sup 3}H)glycerol incorporation, but did not inhibit insulin-stimulated PC hydrolysis. Pertussis toxin also blocked themore » later, but not the initial, increase in DAG production in the myocytes. Phorbol esters activated PC hydrolysis in both myocytes and adipocytes, but insulin-induced stimulation of PC hydrolysis was not dependent upon activation of PKC, since this hydrolysis was not inhibited by 500 {mu}M sangivamycin, an effective PKC inhibitor. The results indicate that insulin increases DAG by pertussis toxin sensitive and insensitive (PC hydrolysis) mechanisms, which are mechanistically separate, but functionally interdependent and integrated. PC hydrolysis may contribute importantly to initial increases in DAG, but later sustained increases are apparently largely dependent on insulin-induced stimulation of the pathway of de novo phospholipid synthesis.« less

Cardiac myocyte diversity and a fibroblast network in the junctional region of the zebrafish heart revealed by transmission and serial block-face scanning electron microscopy.

PubMed

Lafontant, Pascal J; Behzad, Ali R; Brown, Evelyn; Landry, Paul; Hu, Norman; Burns, Alan R

2013-01-01

The zebrafish has emerged as an important model of heart development and regeneration. While the structural characteristics of the developing and adult zebrafish ventricle have been previously studied, little attention has been paid to the nature of the interface between the compact and spongy myocardium. Here we describe how these two distinct layers are structurally and functionally integrated. We demonstrate by transmission electron microscopy that this interface is complex and composed primarily of a junctional region occupied by collagen, as well as a population of fibroblasts that form a highly complex network. We also describe a continuum of uniquely flattened transitional cardiac myocytes that form a circumferential plate upon which the radially-oriented luminal trabeculae are anchored. In addition, we have uncovered within the transitional ring a subpopulation of markedly electron dense cardiac myocytes. At discrete intervals the transitional cardiac myocytes form contact bridges across the junctional space that are stabilized through localized desmosomes and fascia adherentes junctions with adjacent compact cardiac myocytes. Finally using serial block-face scanning electron microscopy, segmentation and volume reconstruction, we confirm the three-dimensional nature of the junctional region as well as the presence of the sheet-like fibroblast network. These ultrastructural studies demonstrate the previously unrecognized complexity with which the compact and spongy layers are structurally integrated, and provide a new basis for understanding development and regeneration in the zebrafish heart.

Remodeling the zonula adherens in response to tension and the role of afadin in this response

PubMed Central

Acharya, Bipul R.; Peyret, Grégoire; Fardin, Marc-Antoine; Mège, René-Marc; Ladoux, Benoit; Yap, Alpha S.; Fanning, Alan S.

2016-01-01

Morphogenesis requires dynamic coordination between cell–cell adhesion and the cytoskeleton to allow cells to change shape and move without losing tissue integrity. We used genetic tools and superresolution microscopy in a simple model epithelial cell line to define how the molecular architecture of cell–cell zonula adherens (ZA) is modified in response to elevated contractility, and how these cells maintain tissue integrity. We previously found that depleting zonula occludens 1 (ZO-1) family proteins in MDCK cells induces a highly organized contractile actomyosin array at the ZA. We find that ZO knockdown elevates contractility via a Shroom3/Rho-associated, coiled-coil containing protein kinase (ROCK) pathway. Our data suggest that each bicellular border is an independent contractile unit, with actin cables anchored end-on to cadherin complexes at tricellular junctions. Cells respond to elevated contractility by increasing junctional afadin. Although ZO/afadin knockdown did not prevent contractile array assembly, it dramatically altered cell shape and barrier function in response to elevated contractility. We propose that afadin acts as a robust protein scaffold that maintains ZA architecture at tricellular junctions. PMID:27114502

Sarcomere length dependence of rat skinned cardiac myocyte mechanical properties: dependence on myosin heavy chain

PubMed Central

Korte, F Steven; McDonald, Kerry S

2007-01-01

The effects of sarcomere length (SL) on sarcomeric loaded shortening velocity, power output and rates of force development were examined in rat skinned cardiac myocytes that contained either α-myosin heavy chain (α-MyHC) or β-MyHC at 12 ± 1°C. When SL was decreased from 2.3 μm to 2.0 μm submaximal isometric force decreased ∼40% in both α-MyHC and β-MyHC myocytes while peak absolute power output decreased 55% in α-MyHC myocytes and 70% in β-MyHC myocytes. After normalization for the fall in force, peak power output decreased about twice as much in β-MyHC as in α-MyHC myocytes (41%versus 20%). To determine whether the fall in normalized power was due to the lower force levels, [Ca2+] was increased at short SL to match force at long SL. Surprisingly, this led to a 32% greater peak normalized power output at short SL compared to long SL in α-MyHC myocytes, whereas in β-MyHC myocytes peak normalized power output remained depressed at short SL. The role that interfilament spacing plays in determining SL dependence of power was tested by myocyte compression at short SL. Addition of 2% dextran at short SL decreased myocyte width and increased force to levels obtained at long SL, and increased peak normalized power output to values greater than at long SL in both α-MyHC and β-MyHC myocytes. The rate constant of force development (ktr) was also measured and was not different between long and short SL at the same [Ca2+] in α-MyHC myocytes but was greater at short SL in β-MyHC myocytes. At short SL with matched force by either dextran or [Ca2+], ktr was greater than at long SL in both α-MyHC and β-MyHC myocytes. Overall, these results are consistent with the idea that an intrinsic length component increases loaded crossbridge cycling rates at short SL and β-MyHC myocytes exhibit a greater sarcomere length dependence of power output. PMID:17347271

Effect of hypokinesia on contractile function of cardiac muscle

NASA Technical Reports Server (NTRS)

Meyerson, F. Z.; Kapelko, V. I.; Trikhpoyeva, A. M.; Gorina, M. S.

1980-01-01

Rats were subjected to hypokinesia for two months and the contractile function of isolated papillary muscle was studied. Hypokinesia reduced significantly the isotonic contraction rate which depended on the ATPase activity of the myofibrils; it also reduced the rate and index of relaxation which depended on the functional capacity of the Ca(++) pump of the sarcoplasmic reticulum. The maximum force of isometric contraction determined by the quantity of actomyosin bridges in the myofibrils did not change after hypokinesia. This complex of changes is contrary to that observed in adaptation to exercise when the rate of isotonic contraction and relaxation increases while the force of isometric contraction does not change. The possible mechanism of this stability of the contractile force during adaptation and readaptation of the heart is discussed.

Cardiomyocytes from late embryos and neonates do optimal work and striate best on substrates with tissue-level elasticity: metrics and mathematics.

PubMed

Majkut, Stephanie F; Discher, Dennis E

2012-11-01

In this review, we discuss recent studies on the mechanosensitive morphology and function of cardiomyocytes derived from embryos and neonates. For early cardiomyocytes cultured on substrates of various stiffnesses, contractile function as measured by force production, work output and calcium handling is optimized when the culture substrate stiffness mimics that of the tissue from which the cells were obtained. This optimal contractile function corresponds to changes in sarcomeric protein conformation and organization that promote contractile ability. In light of current models for myofibillogenesis, a recent mathematical model of striation and alignment on elastic substrates helps to illuminate how substrate stiffness modulates early myofibril formation and organization. During embryonic heart formation and maturation, cardiac tissue mechanics change dynamically. Experiments and models highlighted here have important implications for understanding cardiomyocyte differentiation and function in development and perhaps in regeneration processes.

Extracellular signal-regulated kinase (ERK) activation preserves cardiac function in pressure overload induced hypertrophy.

PubMed

Mutlak, Michael; Schlesinger-Laufer, Michal; Haas, Tali; Shofti, Rona; Ballan, Nimer; Lewis, Yair E; Zuler, Mor; Zohar, Yaniv; Caspi, Lilac H; Kehat, Izhak

2018-05-24

Chronic pressure overload and a variety of mediators induce concentric cardiac hypertrophy. When prolonged, cardiac hypertrophy culminates in decreased myocardial function and heart failure. Activation of the extracellular signal-regulated kinase (ERK) is consistently observed in animal models of hypertrophy and in human patients, but its role in the process is controversial. We generated transgenic mouse lines with cardiomyocyte restricted overexpression of intrinsically active ERK1, which similar to the observations in hypertrophy is phosphorylated on both the TEY and the Thr207 motifs and is overexpressed at pathophysiological levels. The activated ERK1 transgenic mice developed a modest adaptive hypertrophy with increased contractile function and without fibrosis. Following induction of pressure-overload, where multiple pathways are stimulated, this activation did not further increase the degree of hypertrophy but protected the heart through a decrease in the degree of fibrosis and maintenance of ventricular contractile function. The ERK pathway acts to promote a compensated hypertrophic response, with enhanced contractile function and reduced fibrosis. The activation of this pathway may be a therapeutic strategy to preserve contractile function when the pressure overload cannot be easily alleviated. The inhibition of this pathway, which is increasingly being used for cancer therapy on the other hand, should be used with caution in the presence of pressure-overload. Copyright © 2017. Published by Elsevier B.V.

Skeletal muscle contractile properties in a novel murine model for limb girdle muscular dystrophy 2i.

PubMed

Rehwaldt, Jordan D; Rodgers, Buel D; Lin, David C

2017-12-01

Limb-girdle muscular dystrophy (LGMD) 2i results from mutations in fukutin-related protein and aberrant α-dystroglycan glycosylation. Although this significantly compromises muscle function and ambulation, the comprehensive characteristics of contractile dysfunction are unknown. Therefore, we quantified the in situ contractile properties of the medial gastrocnemius in young adult P448L mice, an affected muscle of a novel model of LGMD2i. Normalized maximal twitch force, tetanic force, and power were significantly smaller in P448L mice, compared with sex-matched, wild-type mice. These differences were consistent with the replacement of contractile fibers by passive tissue. The shape of the active force-length relationships were similar in both groups, regardless of sex, consistent with an intact sarcomeric structure in P448L mice. Passive force-length curves normalized to maximal isometric force were steeper in P448L mice, and passive elements contribute disproportionately more to total contractile force in P448L mice. Sex differences were mostly noted in the force-velocity curves, as normalized values for maximal and optimal velocities were significantly slower in P448L males, compared with wild-type, but not in P448L females. This suggests that the dystrophic phenotype, which may include possible changes in cross-bridge kinetics and fiber-type proportions, progresses more quickly in P448L males. These results together indicate that active force and power generation are compromised in both sexes of P448L mice, while passive forces increase. More importantly, the results identified several functional markers of disease pathophysiology that could aid in developing and assessment of novel therapeutics for LGMD2i and possibly other dystroglycanopathies as well. NEW & NOTEWORTHY Comprehensive assessments of muscle contractile function have, until now, never been performed in an animal model for any dystroglycanopathy. This study suggests that skeletal muscle contractile properties are significantly compromised in a recently developed model for limb-girdle muscular dystrophy 2i, the P448L mouse. It further identifies novel pathological markers of muscle function that are suitable for developing therapeutics and for better understanding of disease pathogenesis.

Structure of the Elastin-Contractile Units in the Thoracic Aorta and How Genes That Cause Thoracic Aortic Aneurysms and Dissections Disrupt This Structure.

PubMed

Karimi, Ashkan; Milewicz, Dianna M

2016-01-01

The medial layer of the aorta confers elasticity and strength to the aortic wall and is composed of alternating layers of smooth muscle cells (SMCs) and elastic fibres. The SMC elastin-contractile unit is a structural unit that links the elastin fibres to the SMCs and is characterized by the following: (1) layers of elastin fibres that are surrounded by microfibrils; (2) microfibrils that bind to the integrin receptors in focal adhesions on the cell surface of the SMCs; and (3) SMC contractile filaments that are linked to the focal adhesions on the inner side of the membrane. The genes that are altered to cause thoracic aortic aneurysms and aortic dissections encode proteins involved in the structure or function of the SMC elastin-contractile unit. Included in this gene list are the genes encoding protein that are structural components of elastin fibres and microfibrils, FBN1, MFAP5, ELN, and FBLN4. Also included are genes that encode structural proteins in the SMC contractile unit, including ACTA2, which encodes SMC-specific α-actin and MYH11, which encodes SMC-specific myosin heavy chain, along with MYLK and PRKG1, which encode kinases that control SMC contraction. Finally, mutations in the gene encoding the protein linking integrin receptors to the contractile filaments, FLNA, also predispose to thoracic aortic disease. Thus, these data suggest that functional SMC elastin-contractile units are important for maintaining the structural integrity of the aorta. Copyright © 2016 Canadian Cardiovascular Society. Published by Elsevier Inc. All rights reserved.

Effect of a Periodized Power Training Program on the Functional Performances and Contractile Properties of the Quadriceps in Sprinters

ERIC Educational Resources Information Center

Kamandulis, Sigitas; Skurvydas, Albertas; Brazaitis, Marius; Stanislovaitis, Aleksas; Duchateau, Jacques; Stanislovaitiene, Jurate

2012-01-01

Our purpose was to compare the effect of a periodized preparation consisting of power endurance training and high-intensity power training on the contractile properties of the quadriceps muscle and functional performances in well trained male sprinters (n = 7). After 4 weeks of high-intensity power training, 60-m sprint running time improved by an…

Modulation of Myocardial Mitochondrial Mechanisms during Severe Polymicrobial Sepsis in the Rat

PubMed Central

Chopra, Mani; Golden, Honey B.; Mullapudi, Srinivas; Dowhan, William; Dostal, David E.; Sharma, Avadhesh C.

2011-01-01

Background We tested the hypothesis that 5-Hydroxydecanoic acid (5HD), a putative mitoKATP channel blocker, will reverse sepsis-induced cardiodynamic and adult rat ventricular myocyte (ARVM) contractile dysfunction, restore mitochondrial membrane permeability alterations and improve survival. Methodology/Principal Findings Male Sprague-Dawley rats (350–400 g) were made septic using 400 mg/kg cecal inoculum, ip. Sham animals received 5% dextrose water, ip. The Voltage Dependent Anion Channels (VDAC1), Bax and cytochrome C levels were determined in isolated single ARVMs obtained from sham and septic rat heart. Mitochondria and cytosolic fractions were isolated from ARVMs treated with norepinephrine (NE, 10 µmoles) in the presence/absence of 5HD (100 µmoles). A continuous infusion of 5HD using an Alzet pump reversed sepsis-induced mortality when administered at the time of induction of sepsis (−40%) and at 6 hr post-sepsis (−20%). Electrocardiography revealed that 5HD reversed sepsis-induced decrease in the average ejection fraction, Simpsons+m Mode (53.5±2.5 in sepsis and 69.2±1.2 at 24 hr in sepsis+5HD vs. 79.9±1.5 basal group) and cardiac output (63.3±1.2 mL/min sepsis and 79.3±3.9 mL/min at 24 hr in sepsis+5HD vs. 85.8±1.5 mL/min basal group). The treatment of ARVMs with 5HD also reversed sepsis-induced depressed contractility in both the vehicle and NE-treated groups. Sepsis produced a significant downregulation of VDAC1, and upregulation of Bax levels, along with mitochondrial membrane potential collapse in ARVMs. Pretreatment of septic ARVMs with 5HD blocked a NE-induced decrease in the VDAC1 and release of cytochrome C. Conclusion The data suggest that Bax activation is an upstream event that may precede the opening of the mitoKATP channels in sepsis. We concluded that mitoKATP channel inhibition via decreased mitochondrial membrane potential and reduced release of cytochrome C provided protection against sepsis-induced ARVM and myocardial contractile dysfunction. PMID:21712982

Modulation of myocardial mitochondrial mechanisms during severe polymicrobial sepsis in the rat.

PubMed

Chopra, Mani; Golden, Honey B; Mullapudi, Srinivas; Dowhan, William; Dostal, David E; Sharma, Avadhesh C

2011-01-01

We tested the hypothesis that 5-Hydroxydecanoic acid (5HD), a putative mitoK(ATP) channel blocker, will reverse sepsis-induced cardiodynamic and adult rat ventricular myocyte (ARVM) contractile dysfunction, restore mitochondrial membrane permeability alterations and improve survival. Male Sprague-Dawley rats (350-400 g) were made septic using 400 mg/kg cecal inoculum, ip. Sham animals received 5% dextrose water, ip. The Voltage Dependent Anion Channels (VDAC1), Bax and cytochrome C levels were determined in isolated single ARVMs obtained from sham and septic rat heart. Mitochondria and cytosolic fractions were isolated from ARVMs treated with norepinephrine (NE, 10 µmoles) in the presence/absence of 5HD (100 µmoles). A continuous infusion of 5HD using an Alzet pump reversed sepsis-induced mortality when administered at the time of induction of sepsis (-40%) and at 6 hr post-sepsis (-20%). Electrocardiography revealed that 5HD reversed sepsis-induced decrease in the average ejection fraction, Simpsons+m Mode (53.5±2.5 in sepsis and 69.2±1.2 at 24 hr in sepsis+5HD vs. 79.9±1.5 basal group) and cardiac output (63.3±1.2 mL/min sepsis and 79.3±3.9 mL/min at 24 hr in sepsis+5HD vs. 85.8±1.5 mL/min basal group). The treatment of ARVMs with 5HD also reversed sepsis-induced depressed contractility in both the vehicle and NE-treated groups. Sepsis produced a significant downregulation of VDAC1, and upregulation of Bax levels, along with mitochondrial membrane potential collapse in ARVMs. Pretreatment of septic ARVMs with 5HD blocked a NE-induced decrease in the VDAC1 and release of cytochrome C. The data suggest that Bax activation is an upstream event that may precede the opening of the mitoK(ATP) channels in sepsis. We concluded that mitoK(ATP) channel inhibition via decreased mitochondrial membrane potential and reduced release of cytochrome C provided protection against sepsis-induced ARVM and myocardial contractile dysfunction.

Novel fluorescence resonance energy transfer-based reporter reveals differential calcineurin activation in neonatal and adult cardiomyocytes

PubMed Central

Bazzazi, Hojjat; Sang, Lingjie; Dick, Ivy E; Joshi-Mukherjee, Rosy; Yang, Wanjun; Yue, David T

2015-01-01

Abstract The phosphatase calcineurin is a central component of many calcium signalling pathways, relaying calcium signals from the plasma membrane to the nucleus. It has critical functions in a multitude of systems, including immune, cardiac and neuronal. Given the widespread importance of calcineurin in both normal and pathological conditions, new tools that elucidate the spatiotemporal dynamics of calcineurin activity would be invaluable. Here we develop two separate genetically encoded fluorescence resonance energy transfer (FRET)-based sensors of calcineurin activation, DuoCaN and UniCaN. Both sensors showcase a large dynamic range and rapid response kinetics, differing primarily in the linker structure between the FRET pairs. Both sensors were calibrated in HEK293 cells and their responses correlated well with NFAT translocation to the nucleus, validating the biological relevance of the sensor readout. The sensors were subsequently expressed in neonatal rat ventricular myocytes and acutely isolated adult guinea pig ventricular myocytes. Both sensors demonstrated robust responses in myocytes and revealed kinetic differences in calcineurin activation during changes in pacing rate for neonatal versus adult myocytes. Finally, mathematical modelling combined with quantitative FRET measurements provided novel insights into the kinetics and integration of calcineurin activation in response to myocyte Ca transients. In all, DuoCaN and UniCaN stand as valuable new tools for understanding the role of calcineurin in normal and pathological signalling. Key points Novel fluorescence resonance energy transfer-based genetically encoded reporters of calcineurin are constructed by fusing the two subunits of calcineurin with P2A-based linkers retaining the expected native conformation of calcineurin. Calcineurin reporters display robust responses to calcium transients in HEK293 cells. The sensor responses are correlated with NFATc1 translocation dynamics in HEK293 cells. The sensors are uniformly distributed in neonatal myocytes and respond efficiently to single electrically evoked calcium transients and show cumulative activation at frequencies of 0.5 and 1 Hz. In adult myocytes, the calcineurin sensors appear to be localized to the cardiac z-lines, and respond to cumulative calcium transients at frequencies of 0.5 and 1 Hz. PMID:26096996

Investigating Cardiac MRI Based Right Ventricular Contractility As A Novel Non-Invasive Metric of Pulmonary Arterial Pressure

PubMed Central

Menon, Prahlad G; Adhypak, Srilakshmi M; Williams, Ronald B; Doyle, Mark; Biederman, Robert WW

2014-01-01

BACKGROUND We test the hypothesis that cardiac magnetic resonance (CMR) imaging-based indices of four-dimensional (4D) (three dimensions (3D) + time) right ventricle (RV) function have predictive values in ascertaining invasive pulmonary arterial systolic pressure (PASP) measurements from right heart catheterization (RHC) in patients with pulmonary arterial hypertension (PAH). METHODS We studied five patients with idiopathic PAH and two age and sex-matched controls for RV function using a novel contractility index (CI) for amplitude and phase to peak contraction established from analysis of regional shape variation in the RV endocardium over 20 cardiac phases, segmented from CMR images in multiple orientations. RESULTS The amplitude of RV contractility correlated inversely with RV ejection fraction (RVEF; R2 = 0.64, P = 0.03) and PASP (R2 = 0.71, P = 0.02). Phase of peak RV contractility also correlated inversely to RVEF (R2 = 0.499, P = 0.12) and PASP (R2 = 0.66, P = 0.04). CONCLUSIONS RV contractility analyzed from CMR offers promising non-invasive metrics for classification of PAH, which are congruent with invasive pressure measurements. PMID:25624777

Left atrial volume and function in dogs with naturally occurring myxomatous mitral valve disease.

PubMed

Höllmer, M; Willesen, J L; Tolver, A; Koch, J

2017-02-01

Myxomatous mitral valve disease (MMVD) induces progressive left atrial (LA) enlargement. The LA modulates left ventricular filling and performance through its reservoir, conduit, and contractile function. Assessment of LA size and function may provide valuable information on the level of cardiac compensation. Left atrial function in dogs with naturally occurring MMVD remains largely unexplored. The objective of this study was to evaluate LA volume and function in dogs with naturally occurring MMVD. This prospective study included 205 client-owned dogs of different breeds, 114 healthy dogs, and 91 dogs with MMVD of different disease severities. Using two-dimensional echocardiography, the biplane area-length method was applied to assess LA volume and calculate volumetric indices of LA reservoir, conduit, and contractile function. Left atrial volume and LA stroke volume increased, whereas LA reservoir and contractile function decreased with increasing disease severity. A maximal LA volume <2.25mL/kg was the optimal cut off identified for excluding congestive heart failure in dogs with chronic MMVD with a sensitivity of 96% and a specificity of 100%. An active LA emptying fraction <24% and/or a LA expansion index <126% were suggestive of congestive heart failure in dogs with chronic MMVD with a sensitivity of 77% and a specificity of 89% and a sensitivity of 82% and a specificity of 82%, respectively. Dogs with MMVD appear to have larger LA volumes with poorer LA function. Deteriorating LA function, characterized by a decreasing reservoir and active contractile function, was evident in dogs with MMVD with increasing disease severity. Copyright © 2016 Elsevier B.V. All rights reserved.

Correlation between cardiac remodelling, function, and myocardial contractility in rat hearts 5 weeks after myocardial infarction.

PubMed

Gosselin, H; Qi, X; Rouleau, J L

1998-01-01

Early after infarction, ventricular dysfunction occurs as a result of loss of myocardial tissue. Although papillary muscle studies suggest that reduced myocardial contractility contributes to this ventricular dysfunction, in vivo studies indicate that at rest, cardiac output is normal or near normal, suggesting that contractility of the remaining viable myocardium of the ventricular wall is preserved. However, this has never been verified. To explore this further, 100 rats with various-sized myocardial infarctions had ventricular function assessed by Langendorff preparation or by isolated papillary muscle studies 5 weeks after infarction. Morphologic studies were also done. Rats with large infarctions (54%) had marked ventricular dilatation (dilatation index from 0.23 to 0.75, p < 0.01) and papillary muscle dysfunction (total tension from 6.7 to 3.2 g/mm2, p < 0.01) but only moderate left ventricular dysfunction (maximum developed tension from 206 to 151 mmHg (1 mmHg = 133.3 Pa), p < 0.01), a decrease less than one would expect with an infarct size of 54%. The contractility of the remaining viable myocardium of the ventricle was also moderately depressed (peak systolic midwall stress 91 to 60 mmHg, p < 0.01). Rats with moderate infarctions (32%) had less marked but still moderate ventricular dilatation (dilatation index 0.37, p < 0.001) and moderate papillary muscle dysfunction (total tension 4.2 g/mm2, p < 0.01). However, their decrease in ventricular function was only mild (maximum developed pressure 178 mmHg, p < 0.01) and less than one would expect with an infarct size of 32%. The remaining viable myocardium of the ventricular wall appeared to have normal contractility (peak systolic midwall stress = 86 mmHg, ns). We conclude that in this postinfarction model, in large myocardial infarctions, a loss of contractility of the remaining viable myocardium of the ventricular wall occurs as early as 5 weeks after infarction and that papillary muscle studies slightly overestimate the degree of ventricular dysfunction. In moderate infarctions, the remaining viable myocardium of the ventricular wall has preserved contractility while papillary muscle function is depressed. In this relatively early postinfarction phase, ventricular remodelling appears to help maintain left ventricular function in both moderate and large infarctions.

Mechanically induced orientation of adult rat cardiac myocytes in vitro

NASA Technical Reports Server (NTRS)

Samuel, J.-L.; Vandenburgh, H. H.

1990-01-01

The present study describes the spatial orientation of a population of freshly isolated adult rat cardiac myocytes using a computerized mechanical cell stimulator device for tissue cultured cells. A continuous unidirectional stretch of the substratum at 60 to 400 microns/min for 120 to 30 min, respectively, during the cell attachment period in a serum-free medium was found to induce a significant threefold increase in the number of rod-shaped myocytes oriented parallel to the direction of movement. The myocytes orient less well with unidirectional substratum stretching after their adhesion to the substratum. Adult myocytes plated onto a substratum undergoing continuous 10-percent stretch-relaxation cycling show no significant change in the myocyte orientation or cytoskeletal organization. In addition to the type of mechanical activity, orientation of rod-shaped myocytes is dependent on the speed of the substratum, the final stretch amplitude, and the timing between initiation of substratum stretching and adhesion of myocytes to the substratum.

Pressure overload differentially affects respiratory capacity in interfibrillar and subsarcolemmal mitochondria.

PubMed

Schwarzer, Michael; Schrepper, Andrea; Amorim, Paulo A; Osterholt, Moritz; Doenst, Torsten

2013-02-15

Years ago a debate arose as to whether two functionally different mitochondrial subpopulations, subsarcolemmal mitochondria (SSM) and interfibrillar mitochondria (IFM), exist in heart muscle. Nowadays potential differences are often ignored. Presumably, SSM are providing ATP for basic cell function, whereas IFM provide energy for the contractile apparatus. We speculated that two distinguishable subpopulations exist that are differentially affected by pressure overload. Male Sprague-Dawley rats were subjected to transverse aortic constriction for 20 wk or sham operation. Contractile function was assessed by echocardiography. Heart tissue was analyzed by electron microscopy. Mitochondria were isolated by differential centrifugation, and respiratory capacity was analyzed using a Clark electrode. Pressure overload induced left ventricular hypertrophy with increased posterior wall diameter and impaired contractile function. Mitochondrial state 3 respiration in control was 50% higher in IFM than in SSM. Pressure overload significantly impaired respiratory rates in both IFM and SSM, but in SSM to a lower extent. As a result, there were no differences between SSM and IFM after 20 wk of pressure overload. Pressure overload reduced total citrate synthase activity, suggesting reduced total mitochondrial content. Electron microscopy revealed normal morphology of mitochondria but reduced total mitochondrial volume density. In conclusion, IFM show greater respiratory capacity in the healthy rat heart and a greater depression of respiratory capacity by pressure overload than SSM. The differences in respiratory capacity of cardiac IFM and SSM in healthy hearts are eliminated with pressure overload-induced heart failure. The strong effect of pressure overload on IFM together with the simultaneous appearance of mitochondrial and contractile dysfunction may support the notion of IFM primarily producing ATP for contractile function.

Asymmetrical electrically induced injury of rabbit ventricular myocytes.

PubMed

Knisley, S B; Grant, A O

1995-05-01

Strong defibrillation-type electric field stimulation may injure myocytes when transmembrane potentials during the pulse exceed the threshold for membrane permeabilization. The location of injury may depend on intrinsic transmembrane potential or influx of calcium by "electro-osmosis" during the stimulation pulse in addition to the transmembrane potential changes induced by the pulse. We have studied injury by examining contracture and changes in transmembrane potential-sensitive dye fluorescence induced by electric field stimulation (St) with a duration of 20 ms and strength of 16-400 V/cm in isolated rabbit ventricular myocytes. St of 100-150 V/cm produced injury in myocytes oriented parallel to the St field frequently without injuring myocytes oriented perpendicular to the field. Injury required calcium in the solution and was asymmetric, occurring first at the myocyte and facing the St anode in 100% of injured myocytes in normal Tyrode's solution. Injury depended significantly on whether the product of the electric field strength and myocyte length exceeded a threshold of 1.1 V (P < 0.05). Asymmetric injury at the end facing the anode was still present in 96% of injured myocytes for stimulation after depolarization by an action potential or 20 mM or 125 mM potassium, suggesting that intrinsic transmembrane potential is not responsible for asymmetry. In 125 mM potassium, eliminating calcium from the bathing solution during the St pulse and introducing calcium after the pulse decreased the fraction of injured myocytes in which injury occurred at the end facing the anode to 62%, suggesting that calcium influx by "electro-osmosis" at the myocyte end facing the anode contributes to asymmetry. Asymmetric injury at the end facing the anode was still present in 100% of injured myocytes after adding 1 mM tetraethylammonium chloride, indicating that asymmetry is not sensitive to the potassium channel blockade. For stimulation pulses stronger than 50 V/cm given after depolarization by an action potential, transmembrane potentials at both myocyte ends decayed after the initial deflection indicating that permeabilization occurred at both ends. In conclusion, injury depends on myocyte orientation and is asymmetric occurring first at the myocyte end facing the anode. Asymmetric injury is not explained by asymmetric permeabilization, is independent of the intrinsic transmembrane potential and may result from "electro-osmosis" during the stimulation pulse.

Bone-Derived Stem Cells Repair the Heart after Myocardial Infarction Through Transdifferentiation and Paracrine Signaling Mechanisms

PubMed Central

Duran, Jason M.; Makarewich, Catherine A.; Sharp, Thomas E.; Starosta, Timothy; Fang, Zhu; Hoffman, Nicholas E.; Chiba, Yumi; Madesh, Muniswamy; Berretta, Remus M.; Kubo, Hajime; Houser, Steven R.

2013-01-01

Rationale Autologous bone marrow- or cardiac-derived stem cell therapy for heart disease has demonstrated safety and efficacy in clinical trials but functional improvements have been limited. Finding the optimal stem cell type best suited for cardiac regeneration is key toward improving clinical outcomes. Objective To determine the mechanism by which novel bone-derived stem cells support the injured heart. Methods and Results Cortical bone stem cells (CBSCs) and cardiac-derived stem cells (CDCs) were isolated from EGFP+ transgenic mice and were shown to express c-kit and Sca-1 as well as 8 paracrine factors involved in cardioprotection, angiogenesis and stem cell function. Wild-type C57BL/6 mice underwent sham operation (n=21) or myocardial infarction (MI) with injection of CBSCs (n=67), CDCs (n=36) or saline (n=60). Cardiac function was monitored using echocardiography. Only 2/8 paracrine factors were detected in EGFP+ CBSCs in vivo (basic fibroblast growth factor and vascular endothelial growth factor) and this expression was associated with increased neovascularization of the infarct border zone. CBSC therapy improved survival, cardiac function, regional strain, attenuated remodeling, and decreased infarct size relative to CDC- or saline-treated MI controls. By 6 weeks, EGFP+ cardiomyocytes, vascular smooth muscle and endothelial cells could be identified in CBSC- but not in CDC-treated animals. EGFP+ CBSC-derived isolated myocytes were smaller and more frequently mononucleated, but were functionally indistinguishable from EGFP- myocytes. Conclusions CBSCs improve survival, cardiac function, and attenuate remodeling through two mechanisms:1) secretion of pro-angiogenic factors that stimulate endogenous neovascularization, and 2) differentiation into functional adult myocytes and vascular cells. PMID:23801066

Cytoskeletal role in the transition from compensated to decompensated hypertrophy during adult canine left ventricular pressure overloading

NASA Technical Reports Server (NTRS)

Tagawa, H.; Koide, M.; Sato, H.; Zile, M. R.; Carabello, B. A.; Cooper, G. 4th

1998-01-01

Increased microtubule density causes cardiocyte contractile dysfunction in right ventricular (RV) pressure-overload hypertrophy, and these linked phenotypic and contractile abnormalities persist and progress during the transition to failure. Although more severe in cells from failing than hypertrophied RVs, the mechanical defects are normalized in each case by microtubule depolymerization. To define the role of increased microtubule density in left ventricular (LV) pressure-overload hypertrophy and failure, in a given LV we examined ventricular mechanics, sarcomere mechanics, and free tubulin and microtubule levels in control dogs and in dogs with aortic stenosis both with LV hypertrophy alone and with initially compensated hypertrophy that had progressed to LV muscle failure. In comparing initial values with those at study 8 weeks later, dogs with hypertrophy alone had a very substantial increase in LV mass but preservation of a normal ejection fraction and mean systolic wall stress. Dogs with hypertrophy and associated failure had a substantial but lesser increase in LV mass and a reduction in ejection fraction, as well as a marked increase in mean systolic wall stress. Cardiocyte contractile function was equivalent, and unaffected by microtubule depolymerization, in cells from control LVs and those with compensated hypertrophy. In contrast, cardiocyte contractile function in cells from failing LVs was quite depressed but was normalized by microtubule depolymerization. Microtubules were increased only in failing LVs. These contractile and cytoskeletal changes, when assayed longitudinally in a given dog by biopsy, appeared in failing ventricles only when wall stress began to increase and function began to decrease. Thus, the microtubule-based cardiocyte contractile dysfunction characteristic of pressure-hypertrophied myocardium, originally described in the RV, obtains equally in the LV but is shown here to have a specific association with increased wall stress.

Cardiac Physiology of Aging: Extracellular Considerations.

PubMed

Horn, Margaux A

2015-07-01

Aging is a major risk factor for the development of cardiovascular disease, with the majority of affected patients being elderly. Progressive changes to myocardial structure and function occur with aging, often in concert with underlying pathologies. However, whether chronological aging results in a remodeled "aged substrate" has yet to be established. In addition to myocyte contractility, myocardial performance relies heavily on the cardiac extracellular matrix (ECM), the roles of which are as dynamic as they are significant; including providing structural integrity, assisting in force transmission throughout the cardiac cycle and acting as a signaling medium for communication between cells and the extracellular environment. In the healthy heart, ECM homeostasis must be maintained, and matrix deposition is in balance with degradation. Consequently, alterations to, or misregulation of the cardiac ECM has been shown to occur in both aging and in pathological remodeling with disease. Mounting evidence suggests that age-induced matrix remodeling may occur at the level of ECM control; including collagen synthesis, deposition, maturation, and degradation. Furthermore, experimental studies using aged animal models not only suggest that the aged heart may respond differently to insult than the young, but the identification of key players specific to remodeling with age may hold future therapeutic potential for the treatment of cardiac dysfunction in the elderly. This review will focus on the role of the cardiac interstitium in the physiology of the aging myocardium, with particular emphasis on the implications to age-related remodeling in disease. © 2015 American Physiological Society.

Cardiomyocyte-expressed farnesoid-X-receptor is a novel apoptosis mediator and contributes to myocardial ischaemia/reperfusion injury

PubMed Central

Pu, Jun; Yuan, Ancai; Shan, Peiren; Gao, Erhe; Wang, Xiaoliang; Wang, Yajing; Lau, Wayne Bond; Koch, Walter; Ma, Xin-Liang; He, Ben

2013-01-01

Aims Emerging evidence indicates that nuclear receptors play a critical regulatory role in cardiovascular physiology/pathology. Recently, farnesoid-X-receptor (FXR), a member of the metabolic nuclear receptor superfamily, has been demonstrated to be expressed in vascular cells, with important roles in vascular physiology/pathology. However, the potential cardiac function of FXR remains unclear. We investigated the cardiac expression and biological function of FXR. Methods and results Farnesoid-X-receptor was detected in both isolated neonatal rat cardiac myocytes and fibroblasts. Natural and synthetic FXR agonists upregulated cardiac FXR expression, stimulated myocyte apoptosis, and reduced myocyte viability dose- and time-dependently. Mechanistic studies demonstrated that FXR agonists disrupted mitochondria, characterized by mitochondrial permeability transition pores activation, mitochondrial potential dissipation, cytochrome c release, and both caspase-9 and -3 activation. Such mitochondrial apoptotic responses were abolished by siRNA-mediated silencing of endogenous FXR or pharmacological inhibition of mitochondrial death signalling. Furthermore, low levels of FXR were detected in the adult mouse heart, with significant (∼2.0-fold) upregulation after myocardial ischaemia/reperfusion (MI/R). Pharmacological inhibition or genetic ablation of FXR significantly reduced myocardial apoptosis by 29.0–53.4%, decreased infarct size by 23.4–49.7%, and improved cardiac function in ischaemic/reperfused myocardium. Conclusion These results demonstrate that nuclear receptor FXR acts as a novel functional receptor in cardiac tissue, regulates apoptosis in cardiomyocytes, and contributes to MI/R injury. PMID:22307460

[Subcellular basis of disorders of the contractile capacity of the heart in L-thyroxine-induced thyrotoxicosis].

PubMed

Karsanov, N V; Melashvili, N O; Khugashvili, Z G; Mamulashvili, L D; Azrumelashvili, M I; Khaindrava, G K; Kapanadze, R V

1990-02-01

In experiments on dogs, the authors examined the functional activity of three cardiomyocyte systems responsible for contraction-relaxation (the systems of contractile proteins, calcium transport and energy supply) in the dynamics of L-thyroxine-induced toxicosis. A fall in the capacity of the contractile protein system to generate energy and to perform was shown to play the leading role in decrease of myocardial reserve forces and reduction in cardiac contractility. There was a drop in the intensity of calcium transport through the membranes of the sarcoplasmic reticulum and mitochondria and a deficiency of the direct energy source for contraction only in the late period of the disease.

Contractile function of the myocardium with prolonged hypokinesia in patients with surgical tuberculosis

NASA Technical Reports Server (NTRS)

Zakutayeva, V. P.; Matiks, N. I.

1978-01-01

The changes in the myocardial contractile function with hypokinesia in surgical tuberculosis patients are discussed. The phase nature of the changes is noted, specifically the changes in the various systoles, diastole, and other parts of the cardiac cycle. The data compare these changes during confinement in bed with no motor activity to and with a return to motor activity after leaving the in-bed regimen.

A human in vitro model of Duchenne muscular dystrophy muscle formation and contractility.

PubMed

Nesmith, Alexander P; Wagner, Matthew A; Pasqualini, Francesco S; O'Connor, Blakely B; Pincus, Mark J; August, Paul R; Parker, Kevin Kit

2016-10-10

Tongue weakness, like all weakness in Duchenne muscular dystrophy (DMD), occurs as a result of contraction-induced muscle damage and deficient muscular repair. Although membrane fragility is known to potentiate injury in DMD, whether muscle stem cells are implicated in deficient muscular repair remains unclear. We hypothesized that DMD myoblasts are less sensitive to cues in the extracellular matrix designed to potentiate structure-function relationships of healthy muscle. To test this hypothesis, we drew inspiration from the tongue and engineered contractile human muscle tissues on thin films. On this platform, DMD myoblasts formed fewer and smaller myotubes and exhibited impaired polarization of the cell nucleus and contractile cytoskeleton when compared with healthy cells. These structural aberrations were reflected in their functional behavior, as engineered tongues from DMD myoblasts failed to achieve the same contractile strength as healthy tongue structures. These data suggest that dystrophic muscle may fail to organize with respect to extracellular cues necessary to potentiate adaptive growth and remodeling. © 2016 Nesmith et al.

Recovery in skeletal muscle contractile function after prolonged hindlimb immobilization

NASA Technical Reports Server (NTRS)

Fitts, R. H.; Brimmer, C. J.

1985-01-01

The effect of three-month hindlimb immobilization (IM) in rats on contractile properties of slow-twitch soleus (SOL), fast-twitch extensor digitorum longus, and fast-twitch superficial region of the vastus lateralis were measured after 0, 14, 28, 60, and 90 days of recovery on excized, horizontally suspended muscles stimulated electrically to maximal twitch tension. IM caused decreases in muscle-to-body weight ratios for all muscles, with no complete recovery even after 90 days. The contractile properties of the fast-twitch muscles were less affected by IM than those of the slow-twitch SOL. The SOL isometric twitch duration was shortened, due to reduced contraction and half-relaxation time, both of which returned to control levels after 14 days of recovery. The peak tetanic tension, P(O), g/sq cm,, decreased with IM by 46 percent in the SOL, but recovered by the 28th day. The maximum shortening velocity was not altered by IM in any of the muscles. Thus, normal contractile function could recover after prolonged limb IM.

YY1 Protects Cardiac Myocytes from Pathologic Hypertrophy by Interacting with HDAC5

PubMed Central

Dockstader, Karen; McKinsey, Timothy A.

2008-01-01

YY1 is a transcription factor that can repress or activate the transcription of a variety of genes. Here, we show that the function of YY1 as a repressor in cardiac myocytes is tightly dependent on its ability to interact with histone deacetylase 5 (HDAC5). YY1 interacts with HDAC5, and overexpression of YY1 prevents HDAC5 nuclear export in response to hypertrophic stimuli and the increase in cell size and re-expression of fetal genes that accompany pathological cardiac hypertrophy. Knockdown of YY1 results in up-regulation of all genes present during fetal development and increases the cell size of neonatal cardiac myocytes. Moreover, overexpression of a YY1 deletion construct that does not interact with HDAC5 results in transcription activation, suggesting that HDAC5 is necessary for YY1 function as a transcription repressor. In support of this relationship, we show that knockdown of HDAC5 results in transcription activation by YY1. Finally, we show that YY1 interaction with HDAC5 is dependent on the HDAC5 phosphorylation domain and that overexpression of YY1 reduces HDAC5 phosphorylation in response to hypertrophic stimuli. Our results strongly suggest that YY1 functions as an antihypertrophic factor by preventing HDAC5 nuclear export and that up-regulation of YY1 in human heart failure may be a protective mechanism against pathological hypertrophy. PMID:18632988

Doxorubicin: Comparison between 3-h continuous and bolus intravenous administration paradigms on cardio-renal axis, mitochondrial sphingolipids and pathology

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kamendi, Harriet, E-mail: harriet_kamendi@kandih.com; Zhou, Ying, E-mail: yingzhou526@gmail.com; Crosby, Meredith, E-mail: Meredith.crosby@astrazeneca.com

Doxorubicin (DOX) is a potent and effective broad-spectrum anthracycline antitumor agent, but its clinical usefulness is restricted by cardiotoxicity. This study compared pharmacokinetic, functional, structural and biochemical effects of single dose DOX bolus or 3-h continuous iv infusion (3-h iv) in the Han–Wistar rat to characterize possible treatment-related differences in drug safety over a 72 h observation period. Both DOX dosing paradigms significantly altered blood pressure, core body temperature and QA interval (indirect measure of cardiac contractility); however, there was no recovery observed in the bolus iv treatment group. Following the 3-h iv treatment, blood pressures and QA interval normalizedmore » by 36 h then rose above baseline levels over 72 h. Both treatments induced biphasic changes in heart rate with initial increases followed by sustained decreases. Cardiac injury biomarkers in plasma were elevated only in the bolus iv treatment group. Tissue cardiac injury biomarkers, cardiac mitochondrial complexes I, III and V and cardiac mitochondrial sphingolipids were decreased only in the bolus iv treatment group. Results indicate that each DOX dosing paradigm deregulates sinus rhythm. However, slowing the rate of infusion allows for functional compensation of blood pressure and may decrease the likelihood of cardiac myocyte necrosis via a mechanism associated with reduced mitochondrial damage. - Highlights: • Despite damaging cardiomyocytes, continuous iv doxorubicin improves cardiovascular outcomes. • This study supports administration of doxorubicin via slow continuous iv infusion limits acute cardio-toxicity. • This study supports use of metabolomic-derived lipid biomarkers for improved quantification of cardiovascular risk. • This study supports systems-based physiological approach to generate a data that can greatly inform risk assessments.« less

LRRC10 is required to maintain cardiac function in response to pressure overload

PubMed Central

Brody, Matthew J.; Feng, Li; Grimes, Adrian C.; Hacker, Timothy A.; Olson, Timothy M.; Kamp, Timothy J.

2015-01-01

We previously reported that the cardiomyocyte-specific leucine-rich repeat containing protein (LRRC)10 has critical functions in the mammalian heart. In the present study, we tested the role of LRRC10 in the response of the heart to biomechanical stress by performing transverse aortic constriction on Lrrc10-null (Lrrc10−/−) mice. Mild pressure overload induced severe cardiac dysfunction and ventricular dilation in Lrrc10−/− mice compared with control mice. In addition to dilation and cardiomyopathy, Lrrc10−/− mice showed a pronounced increase in heart weight with pressure overload stimulation and a more dramatic loss of cardiac ventricular performance, collectively suggesting that the absence of LRRC10 renders the heart more disease prone with greater hypertrophy and structural remodeling, although rates of cardiac fibrosis and myocyte dropout were not different from control mice. Lrrc10−/− cardiomyocytes also exhibited reduced contractility in response to β-adrenergic stimulation, consistent with loss of cardiac ventricular performance after pressure overload. We have previously shown that LRRC10 interacts with actin in the heart. Here, we show that His150 of LRRC10 was required for an interaction with actin, and this interaction was reduced after pressure overload, suggesting an integral role for LRRC10 in the response of the heart to mechanical stress. Importantly, these experiments demonstrated that LRRC10 is required to maintain cardiac performance in response to pressure overload and suggest that dysregulated expression or mutation of LRRC10 may greatly sensitize human patients to more severe cardiac disease in conditions such as chronic hypertension or aortic stenosis. PMID:26608339

Altered sarcoplasmic reticulum calcium cycling—targets for heart failure therapy

PubMed Central

Kho, Changwon; Lee, Ahyoung; Hajjar, Roger J.

2013-01-01

Cardiac myocyte function is dependent on the synchronized movements of Ca2+ into and out of the cell, as well as between the cytosol and sarcoplasmic reticulum. These movements determine cardiac rhythm and regulate excitation–contraction coupling. Ca2+ cycling is mediated by a number of critical Ca2+-handling proteins and transporters, such as L-type Ca2+ channels (LTCCs) and sodium/calcium exchangers in the sarcolemma, and sarcoplasmic/endoplasmic reticulum calcium ATPase 2a (SERCA2a), ryanodine receptors, and cardiac phospholamban in the sarcoplasmic reticulum. The entry of Ca2+ into the cytosol through LTCCs activates the release of Ca2+ from the sarcoplasmic reticulum through ryanodine receptor channels and initiates myocyte contraction, whereas SERCA2a and cardiac phospholamban have a key role in sarcoplasmic reticulum Ca2+ sequesteration and myocyte relaxation. Excitation–contraction coupling is regulated by phosphorylation of Ca2+-handling proteins. Abnormalities in sarcoplasmic reticulum Ca2+ cycling are hallmarks of heart failure and contribute to the pathophysiology and progression of this disease. Correcting impaired intracellular Ca2+ cycling is a promising new approach for the treatment of heart failure. Novel therapeutic strategies that enhance myocyte Ca2+ homeostasis could prevent and reverse adverse cardiac remodeling and improve clinical outcomes in patients with heart failure. PMID:23090087

Calcineurin Regulates Myocardial Function during Acute Endotoxemia

PubMed Central

Joshi, Mandar S.; Julian, Mark W.; Huff, Jennifer E.; Bauer, John A.; Xia, Yong; Crouser, Elliott D.

2006-01-01

Rationale: Cyclosporin A (CsA) is known to preserve cardiac contractile function during endotoxemia, but the mechanism is unclear. Increased nitric oxide (NO) production and altered mitochondrial function are implicated as mechanisms contributing to sepsis-induced cardiac dysfunction, and CsA has the capacity to reduce NO production and inhibit mitochondrial dysfunction relating to the mitochondrial permeability transition (MPT). Objectives: We hypothesized that CsA would protect against endotoxin-mediated cardiac contractile dysfunction by attenuating NO production and preserving mitochondrial function. Methods: Left ventricular function was measured continuously over 4 h in cats assigned as follows: control animals (n = 7); LPS alone (3 mg/kg, n = 8); and CsA (6 mg/kg, n = 7), a calcineurin inhibitor that blocks the MPT, or tacrolimus (FK506, 0.1 mg/kg, n = 7), a calcineurin inhibitor lacking MPT activity, followed in 30 min by LPS. Myocardial tissue was then analyzed for NO synthase-2 expression, tissue nitration, protein carbonylation, and mitochondrial morphology and function. Measurements and Main Results: LPS treatment resulted in impaired left ventricular contractility, altered mitochondrial morphology and function, and increased protein nitration. As hypothesized, CsA pretreatment normalized cardiac performance and mitochondrial respiration and reduced myocardial protein nitration. Unexpectedly, FK506 pretreatment had similar effects, normalizing both cardiac and mitochondrial parameters. However, CsA and FK506 pretreatments markedly increased protein carbonylation in the myocardium despite elevated manganese superoxide dismutase activity during endotoxemia. Conclusions: Our data indicate that calcineurin is a critical regulator of mitochondrial respiration, tissue nitration, protein carbonylation, and contractile function in the heart during acute endotoxemia. PMID:16424445

Effects of ANP receptor antagonists on ANP secretion from adult rat cultured atrial myocytes.

PubMed

Nachshon, S; Zamir, O; Matsuda, Y; Zamir, N

1995-03-01

Atrial natriuretic peptide (ANP) is a hormone-secreted predominantly by atrial myocytes. ANP exerts many of its actions via activation of the particulate guanylyl cyclase receptor ANPR-A and the formation of guanosine 3',5'-cyclic monophosphate (cGMP), which serves as a second messenger in the target cells. Using membrane-permeable cGMP analogues (8-bromo-cGMP and dibutyryl- cGMP), we first tested the hypothesis that ANP secretion by adult rat cultured atrial myocytes can be modulated through the second messenger cGMP. Second, we examined the effects of two competitive ANPR-A receptor antagonists, namely HS-142-1 and anantin, on cGMP formation and ANP secretion from cultured atrial myocytes. Cultured atrial myocytes secreted large quantities of immunoreactive (ir) ANP under basal conditions. We found that cGMP analogues inhibited basal irANP secretion from cultured atrial myocytes, whereas HS-142-1 and anantin had stimulating effects. HS-142-1 and anantin reduced cGMP formation in cultured atrial myocytes at basal conditions. These results suggest an autoregulatory mechanism of ANP secretion by atrial myocytes in an autocrine/paracrine fashion.

Store-operated Ca2+ entry supports contractile function in hearts of hibernators

PubMed Central

Nakipova, Olga V.; Averin, Alexey S.; Evdokimovskii, Edward V.; Pimenov, Oleg Yu.; Kosarski, Leonid; Ignat’ev, Dmitriy; Anufriev, Andrey; Kokoz, Yuri M.; Reyes, Santiago; Terzic, Andre; Alekseev, Alexey E.

2017-01-01

Hibernators have a distinctive ability to adapt to seasonal changes of body temperature in a range between 37°C and near freezing, exhibiting, among other features, a unique reversibility of cardiac contractility. The adaptation of myocardial contractility in hibernation state relies on alterations of excitation contraction coupling, which becomes less-dependent from extracellular Ca2+ entry and is predominantly controlled by Ca2+ release from sarcoplasmic reticulum, replenished by the Ca2+-ATPase (SERCA). We found that the specific SERCA inhibitor cyclopiazonic acid (CPA), in contrast to its effect in papillary muscles (PM) from rat hearts, did not reduce but rather potentiated contractility of PM from hibernating ground squirrels (GS). In GS ventricles we identified drastically elevated, compared to rats, expression of Orai1, Stim1 and Trpc1/3/4/5/6/7 mRNAs, putative components of store operated Ca2+ channels (SOC). Trpc3 protein levels were found increased in winter compared to summer GS, yet levels of Trpc5, Trpc6 or Trpc7 remained unchanged. Under suppressed voltage-dependent K+, Na+ and Ca2+ currents, the SOC inhibitor 2-aminoethyl diphenylborinate (2-APB) diminished whole-cell membrane currents in isolated cardiomyocytes from hibernating GS, but not from rats. During cooling-reheating cycles (30°C–7°C–30°C) of ground squirrel PM, 2-APB did not affect typical CPA-sensitive elevation of contractile force at low temperatures, but precluded the contractility at 30°C before and after the cooling. Wash-out of 2-APB reversed PM contractility to control values. Thus, we suggest that SOC play a pivotal role in governing the ability of hibernator hearts to maintain their function during the transition in and out of hibernating states. PMID:28531217

Skeletal muscle morphology and contractile function in relation to muscle denervation in diabetic neuropathy

PubMed Central

Major, Brendan; Kimpinski, Kurt; Doherty, Timothy J.; Rice, Charles L.

2013-01-01

The objective of the study was to assess the effects of diabetic polyneuropathy (DPN) on muscle contractile properties in humans, and how these changes are related to alterations in muscle morphology and denervation. Patients with DPN (n = 12) were compared with age- and sex-matched controls (n = 12). Evoked and voluntary contractile properties, including stimulated twitch responses and maximal voluntary contractions, of the dorsiflexor muscles were assessed using an isometric ankle dynamometer. Motor unit number estimates (MUNE) of the tibialis anterior (TA) were performed via quantitative electromyography and decomposition-enhanced spike-triggered averaging. Peak tibialis anterior (TA) cross-sectional area (CSA; cm2), and relative proportion of contractile to noncontractile tissue (%) was determined from magnetic resonance images. Patients with DPN demonstrated decreased strength (−35%) and slower (−45%) dorsiflexion contractile properties for both evoked and voluntary contractions (P < 0.05). These findings were not accounted for by differences in voluntary activation (P > 0.05) or antagonist coactivation (P > 0.05). Additionally, patients with DPN were weaker when strength was normalized to TA total CSA (−30%; P < 0.05) or contractile tissue CSA (−26%; P < 0.05). In the DPN patient group, TA MUNEs were negatively related to both % noncontractile tissue (P < 0.05; r = 0.72) and twitch half-relaxation time (P < 0.05; r = 0.60), whereas no relationships were found between these variables in controls (P > 0.05). We conclude that patients with DPN demonstrated reduced strength and muscle quality as well as contractile slowing. This process may contribute to muscle power loss and functional impairments reported in patients with DPN, beyond the loss of strength commonly observed. PMID:24356519

LncRNA ZFAS1 as a SERCA2a Inhibitor to Cause Intracellular Ca2+ Overload and Contractile Dysfunction in a Mouse Model of Myocardial Infarction.

PubMed

Zhang, Ying; Jiao, Lei; Sun, Lihua; Li, Yanru; Gao, Yuqiu; Xu, Chaoqian; Shao, Yingchun; Li, Mengmeng; Li, Chunyan; Lu, Yanjie; Pan, Zhenwei; Xuan, Lina; Zhang, Yiyuan; Li, Qingqi; Yang, Rui; Zhuang, Yuting; Zhang, Yong; Yang, Baofeng

2018-05-11

Ca 2+ homeostasis-a critical determinant of cardiac contractile function-is critically regulated by SERCA2a (sarcoplasmic reticulum Ca 2+ -ATPase 2a). Our previous study has identified ZFAS1 as a new lncRNA biomarker of acute myocardial infarction (MI). To evaluate the effects of ZFAS1 on SERCA2a and the associated Ca 2+ homeostasis and cardiac contractile function in the setting of MI. ZFAS1 expression was robustly increased in cytoplasm and sarcoplasmic reticulum in a mouse model of MI and a cellular model of hypoxia. Knockdown of endogenous ZFAS1 by virus-mediated silencing shRNA partially abrogated the ischemia-induced contractile dysfunction. Overexpression of ZFAS1 in otherwise normal mice created similar impairment of cardiac function as that observed in MI mice. Moreover, at the cellular level, ZFAS1 overexpression weakened the contractility of cardiac muscles. At the subcellular level, ZFAS1 deleteriously altered the Ca 2+ transient leading to intracellular Ca 2+ overload in cardiomyocytes. At the molecular level, ZFAS1 was found to directly bind SERCA2a protein and to limit its activity, as well as to repress its expression. The effects of ZFAS1 were readily reversible on knockdown of this lncRNA. Notably, a sequence domain of ZFAS1 gene that is conserved across species mimicked the effects of the full-length ZFAS1 . Mutation of this domain or application of an antisense fragment to this conserved region efficiently canceled out the deleterious actions of ZFAS1 . ZFAS1 had no significant effects on other Ca 2+ -handling regulatory proteins. ZFAS1 is an endogenous SERCA2a inhibitor, acting by binding to SERCA2a protein to limit its intracellular level and inhibit its activity, and a contributor to the impairment of cardiac contractile function in MI. Therefore, anti- ZFAS1 might be considered as a new therapeutic strategy for preserving SERCA2a activity and cardiac function under pathological conditions of the heart. © 2018 The Authors.

Reversal of cardiac myocyte dysfunction as a unique mechanism of rescue by P2X4 receptors in cardiomyopathy.

PubMed

Shen, Jian-Bing; Shutt, Robin; Agosto, Mariela; Pappano, Achilles; Liang, Bruce T

2009-04-01

Binary cardiac transgenic (Tg) overexpression of P2X(4) receptors (P2X(4)R) improved the survival of the cardiomyopathic calsequestrin (CSQ) mice. Here we studied the mechanism of rescue using binary P2X(4)R/CSQ Tg and CSQ Tg mice as models. Cellular and intact heart properties were determined by simultaneous sarcomere shortening (SS) and Ca(2+) transients in vitro and echocardiography in vivo. Similar to a delay in death, binary mice exhibited a slowed heart failure progression with a greater left ventricular (LV) fractional shortening (FS) and thickness and a concomitant lesser degree of LV dilatation in both systole and diastole at 8 or 12 wk. By 16 wk, binary hearts showed similarly depressed FS and thinned out LV and equal enlargement of LV as did 12-wk-old CSQ hearts. Binary cardiac myocytes showed higher peak basal cell shortening (CS) and SS as well as greater basal rates of shortening and relaxation than did the CSQ myocytes at either 8 or 12 wk. Similar data were obtained in comparing the Ca(2+) transient. At 16 wk, binary myocytes were like the 12-wk-old CSQ myocytes with equally depressed CS, SS, and Ca(2+) transient. CSQ myocytes were longer than myocytes from wild-type and binary mice at 12 wk of age. At 16 wk, the binary myocyte length increased to that of the 12-wk-old CSQ myocyte, parallel to LV dilatation. The data suggest a unique mechanism, which involves a reversal of cardiac myocyte dysfunction and a delay in heart failure progression. It represents an example of targeting the abnormal failing myocyte in treating heart failure.

Cardiac-Specific Knockout of ETA Receptor Mitigates Paraquat-Induced Cardiac Contractile Dysfunction.

PubMed

Wang, Jiaxing; Lu, Songhe; Zheng, Qijun; Hu, Nan; Yu, Wenjun; Li, Na; Liu, Min; Gao, Beilei; Zhang, Guoyong; Zhang, Yingmei; Wang, Haichang

2016-07-01

Paraquat (1,1'-dim ethyl-4-4'-bipyridinium dichloride), a highly toxic quaternary ammonium herbicide widely used in agriculture, exerts potent toxic prooxidant effects resulting in multi-organ failure including the lung and heart although the underlying mechanism remains elusive. Recent evidence suggests possible involvement of endothelin system in paraquat-induced acute lung injury. This study was designed to examine the role of endothelin receptor A (ETA) in paraquat-induced cardiac contractile and mitochondrial injury. Wild-type (WT) and cardiac-specific ETA receptor knockout mice were challenged to paraquat (45 mg/kg, i.p.) for 48 h prior to the assessment of echocardiographic, cardiomyocyte contractile and intracellular Ca(2+) properties, as well as apoptosis and mitochondrial damage. Levels of the mitochondrial proteins for biogenesis and oxidative phosphorylation including UCP2, HSP90 and PGC1α were evaluated. Our results revealed that paraquat elicited cardiac enlargement, mechanical anomalies including compromised echocardiographic parameters (elevated left ventricular end-systolic and end-diastolic diameters as well as reduced factional shortening), suppressed cardiomyocyte contractile function, intracellular Ca(2+) handling, overt apoptosis and mitochondrial damage. ETA receptor knockout itself failed to affect myocardial function, apoptosis, mitochondrial integrity and mitochondrial protein expression. However, ETA receptor knockout ablated or significantly attenuated paraquat-induced cardiac contractile and intracellular Ca(2+) defect, apoptosis and mitochondrial damage. Taken together, these findings revealed that endothelin system in particular the ETA receptor may be involved in paraquat-induced toxic myocardial contractile anomalies possibly related to apoptosis and mitochondrial damage.

The Reliability of Pharyngeal High Resolution Manometry with Impedance for Derivation of Measures of Swallowing Function in Healthy Volunteers

PubMed Central

Omari, Taher I.; Savilampi, Johanna; Kokkinn, Karmen; Schar, Mistyka; Lamvik, Kristin; Doeltgen, Sebastian; Cock, Charles

2016-01-01

Purpose. We evaluated the intra- and interrater agreement and test-retest reliability of analyst derivation of swallow function variables based on repeated high resolution manometry with impedance measurements. Methods. Five subjects swallowed 10 × 10 mL saline on two occasions one week apart producing a database of 100 swallows. Swallows were repeat-analysed by six observers using software. Swallow variables were indicative of contractility, intrabolus pressure, and flow timing. Results. The average intraclass correlation coefficients (ICC) for intra- and interrater comparisons of all variable means showed substantial to excellent agreement (intrarater ICC 0.85–1.00; mean interrater ICC 0.77–1.00). Test-retest results were less reliable. ICC for test-retest comparisons ranged from slight to excellent depending on the class of variable. Contractility variables differed most in terms of test-retest reliability. Amongst contractility variables, UES basal pressure showed excellent test-retest agreement (mean ICC 0.94), measures of UES postrelaxation contractile pressure showed moderate to substantial test-retest agreement (mean Interrater ICC 0.47–0.67), and test-retest agreement of pharyngeal contractile pressure ranged from slight to substantial (mean Interrater ICC 0.15–0.61). Conclusions. Test-retest reliability of HRIM measures depends on the class of variable. Measures of bolus distension pressure and flow timing appear to be more test-retest reliable than measures of contractility. PMID:27190520

Nanotized PPARα Overexpression Targeted to Hypertrophied Myocardium Improves Cardiac Function by Attenuating the p53-GSK3β-Mediated Mitochondrial Death Pathway.

PubMed

Rana, Santanu; Datta, Ritwik; Chaudhuri, Ratul Datta; Chatterjee, Emeli; Chawla-Sarkar, Mamta; Sarkar, Sagartirtha

2018-05-09

Metabolic remodeling of cardiac muscles during pathological hypertrophy is characterized by downregulation of fatty acid oxidation (FAO) regulator, peroxisome proliferator-activated receptor alpha (PPARα). Thereby, we hypothesized that a cardiac-specific induction of PPARα might restore the FAO-related protein expression and resultant energy deficit. In the present study, consequences of PPARα augmentation were evaluated for amelioration of chronic oxidative stress, myocyte apoptosis, and cardiac function during pathological cardiac hypertrophy. Nanotized PPARα overexpression targeted to myocardium was done by a stearic acid-modified carboxymethyl-chitosan (CMC) conjugated to a 20-mer myocyte-targeted peptide (CMCP). Overexpression of PPARα ameliorated pathological hypertrophy and improved cardiac function. Augmented PPARα in hypertrophied myocytes revealed downregulated p53 acetylation (lys 382), leading to reduced apoptosis. Such cells showed increased binding of PPARα with p53 that in turn reduced interaction of p53 with glycogen synthase kinase-3β (GSK3β), which upregulated inactive phospho-GSK3β (serine [Ser]9) expression within mitochondrial protein fraction. Altogether, the altered molecular milieu in PPARα-overexpressed hypertrophy groups restored mitochondrial structure and function both in vitro and in vivo. Cardiomyocyte-targeted overexpression of a protein of interest (PPARα) by nanotized plasmid has been described for the first time in this study. Our data provide a novel insight towards regression of pathological hypertrophy by ameliorating mitochondrial oxidative stress in targeted PPARα-overexpressed myocardium. PPARα-overexpression during pathological hypertrophy showed substantial betterment of mitochondrial structure and function, along with downregulated apoptosis. Myocardium-targeted overexpression of PPARα during pathological cardiac hypertrophy led to an overall improvement of cardiac energy deficit and subsequent cardiac function, thereby, opening up a potential avenue for cardiac tissue engineering during hypertrophic cardiac pathophysiology.

Frequent premature atrial contractions impair left atrial contractile function and promote adverse left atrial remodeling.

PubMed

John, Anub G; Hirsch, Glenn A; Stoddard, Marcus F

2018-06-10

This study assessed if frequent premature atrial contractions (PACs) were associated with decreased left atrial (LA) strain and adverse remodeling. Left atrial dysfunction and enlargement increases risk of stroke. If frequent PACs cause LA dysfunction and remodeling, PAC suppressive therapy may be beneficial. Inclusion criteria were age ≥18 years and sinus rhythm. Exclusion criteria were atrial fibrillation or any etiology for LA enlargement. Hundred and thirty-two patients with frequent PACs (≥100/24 hours) by Holter were matched to controls. Speckle tracking strain of the left atrium was performed from the 4-chamber view. Strain measurements were LA peak contractile, reservoir and conduit strain and strain rates. In the frequent PAC vs control group, PACs were more frequent (1959 ± 3796 vs 28 ± 25/24 hours, P < .0001). LA peak contractile strain was reduced in the group with frequent PACs vs controls (-7.85 ± 4.12% vs -9.33 ± 4.45%, P = .006). LA peak late negative contractile strain rate was less negative in the frequent PAC vs control group (-0.63 ± 0.27 s -1 vs -0.69 ± 0.32 s -1 , P = .051). LA reservoir and conduit strain and strain rates did not differ. LA volume index (LAVI) was larger in the frequent PAC vs control group (26.6 ± 7.8 vs 24.6 ± 8.8 mL/m 2 , P < .05). Frequent PACs were an independent predictor of reduced LA peak contractile strain and reduced LA peak late negative contractile strain rate. Patients with frequent PACs have reduced LA peak contractile strain and strain rates and larger LAVI compared to controls. Frequent PACs are an independent predictor of reduced LA peak contractile strain and strain rate. These findings support the hypothesis that frequent PACs impair LA contractile function and promote adverse LA remodeling. © 2018 Wiley Periodicals, Inc.

Nonesterified fatty acid accumulation and release during heart muscle-cell (myocyte) injury: modulation by extracellular "acceptor".

PubMed

Janero, D R; Burghardt, C

1989-07-01

Long-chain nonesterified fatty acid (NEFA) accumulation in the heart muscle cell (myocyte) and NEFA release to the extracellular milieu are considered contributors to the pathogenesis of myocardial injury in a number of cardiovascular disease states. Reported here is a study of the factors which influence and control the interactions among NEFA formation, intracellular NEFA accumulation, and NEFA release to the extracellular compartment by the irreversibly injured myocyte. Under conditions of metabolic inhibition, neonatal rat myocytes in primary monolayer culture became virtually depleted of ATP within 8 h. The metabolically inhibited myocytes evidenced membrane phospholipid degradation and a resultant net accumulation of NEFA produced thereby in the extracellular medium. However, under conditions of nutrient deprivation, the injured myocytes retained the NEFA produced from phospholipid catabolism intracellularly and did not release it to the culture medium, although the extent of myocyte ATP depletion was the same as it had been from metabolic inhibition. Serum could elicit, in a concentration-dependent fashion, the quantitative release of NEFA from metabolically inhibited myocytes to the culture medium but did not influence the net production of NEFA by the injured cells. Similarly, NEFA release from nutrient-deprived myocytes incubated in serum-free, substrate-free medium or in physiological buffer could be induced by supplementing the medium or buffer with bovine serum albumin (BSA), and the extent of NEFA release, but not NEFA formation, was dependent upon the extracellular BSA concentration. No manipulations to media other than changing their serum content or supplementing them with BSA were found to influence the disposition of NEFA produced during phospholipid catabolism in the irreversibly injured, ATP-depleted myocyte. Therefore, although progressive metabolic compromise in the myocyte was correlated with increasing, net NEFA formation, the distribution of the NEFA between the intracellular and the extracellular compartments was not determined by the magnitude of ATP loss or by the nature or duration of at least two injury stimuli, metabolic inhibition and nutrient deprivation. Rather, the net release of NEFA from the ATP-depleted myocyte to the culture medium and the consequent reduction of intracellular myocyte NEFA overload were critically and causally dependent upon the presence and concentration of extracellular NEFA "acceptor". The influence of acceptor on the mobilization of NEFA from the injured myocyte has implications regarding the use of NEFA release as an index of myocyte pathology and could serve to modify the progression and extent of myocardial injury in vivo.

Chronic N(G)-nitro-L-arginine methyl ester-induced hypertension : novel molecular adaptation to systolic load in absence of hypertrophy

NASA Technical Reports Server (NTRS)

Bartunek, J.; Weinberg, E. O.; Tajima, M.; Rohrbach, S.; Katz, S. E.; Douglas, P. S.; Lorell, B. H.; Schneider, M. (Principal Investigator)

2000-01-01

BACKGROUND: Chronic N(G)-nitro-L-arginine methyl ester (L-NAME), which inhibits nitric oxide synthesis, causes hypertension and would therefore be expected to induce robust cardiac hypertrophy. However, L-NAME has negative metabolic effects on protein synthesis that suppress the increase in left ventricular (LV) mass in response to sustained pressure overload. In the present study, we used L-NAME-induced hypertension to test the hypothesis that adaptation to pressure overload occurs even when hypertrophy is suppressed. METHODS AND RESULTS: Male rats received L-NAME (50 mg. kg(-1). d(-1)) or no drug for 6 weeks. Rats with L-NAME-induced hypertension had levels of systolic wall stress similar to those of rats with aortic stenosis (85+/-19 versus 92+/-16 kdyne/cm). Rats with aortic stenosis developed a nearly 2-fold increase in LV mass compared with controls. In contrast, in the L-NAME rats, no increase in LV mass (1. 00+/-0.03 versus 1.04+/-0.04 g) or hypertrophy of isolated myocytes occurred (3586+/-129 versus 3756+/-135 microm(2)) compared with controls. Nevertheless, chronic pressure overload was not accompanied by the development of heart failure. LV systolic performance was maintained by mechanisms of concentric remodeling (decrease of in vivo LV chamber dimension relative to wall thickness) and augmented myocardial calcium-dependent contractile reserve associated with preserved expression of alpha- and beta-myosin heavy chain isoforms and sarcoplasmic reticulum Ca(2+) ATPase (SERCA-2). CONCLUSIONS: When the expected compensatory hypertrophic response is suppressed during L-NAME-induced hypertension, severe chronic pressure overload is associated with a successful adaptation to maintain systolic performance; this adaptation depends on both LV remodeling and enhanced contractility in response to calcium.

Block of Na(+)/Ca(2+) exchanger by SEA0400 in human right atrial preparations from patients in sinus rhythm and in atrial fibrillation.

PubMed

Christ, Torsten; Kovács, Peter P; Acsai, Karoly; Knaut, Michael; Eschenhagen, Thomas; Jost, Norbert; Varró, András; Wettwer, Erich; Ravens, Ursula

2016-10-05

The Na(+)/Ca(2+) exchanger (NCX) plays a major role in myocardial Ca(2+) homoeostasis, but is also considered to contribute to the electrical instability and contractile dysfunction in chronic atrial fibrillation (AF). Here we have investigated the effects of the selective NCX blocker SEA0400 in human right atrial cardiomyocytes from patients in sinus rhythm (SR) and AF in order to obtain electrophysiological evidence for putative antiarrhythmic activity of this new class of drugs. Action potentials were measured in right atrial trabeculae using conventional microelectrodes. Human myocytes were enzymatically isolated. Rat atrial and ventricular cardiomyocytes were used for comparison. Using perforated-patch, NCX was measured as Ni(2+)-sensitive current during ramp pulses. In ruptured-patch experiments, NCX current was activated by changing the extracellular Ca(2+) concentration from 0 to 1mM in Na(+)-free bath solution (100mM Na(+) intracellular, "Hilgemann protocol"). Although SEA0400 was effective in rat cardiomyocytes, 10µM did not influence action potentials and contractility, neither in SR nor AF. SEA0400 (10μM) also failed to affect human atrial NCX current measured with perforated patch. With the "Hilgemann protocol" SEA0400 concentration-dependently suppressed human atrial NCX current, and its amplitude was larger in AF than in SR cardiomyocytes. Our results confirm higher NCX activity in AF than SR. SEA0400 fails to block Ni(2+)-sensitive current in human atrial cells unless unphysiological conditions are used. We speculate that block of NCX with SEA0400 depends on intracellular Na(+) concentration. Copyright © 2016 Elsevier B.V. All rights reserved.

Spiral-wave dynamics in a mathematical model of human ventricular tissue with myocytes and fibroblasts.

PubMed

Nayak, Alok Ranjan; Shajahan, T K; Panfilov, A V; Pandit, Rahul

2013-01-01

Cardiac fibroblasts, when coupled functionally with myocytes, can modulate the electrophysiological properties of cardiac tissue. We present systematic numerical studies of such modulation of electrophysiological properties in mathematical models for (a) single myocyte-fibroblast (MF) units and (b) two-dimensional (2D) arrays of such units; our models build on earlier ones and allow for zero-, one-, and two-sided MF couplings. Our studies of MF units elucidate the dependence of the action-potential (AP) morphology on parameters such as [Formula: see text], the fibroblast resting-membrane potential, the fibroblast conductance [Formula: see text], and the MF gap-junctional coupling [Formula: see text]. Furthermore, we find that our MF composite can show autorhythmic and oscillatory behaviors in addition to an excitable response. Our 2D studies use (a) both homogeneous and inhomogeneous distributions of fibroblasts, (b) various ranges for parameters such as [Formula: see text], and [Formula: see text], and (c) intercellular couplings that can be zero-sided, one-sided, and two-sided connections of fibroblasts with myocytes. We show, in particular, that the plane-wave conduction velocity [Formula: see text] decreases as a function of [Formula: see text], for zero-sided and one-sided couplings; however, for two-sided coupling, [Formula: see text] decreases initially and then increases as a function of [Formula: see text], and, eventually, we observe that conduction failure occurs for low values of [Formula: see text]. In our homogeneous studies, we find that the rotation speed and stability of a spiral wave can be controlled either by controlling [Formula: see text] or [Formula: see text]. Our studies with fibroblast inhomogeneities show that a spiral wave can get anchored to a local fibroblast inhomogeneity. We also study the efficacy of a low-amplitude control scheme, which has been suggested for the control of spiral-wave turbulence in mathematical models for cardiac tissue, in our MF model both with and without heterogeneities.

The natriuretic peptides BNP and CNP increase heart rate and electrical conduction by stimulating ionic currents in the sinoatrial node and atrial myocardium following activation of guanylyl cyclase-linked natriuretic peptide receptors.

PubMed

Springer, Jeremy; Azer, John; Hua, Rui; Robbins, Courtney; Adamczyk, Andrew; McBoyle, Sarah; Bissell, Mary Beth; Rose, Robert A

2012-05-01

Natriuretic peptides (NPs) are best known for their ability to regulate blood vessel tone and kidney function whereas their electrophysiological effects on the heart are less clear. Here, we measured the effects of BNP and CNP on sinoatrial node (SAN) and atrial electrophysiology in isolated hearts as well as isolated SAN and right atrial myocytes from mice. BNP and CNP dose-dependently increased heart rate and conduction through the heart as indicated by reductions in R-R interval, P wave duration and P-R interval on ECGs. In conjunction with these ECG changes BNP and CNP (100 nM) increased spontaneous action potential frequency in isolated SAN myocytes by increasing L-type Ca(2+) current (I(Ca,L)) and the hyperpolarization-activated current (I(f)). BNP had no effect on right atrial myocyte APs in basal conditions; however, in the presence of isoproterenol (10nM), BNP increased atrial AP duration and I(Ca,L). Quantitative gene expression and immunocytochemistry data show that all three NP receptors (NPR-A, NPR-B and NPR-C) are expressed in the SAN and atrium. The effects of BNP and CNP on SAN and right atrial myocytes were maintained in mutant mice lacking functional NPR-C receptors and blocked by the NPR-A antagonist A71915 indicating that BNP and CNP function through their guanylyl cyclase-linked receptors. Our data also show that the effects of BNP and CNP are completely absent in the presence of the phosphodiesterase 3 inhibitor milrinone. Based on these data we conclude that NPs can increase heart rate and electrical conduction by activating the guanylyl cyclase-linked NPR-A and NPR-B receptors and inhibiting PDE3 activity. Copyright © 2012 Elsevier Ltd. All rights reserved.

Shear fluid-induced Ca2+ release and the role of mitochondria in rat cardiac myocytes.

PubMed

Belmonte, Steve; Morad, Martin

2008-03-01

Cardiac myocyte contraction occurs when Ca2+ influx through voltage-gated L-type Ca2+ channels causes Ca2+ release from ryanodine receptors of the sarcoplasmic reticulum (SR). Although mitochondria occupy about 35% of the cell volume in rat cardiac myocytes, and are thought to be located <300 nm from the junctional SR, their role in the beat-to-beat regulation of cardiac Ca2+ signaling remains unclear. We have recently shown that rapid ( approximately 20 ms) application of shear fluid forces ( approximately 25 dynes/cm2) to rat cardiac myocytes triggers slowly ( approximately 300 ms) developing Cai transients that were independent of activation of all transmembrane Ca2+ transporting pathways, but were suppressed by FCCP, CCCP, and Ru360, all of which are known to disrupt mitochondrial function. We have here used rapid 2-D confocal microscopy to monitor fluctuations in mitochondrial Ca2+ levels ([Ca2+]m) and mitochondrial membrane potential (Delta Psi m) in rat cardiac myocytes loaded either with rhod-2 AM or tetramethylrhodamine methyl ester (TMRM), respectively. Freshly isolated intact rat cardiac myocytes were plated on glass coverslips and incubated in 5 mM Ca2+ containing Tyrode's solution and 40 mM 2,3-butanedione monoxime (BDM) to inhibit cell contraction. Alternatively, myocytes were permeabilized with 10 microM digitonin and perfused with an "intracellular" solution containing 10 microM free [Ca2+], 5 mM EGTA, and 15 mM BDM. Direct [Ca2+]m measurements showed transient mitochondrial Ca2+ accumulation after exposure to 10 mM caffeine, as revealed by a 66% increase in the rhod-2 fluorescence intensity. Shear fluid forces, however, produced a 12% decrease in signal, suggesting that application of a mechanical force releases Ca2+ from the mitochondria. In addition, caffeine and CCCP or FCCP strongly reduced Delta Psi m, while application of a pressurized solution produced a transient Delta Psi m hyperpolarization in intact ventricular myocytes loaded with TMRM. The close proximity of mitochondria to ryanodine receptors and large [Ca2+] that develop in microdomains following calcium release are likely to play a critical role in regulating cytosolic Ca2+ signaling. We suggest that mitochondria may accumulate and release Ca2+ in response to mechanical forces generated by blood flow, independent of surface membrane-regulated CICR. The extent to which such a signaling mechanism contributes to stretch-induced increase in myocardial force and pathogenesis of arrhythmias remains to be assessed.

Modeling regulation of cardiac KATP and L-type Ca2+ currents by ATP, ADP, and Mg2+.

PubMed

Michailova, Anushka; Saucerman, Jeffrey; Belik, Mary Ellen; McCulloch, Andrew D

2005-03-01

Changes in cytosolic free Mg(2+) and adenosine nucleotide phosphates affect cardiac excitability and contractility. To investigate how modulation by Mg(2+), ATP, and ADP of K(ATP) and L-type Ca(2+) channels influences excitation-contraction coupling, we incorporated equations for intracellular ATP and MgADP regulation of the K(ATP) current and MgATP regulation of the L-type Ca(2+) current in an ionic-metabolic model of the canine ventricular myocyte. The new model: 1), quantitatively reproduces a dose-response relationship for the effects of changes in ATP on K(ATP) current, 2), simulates effects of ADP in modulating ATP sensitivity of K(ATP) channel, 3), predicts activation of Ca(2+) current during rapid increase in MgATP, and 4), demonstrates that decreased ATP/ADP ratio with normal total Mg(2+) or increased free Mg(2+) with normal ATP and ADP activate K(ATP) current, shorten action potential, and alter ionic currents and intracellular Ca(2+) signals. The model predictions are in agreement with experimental data measured under normal and a variety of pathological conditions.

Modeling regulation of cardiac KATP and L-type Ca2+ currents by ATP, ADP, and Mg2+

NASA Technical Reports Server (NTRS)

Michailova, Anushka; Saucerman, Jeffrey; Belik, Mary Ellen; McCulloch, Andrew D.

2005-01-01

Changes in cytosolic free Mg(2+) and adenosine nucleotide phosphates affect cardiac excitability and contractility. To investigate how modulation by Mg(2+), ATP, and ADP of K(ATP) and L-type Ca(2+) channels influences excitation-contraction coupling, we incorporated equations for intracellular ATP and MgADP regulation of the K(ATP) current and MgATP regulation of the L-type Ca(2+) current in an ionic-metabolic model of the canine ventricular myocyte. The new model: 1), quantitatively reproduces a dose-response relationship for the effects of changes in ATP on K(ATP) current, 2), simulates effects of ADP in modulating ATP sensitivity of K(ATP) channel, 3), predicts activation of Ca(2+) current during rapid increase in MgATP, and 4), demonstrates that decreased ATP/ADP ratio with normal total Mg(2+) or increased free Mg(2+) with normal ATP and ADP activate K(ATP) current, shorten action potential, and alter ionic currents and intracellular Ca(2+) signals. The model predictions are in agreement with experimental data measured under normal and a variety of pathological conditions.

Prostacyclin primes pregnant human myometrium for an enhanced contractile response in parturition

PubMed Central

Fetalvero, Kristina M.; Zhang, Peisheng; Shyu, Maureen; Young, Benjamin T.; Hwa, John; Young, Roger C.; Martin, Kathleen A.

2008-01-01

An incomplete understanding of the molecular events that regulate the myometrial transition from the quiescent pregnant state to the active contractile state during labor has hindered the development of improved therapies for preterm labor. During myometrial activation, proteins that prime the smooth muscle for contraction are upregulated, allowing maximal responsiveness to contractile agonists and thereby producing strong phasic contractions. Upregulation of one such protein, COX-2, generates PGs that induce contractions. Intriguingly, the predominant myometrial PG produced just prior to labor is prostacyclin (PGI2), a smooth muscle relaxant. However, here we have shown that activation of PGI2 receptor (IP) upregulated the expression of several contractile proteins and the gap junction protein connexin 43 through cAMP/PKA signaling in human myometrial tissue in organ and cell culture. Functionally, these IP-dependent changes in gene expression promoted an enhanced contractile response to oxytocin in pregnant human myometrial tissue strips, which was inhibited by the IP antagonist RO3244794. Furthermore, contractile protein induction was dependent on the concentration and time of exposure to the PGI2 analog iloprost and was blocked by both RO3244794 and PKA knockdown. We therefore propose that PGI2-mediated upregulation of contractile proteins and connexin 43 is a critical step in myometrial activation, allowing for a maximal contractile response. Our observations have important implications regarding activation of the myometrium prior to the onset of labor. PMID:19033666

Identification of biochemical adaptations in hyper- or hypocontractile hearts from phospholamban mutant mice by expression proteomics.

PubMed

Pan, Yan; Kislinger, Thomas; Gramolini, Anthony O; Zvaritch, Elena; Kranias, Evangelia G; MacLennan, David H; Emili, Andrew

2004-02-24

Phospholamban (PLN) is a critical regulator of cardiac contractility through its binding to and regulation of the activity of the sarco(endo)plasmic reticulum Ca2+ ATPase. To uncover biochemical adaptations associated with extremes of cardiac muscle contractility, we used high-throughput gel-free tandem MS to monitor differences in the relative abundance of membrane proteins in standard microsomal fractions isolated from the hearts of PLN-null mice (PLN-KO) with high contractility and from transgenic mice overexpressing a superinhibitory PLN mutant in a PLN-null background (I40A-KO) with diminished contractility. Significant differential expression was detected for a subset of the 782 proteins identified, including known membrane-associated biomarkers, components of signaling pathways, and previously uninvestigated proteins. Proteins involved in fat and carbohydrate metabolism and proteins linked to G protein-signaling pathways activating protein kinase C were enriched in I40A-KO cardiac muscle, whereas proteins linked to enhanced contractile function were enriched in PLN-KO mutant hearts. These data demonstrate that Ca2+ dysregulation, leading to elevated or depressed cardiac contractility, induces compensatory biochemical responses.

Use of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes (hiPSC-CMs) to Monitor Compound Effects on Cardiac Myocyte Signaling Pathways.

PubMed

Guo, Liang; Eldridge, Sandy; Furniss, Mike; Mussio, Jodie; Davis, Myrtle

2015-09-01

There is a need to develop mechanism-based assays to better inform risk of cardiotoxicity. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are rapidly gaining acceptance as a biologically relevant in vitro model for use in drug discovery and cardiotoxicity screens. Utilization of hiPSC-CMs for mechanistic investigations would benefit from confirmation of the expression and activity of cellular pathways that are known to regulate cardiac myocyte viability and function. This unit describes an approach to demonstrate the presence and function of signaling pathways in hiPSC-CMs and the effects of treatments on these pathways. We present a workflow that employs protocols to demonstrate protein expression and functional integrity of signaling pathway(s) of interest and to characterize biological consequences of signaling modulation. These protocols utilize a unique combination of structural, functional, and biochemical endpoints to interrogate compound effects on cardiomyocytes. Copyright © 2015 John Wiley & Sons, Inc.

Integrins and Integrin-Associated Proteins in the Cardiac Myocyte

PubMed Central

Ross, Robert S.

2014-01-01

Integrins are heterodimeric, transmembrane receptors that are expressed in all cells, including those in the heart. They participate in multiple critical cellular processes including adhesion, extracellular matrix organization, signaling, survival, and proliferation. Particularly relevant for a contracting muscle cell, integrins are mechanotransducers, translating mechanical to biochemical information. While it is likely that cardiovascular clinicians and scientists have highest recognition of integrins in the cardiovascular system from drugs used to inhibit platelet aggregation, the focus of this article will be on the role of integrins specifically in the cardiac myocyte. Following a general introduction to integrin biology, the manuscript will discuss important work on integrin signaling, mechanotransduction, and lessons learned about integrin function from a range of model organisms. Then we will detail work on integrin-related proteins in the myocyte, how integrins may interact with ion channels and mediate viral uptake into cells, and also play a role in stem cell biology. Finally, we will discuss directions for future study. PMID:24481847

Signaling Pathways in Cardiac Myocyte Apoptosis

PubMed Central

Xia, Peng; Liu, Yuening

2016-01-01

Cardiovascular diseases, the number 1 cause of death worldwide, are frequently associated with apoptotic death of cardiac myocytes. Since cardiomyocyte apoptosis is a highly regulated process, pharmacological intervention of apoptosis pathways may represent a promising therapeutic strategy for a number of cardiovascular diseases and disorders including myocardial infarction, ischemia/reperfusion injury, chemotherapy cardiotoxicity, and end-stage heart failure. Despite rapid growth of our knowledge in apoptosis signaling pathways, a clinically applicable treatment targeting this cellular process is currently unavailable. To help identify potential innovative directions for future research, it is necessary to have a full understanding of the apoptotic pathways currently known to be functional in cardiac myocytes. Here, we summarize recent progress in the regulation of cardiomyocyte apoptosis by multiple signaling molecules and pathways, with a focus on the involvement of these pathways in the pathogenesis of heart disease. In addition, we provide an update regarding bench to bedside translation of this knowledge and discuss unanswered questions that need further investigation. PMID:28101515

Sarcolemmal localisation of Na+/H+ exchange and Na+–HCO3− co-transport influences the spatial regulation of intracellular pH in rat ventricular myocytes

PubMed Central

Garciarena, Carolina D; Ma, Yu-ling; Swietach, Pawel; Huc, Laurence; Vaughan-Jones, Richard D

2013-01-01

Membrane acid extrusion by Na+/H+ exchange (NHE1) and Na+–HCO3− co-transport (NBC) is essential for maintaining a low cytoplasmic [H+] (∼60 nm, equivalent to an intracellular pH (pHi) of 7.2). This protects myocardial function from the high chemical reactivity of H+ ions, universal end-products of metabolism. We show here that, in rat ventricular myocytes, fluorescent antibodies map the NBC isoforms NBCe1 and NBCn1 to lateral sarcolemma, intercalated discs and transverse tubules (t-tubules), while NHE1 is absent from t-tubules. This unexpected difference matches functional measurements of pHi regulation (using AM-loaded SNARF-1, a pH fluorophore). Thus, myocyte detubulation (by transient exposure to 1.5 m formamide) reduces global acid extrusion on NBC by 40%, without affecting NHE1. Similarly, confocal pHi imaging reveals that NBC stimulation induces spatially uniform pHi recovery from acidosis, whereas NHE1 stimulation induces pHi non-uniformity during recovery (of ∼0.1 units, for 2–3 min), particularly at the ends of the cell where intercalated discs are commonly located, and where NHE1 immunostaining is prominent. Mathematical modelling shows that this induction of local pHi microdomains is favoured by low cytoplasmic H+ mobility and long H+ diffusion distances, particularly to surface NHE1 transporters mediating high membrane flux. Our results provide the first evidence for a spatial localisation of [H+]i regulation in ventricular myocytes, suggesting that, by guarding pHi, NHE1 preferentially protects gap junctional communication at intercalated discs, while NBC locally protects t-tubular excitation–contraction coupling. PMID:23420656

Neuronal Ca2+ sensor-1 contributes to stress tolerance in cardiomyocytes via activation of mitochondrial detoxification pathways.

PubMed

Nakamura, Tomoe Y; Nakao, Shu; Wakabayashi, Shigeo

2016-10-01

Identification of the molecules involved in cell death/survival pathways is important for understanding the mechanisms of cell loss in cardiac disease, and thus is clinically relevant. Ca 2+ -dependent signals are often involved in these pathways. Here, we found that neuronal Ca 2+ -sensor-1 (NCS-1), a Ca 2+ -binding protein, has an important role in cardiac survival during stress. Cardiomyocytes derived from NCS-1-deficient (Ncs1 -/- ) mice were more susceptible to oxidative and metabolic stress than wild-type (WT) myocytes. Cellular ATP levels and mitochondrial respiration rates, as well as the levels of mitochondrial marker proteins, were lower in Ncs1 -/- myocytes. Although oxidative stress elevated mitochondrial proton leak, which exerts a protective effect by inhibiting the production of reactive oxygen species in WT myocytes, this response was considerably diminished in Ncs1 -/- cardiomyocytes, and this would be a major reason for cell death. Consistently, H 2 O 2 -induced loss of mitochondrial membrane potential, a critical early event in cell death, was accelerated in Ncs1 -/- myocytes. Furthermore, NCS-1 was upregulated in hearts subjected to ischemia-reperfusion, and ischemia-reperfusion injury was more severe in Ncs1 -/- hearts. Activation of stress-induced Ca 2+ -dependent survival pathways, such as Akt and PGC-1α (which promotes mitochondrial biogenesis and function), was diminished in Ncs1 -/- hearts. Overall, these data demonstrate that NCS-1 contributes to stress tolerance in cardiomyocytes at least in part by activating certain Ca 2+ -dependent survival pathways that promote mitochondrial biosynthesis/function and detoxification pathways. Copyright © 2016 Elsevier Ltd. All rights reserved.

Qiliqiangxin Affects L Type Ca2+ Current in the Normal and Hypertrophied Rat Heart

PubMed Central

Wei, Yidong; Liu, Xiaoyu; Hou, Lei; Che, Wenliang; The, Erlinda; Jhummon, Muktanand Vikash

2012-01-01

Qiliqiangxin capsule is newly developed Chinese patent drug and proved to be effective and safe for the treatment of patients with chronic heart failure. We compared the effects of different dose Qiliqiangxin on L type Ca2+ current (I Ca-L) between normal and hypertrophied myocytes. A total of 40 healthy Sprague—Dawley rats were used in the study. The rats were randomly divided into two groups (control group and hypertrophy group). Cardiac hypertrophy was induced by pressure overload produced by partial ligation of the abdominal aorta. The control group was the sham-operated group. After 1 month, cardiac ventricular myocytes were isolated from the hearts of rats. Ventricular myocytes were exposed to 10 and 50 μmol/L Qiliqiangxin, and whole cell patch-clamp technique was used to study the effects of Qiliqiangxin on I Ca-L. The current densities of I Ca-L were similar in control group (−12.70 ± 0.53 pA/pF, n = 12) and in hypertrophy group (−12.39 ± 0.62 pA/pF, n = 10). They were not statistically significant. 10 and 50 μmol/L Qiliqiangxin can decrease I Ca-L peak current 48.6%±16.8% and 59.0%±4.4% in control group. However, the peak current was only reduced 16.73%±8.03% by 50 μmol/L Qiliqiangxin in hypertrophied myocytes. The inhibited action of Qiliqiangxin on I Ca-L of hypertrophy group was lower than in control group. Qiliqiangxin affected L-type Ca2+ channel and blocked I Ca-L, as well as affected cardiac function finally. Qiliqiangxin has diphasic action that is either class IV antiarrhythmic agent or the agent of effect cardiac function. PMID:22536279

Functional vascular smooth muscle cells derived from human induced pluripotent stem cells via mesenchymal stem cell intermediates

PubMed Central

Bajpai, Vivek K.; Mistriotis, Panagiotis; Loh, Yuin-Han; Daley, George Q.; Andreadis, Stelios T.

2012-01-01

Aims Smooth muscle cells (SMC) play an important role in vascular homeostasis and disease. Although adult mesenchymal stem cells (MSC) have been used as a source of contractile SMC, they suffer from limited proliferation potential and culture senescence, particularly when originating from older donors. By comparison, human induced pluripotent stem cells (hiPSC) can provide an unlimited source of functional SMC for autologous cell-based therapies and for creating models of vascular disease. Our goal was to develop an efficient strategy to derive functional, contractile SMC from hiPSC. Methods and results We developed a robust, stage-wise, feeder-free strategy for hiPSC differentiation into functional SMC through an intermediate stage of multipotent MSC, which could be coaxed to differentiate into fat, bone, cartilage, and muscle. At this stage, the cells were highly proliferative and displayed higher clonogenic potential and reduced senescence when compared with parental hair follicle mesenchymal stem cells. In addition, when exposed to differentiation medium, the myogenic proteins such as α-smooth muscle actin, calponin, and myosin heavy chain were significantly upregulated and displayed robust fibrillar organization, suggesting the development of a contractile phenotype. Indeed, tissue constructs prepared from these cells exhibited high levels of contractility in response to receptor- and non-receptor-mediated agonists. Conclusion We developed an efficient stage-wise strategy that enabled hiPSC differentiation into contractile SMC through an intermediate population of clonogenic and multipotent MSC. The high yield of MSC and SMC derivation suggests that our strategy may facilitate an acquisition of the large numbers of cells required for regenerative medicine or for studying vascular disease pathophysiology. PMID:22941255

Glucose Regulation of Load‐Induced mTOR Signaling and ER Stress in Mammalian Heart

PubMed Central

Sen, Shiraj; Kundu, Bijoy K.; Wu, Henry Cheng‐Ju; Hashmi, S. Shahrukh; Guthrie, Patrick; Locke, Landon W.; Roy, R. Jack; Matherne, G. Paul; Berr, Stuart S.; Terwelp, Matthew; Scott, Brian; Carranza, Sylvia; Frazier, O. Howard; Glover, David K.; Dillmann, Wolfgang H.; Gambello, Michael J.; Entman, Mark L.; Taegtmeyer, Heinrich

2013-01-01

Background Changes in energy substrate metabolism are first responders to hemodynamic stress in the heart. We have previously shown that hexose‐6‐phosphate levels regulate mammalian target of rapamycin (mTOR) activation in response to insulin. We now tested the hypothesis that inotropic stimulation and increased afterload also regulate mTOR activation via glucose 6‐phosphate (G6P) accumulation. Methods and Results We subjected the working rat heart ex vivo to a high workload in the presence of different energy‐providing substrates including glucose, glucose analogues, and noncarbohydrate substrates. We observed an association between G6P accumulation, mTOR activation, endoplasmic reticulum (ER) stress, and impaired contractile function, all of which were prevented by pretreating animals with rapamycin (mTOR inhibition) or metformin (AMPK activation). The histone deacetylase inhibitor 4‐phenylbutyrate, which relieves ER stress, also improved contractile function. In contrast, adding the glucose analogue 2‐deoxy‐d‐glucose, which is phosphorylated but not further metabolized, to the perfusate resulted in mTOR activation and contractile dysfunction. Next we tested our hypothesis in vivo by transverse aortic constriction in mice. Using a micro‐PET system, we observed enhanced glucose tracer analog uptake and contractile dysfunction preceding dilatation of the left ventricle. In contrast, in hearts overexpressing SERCA2a, ER stress was reduced and contractile function was preserved with hypertrophy. Finally, we examined failing human hearts and found that mechanical unloading decreased G6P levels and ER stress markers. Conclusions We propose that glucose metabolic changes precede and regulate functional (and possibly also structural) remodeling of the heart. We implicate a critical role for G6P in load‐induced mTOR activation and ER stress. PMID:23686371

Impaired M3 and enhanced M2 muscarinic receptor contractile function in a streptozotocin model of mouse diabetic urinary bladder

PubMed Central

Pak, K. J.; Ostrom, R. S.; Matsui, M.

2010-01-01

We investigated the contractile roles of M2 and M3 muscarinic receptors in urinary bladder from streptozotocin-treated mice. Wild-type and M2 muscarinic receptor knockout (M2 KO) mice were given a single injection of vehicle or streptozotocin (125 mg kg−1) 2–24 weeks prior to bladder assays. The effect of forskolin on contractions elicited to the muscarinic agonist, oxotremorine-M, was measured in isolated urinary bladder (intact or denuded of urothelium). Denuded urinary bladder from vehicle-treated wild-type and M2 KO mice exhibited similar contractile responses to oxotremorine-M, when contraction was normalized relative to that elicited by KCl (50 mM). Eight to 9 weeks after streptozotocin treatment, the EC50 value of oxotremorine-M increased 3.1-fold in urinary bladder from the M2 KO mouse (N = 5) compared to wild type (N = 6; P < 0.001). Analogous changes were observed in intact bladder. In denuded urinary bladder from vehicle-treated mice, forskolin (5 µM) caused a much greater inhibition of contraction in M2 KO bladder compared to wild type. Following streptozotocin treatment, this forskolin effect increased 1.6-fold (P = 0.032). At the 20- to 24-week time point, the forskolin effect increased 1.7-fold for denuded as well as intact bladders (P = 0.036, 0.01, respectively). Although streptozotocin treatment inhibits M3 receptor-mediated contraction in denuded urinary bladder, muscarinic contractile function is maintained in wild-type bladder by enhanced M2 contractile function. M2 receptor activation opposes forskolin-induced relaxation of the urinary bladder, and this M2 function is enhanced following streptozotocin treatment. PMID:20349044

Impaired M3 and enhanced M2 muscarinic receptor contractile function in a streptozotocin model of mouse diabetic urinary bladder.

PubMed

Pak, K J; Ostrom, R S; Matsui, M; Ehlert, F J

2010-05-01

We investigated the contractile roles of M2 and M3 muscarinic receptors in urinary bladder from streptozotocin-treated mice. Wild-type and M2 muscarinic receptor knockout (M2 KO) mice were given a single injection of vehicle or streptozotocin (125 mg kg(-1)) 2-24 weeks prior to bladder assays. The effect of forskolin on contractions elicited to the muscarinic agonist, oxotremorine-M, was measured in isolated urinary bladder (intact or denuded of urothelium). Denuded urinary bladder from vehicle-treated wild-type and M2 KO mice exhibited similar contractile responses to oxotremorine-M, when contraction was normalized relative to that elicited by KCl (50 mM). Eight to 9 weeks after streptozotocin treatment, the EC(50) value of oxotremorine-M increased 3.1-fold in urinary bladder from the M2 KO mouse (N = 5) compared to wild type (N = 6; P < 0.001). Analogous changes were observed in intact bladder. In denuded urinary bladder from vehicle-treated mice, forskolin (5 microM) caused a much greater inhibition of contraction in M2 KO bladder compared to wild type. Following streptozotocin treatment, this forskolin effect increased 1.6-fold (P = 0.032). At the 20- to 24-week time point, the forskolin effect increased 1.7-fold for denuded as well as intact bladders (P = 0.036, 0.01, respectively). Although streptozotocin treatment inhibits M3 receptor-mediated contraction in denuded urinary bladder, muscarinic contractile function is maintained in wild-type bladder by enhanced M2 contractile function. M2 receptor activation opposes forskolin-induced relaxation of the urinary bladder, and this M(2) function is enhanced following streptozotocin treatment.

Effect of estrogen on molecular and functional characteristics of the rodent vaginal muscularis

PubMed Central

Basha, Maureen E.; Chang, Shaohua; Burrows, Lara J.; Lassmann, Jenny; Wein, Alan J.; Moreland, Robert S.; Chacko, Samuel K.

2013-01-01

Introduction Vaginal atrophy is a consequence of menopause however little is known concerning the effect of a decrease in systemic estrogen on vaginal smooth muscle structure and function. As the incidence of pelvic floor disorders increases with age, it is important to determine if estrogen regulates the molecular composition and contractility of the vaginal muscularis. Aim The goal of this study was to determine the effect of estrogen on molecular and functional characteristics of the vaginal muscularis utilizing a rodent model of surgical menopause. Methods 3–4 month old Sprague Dawley rats underwent sham laparotomy (Sham, n=18) or ovariectomy (Ovx, n=39). Two weeks following surgery, animals received a subcutaneous osmotic pump containing vehicle (Sham, Ovx) or 17- β estradiol (Ovx). Animals were euthanized one week later and the proximal vagina was collected for analysis of contractile protein expression and in vitro studies of contractility. Measurements were analyzed using a one-way ANOVA followed by Tukey's post hoc analysis (α= 0.05). Main Outcome Measures Protein and mRNA transcript expression levels of contractile proteins, in vitro measurements of vaginal contractility Results Ovariectomy decreased the expression of carboxyl-terminal myosin heavy chain isoform SM1 and h-caldesmon and reduced the amplitude of contraction of the vaginal muscularis in response to KCl. Estradiol replacement reversed these changes. No differences were detected in the % vaginal muscularis, mRNA transcript expression of amino terminal MHC isoforms, l-caldesmon expression and maximal velocity of shortening. Conclusion Systemic estrogen replacement restores functional and molecular characteristics of the vaginal muscularis of ovariectomized rats. Our results indicate that menopause is associated with changes in the vaginal muscularis, which may contribute to the increased incidence of pelvic floor disorders with age. PMID:23438289

Increased Response to β2-Adrenoreceptor Stimulation Augments Inhibition of IKr in Heart Failure Ventricular Myocytes

PubMed Central

Wang, Hegui; Chen, Yanhong; Zhu, Hongjun; Wang, Sen; Zhang, Xiwen; Xu, Dongjie; Cao, Kejiang; Zou, Jiangang

2012-01-01

Background Increasing evidence indicates that the rapid component of delayed rectifier potassium current (IKr) is modulated by α- and β-adrenergic stimulation. However, the role and mechanism regulating IKr through β2-adrenoreceptor (β-AR) stimulation in heart failure (HF) are unclear. Methodology/Principal Findings In the present study, we investigated the effects of fenoterol, a highly selective β2-AR agonist, on IKr in left ventricular myocytes obtained from control and guinea pigs with HF induced by descending aortic banding. IKr was measured by using whole cell patch clamp technique. In control myocytes, superfusion of fenoterol (10 µM) caused a 17% decrease in IKr. In HF myocytes, the same concentration of fenoterol produced a significantly greater decrease (33%) in IKr. These effects were not modified by the incubation of myocytes with CGP-20712A, a β1-AR antagonist, but were abolished by pretreatment of myocytes with ICI-118551, a β2-AR antagonist. An inhibitory cAMP analog, Rp-cAMPS and PKA inhibitor significantly attenuated fenoterol-induced inhibition of IKr in HF myocytes. Moreover, fenoterol markedly prolonged action potential durations at 90% (APD90) repolarization in HF ventricular myocytes. Conclusions The results indicate that inhibition of IKr induced by β2-AR stimulation is increased in HF. The inhibitory effect is likely to be mediated through a cAMP/PKA pathway in HF ventricular myocytes. PMID:23029432

An Experimental Model Using Cultured Cardiac Myocytes for a Study of the Generation of Premature Ventricular Contractions Under Ultrasound Exposure

NASA Astrophysics Data System (ADS)

Kudo, Nobuki; Yamamoto, Masaya

2011-09-01

It is known that use of a contrast agents in echocardiography increases the probability of generation of premature ventricular contractions (PVCs). As a basic study to elucidate the mechanisms and to reduce adverse effects, the generation of PVCs was investigated using cultured cardiac myocytes instead of the intact heart in vivo. Cardiac myocytes were isolated from neonatal rats and cultured on a cover slip. The myocyte sample was exposed to pulsed ultrasound with microbubbles adjacent to the myocytes, and generation of PVCs was examined with ultrasound exposure at various delay times after onset of myocyte contraction. The experimental results showed that generation of PVCs had a stable threshold delay time and that PVCs were generated only when myocytes were exposed to ultrasound with delay times longer than the threshold. The results indicate that the model used in this study is useful for revealing the mechanisms by which PVCs are induced by ultrasound exposure.

Temporally distinct transcriptional regulation of myocyte dedifferentiation and Myofiber growth during muscle regeneration.

PubMed

Louie, Ke'ale W; Saera-Vila, Alfonso; Kish, Phillip E; Colacino, Justin A; Kahana, Alon

2017-11-09

Tissue regeneration requires a series of steps, beginning with generation of the necessary cell mass, followed by cell migration into damaged area, and ending with differentiation and integration with surrounding tissues. Temporal regulation of these steps lies at the heart of the regenerative process, yet its basis is not well understood. The ability of zebrafish to dedifferentiate mature "post-mitotic" myocytes into proliferating myoblasts that in turn regenerate lost muscle tissue provides an opportunity to probe the molecular mechanisms of regeneration. Following subtotal excision of adult zebrafish lateral rectus muscle, dedifferentiating residual myocytes were collected at two time points prior to cell cycle reentry and compared to uninjured muscles using RNA-seq. Functional annotation (GAGE or K-means clustering followed by GO enrichment) revealed a coordinated response encompassing epigenetic regulation of transcription, RNA processing, and DNA replication and repair, along with protein degradation and translation that would rewire the cellular proteome and metabolome. Selected candidate genes were phenotypically validated in vivo by morpholino knockdown. Rapidly induced gene products, such as the Polycomb group factors Ezh2 and Suz12a, were necessary for both efficient dedifferentiation (i.e. cell reprogramming leading to cell cycle reentry) and complete anatomic regeneration. In contrast, the late activated gene fibronectin was important for efficient anatomic muscle regeneration but not for the early step of myocyte cell cycle reentry. Reprogramming of a "post-mitotic" myocyte into a dedifferentiated myoblast requires a complex coordinated effort that reshapes the cellular proteome and rewires metabolic pathways mediated by heritable yet nuanced epigenetic alterations and molecular switches, including transcription factors and non-coding RNAs. Our studies show that temporal regulation of gene expression is programmatically linked to distinct steps in the regeneration process, with immediate early expression driving dedifferentiation and reprogramming, and later expression facilitating anatomical regeneration.

Deletion of Interleukin-6 Attenuates Pressure Overload-Induced Left Ventricular Hypertrophy and Dysfunction

PubMed Central

Afzal, Muhammad R.; Samanta, Anweshan; Xuan, Yu-Ting; Girgis, Magdy; Elias, Harold K; Zhu, Yanqing; Davani, Arash; Yang, Yanjuan; Chen, Xing; Ye, Sheng; Wang, Ou-Li; Chen, Lei; Hauptman, Jeryl; Vincent, Robert J.; Dawn, Buddhadeb

2016-01-01

Rationale The role of interleukin (IL)-6 in the pathogenesis of cardiac myocyte hypertrophy remains controversial. Objective To conclusively determine whether IL-6 signaling is essential for the development of pressure overload-induced left ventricular (LV) hypertrophy, and to elucidate the underlying molecular pathways. Methods and Results Wild-type (WT) and IL-6 knockout (IL-6−/−) mice underwent sham surgery or transverse aortic constriction (TAC) to induce pressure overload. Serial echocardiograms and terminal hemodynamic studies revealed attenuated LV hypertrophy and superior preservation of LV function in IL-6−/− mice after TAC. The extents of LV remodeling, fibrosis, and apoptosis were reduced in IL-6−/− hearts after TAC. Transcriptional and protein assays of myocardial tissue identified CaMKII and STAT3 activation as important underlying mechanisms during cardiac hypertrophy induced by TAC. The involvement of these pathways in myocyte hypertrophy was verified in isolated cardiac myocytes from WT and IL-6−/− mice exposed to pro-hypertrophy agents. Furthermore, overexpression of CaMKII in H9c2 cells increased STAT3 phosphorylation, and exposure of H9c2 cells to IL-6 resulted in STAT3 activation that was attenuated by CaMKII inhibition. Together these results identify the importance of CaMKII-dependent activation of STAT3 during cardiac myocyte hypertrophy via IL-6 signaling. Conclusions Genetic deletion of IL-6 attenuates TAC-induced LV hypertrophy and dysfunction, indicating a critical role played by IL-6 in the pathogenesis of LV hypertrophy in response to pressure overload. CaMKII plays an important role in IL-6-induced STAT3 activation and consequent cardiac myocyte hypertrophy. These findings may have significant therapeutic implications for LV hypertrophy and failure in patients with hypertension. PMID:27126808

Generation of a constitutive Na+-dependent inward-rectifier current in rat adult atrial myocytes by overexpression of Kir3.4.

PubMed

Mintert, Elisa; Bösche, Leif I; Rinne, Andreas; Timpert, Mathias; Kienitz, Marie-Cécile; Pott, Lutz; Bender, Kirsten

2007-11-15

Apart from gating by interaction with betagamma subunits from heterotrimeric G proteins upon stimulation of appropriate receptors, Kir.3 channels have been shown to be gated by intracellular Na+. However, no information is available on how Na+-dependent gating affects endogenous Kir3.1/Kir3.4 channels in mammalian atrial myocytes. We therefore studied how loading of adult atrial myocytes from rat hearts via the patch pipette filling solution with different concentrations of Na+ ([Na+]pip) affects Kir3 current. Surprisingly, in a range between 0 and 60 mm, Na+ neither had an effect on basal inward-rectifier current nor on the current activated by acetylcholine. Overexpression of Kir3.4 in adult atrial myocytes forced by adenoviral gene transfer results in formation of functional homomeric channels that interact with betagamma subunits upon activation of endogenous muscarinic receptors. These channels are activated at [Na+]pip >or= 15 mm, resulting in a receptor-independent basal inward rectifier current (I bir). I bir was neither affected by pertussis toxin nor by GDP-beta-S, suggesting G-protein-independent activation. PIP(2) depletion via endogenous PLC-coupled alpha1 adrenergic receptors causes inhibition of endogenous Kir3.1/3.4 channel currents by about 75%. In contrast, inhibition of Na+-activated I bir amounts to < 20%. The effect of the Kir3 channel blocker tertiapin-Q can be described using an IC50 of 12 nm (endogenous I K(ACh)) and 0.61 nm (I bir). These data clearly identify I bir as a homotetrameric Kir3.4 channel current with novel properties of regulation and pharmacology. Ibir shares some properties with a basal current recently described in atrial myocytes from an animal model of atrial fibrillation (AF) and AF patients.

Adrenergic Receptors in Individual Ventricular Myocytes: The Beta-1 and Alpha-1B Are in All Cells, the Alpha-1A Is in a Subpopulation, and the Beta-2 and Beta-3 Are Mostly Absent.

PubMed

Myagmar, Bat-Erdene; Flynn, James M; Cowley, Patrick M; Swigart, Philip M; Montgomery, Megan D; Thai, Kevin; Nair, Divya; Gupta, Rumita; Deng, David X; Hosoda, Chihiro; Melov, Simon; Baker, Anthony J; Simpson, Paul C

2017-03-31

It is unknown whether every ventricular myocyte expresses all 5 of the cardiac adrenergic receptors (ARs), β1, β2, β3, α1A, and α1B. The β1 and β2 are thought to be the dominant myocyte ARs. Quantify the 5 cardiac ARs in individual ventricular myocytes. We studied ventricular myocytes from wild-type mice, mice with α1A and α1B knockin reporters, and β1 and β2 knockout mice. Using individual isolated cells, we measured knockin reporters, mRNAs, signaling (phosphorylation of extracellular signal-regulated kinase and phospholamban), and contraction. We found that the β1 and α1B were present in all myocytes. The α1A was present in 60%, with high levels in 20%. The β2 and β3 were detected in only ≈5% of myocytes, mostly in different cells. In intact heart, 30% of total β-ARs were β2 and 20% were β3, both mainly in nonmyocytes. The dominant ventricular myocyte ARs present in all cells are the β1 and α1B. The β2 and β3 are mostly absent in myocytes but are abundant in nonmyocytes. The α1A is in just over half of cells, but only 20% have high levels. Four distinct myocyte AR phenotypes are defined: 30% of cells with β1 and α1B only; 60% that also have the α1A; and 5% each that also have the β2 or β3. The results raise cautions in experimental design, such as receptor overexpression in myocytes that do not express the AR normally. The data suggest new paradigms in cardiac adrenergic signaling mechanisms. © 2017 American Heart Association, Inc.

The effect of N-acetylcysteine on cardiac contractility to dobutamine in rats with streptozotocin-induced diabetes.

PubMed

Cheng, Xing; Xia, Zhengyuan; Leo, Joyce M; Pang, Catherine C Y

2005-09-05

We examined if myocardial depression at the acute phase of diabetes (3 weeks after injection of streptozotocin, 60 mg/kg i.v.) is due to activation of inducible nitric oxide synthase and production of peroxynitrite, and if treatment with N-acetylcysteine (1.2 g/day/kg for 3 weeks, antioxidant) improves cardiac function. Four groups of rats were used: control, N-acetylcysteine-treated control, diabetic and N-acetylcysteine-treated diabetic. Pentobarbital-anaesthetized diabetic rats, relative to the controls, had reduced left ventricular contractility to dobutamine (1-57 microg/min/kg). The diabetic rats also had increased myocardial levels of thiobarbituric acid reactive substances, immunostaining of inducible nitric oxide synthase and nitrotyrosine, and similar baseline 15-F2t-isoprostane. N-acetylcysteine did not affect responses in the control rats; but increased cardiac contractility to dobutamine, reduced myocardial immunostaining of inducible nitric oxide synthase and nitrotyrosine and level of 15-F2t-isoprostane, and increased cardiac contractility to dobutamine in the diabetic rats. Antioxidant supplementation in diabetes reduces oxidative stress and improves cardiac function.

Thrombopoietin modulates cardiac contractility in vitro and contributes to myocardial depressing activity of septic shock serum.

PubMed

Lupia, Enrico; Spatola, Tiziana; Cuccurullo, Alessandra; Bosco, Ornella; Mariano, Filippo; Pucci, Angela; Ramella, Roberta; Alloatti, Giuseppe; Montrucchio, Giuseppe

2010-09-01

Thrombopoietin (TPO) is a humoral growth factor that has been shown to increase platelet activation in response to several agonists. Patients with sepsis have increased circulating TPO levels, which may enhance platelet activation, potentially participating to the pathogenesis of multi-organ failure. Aim of this study was to investigate whether TPO affects myocardial contractility and participates to depress cardiac function during sepsis. We showed the expression of the TPO receptor c-Mpl on myocardial cells and tissue by RT-PCR, immunofluorescence and western blotting. We then evaluated the effect of TPO on the contractile function of rat papillary muscle and isolated heart. TPO did not change myocardial contractility in basal conditions, but, when followed by epinephrine (EPI) stimulation, it blunted the enhancement of contractile force induced by EPI both in papillary muscle and isolated heart. An inhibitor of TPO prevented TPO effect on cardiac inotropy. Treatment of papillary muscle with pharmacological inhibitors of phosphatidylinositol 3-kinase, NO synthase, and guanilyl cyclase abolished TPO effect, indicating NO as the final mediator. We finally studied the role of TPO in the negative inotropic effect exerted by human septic shock (HSS) serum and TPO cooperation with TNF-alpha and IL-1beta. Pre-treatment with the TPO inhibitor prevented the decrease in contractile force induced by HSS serum. Moreover, TPO significantly amplified the negative inotropic effect induced by TNF-alpha and IL-1beta in papillary muscle. In conclusion, TPO negatively modulates cardiac inotropy in vitro and contributes to the myocardial depressing activity of septic shock serum.

Post-exercise contractility, diastolic function, and pressure: Operator-independent sensor-based intelligent monitoring for heart failure telemedicine

PubMed Central

Bombardini, Tonino; Gemignani, Vincenzo; Bianchini, Elisabetta; Pasanisi, Emilio; Pratali, Lorenza; Pianelli, Mascia; Faita, Francesco; Giannoni, Massimo; Arpesella, Giorgio; Sicari, Rosa; Picano, Eugenio

2009-01-01

Background New sensors for intelligent remote monitoring of the heart should be developed. Recently, a cutaneous force-frequency relation recording system has been validated based on heart sound amplitude and timing variations at increasing heart rates. Aim To assess sensor-based post-exercise contractility, diastolic function and pressure in normal and diseased hearts as a model of a wireless telemedicine system. Methods We enrolled 150 patients and 22 controls referred for exercise-stress echocardiography, age 55 ± 18 years. The sensor was attached in the precordial region by an ECG electrode. Stress and recovery contractility were derived by first heart sound amplitude vibration changes; diastolic times were acquired continuously. Systemic pressure changes were quantitatively documented by second heart sound recording. Results Interpretable sensor recordings were obtained in all patients (feasibility = 100%). Post-exercise contractility overshoot (defined as increase > 10% of recovery contractility vs exercise value) was more frequent in patients than controls (27% vs 8%, p < 0.05). At 100 bpm stress heart rate, systolic/diastolic time ratio (normal, < 1) was > 1 in 20 patients and in none of the controls (p < 0.01); at recovery systolic/diastolic ratio was > 1 in only 3 patients (p < 0.01 vs stress). Post-exercise reduced arterial pressure was sensed. Conclusion Post-exercise contractility, diastolic time and pressure changes can be continuously measured by a cutaneous sensor. Heart disease affects not only exercise systolic performance, but also post-exercise recovery, diastolic time intervals and blood pressure changes – in our study, all of these were monitored by a non-invasive wearable sensor. PMID:19442285

Direct toxic effects of aqueous extract of cigarette smoke on cardiac myocytes at clinically relevant concentrations

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yamada, Shigeyuki; Zhang Xiuquan; Kadono, Toshie

Aims: Our goal was to determine if clinically relevant concentrations of aqueous extract of cigarette smoke (CSE) have direct deleterious effects on ventricular myocytes during simulated ischemia, and to investigate the mechanisms involved. Methods: CSE was prepared with a smoking chamber. Ischemia was simulated by metabolic inhibition (MI) with cyanide (CN) and 0 glucose. Adult rabbit and mouse ventricular myocyte [Ca{sup 2+}]{sub i} was measured by flow cytometry using fluo-3. Mitochondrial [Ca{sup 2+}] was measured with confocal microscopy, and Rhod-2 fluorescence. The mitochondrial permeability transition (MPT) was detected by TMRM fluorescence and myocyte contracture. Myocyte oxidative stress was quantified bymore » dichlorofluorescein (DCF) fluorescence with confocal microscopy. Results: CSE 0.1% increased myocyte contracture caused by MI. The nicotine concentration (HPLC) in 0.1% CSE was 15 ng/ml, similar to that in humans after smoking cigarettes. CSE 0.1% increased mitochondrial Ca{sup 2+} uptake, and increased the susceptibility of mitochondria to the MPT. CSE 0.1% increased DCF fluorescence in isolated myocytes, and increased [Ca{sup 2+}]{sub i} in paced myocytes exposed to 2.0 mM CN, 0 glucose (P-MI). These effects were inhibited by the superoxide scavenger Tiron. The effect of CSE on [Ca{sup 2+}]{sub i} during P-MI was also prevented by ranolazine. Conclusions: CSE in clinically relevant concentrations increases myocyte [Ca{sup 2+}]{sub i} during simulated ischemia, and increases myocyte susceptibility to the MPT. These effects appear to be mediated at least in part by oxidative radicals in CSE, and likely contribute to the effects of cigarette smoke to increase myocardial infarct size, and to decrease angina threshold.« less

Enhanced basal late sodium current appears to underlie the age-related prolongation of action potential duration in guinea pig ventricular myocytes.

PubMed

Song, Yejia; Belardinelli, Luiz

2017-12-14

Aging hearts have prolonged QT interval and are vulnerable to oxidative stress. Because the QT interval indirectly reflects the action potential duration (APD), we examined the hypotheses that 1) the APD of ventricular myocytes increases with age; 2) the age-related prolongation of APD is due to an enhancement of basal late Na + current (I NaL ); 3) inhibition of I NaL may protect aging hearts from arrhythmogenic effects of hydrogen peroxide (H 2 O 2 ). Experiments were performed on ventricular myocytes isolated from one-month (young) and one-year (old) guinea pigs (GPs). The APD of myocytes from old GPs was significantly longer than that from young GPs and was shortened by the I NaL inhibitors GS967 and tetrodotoxin. The magnitude of I NaL was significantly larger in myocytes from old than from young GPs. The CaMKII inhibitors KN-93 and AIP and the Na V 1.5-channel blocker MTSEA blocked the I NaL . There were no significant differences between myocytes from young and old GPs in L-type Ca 2+ current and the rapidly- and slowly-activating delayed rectifier K + currents, although the inward rectifier K + current was slightly decreased in myocytes from old GPs. H 2 O 2 induced more early afterdepolarizations in myocytes from old than from young GPs. The effect of H 2 O 2 was attenuated by GS967. The results suggest that 1) the APD of myocytes from old GPs is prolonged, 2) a CaMKII-mediated increase in Na V 1.5-channel I NaL is responsible for the prolongation of APD, and 3) Inhibition of I NaL may be beneficial for maintaining electrical stability under oxidative stress in myocytes of old GPs.

Growth and Electrophysiological Properties of Rat Embryonic Cardiomyocytes on Hydroxyl- and Carboxyl-Modified Surfaces

PubMed Central

NATARAJAN, ANUPAMA; CHUN, CHANGJU; HICKMAN, JAMES J.; MOLNAR, PETER

2010-01-01

Biodegradable scaffolds such as poly(lactic acid) (PLA), poly(lactic-co-glycolic acid) (PLGA) or poly(glycolic acid) (PGA) are commonly used materials in tissue engineering. The chemical composition of these scaffolds changes during degradation which provides a changing environment for the seeded cells. In this study we have developed a simple and relatively high-throughput method in order to test the physiological effects of this varying chemical environment on rat embryonic cardiac myocytes. In order to model the different degradation stages of the scaffold, glass coverslips were functionalized with 11-mercaptoundecanoic acid (MUA) and 11-mercapto-1-undecanol (MUL) as carboxyl- and hydroxyl-group presenting surfaces and also with trimethoxysilylpropyldiethylenetriamine (DETA) and (3-aminopropyl)triethoxysilane (APTES) as controls. Embryonic cardiac myocytes formed beating islands on all tested surfaces but the number of attached cells and beating patches was significantly lower on MUL compared to any of the other functionalized surfaces. Moreover, whole cell patch clamp experiments showed that the average length of action potentials generated by the beating cardiac myocytes were significantly longer on MUL compared to the other surfaces. Our results, using our simple test system, are in agreement with earlier observations that utilized the complex 3D biodegradable scaffold. Thus, surface functionalization with self-assembled monolayers combined with histological/physiological testing could be a relatively high throughput method for biocompatibility studies and for the optimization of the material/tissue interface in tissue engineering. PMID:18854125

Increased response to β₂-adrenoreceptor stimulation augments inhibition of IKr in heart failure ventricular myocytes.

PubMed

Wang, Hegui; Chen, Yanhong; Zhu, Hongjun; Wang, Sen; Zhang, Xiwen; Xu, Dongjie; Cao, Kejiang; Zou, Jiangang

2012-01-01

Increasing evidence indicates that the rapid component of delayed rectifier potassium current (I(Kr)) is modulated by α- and β-adrenergic stimulation. However, the role and mechanism regulating I(Kr) through β(2)-adrenoreceptor (β-AR) stimulation in heart failure (HF) are unclear. In the present study, we investigated the effects of fenoterol, a highly selective β(2)-AR agonist, on I(Kr) in left ventricular myocytes obtained from control and guinea pigs with HF induced by descending aortic banding. I(Kr) was measured by using whole cell patch clamp technique. In control myocytes, superfusion of fenoterol (10 µM) caused a 17% decrease in I(Kr). In HF myocytes, the same concentration of fenoterol produced a significantly greater decrease (33%) in I(Kr). These effects were not modified by the incubation of myocytes with CGP-20712A, a β(1)-AR antagonist, but were abolished by pretreatment of myocytes with ICI-118551, a β(2)-AR antagonist. An inhibitory cAMP analog, Rp-cAMPS and PKA inhibitor significantly attenuated fenoterol-induced inhibition of I(Kr) in HF myocytes. Moreover, fenoterol markedly prolonged action potential durations at 90% (APD(90)) repolarization in HF ventricular myocytes. The results indicate that inhibition of I(Kr) induced by β(2)-AR stimulation is increased in HF. The inhibitory effect is likely to be mediated through a cAMP/PKA pathway in HF ventricular myocytes.

The contractile ring coordinates curvature-dependent septum assembly during fission yeast cytokinesis

PubMed Central

Zhou, Zhou; Munteanu, Emilia Laura; He, Jun; Ursell, Tristan; Bathe, Mark; Huang, Kerwyn Casey; Chang, Fred

2015-01-01

The functions of the actin-myosin–based contractile ring in cytokinesis remain to be elucidated. Recent findings show that in the fission yeast Schizosaccharomyces pombe, cleavage furrow ingression is driven by polymerization of cell wall fibers outside the plasma membrane, not by the contractile ring. Here we show that one function of the ring is to spatially coordinate septum cell wall assembly. We develop an improved method for live-cell imaging of the division apparatus by orienting the rod-shaped cells vertically using microfabricated wells. We observe that the septum hole and ring are circular and centered in wild-type cells and that in the absence of a functional ring, the septum continues to ingress but in a disorganized and asymmetric manner. By manipulating the cleavage furrow into different shapes, we show that the ring promotes local septum growth in a curvature-dependent manner, allowing even a misshapen septum to grow into a more regular shape. This curvature-dependent growth suggests a model in which contractile forces of the ring shape the septum cell wall by stimulating the cell wall machinery in a mechanosensitive manner. Mechanical regulation of the cell wall assembly may have general relevance to the morphogenesis of walled cells. PMID:25355954

Analysis of Tyrosine Kinase Inhibitor-Mediated Decline in Contractile Force in Rat Engineered Heart Tissue.

PubMed

Jacob, Fabian; Yonis, Amina Y; Cuello, Friederike; Luther, Pradeep; Schulze, Thomas; Eder, Alexandra; Streichert, Thomas; Mannhardt, Ingra; Hirt, Marc N; Schaaf, Sebastian; Stenzig, Justus; Force, Thomas; Eschenhagen, Thomas; Hansen, Arne

2016-01-01

Left ventricular dysfunction is a frequent and potentially severe side effect of many tyrosine kinase inhibitors (TKI). The mode of toxicity is not identified, but may include impairment of mitochondrial or sarcomeric function, autophagy or angiogenesis, either as an on-target or off-target mechanism. We studied concentration-response curves and time courses for nine TKIs in three-dimensional, force generating engineered heart tissue (EHT) from neonatal rat heart cells. We detected a concentration- and time-dependent decline in contractile force for gefitinib, lapatinib, sunitinib, imatinib, sorafenib, vandetanib and lestaurtinib and no decline in contractile force for erlotinib and dasatinib after 96 hours of incubation. The decline in contractile force was associated with an impairment of autophagy (LC3 Western blot) and appearance of autophagolysosomes (transmission electron microscopy). This study demonstrates the feasibility to study TKI-mediated force effects in EHTs and identifies an association between a decline in contractility and inhibition of autophagic flux.

Analysis of Tyrosine Kinase Inhibitor-Mediated Decline in Contractile Force in Rat Engineered Heart Tissue

PubMed Central

Cuello, Friederike; Luther, Pradeep; Schulze, Thomas; Eder, Alexandra; Streichert, Thomas; Mannhardt, Ingra; Hirt, Marc N.; Schaaf, Sebastian; Stenzig, Justus; Force, Thomas

2016-01-01

Introduction Left ventricular dysfunction is a frequent and potentially severe side effect of many tyrosine kinase inhibitors (TKI). The mode of toxicity is not identified, but may include impairment of mitochondrial or sarcomeric function, autophagy or angiogenesis, either as an on-target or off-target mechanism. Methods and Results We studied concentration-response curves and time courses for nine TKIs in three-dimensional, force generating engineered heart tissue (EHT) from neonatal rat heart cells. We detected a concentration- and time-dependent decline in contractile force for gefitinib, lapatinib, sunitinib, imatinib, sorafenib, vandetanib and lestaurtinib and no decline in contractile force for erlotinib and dasatinib after 96 hours of incubation. The decline in contractile force was associated with an impairment of autophagy (LC3 Western blot) and appearance of autophagolysosomes (transmission electron microscopy). Conclusion This study demonstrates the feasibility to study TKI-mediated force effects in EHTs and identifies an association between a decline in contractility and inhibition of autophagic flux. PMID:26840448

NADPH Oxidase 5 Is a Pro-Contractile Nox Isoform and a Point of Cross-Talk for Calcium and Redox Signaling-Implications in Vascular Function.

PubMed

Montezano, Augusto C; De Lucca Camargo, Livia; Persson, Patrik; Rios, Francisco J; Harvey, Adam P; Anagnostopoulou, Aikaterini; Palacios, Roberto; Gandara, Ana Caroline P; Alves-Lopes, Rheure; Neves, Karla B; Dulak-Lis, Maria; Holterman, Chet E; de Oliveira, Pedro Lagerblad; Graham, Delyth; Kennedy, Christopher; Touyz, Rhian M

2018-06-15

NADPH Oxidase 5 (Nox5) is a calcium-sensitive superoxide-generating Nox. It is present in lower forms and higher mammals, but not in rodents. Nox5 is expressed in vascular cells, but the functional significance remains elusive. Given that contraction is controlled by calcium and reactive oxygen species, both associated with Nox5, we questioned the role of Nox5 in pro-contractile signaling and vascular function. Transgenic mice expressing human Nox5 in a vascular smooth muscle cell-specific manner (Nox5 mice) and Rhodnius prolixus , an arthropod model that expresses Nox5 endogenoulsy, were studied. Reactive oxygen species generation was increased systemically and in the vasculature and heart in Nox5 mice. In Nox5-expressing mice, agonist-induced vasoconstriction was exaggerated and endothelium-dependent vasorelaxation was impaired. Vascular structural and mechanical properties were not influenced by Nox5. Vascular contractile responses in Nox5 mice were normalized by N -acetylcysteine and inhibitors of calcium channels, calmodulin, and endoplasmic reticulum ryanodine receptors, but not by GKT137831 (Nox1/4 inhibitor). At the cellular level, vascular changes in Nox5 mice were associated with increased vascular smooth muscle cell [Ca 2+ ] i , increased reactive oxygen species and nitrotyrosine levels, and hyperphosphorylation of pro-contractile signaling molecules MLC20 (myosin light chain 20) and MYPT1 (myosin phosphatase target subunit 1). Blood pressure was similar in wild-type and Nox5 mice. Nox5 did not amplify angiotensin II effects. In R. prolixus , gastrointestinal smooth muscle contraction was blunted by Nox5 silencing, but not by VAS2870 (Nox1/2/4 inhibitor). Nox5 is a pro-contractile Nox isoform important in redox-sensitive contraction. This involves calcium-calmodulin and endoplasmic reticulum-regulated mechanisms. Our findings define a novel function for vascular Nox5, linking calcium and reactive oxygen species to the pro-contractile molecular machinery in vascular smooth muscle cells. © 2018 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley.

Genetic engineering of somatic cells to study and improve cardiac function.

PubMed

Kirkton, Robert D; Bursac, Nenad

2012-11-01

To demonstrate the utility of genetically engineered excitable cells for studies of basic electrophysiology and cardiac cell therapy. 'Zig-zag' networks of neonatal rat ventricular myocytes (NRVMs) were micropatterned onto thin elastomeric films to mimic the slow action potential (AP) conduction found in fibrotic myocardium. Addition of genetically engineered excitable human embryonic kidney cells (HEK-293 cells) ('Ex-293' cells stably expressing Kir2.1, Na(v)1.5, and Cx43 channels) increased both cardiac conduction velocity by 370% and twitch force amplitude by 64%. Furthermore, we stably expressed mutant Na(v)1.5 [A1924T (fast sodium channel mutant (substitution of alanine by threonine at amino acid 1924)] channels with hyperpolarized steady-state activation and showed that, despite a 71.6% reduction in peak I(Na), these cells propagated APs at the same velocity as the wild-type Na(v)1.5-expressing Ex-293 cells. Stable expression of Ca(v)3.3 (T-type voltage-gated calcium) channels in Ex-293 cells (to generate an 'ExCa-293' line) significantly increased their AP duration and reduced repolarization gradients in cocultures of these cells and NRVMs. Additional expression of an optogenetic construct [ChIEF (light-gated Channelrhodopsin mutant)]enabled light-based control of AP firing in ExCa-293 cells. We show that, despite being non-contractile, genetically engineered excitable cells can significantly improve both electrical and mechanical function of engineered cardiac tissues in vitro. We further demonstrate the utility of engineered cells for tissue-level studies of basic electrophysiology and cardiac channelopathies. In the future, this novel platform could be utilized in the high-throughput design of new genetically encoded indicators of cell electrical function, validation, and improvement of computer models of AP conduction, and development of novel engineered somatic cell therapies for the treatment of cardiac infarction and arrhythmias.

Intracellular pH regulatory mechanism in human atrial myocardium: functional evidence for Na(+)/H(+) exchanger and Na(+)/HCO(3)(-) symporter.

PubMed

Loh, Shih-Hurng; Chen, Wei-Hwa; Chiang, Cheng-Hsien; Tsai, Chien-Sung; Lee, Guo-Chen; Jin, Jong-Shiaw; Cheng, Tzu-Hurng; Chen, Jin-Jer

2002-01-01

Intracellular pH (pH(i)) exerts considerable influence on cardiac contractility and rhythm. Over the last few years, extensive progress has been made in understanding the system that controls pH(i) in animal cardiomyocytes. In addition to the housekeeping Na(+)-H(+) exchanger (NHE), the Na(+)-HCO(3)(-) symporter (NHS) has been demonstrated in animal cardiomyocytes as another acid extruder. However, whether the NHE and NHS functions exist in human atrial cardiomyocytes remains unclear. We therefore investigated the mechanism of pH(i) recovery from intracellular acidosis (induced by NH(4)Cl prepulse) using intracellular 2',7'-bis(2-carboxethyl)-5(6)-carboxy-fluorescein fluorescence in human atrial myocardium. In HEPES (nominally HCO(3)(-)-free) Tyrode solution, pH(i) recovery from induced intracellular acidosis could be blocked completely by 30 microM 3-methylsulfonyl-4-piperidinobenzoyl, guanidine hydrochloride (HOE 694), a specific NHE inhibitor, or by removing extracellular Na(+). In 3% CO(2)-HCO(3)(-) Tyrode solution, HOE 694 only slowed the pH(i) recovery, while addition of HOE 694 together with 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (an NHS inhibitor) or removal of extracellular Na(+) inhibited the acid extrusion entirely. Therefore, in the present study, we provided evidence that two acid extruders involved in acid extrusion in human atrial myocytes, one which is HCO(3)(-) independent and one which is HCO(3)(-) dependent, are mostly likely NHE and NHS, respectively. When we checked the percentage of contribution of these two carriers to pH(i) recovery following induced acidosis, we found that the activity of NHE increased steeply in the acid direction, while that of NHS did not change. Our present data indicate for the first time that two acid extruders, NHE and NHS, exist functionally and pH(i) dependently in human atrial cardiomyocytes. Copyright 2002 National Science Council, ROC and S. Karger AG, Basel

Soluble activin receptor type IIB decoy receptor differentially impacts murine osteogenesis imperfecta muscle function.

PubMed

Jeong, Youngjae; Daghlas, Salah A; Kahveci, Alp S; Salamango, Daniel; Gentry, Bettina A; Brown, Marybeth; Rector, R Scott; Pearsall, R Scott; Phillips, Charlotte L

2018-02-01

Osteogenesis imperfecta (OI) is characterized by skeletal fragility and muscle weakness. In this study we investigated the effects of soluble activin type IIB receptor (sActRIIB-mFc) on muscle mass and function in 2 distinct mouse models of OI: osteogenesis imperfecta murine (oim) and +/G610C. Wild-type (WT), +/G610C, and oim/oim mice were treated from 2 to 4 months of age with Tris-buffered saline (vehicle) or sActRIIB-mFc and their hindlimb muscles evaluated for mass, morphology, and contractile function. sActRIIB-mFc-treated WT, +/G610C, and oim/oim mice had increased hindlimb muscle weights and myofiber cross-sectional area compared with vehicle-treated counterparts. sActRIIB-mFc-treated oim/oim mice also exhibited increased contractile function relative to vehicle-treated counterparts. Blocking endogenous ActRIIB was effective at increasing muscle size in mouse models of OI, and increasing contractile function in oim/oim mice. ActRIIB inhibitors may provide a potential mutation-specific therapeutic option for compromised muscle function in OI. Muscle Nerve 57: 294-304, 2018. © 2017 Wiley Periodicals, Inc.

Effect of oxidative insult on young and adult cardiac muscle cells in vitro.

PubMed

Nag, A C; Sreepathi, P; Lee, M L; Reddan, J R

1996-01-01

The effect of hydrogen peroxide on cultured neonatal and adult cardiac myocytes was investigated. On neonatal cardiac myocytes the effect was very pronounced at a low concentration (0.03 mM), whereas the adult cardiac myocytes were resistant to the same concentration of H2O2. Dividing neonatal cardiac myocytes were more susceptible to H2O2 insult than the non-dividing adult cardiac myocytes. At a concentration of 0.1 mM H2O2, the neonatal cardiac myocytes were significantly damaged compared with the adult cardiac myocytes. Cardiac muscle cells from neonatal and adult hearts at high density culture were more tolerant to the oxidative insult by H2O2 than cells in low density culture. The damaging effect of H2O2 was very selective on F-actin in neonatal and adult cardiac muscle cells. The effect of H2O2 on myosin, titin, alpha-actinin, desmin or tubulin was not pronounced. Microscopical studies suggested a more marked protection by catalase than by glutathione reductase in the neonatal cells.

Contractile ring stability in S. pombe depends on F-BAR protein Cdc15p and Bgs1p transport from the Golgi complex.

PubMed

Arasada, Rajesh; Pollard, Thomas D

2014-09-11

Cdc15p is known to contribute to cytokinesis in fission yeast; however, the protein is not required to assemble the contractile ring of actin and myosin, but it helps to anchor the ring to the plasma membrane. Cdc15p has a lipid-binding F-BAR domain, suggesting that it provides a physical link between the plasma membrane and contractile ring proteins. However, we find that a more important function of Cdc15p during cytokinesis is to help deliver a transmembrane enzyme, Bgs1p (also called Cps1p), from the Golgi apparatus to the plasma membrane, where it appears to anchor the contractile ring. Bgs1p synthesizes the cell wall in the cleavage furrow, but its enzyme activity is not required to anchor the contractile ring. We estimate that ∼ 2,000 Bgs1p molecules are required to anchor the ring. Without Bgs1p anchors, contractile rings slide along the plasma membrane, a phenomenon that depends on an unconventional type II myosin called Myp2p. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

Use of arginine-glycine-aspartic acid adhesion peptides coupled with a new collagen scaffold to engineer a myocardium-like tissue graft.

PubMed

Schussler, O; Coirault, C; Louis-Tisserand, M; Al-Chare, W; Oliviero, P; Menard, C; Michelot, R; Bochet, P; Salomon, D R; Chachques, J C; Carpentier, A; Lecarpentier, Y

2009-03-01

Cardiac tissue engineering might be useful in treatment of diseased myocardium or cardiac malformations. The creation of functional, biocompatible contractile tissues, however, remains challenging. We hypothesized that coupling of arginine-glycine-aspartic acid-serine (RGD+) adhesion peptides would improve cardiomyocyte viability and differentiation and contractile performance of collagen-cell scaffolds. Clinically approved collagen scaffolds were functionalized with RGD+ cells and seeded with cardiomyocytes. Contractile performance, cardiomyocyte viability and differentiation were analyzed at days 1 and 8 and/or after culture for 1 month. The method used for the RGD+ cell-collagen scaffold coupling enabled the following features: high coupling yields and complete washout of excess reagent and by-products with no need for chromatography; spectroscopic quantification of RGD+ coupling; a spacer arm of 36 A, a length reported as optimal for RGD+-peptide presentation and favorable for integrin-receptor clustering and subsequent activation. Isotonic and isometric mechanical parameters, either spontaneous or electrostimulated, exhibited good performance in RGD+ constructs. Cell number and viability was increased in RGD+ scaffolds, and we saw good organization of cell contractile apparatus with occurrence of cross-striation. We report a novel method of engineering a highly effective collagen-cell scaffold based on RGD+ peptides cross-linked to a clinically approved collagen matrix. The main advantages were cell contractile performance, cardiomyocyte viability and differentiation.

Caveolin 3-dependent loss of t-tubular ICa during hypertrophy and heart failure in mice.

PubMed

Bryant, Simon M; Kong, Cherrie H T; Watson, Judy J; Gadeberg, Hanne C; James, Andrew F; Cannell, Mark B; Orchard, Clive H

2018-05-01

What is the central question of this study? Heart failure is associated with redistribution of L-type Ca 2+ current (I Ca ) away from the t-tubule membrane to the surface membrane of cardiac ventricular myocytes. However, the underlying mechanism and its dependence on severity of pathology (hypertrophy versus failure) are unclear. What is the main finding and its importance? Increasing severity of response to transverse aortic constriction, from hypertrophy to failure, was accompanied by graded loss of t-tubular I Ca and loss of regulation of I Ca by caveolin 3. Thus, the pathological loss of t-tubular I Ca , which contributes to impaired excitation-contraction coupling and thereby cardiac function in vivo, appears to be attributable to loss of caveolin 3-dependent stimulation of t-tubular I Ca . Previous work has shown redistribution of L-type Ca 2+ current (I Ca ) from the t-tubules to the surface membrane of rat ventricular myocytes after myocardial infarction. However, whether this occurs in all species and in response to other insults, the relationship of this redistribution to the severity of the pathology, and the underlying mechanism, are unknown. We have therefore investigated the response of mouse hearts and myocytes to pressure overload induced by transverse aortic constriction (TAC). Male C57BL/6 mice underwent TAC or equivalent sham operation 8 weeks before use. I Ca and Ca 2+ transients were measured in isolated myocytes, and expression of caveolin 3 (Cav3), junctophilin 2 (Jph2) and bridging integrator 1 (Bin1) was determined. C3SD peptide was used to disrupt Cav3 binding to its protein partners. Some animals showed cardiac hypertrophy in response to TAC with little evidence of heart failure, whereas others showed greater hypertrophy and pulmonary congestion. These graded changes were accompanied by graded cellular hypertrophy, t-tubule disruption, decreased expression of Jph2 and Cav3, and decreased t-tubular I Ca density, with no change at the cell surface, and graded impairment of Ca 2+ release at t-tubules. C3SD decreased I Ca density in control but not in TAC myocytes. These data suggest that the graded changes in cardiac function and size that occur in response to TAC are paralleled by graded changes in cell structure and function, which will contribute to the impaired function observed in vivo. They also suggest that loss of t-tubular I Ca is attributable to loss of Cav3-dependent stimulation of I Ca . © 2018 The Authors. Experimental Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.

Comparative Efficacy of Intracoronary Allogeneic Mesenchymal Stem Cells and Cardiosphere-Derived Cells in Swine with Hibernating Myocardium

PubMed Central

Weil, Brian R.; Suzuki, Gen; Leiker, Merced M.; Fallavollita, James A.; Canty, John M.

2015-01-01

Rationale Allogeneic bone marrow-derived mesenchymal stem cells (MSCs) and cardiosphere-derived cells (CDCs) have each entered clinical trials but a direct comparison of these cell types has not been performed in a large animal model of hibernating myocardium. Objective Using completely blinded methodology, compare the efficacy of global intracoronary allogeneic MSCs (icMSCs, ~35×106) and CDCs (icCDCs, ~35×106) vs. vehicle in cyclosporine-immunosuppressed swine with a chronic LAD stenosis (n=26). Methods and Results Studies began 3-months after instrumentation when wall-thickening (%WT) was reduced (LAD%WT 38±11% (mean ± SD) vs. 83±26% in remote, p<0.01) and similar among groups. Four-weeks after treatment, LAD%WT increased similarly following icCDCs and icMSCs, while it remained depressed in vehicle-treated controls (icMSCs: 51±13%; icCDCs: 51±17%; vehicle: 34±3%, treatments p<0.05 vs. vehicle). There was no change in myocardial perfusion. Both icMSCs and icCDCs increased LAD myocyte nuclear density (icMSCs: 1601±279 nuclei/mm2, icCDCs: 1569±294 nuclei/mm2, vehicle: 973±181 nuclei/mm2, treatments p<0.05 vs. vehicle) and reduced myocyte diameter (icMSCs: 16.4±1.5 μm, icCDCs: 16.8±1.2 μm, vehicle: 20.2±3.7 μm, treatments p<0.05 vs. vehicle) to the same extent. Similar changes in myocyte nuclear density and diameter were observed in the remote region of cell-treated animals. Cell fate analysis using Y-FISH demonstrated rare cells from sex-mismatched donors. Conclusions Allogeneic icMSCs and icCDCs exhibit comparable therapeutic efficacy in a large animal model of hibernating myocardium. Both cell types produced equivalent increases in regional function and stimulated myocyte regeneration in ischemic and remote myocardium. The activation of endogenous myocyte proliferation and regression of myocyte cellular hypertrophy support a common mechanism of cardiac repair. PMID:26271689

Transcriptional Reversion of Cardiac Myocyte Fate During Mammalian Cardiac Regeneration

PubMed Central

O’Meara, Caitlin C.; Wamstad, Joseph A.; Gladstone, Rachel; Fomovsky, Gregory M.; Butty, Vincent L.; Shrikumar, Avanti; Gannon, Joseph; Boyer, Laurie A.; Lee, Richard T.

2014-01-01

Rationale Neonatal mice have the capacity to regenerate their hearts in response to injury, but this potential is lost after the first week of life. The transcriptional changes that underpin mammalian cardiac regeneration have not been fully characterized at the molecular level. Objective The objectives of our study were to determine if myocytes revert the transcriptional phenotype to a less differentiated state during regeneration and to systematically interrogate the transcriptional data to identify and validate potential regulators of this process. Methods and Results We derived a core transcriptional signature of injury-induced cardiac myocyte regeneration in mouse by comparing global transcriptional programs in a dynamic model of in vitro and in vivo cardiac myocyte differentiation, in vitro cardiac myocyte explant model, as well as a neonatal heart resection model. The regenerating mouse heart revealed a transcriptional reversion of cardiac myocyte differentiation processes including reactivation of latent developmental programs similar to those observed during de-stabilization of a mature cardiac myocyte phenotype in the explant model. We identified potential upstream regulators of the core network, including interleukin 13 (IL13), which induced cardiac myocyte cell cycle entry and STAT6/STAT3 signaling in vitro. We demonstrate that STAT3/periostin and STAT6 signaling are critical mediators of IL13 signaling in cardiac myocytes. These downstream signaling molecules are also modulated in the regenerating mouse heart. Conclusions Our work reveals new insights into the transcriptional regulation of mammalian cardiac regeneration and provides the founding circuitry for identifying potential regulators for stimulating heart regeneration. PMID:25477501

Single-Cell Functional Analysis of Stem-Cell Derived Cardiomyocytes on Micropatterned Flexible Substrates.

PubMed

Kijlstra, Jan David; Hu, Dongjian; van der Meer, Peter; Domian, Ibrahim J

2017-11-15

Human pluripotent stem-cell derived cardiomyocytes (hPSC-CMs) hold great promise for applications in human disease modeling, drug discovery, cardiotoxicity screening, and, ultimately, regenerative medicine. The ability to study multiple parameters of hPSC-CM function, such as contractile and electrical activity, calcium cycling, and force generation, is therefore of paramount importance. hPSC-CMs cultured on stiff substrates like glass or polystyrene do not have the ability to shorten during contraction, making them less suitable for the study of hPSC-CM contractile function. Other approaches require highly specialized hardware and are difficult to reproduce. Here we describe a protocol for the preparation of hPSC-CMs on soft substrates that enable shortening, and subsequently the simultaneous quantitative analysis of their contractile and electrical activity, calcium cycling, and force generation at single-cell resolution. This protocol requires only affordable and readily available materials and works with standard imaging hardware. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

Neutrophil adherence to isolated adult canine myocytes. Evidence for a CD18-dependent mechanism.

PubMed

Entman, M L; Youker, K; Shappell, S B; Siegel, C; Rothlein, R; Dreyer, W J; Schmalstieg, F C; Smith, C W

1990-05-01

Cardiac myocytes were isolated from adult dogs and incubated with isolated canine neutrophils (PMN). Intercellular adhesion was low and unchanged by stimulation of the PMN with zymosan activated serum or platelet activating factor (PAF) at concentrations that significantly enhance PMN adhesion to protein-coated glass and canine endothelial cell monolayers. Intercellular adhesion was significantly increased only when both myocytes and PMN were stimulated (e.g., myocytes incubated with IL-1, tumor necrosis factor, or phorbol myristate acetate, and PMN were chemotactically stimulated). Inhibitors of protein synthesis diminished the IL-1 beta-induced effect by greater than 80%. The IL-1 beta, PAF-stimulated PMN-myocyte adhesion was associated with substantial H2O2 production. Under conditions with low PMN-myocyte adhesion (i.e., IL-1 beta alone, PAF alone, or no stimulus) H2O2 production was generally less than 5% of that occurring with high adhesion. An anti-CD18 monoclonal antibody (R15.7) inhibited stimulated PMN-myocyte adhesion by greater than 95% and reduced H2O2 production by greater than 90%. Control isotype-matched, binding, and nonbinding antibodies were without effect on adherence or H2O2 production. The results indicate that cytokine stimulation of adult myocytes induces expression of a ligand involved in CD18-dependent adherence of canine neutrophils.

Connexin43 contributes to electrotonic conduction across scar tissue in the intact heart

NASA Astrophysics Data System (ADS)

Mahoney, Vanessa M.; Mezzano, Valeria; Mirams, Gary R.; Maass, Karen; Li, Zhen; Cerrone, Marina; Vasquez, Carolina; Bapat, Aneesh; Delmar, Mario; Morley, Gregory E.

2016-05-01

Studies have demonstrated non-myocytes, including fibroblasts, can electrically couple to myocytes in culture. However, evidence demonstrating current can passively spread across scar tissue in the intact heart remains elusive. We hypothesize electrotonic conduction occurs across non-myocyte gaps in the heart and is partly mediated by Connexin43 (Cx43). We investigated whether non-myocytes in ventricular scar tissue are electrically connected to surrounding myocardial tissue in wild type and fibroblast-specific protein-1 driven conditional Cx43 knock-out mice (Cx43fsp1KO). Electrical coupling between the scar and uninjured myocardium was demonstrated by injecting current into the myocardium and recording depolarization in the scar through optical mapping. Coupling was significantly reduced in Cx43fsp1KO hearts. Voltage signals were recorded using microelectrodes from control scars but no signals were obtained from Cx43fsp1KO hearts. Recordings showed significantly decreased amplitude, depolarized resting membrane potential, increased duration and reduced upstroke velocity compared to surrounding myocytes, suggesting that the non-excitable cells in the scar closely follow myocyte action potentials. These results were further validated by mathematical simulations. Optical mapping demonstrated that current delivered within the scar could induce activation of the surrounding myocardium. These data demonstrate non-myocytes in the scar are electrically coupled to myocytes, and coupling depends on Cx43 expression.

HD Exchange and PLIMSTEX Determine the Affinities and Order of Binding of Ca2+ with Troponin C

PubMed Central

Huang, Richard Y-C.; Rempel, Don L.; Gross, Michael L.

2011-01-01

Troponin C (TnC), present in all striated muscle, is the Ca2+-activated trigger that initiates myocyte contraction. The binding of Ca2+ to TnC initiates a cascade of conformational changes involving the constituent proteins of the thin filament. The functional properties of TnC and its ability to bind Ca2+ have significant regulatory influence on the contractile reaction of muscle. Changes in TnC may also correlate with cardiac and various other muscle-related diseases. We report here the implementation of the PLIMSTEX strategy (Protein Ligand Interaction by Mass Spectrometry, Titration and H/D Exchange) to elucidate the binding affinity of TnC with Ca2+ and, more importantly, to determine the order of Ca2+ binding of the four EF hands of the protein. The four equilibrium constants, K1 = (5 ± 5) × 10 M-1, K2 = (1.8 ± 0.8) × 107 M-1, K3 = (4.2 ± 0.9) × 106 M-1, and K4 = (1.6 ± 0.6) × 106 M-1, agree well with determinations by other methods and serve to increase our confidence in the PLIMSTEX approach. We determined the order of binding to the four EF hands to be III, IV, II, and I by extracting from the H/DX results the deuterium patterns for each EF hand for each state of the protein (apo through fully Ca2+ bound). This approach, demonstrated for the first time, may be general for determining binding orders of metal ions and other ligands to proteins. PMID:21574565

Protective effects of anisodamine on cigarette smoke extract-induced airway smooth muscle cell proliferation and tracheal contractility

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xu, Guang-Ni; Yang, Kai; Xu, Zu-Peng

2012-07-01

Anisodamine, an antagonist of muscarinic acetylcholine receptors (mAChRs), has been used therapeutically to improve smooth muscle function, including microvascular, intestinal and airway spasms. Our previous studies have revealed that airway hyper-reactivity could be prevented by anisodamine. However, whether anisodamine prevents smoking-induced airway smooth muscle (ASM) cell proliferation remained unclear. In this study, a primary culture of rat ASM cells was used to evaluate an ASM phenotype through the ability of the cells to proliferate and express contractile proteins in response to cigarette smoke extract (CSE) and intervention of anisodamine. Our results showed that CSE resulted in an increase in cyclinmore » D1 expression concomitant with the G0/G1-to-S phase transition, and high expression of M2 and M3. Functional studies showed that tracheal hyper-contractility accompanied contractile marker α-SMA high-expression. These changes, which occur only after CSE stimulation, were prevented and reversed by anisodamine, and CSE-induced cyclin D1 expression was significantly inhibited by anisodamine and the specific inhibitor U0126, BAY11-7082 and LY294002. Thus, we concluded that the protective and reversal effects and mechanism of anisodamine on CSE-induced events might involve, at least partially, the ERK, Akt and NF-κB signaling pathways associated with cyclin D1 via mAChRs. Our study validated that anisodamine intervention on ASM cells may contribute to anti-remodeling properties other than bronchodilation. -- Highlights: ► CSE induces tracheal cell proliferation, hyper-contractility and α-SMA expression. ► Anisodamine reverses CSE-induced tracheal hyper-contractility and cell proliferation. ► ERK, PI3K, and NF-κB pathways and cyclin D1 contribute to the reversal effect.« less

Activation of Akt rescues endoplasmic reticulum stress-impaired murine cardiac contractile function via glycogen synthase kinase-3β-mediated suppression of mitochondrial permeation pore opening.

PubMed

Zhang, Yingmei; Xia, Zhi; La Cour, Karissa H; Ren, Jun

2011-11-01

The present study was designed to examine the impact of chronic Akt activation on endoplasmic reticulum (ER) stress-induced cardiac mechanical anomalies, if any, and the underlying mechanism involved. Wild-type and transgenic mice with cardiac-specific overexpression of the active mutant of Akt (Myr-Akt) were subjected to the ER stress inducer tunicamycin (1 or 3 mg/kg). ER stress led to compromised echocardiographic (elevated left ventricular end-systolic diameter and reduced fractional shortening) and cardiomyocyte contractile function, intracellular Ca(2+) mishandling, and cell survival in wild-type mice associated with mitochondrial damage. In vitro ER stress induction in murine cardiomyocytes upregulated the ER stress proteins Gadd153, GRP78, and phospho-eIF2α, and promoted reactive oxygen species production, carbonyl formation, apoptosis, mitochondrial membrane potential loss, and mitochondrial permeation pore (mPTP) opening associated with overtly impaired cardiomyocyte contractile and intracellular Ca(2+) properties. Interestingly, these anomalies were mitigated by chronic Akt activation or the ER chaperon tauroursodeoxycholic acid (TUDCA). Treatment with tunicamycin also dephosphorylated Akt and its downstream signal glycogen synthase kinase 3β (GSK3β) (leading to activation of GSK3β), the effect of which was abrogated by Akt activation and TUDCA. The ER stress-induced cardiomyocyte contractile and mitochondrial anomalies were obliterated by the mPTP inhibitor cyclosporin A, GSK3β inhibitor SB216763, and ER stress inhibitor TUDCA. This research reported the direct relationship between ER stress and cardiomyocyte contractile and mitochondrial anomalies for the first time. Taken together, these data suggest that ER stress may compromise cardiac contractile and intracellular Ca(2+) properties, possibly through the Akt/GSK3β-dependent impairment of mitochondrial integrity.

T-tubule disease: Relationship between t-tubule organization and regional contractile performance in human dilated cardiomyopathy.

PubMed

Crossman, David J; Young, Alistair A; Ruygrok, Peter N; Nason, Guy P; Baddelely, David; Soeller, Christian; Cannell, Mark B

2015-07-01

Evidence from animal models suggest that t-tubule changes may play an important role in the contractile deficit associated with heart failure. However samples are usually taken at random with no regard as to regional variability present in failing hearts which leads to uncertainty in the relationship between contractile performance and possible t-tubule derangement. Regional contraction in human hearts was measured by tagged cine MRI and model fitting. At transplant, failing hearts were biopsy sampled in identified regions and immunocytochemistry was used to label t-tubules and sarcomeric z-lines. Computer image analysis was used to assess 5 different unbiased measures of t-tubule structure/organization. In regions of failing hearts that showed good contractile performance, t-tubule organization was similar to that seen in normal hearts, with worsening structure correlating with the loss of regional contractile performance. Statistical analysis showed that t-tubule direction was most highly correlated with local contractile performance, followed by the amplitude of the sarcomeric peak in the Fourier transform of the t-tubule image. Other area based measures were less well correlated. We conclude that regional contractile performance in failing human hearts is strongly correlated with the local t-tubule organization. Cluster tree analysis with a functional definition of failing contraction strength allowed a pathological definition of 't-tubule disease'. The regional variability in contractile performance and cellular structure is a confounding issue for analysis of samples taken from failing human hearts, although this may be overcome with regional analysis by using tagged cMRI and biopsy mapping. Copyright © 2015 Elsevier Ltd. All rights reserved.

PDE4 and mAKAPβ are nodal organizers of β2-ARs nuclear PKA signaling in cardiac myocytes.

PubMed

Bedioune, Ibrahim; Lefebvre, Florence; Lechêne, Patrick; Varin, Audrey; Domergue, Valérie; Kapiloff, Michael S; Fischmeister, Rodolphe; Vandecasteele, Grégoire

2018-05-03

β1- and β2-adrenergic receptors (β-ARs) produce different acute contractile effects on the heart partly because they impact on different cytosolic pools of cAMP-dependent protein kinase (PKA). They also exert different effects on gene expression but the underlying mechanisms remain unknown. The aim of this study was to understand the mechanisms by which β1- and β2-ARs regulate nuclear PKA activity in cardiomyocytes. We used cytoplasmic and nuclear targeted biosensors to examine cAMP signals and PKA activity in adult rat ventricular myocytes upon selective β1- or β2-ARs stimulation. Both β1- and β2-AR stimulation increased cAMP and activated PKA in the cytoplasm. While the two receptors also increased cAMP in the nucleus, only β1-ARs increased nuclear PKA activity and up-regulated the PKA target gene and pro-apoptotic factor, inducible cAMP element repressor (ICER). Inhibition of PDE4, but not Gi, PDE3, GRK2 nor caveolae disruption disclosed nuclear PKA activation and ICER induction by β2-ARs. Both nuclear and cytoplasmic PKI prevented nuclear PKA activation and ICER induction by β1-ARs, indicating that PKA activation outside the nucleus is required for subsequent nuclear PKA activation and ICER mRNA expression. Cytoplasmic PKI also blocked ICER induction by β2-AR stimulation (with concomitant PDE4 inhibition). However, in this case nuclear PKI decreased ICER up-regulation by only 30%, indicating that other mechanisms are involved. Down-regulation of mAKAPβ partially inhibited nuclear PKA activation upon β1-AR stimulation, and drastically decreased nuclear PKA activation upon β2-AR stimulation in the presence of PDE4 inhibition. β1- and β2-ARs differentially regulate nuclear PKA activity and ICER expression in cardiomyocytes. PDE4 insulates a mAKAPβ-targeted PKA pool at the nuclear envelope that prevents nuclear PKA activation upon β2-AR stimulation.

Pressure-volume analysis reveals characteristic sex-related differences in cardiac function in a rat model of aortic banding-induced myocardial hypertrophy.

PubMed

Ruppert, Mihály; Korkmaz-Icöz, Sevil; Loganathan, Sivakkanan; Jiang, Weipeng; Lehmann, Lorenz H; Oláh, Attila; Sayour, Alex Ali; Barta, Bálint András; Merkely, Béla; Karck, Matthias; Radovits, Tamás; Szabó, Gábor

2018-05-25

Sex differences in pressure overload (PO)-induced left ventricular (LV) myocardial hypertrophy (LVH) have been intensely investigated. Nevertheless, sex-related disparities of LV hemodynamics in LVH were not examined in detail. Therefore, we aimed to provide a detailed characterization of distinct aspects of LV function in male and female rats during different stages of LVH. Banding of the abdominal aorta (AB) was performed to induce PO for 6 or 12 weeks in male and female rats. Control animals underwent sham operation. The development of LVH was followed by serial echocardiography. Cardiac function was assessed by pressure-volume analysis. Cardiomyocyte hypertrophy and fibrosis were evaluated by histology. At week 6, increased LV mass index, heart weight-to-tibial length, cardiomyocyte diameter, concentric LV geometry and moderate interstitial fibrosis were detected in both male and female AB rats, indicating the development of an early stage of LVH. Functionally, at this time point, impaired active relaxation, increased contractility and preserved ventricular-arterial coupling were observed in the AB groups in both genders. In contrast, at week 12, progressive deterioration of LVH-associated structural and functional alterations occurred in male but not in female animals with sustained PO. Accordingly, at this later stage, LVH was associated with eccentric remodeling, exacerbated fibrosis and increased chamber stiffness in male AB rats. Furthermore, augmented contractility declined in male and not in female AB animals, resulting in contractility-afterload mismatch. Maintained contractility augmentation, preserved ventricular-arterial coupling and better myocardial compliance in female rats contribute to sex differences in LV function during the progression of PO-induced LVH.

Does vector-free gravity simulate microgravity? Functional and morphologic attributes of clinorotated nerve and muscle grown in cell culture

NASA Technical Reports Server (NTRS)

Gruener, R.; Hoeger, G.

1988-01-01

Cocultured Xenopus neurons and myocytes were subjected to non-vectorial gravity by clinostat rotation to determine if microgravity, during space flights, may affect cell development and communications. Clinorotated cells showed changes consistent with the hypothesis that cell differentiation, in microgravity, is altered by interference with cytoskeleton-related mechanisms. We found: increases in the myocyte and its nuclear area, "fragmentation" of nucleoli, appearance of neuritic "aneurysms", decreased growth in the presence of "trophic" factors, and decreased yolk utilization. The effects were most notable at 1-10 rpm and depended on the onset and duration of rotation. Some parameters returned to near control values within 48 hrs after cessation of rotation. Cells from cultures rotated at higher speeds (>50 rpm) appeared comparable to controls. Compensation by centrifugal forces may account for this finding. Our data are consistent, in principle, with effects on other, flighted cells and suggest that "vector-free" gravity may simulate certain aspects of microgravity. The distribution of acetylcholine receptor aggregates, on myocytes, was also altered. This indicates that brain development, in microgravity, may also be affected.

Compensatory Hypertrophy Induced by Ventricular Cardiomyocyte Specific COX-2 Expression in Mice

PubMed Central

Streicher, John M.; Kamei, Kenichiro; Ishikawa, Tomo-o; Herschman, Harvey; Wang, Yibin

2010-01-01

Cyclooxygenase-2 (COX-2) is an important mediator of inflammation in stress and disease states. Recent attention has focused on the role of COX-2 in human heart failure and diseases, due to the finding that highly specific COX-2 inhibitors (i.e. Vioxx) increased the risk of myocardial infarction and stroke in chronic users. However, the specific impact of COX-2 expression in the intact heart remains to be determined. We report here the development of a transgenic mouse model, using a loxP-Cre approach, that displays robust COX-2 overexpression and subsequent prostaglandin synthesis specifically in ventricular myocytes. Histological, functional and molecular analyses showed that ventricular myocyte specific COX-2 overexpression led to cardiac hypertrophy and fetal gene marker activation, but with preserved cardiac function. Therefore, specific induction of COX-2 and prostaglandin in vivo is sufficient to induce compensated hypertrophy and molecular remodeling. PMID:20170663

Dynamic Action Potential Restitution Contributes to Mechanical Restitution in Right Ventricular Myocytes From Pulmonary Hypertensive Rats.

PubMed

Hardy, Matthew E L; Pervolaraki, Eleftheria; Bernus, Olivier; White, Ed

2018-01-01

We investigated the steepened dynamic action potential duration (APD) restitution of rats with pulmonary artery hypertension (PAH) and right ventricular (RV) failure and tested whether the observed APD restitution properties were responsible for negative mechanical restitution in these myocytes. PAH and RV failure were provoked in male Wistar rats by a single injection of monocrotaline (MCT) and compared with saline-injected animals (CON). Action potentials were recorded from isolated RV myocytes at stimulation frequencies between 1 and 9 Hz. Action potential waveforms recorded at 1 Hz were used as voltage clamp profiles (action potential clamp) at stimulation frequencies between 1 and 7 Hz to evoke rate-dependent currents. Voltage clamp profiles mimicking typical CON and MCT APD restitution were applied and cell shortening simultaneously monitored. Compared with CON myocytes, MCT myocytes were hypertrophied; had less polarized diastolic membrane potentials; had action potentials that were triggered by decreased positive current density and shortened by decreased negative current density; APD was longer and APD restitution steeper. APD90 restitution was unchanged by exposure to the late Na + -channel blocker (5 μM) ranolazine or the intracellular Ca 2+ buffer BAPTA. Under AP clamp, stimulation frequency-dependent inward currents were smaller in MCT myocytes and were abolished by BAPTA. In MCT myocytes, increasing stimulation frequency decreased contraction amplitude when depolarization duration was shortened, to mimic APD restitution, but not when depolarization duration was maintained. We present new evidence that the membrane potential of PAH myocytes is less stable than normal myocytes, being more easily perturbed by external currents. These observations can explain increased susceptibility to arrhythmias. We also present novel evidence that negative APD restitution is at least in part responsible for the negative mechanical restitution in PAH myocytes. Thus, our study links electrical restitution remodeling to a defining mechanical characteristic of heart failure, the reduced ability to respond to an increase in demand.

Phosphodiesterase-5 inhibitor sildenafil preconditions adult cardiac myocytes against necrosis and apoptosis. Essential role of nitric oxide signaling.

PubMed

Das, Anindita; Xi, Lei; Kukreja, Rakesh C

2005-04-01

We investigated the effect of sildenafil in protection against necrosis or apoptosis in cardiomyocytes. Adult mouse ventricular myocytes were treated with sildenafil (1 or 10 microM) for 1 h before 40 min of simulated ischemia (SI). Necrosis was determined by trypan blue exclusion and lactate dehydrogenase release following SI alone or plus 1 or 18 h of reoxygenation (RO). Apoptosis was assessed by terminal deoxynucleotidyl transferase-mediated nick end labeling assay and mitochondrial membrane potential measured using a fluorescent probe 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl-carbocyanine iodide (JC-1). Sildenafil reduced necrosis as indicated by decrease in trypan blue-positive myocytes and leakage of lactate dehydrogenase compared with untreated cells after either SI or SI-RO. The number of terminal deoxynucleotidyl transferase-mediated nick end labeling-positive myocytes or loss of JC-1 fluorescence following SI and 18 h of RO was attenuated in the sildenafil-treated group with concomitant inhibition of caspase 3 activity. An early increase in Bcl-2 to Bax ratio with sildenafil treatment was also observed in myocytes after SI-RO. The increase of Bcl-2 expression by sildenafil was inhibited by nitric-oxide synthase (NOS) inhibitor, L-nitro-amino-methyl-ester. The drug also enhanced mRNA and protein content of inducible NOS (iNOS) and endothelial NOS (eNOS) in the myocytes. Sildenafil-induced protection against necrosis and apoptosis was absent in the myocytes derived from iNOS knock-out mice and was attenuated in eNOS knock-out myocytes. The up-regulation of Bcl-2 expression by sildenafil was also absent in iNOS-deficient myocytes. Reverse transcription-PCR, Western blots, and immunohistochemical assay confirmed the expression of phosphodiesterase-5 in mouse cardiomyocytes. These data provide strong evidence for a direct protective effect of sildenafil against necrosis and apoptosis through NO signaling pathway. The results may have possible therapeutic potential in preventing myocyte cell death following ischemia/reperfusion.

Identification of the functional domain in the transcription factor RTEF-1 that mediates alpha 1-adrenergic signaling in hypertrophied cardiac myocytes.

PubMed

Ueyama, T; Zhu, C; Valenzuela, Y M; Suzow, J G; Stewart, A F

2000-06-09

Cardiac myocytes respond to alpha(1)-adrenergic receptor stimulation by a progressive hypertrophy accompanied by the activation of many fetal genes, including skeletal muscle alpha-actin. The skeletal muscle alpha-actin gene is activated by signaling through an MCAT element, the binding site of the transcription enhancer factor-1 (TEF-1) family of transcription factors. Previously, we showed that overexpression of the TEF-1-related factor (RTEF-1) increased the alpha(1)-adrenergic response of the skeletal muscle alpha-actin promoter, whereas TEF-1 overexpression did not. Here, we identified the functional domains and specific sequences in RTEF-1 that mediate the alpha(1)-adrenergic response. Chimeric TEF-1 and RTEF-1 expression constructs localized the region responsible for the alpha(1)-adrenergic response to the carboxyl-terminal domain of RTEF-1. Site-directed mutagenesis was used to inactivate eight serine residues of RTEF-1, not present in TEF-1, that are putative targets of alpha(1)-adrenergic-dependent kinases. Mutation of a single serine residue, Ser-322, reduced the alpha(1)-adrenergic activation of RTEF-1 by 70% without affecting protein stability, suggesting that phosphorylation at this serine residue accounts for most of the alpha(1)-adrenergic response. Thus, these results demonstrate that RTEF-1 is a direct target of alpha(1)-adrenergic signaling in hypertrophied cardiac myocytes.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Allen, I.S.; Gaa, S.T.; Rogers, T.B.

The muscarinic cholinergic agonist, carbachol, and pertussis toxin were used to examine the functional status of the guanine nucleotide-binding protein that inhibits adenylate cyclase (G{sub i}) in cultured neonatal rat heart myocytes. The isoproterenol stimulation of adenylate cyclase activity in myocyte membranes and adenosine 3{prime},5{prime}-cyclic monophosphate (cAMP) accumulation in intact cells (4 days in culture) were insensitive to carbachol. However, in cells cultured for 11 days, carbachol inhibited isoproterenol-stimulated cAMP accumulation by 30%. Angiotensin II (ANG II) was also found to inhibit isoproterenol-stimulated cAMP accumulation in day 11 cells in a dose-dependent manner. Pertussis toxin treatment reversed the inhibitory effectsmore » of both ANG II and carbachol, suggesting a role for G{sub i} in the process. Carbachol binding to membranes from day 4 cells was relatively insensitive to guanine nucleotides when compared with binding to membranes from day 11 or adult cells. Furthermore, pertussis toxin-mediated {sup 32}P incorporation into a 39- to 41-kDa substrate in day 11 membranes was increased 3.2-fold over that measured in day 4 membranes. These findings support the view that, although G{sub i} is expressed, it is nonfunctional in 4-day-old cultured neonatal rat heart myocytes and acquisition of functional G{sub i} is dependent on culture conditions. Furthermore, the ANG II receptor can couple to G{sub i} in heart.« less

The effect of PKA-mediated phosphorylation of ryanodine receptor on SR Ca2+ leak in ventricular myocytes.

PubMed

Bovo, Elisa; Huke, Sabine; Blatter, Lothar A; Zima, Aleksey V

2017-03-01

Functional impact of cardiac ryanodine receptor (type 2 RyR or RyR2) phosphorylation by protein kinase A (PKA) remains highly controversial. In this study, we characterized a functional link between PKA-mediated RyR2 phosphorylation level and sarcoplasmic reticulum (SR) Ca 2+ release and leak in permeabilized rabbit ventricular myocytes. Changes in cytosolic [Ca 2+ ] and intra-SR [Ca 2+ ] SR were measured with Fluo-4 and Fluo-5N, respectively. Changes in RyR2 phosphorylation at two PKA sites, serine-2031 and -2809, were measured with phospho-specific antibodies. cAMP (10μM) increased Ca 2+ spark frequency approximately two-fold. This effect was associated with an increase in SR Ca 2+ load from 0.84 to 1.24mM. PKA inhibitory peptide (PKI; 10μM) abolished the cAMP-dependent increase of SR Ca 2+ load and spark frequency. When SERCA was completely blocked by thapsigargin, cAMP did not affect RyR2-mediated Ca 2+ leak. The lack of a cAMP effect on RyR2 function can be explained by almost maximal phosphorylation of RyR2 at serine-2809 after sarcolemma permeabilization. This high RyR2 phosphorylation level is likely the consequence of a balance shift between protein kinase and phosphatase activity after permeabilization. When RyR2 phosphorylation at serine-2809 was reduced to its "basal" level (i.e. RyR2 phosphorylation level in intact myocytes) using kinase inhibitor staurosporine, SR Ca 2+ leak was significantly reduced. Surprisingly, further dephosphorylation of RyR2 with protein phosphatase 1 (PP1) markedly increased SR Ca 2+ leak. At the same time, phosphorylation of RyR2 at serine 2031 did not significantly change under identical experimental conditions. These results suggest that RyR2 phosphorylation by PKA has a complex effect on SR Ca 2+ leak in ventricular myocytes. At an intermediate level of RyR2 phosphorylation SR Ca 2+ leak is minimal. However, complete dephosphorylation and maximal phosphorylation of RyR2 increases SR Ca 2+ leak. Copyright © 2017 Elsevier Ltd. All rights reserved.

Molecular Regulation of Parturition: A Myometrial Perspective

PubMed Central

Renthal, Nora E.; Williams, Koriand’r C.; Montalbano, Alina P.; Chen, Chien-Cheng; Gao, Lu; Mendelson, Carole R.

2015-01-01

The molecular mechanisms that maintain quiescence of the myometrium throughout most of pregnancy and promote its transformation to a highly coordinated contractile unit culminating in labor are complex and intertwined. During pregnancy, progesterone (P4) produced by the placenta and/or ovary serves a dominant role in maintaining myometrial quiescence by blocking proinflammatory response pathways and expression of so-called “contractile” genes. In the majority of placental mammals, increased uterine contractility near term is heralded by an increase in circulating estradiol-17β (E2) and/or increased estrogen receptor α (ERα) activity and a sharp decline in circulating P4 levels. However, in women, circulating levels of P4 and progesterone receptors (PR) in myometrium remain elevated throughout pregnancy and into labor. This has led to the concept that increased uterine contractility leading to term and preterm labor is mediated, in part, by a decline in PR function. The biochemical mechanisms for this decrease in PR function are also multifaceted and interwoven. In this paper, we focus on the molecular mechanisms that mediate myometrial quiescence and contractility and their regulation by the two central hormones of pregnancy, P4 and estradiol-17β. The integrative roles of microRNAs also are considered. PMID:26337112

Probing the contractile vacuole as Achilles' heel of the biotrophic grapevine pathogen Plasmopara viticola.

PubMed

Tröster, Viktoria; Setzer, Tabea; Hirth, Thomas; Pecina, Anna; Kortekamp, Andreas; Nick, Peter

2017-09-01

The causative agent of Grapevine Downy Mildew, the oomycete Plasmopara viticola, poses a serious threat to viticulture. In the current work, the contractile vacuole of the zoospore is analysed as potential target for novel plant protection strategies. Using a combination of electron microscopy, spinning disc confocal microscopy, and video differential interference contrast microscopy, we have followed the genesis and dynamics of this vacuole required during the search for the stomata, when the non-walled zoospore is exposed to hypotonic conditions. This subcellular description was combined with a pharmacological study, where the functionality of the contractile vacuole was blocked by manipulation of actin, by Na, Cu, and Al ions or by inhibition of the NADPH oxidase. We further observe that RGD peptides (mimicking binding sites for integrins at the extracellular matrix) can inhibit the function of the contractile vacuole as well. Finally, we show that an extract from Chinese liquorice (Glycyrrhiza uralensis) proposed as biocontrol for Downy Mildews can efficiently induce zoospore burst and that this activity depends on the activity of NADPH oxidase. The effect of the extract can be phenocopied by its major compound, glycyrrhizin, suggesting a mode of action for this biologically safe alternative to copper products.

Moderate ethanol administration accentuates cardiomyocyte contractile dysfunction and mitochondrial injury in high fat diet-induced obesity.

PubMed

Yuan, Fang; Lei, Yonghong; Wang, Qiurong; Esberg, Lucy B; Huang, Zaixing; Scott, Glenda I; Li, Xue; Ren, Jun

2015-03-18

Light to moderate drinking confers cardioprotection although it remains unclear with regards to the role of moderate drinking on cardiac function in obesity. This study was designed to examine the impact of moderate ethanol intake on myocardial function in high fat diet intake-induced obesity and the mechanism(s) involved with a focus on mitochondrial integrity. C57BL/6 mice were fed low or high fat diet for 16 weeks prior to ethanol challenge (1g/kg/d for 3 days). Cardiac contractile function, intracellular Ca(2+) homeostasis, myocardial histology, and mitochondrial integrity [aconitase activity and the mitochondrial proteins SOD1, UCP-2 and PPARγ coactivator 1α (PGC-1α)] were assessed 24h after the final ethanol challenge. Fat diet intake compromised cardiomyocyte contractile and intracellular Ca(2+) properties (depressed peak shortening and maximal velocities of shortening/relengthening, prolonged duration of relengthening, dampened intracellular Ca(2+) rise and clearance without affecting duration of shortening). Although moderate ethanol challenge failed to alter cardiomyocyte mechanical property under low fat diet intake, it accentuated high fat diet intake-induced changes in cardiomyocyte contractile function and intracellular Ca(2+) handling. Moderate ethanol challenge failed to affect fat diet intake-induced cardiac hypertrophy as evidenced by H&E staining. High fat diet intake reduced myocardial aconitase activity, downregulated levels of mitochondrial protein UCP-2, PGC-1α, SOD1 and interrupted intracellular Ca(2+) regulatory proteins, the effect of which was augmented by moderate ethanol challenge. Neither high fat diet intake nor moderate ethanol challenge affected protein or mRNA levels as well as phosphorylation of Akt and GSK3β in mouse hearts. Taken together, our data revealed that moderate ethanol challenge accentuated high fat diet-induced cardiac contractile and intracellular Ca(2+) anomalies as well as mitochondrial injury. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Divergent Requirements for EZH1 in Heart Development Versus Regeneration.

PubMed

Ai, Shanshan; Yu, Xianhong; Li, Yumei; Peng, Yong; Li, Chen; Yue, Yanzhu; Tao, Ge; Li, Chuanyun; Pu, William T; He, Aibin

2017-07-07

Polycomb repressive complex 2 is a major epigenetic repressor that deposits methylation on histone H3 on lysine 27 (H3K27me) and controls differentiation and function of many cells, including cardiac myocytes. EZH1 and EZH2 are 2 alternative catalytic subunits with partial functional redundancy. The relative roles of EZH1 and EZH2 in heart development and regeneration are unknown. We compared the roles of EZH1 versus EZH2 in heart development and neonatal heart regeneration. Heart development was normal in Ezh1 -/- ( Ezh 1 knockout) and Ezh2 f/f ::cTNT -Cre ( Ezh 2 knockout) embryos. Ablation of both genes in Ezh1 -/- ::Ezh2 f/f ::cTNT -Cre embryos caused lethal heart malformations, including hypertrabeculation, compact myocardial hypoplasia, and ventricular septal defect. Epigenome and transcriptome profiling showed that derepressed genes were upregulated in a manner consistent with total EZH dose. In neonatal heart regeneration, Ezh1 was required, but Ezh2 was dispensable. This finding was further supported by rescue experiments: cardiac myocyte-restricted re-expression of EZH1 but not EZH2 restored neonatal heart regeneration in Ezh 1 knockout. In myocardial infarction performed outside of the neonatal regenerative window, EZH1 but not EZH2 likewise improved heart function and stimulated cardiac myocyte proliferation. Mechanistically, EZH1 occupied and activated genes related to cardiac growth. Our work unravels divergent mechanisms of EZH1 in heart development and regeneration, which will empower efforts to overcome epigenetic barriers to heart regeneration. © 2017 American Heart Association, Inc.

[Heart functions in monkeys during a 2-week antiorthostatic hypokinesia

NASA Technical Reports Server (NTRS)

Krotov, V. P.; Convertino, V.; Korol'kov, V. I.; Latham, R.; Trambovetskii, E. V.; Fanton, J.; Crisman, R.; Truzhennikov, A. N.; Evert, D.; Nosovskii, A. M.;

Developmental potential of muscle cell progenitors and the myogenic factor SUM-1 in the sea urchin embryo.

PubMed

Venuti, J M; Gan, L; Kozlowski, M T; Klein, W H

1993-04-01

During sea urchin development, esophageal muscle arises from secondary mesenchyme cells, descendants of the vegetal plate that delaminate from the coelomic epithelium at the end of gastrulation. In lithium-induced exogastrulae, where vegetal plate descendants evert rather than invaginate, myogenesis occurs normally, indicating that myocyte progenitors do not have to be near the future stomodeum for differentiation to occur. Vegetal plate descendants isolated along with the extracellular matrix at different times during gastrulation produce differentiated myocytes in culture as monitored by staining with a myosin heavy chain antibody. Vegetal isolates prepared at mid-gastrulation or later consistently produce differentiated myocytes whose form and position resembled their counterparts in the intact embryo, whereas vegetal isolates prepared a few hours earlier while capable of gut differentiation, as evidenced by the de novo synthesis of the endodermal surface marker Endo 1, did not produce differentiated myocytes. These results suggest that sometime after early gastrulation, a subset of secondary mesenchyme cells are competent to differentiate into muscle cells. RNase protection assays showed that the accumulation of sea urchin myogenic factor (SUM-1) mRNA is likely to be coincident with the earliest demonstrable commitment of myogenic precursors. Premature expression of SUM-1 coding sequences in mesenchyme blastulae resulted in the activation of muscle-specific enhancer elements, demonstrating that SUM-1 can function precociously in the early embryo. However, SUM-1 expressed in this manner did not activate the endogenous MHC gene, nor induce premature or ectopic production of muscle cells.

Wnt/β-catenin signaling directs the regional expansion of first and second heart field-derived ventricular cardiomyocytes

PubMed Central

Buikema, Jan Willem; Mady, Ahmed S.; Mittal, Nikhil V.; Atmanli, Ayhan; Caron, Leslie; Doevendans, Pieter A.; Sluijter, Joost P. G.; Domian, Ibrahim J.

2013-01-01

In mammals, cardiac development proceeds from the formation of the linear heart tube, through complex looping and septation, all the while increasing in mass to provide the oxygen delivery demands of embryonic growth. The developing heart must orchestrate regional differences in cardiomyocyte proliferation to control cardiac morphogenesis. During ventricular wall formation, the compact myocardium proliferates more vigorously than the trabecular myocardium, but the mechanisms controlling such regional differences among cardiomyocyte populations are not understood. Control of definitive cardiomyocyte proliferation is of great importance for application to regenerative cell-based therapies. We have used murine and human pluripotent stem cell systems to demonstrate that, during in vitro cellular differentiation, early ventricular cardiac myocytes display a robust proliferative response to β-catenin-mediated signaling and conversely accelerate differentiation in response to inhibition of this pathway. Using gain- and loss-of-function murine genetic models, we show that β-catenin controls ventricular myocyte proliferation during development and the perinatal period. We further demonstrate that the differential activation of the Wnt/β-catenin signaling pathway accounts for the observed differences in the proliferation rates of the compact versus the trabecular myocardium during normal cardiac development. Collectively, these results provide a mechanistic explanation for the differences in localized proliferation rates of cardiac myocytes and point to a practical method for the generation of the large numbers of stem cell-derived cardiac myocytes necessary for clinical applications. PMID:24026118

Multiparity modifies contractile properties of pelvic muscles affecting the genesis of vaginal pressure in rabbits.

PubMed

López-Juárez, Rhode; Zempoalteca, René; Corona-Quintanilla, Dora Luz; Jiménez-Estrada, Ismael; Castelán, Francisco; Martínez-Gómez, Margarita

2018-01-01

To characterize the contractile properties of the bulbospongiosus (Bsm), isquiocavernosus (Ism), and pubococcygeus muscles (Pcm), and their involvement in the genesis of vaginal pressure in nulliparous and multiparous rabbits. Age-matched nulliparous and multiparous rabbits were used to record the isometric contractile responses of each muscle as well as the intravaginal pressure evoked by single square electrical pulses and stimulation trains of ascending frequency. To establish significant differences between groups, two-tail unpaired Student t tests were carried out. The linear correlation between intravaginal pressure and muscle contractile force was analyzed with Pearson correlation tests. For all cases, a P ≤ 0.05 was set as statistically significant. Multiparity decreased the contractile force of Bsm and Ism generated by high-frequency stimulation trains. The normalized force of the Pcm increased when evoked at 1, 4, and 10 Hz while this decreased at higher frequencies (20, 50, and 100 Hz). The contraction of both Bsm and Ism raised particularly the pressure on the perineal vagina while that of the Pcm increased the pressure in the pelvic vagina. Such a functional segregation is still present in multiparous rabbits albeit it was modified. Multiparity induces changes in the contractile responses of Bsm, Ism, and Pcm, which alterates the vaginal pressure. © 2017 Wiley Periodicals, Inc.

Slack length reduces the contractile phenotype of the Swine carotid artery.

PubMed

Rembold, Christopher M; Garvey, Sean M; Tejani, Ankit D

2013-01-01

Contraction is the primary function of adult arterial smooth muscle. However, in response to vessel injury or inflammation, arterial smooth muscle is able to phenotypically modulate from the contractile state to several 'synthetic' states characterized by proliferation, migration and/or increased cytokine secretion. We examined the effect of tissue length (L) on the phenotype of intact, isometrically held, initially contractile swine carotid artery tissues. Tissues were studied (1) without prolonged incubation at the optimal length for force generation (1.0 Lo, control), (2) with prolonged incubation for 17 h at 1.0 Lo, or (3) with prolonged incubation at slack length (0.6 Lo) for 16 h and then restoration to 1.0 Lo for 1 h. Prolonged incubation at 1.0 Lo minimally reduced the contractile force without substantially altering the mediators of contraction (crossbridge phosphorylation, shortening velocity or stimulated actin polymerization). Prolonged incubation of tissues at slack length (0.6 Lo), despite return of length to 1.0 Lo, substantially reduced contractile force, reduced crossbridge phosphorylation, nearly abolished crossbridge cycling (shortening velocity) and abolished stimulated actin polymerization. These data suggest that (1) slack length treatment significantly alters the contractile phenotype of arterial tissue, and (2) slack length treatment is a model to study acute phenotypic modulation of intact arterial smooth muscle. Copyright © 2013 S. Karger AG, Basel.

The benefits of endurance training in cardiomyocyte function in hypertensive rats are reversed within four weeks of detraining.

PubMed

Carneiro-Júnior, Miguel Araujo; Quintão-Júnior, Judson Fonseca; Drummond, Lucas Rios; Lavorato, Victor Neiva; Drummond, Filipe Rios; da Cunha, Daise Nunes Queiroz; Amadeu, Marco Aurélio; Felix, Leonardo Bonato; de Oliveira, Edilamar Menezes; Cruz, Jader Santos; Prímola-Gomes, Thales Nicolau; Mill, José Geraldo; Natali, Antonio José

2013-04-01

The aim of the present study was to verify the effects of low-intensity endurance training and detraining on the mechanical and molecular properties of cardiomyocytes from spontaneously hypertensive rats (SHRs). Male SHRs and normotensive control Wistar rats at 16-weeks of age were randomly divided into eight groups of eight animals: NC8 and HC8 (normotensive and hypertensive control for 8weeks); NT8 and HT8 (normotensive and hypertensive trained at 50-60% of maximal exercise capacity for 8weeks); NC12 and HC12 (normotensive and hypertensive control for 12weeks); NDT and HDT (normotensive and hypertensive trained for 8weeks and detrained for 4weeks). The total exercise time until fatigue (TTF) was determined by a maximal exercise capacity test. Resting heart rate (RHR) and systolic arterial pressure (SAP) were measured. After the treatments, animals were killed by cervical dislocation and left ventricular myocytes were isolated by enzymatic dispersion. Isolated cells were used to determine intracellular global Ca(2+) ([Ca(2+)]i) transient and cardiomyocyte contractility (1Hz; ~25°C). [Ca(2+)]i regulatory proteins were measured by Western blot, and the markers of pathologic cardiac hypertrophy by quantitative real-time polymerase chain reaction (q-RT-PCR). Exercise training augmented the TTF (NC8, 11.4±1.5min vs. NT8, 22.5±1.4min; HC8, 11.7±1.4min vs. HT8, 24.5±1.3min; P<0.05), reduced RHR (NT8initial, 340±8bpm vs. NT8final, 322±10bpm; HT8initial, 369±8bpm vs. HT8final, 344±10bpm; P<0.05), and SBP in SHR animals (HC8, 178±3mmHg vs. HT8, 161±4mmHg; P<0.05). HC8 rats showed a slower [Ca(2+)]i transient (Tpeak, 83.7±1.8ms vs. 71.7±2.4ms; T50%decay, 284.0±4.3ms vs. 264.0±4.1ms; P<0.05) and cell contractility (Vshortening, 86.1±6.7μm/s vs. 118.6±6.7μm/s; Vrelengthening, 57.5±7.4μm/s vs. 101.3±7.4μm/s; P<0.05), and higher expression of ANF (300%; P<0.05), skeletal α-actin (250%; P<0.05) and a decreased α/β-MHC ratio (70%; P<0.05) compared to NC8. Exercise training increased [Ca(2+)]i transient (NC8, 2.39±0.06F/F0 vs. NT8, 2.72±0.06F/F0; HC8, 2.28±0.05F/F0 vs. HT8, 2.82±0.05F/F0; P<0.05), and cell contractility (NC8, 7.4±0.3% vs. NT8, 8.4±0.3%; HC8, 6.8±0.3% vs. HT8, 7.8±0.3%; P<0.05). Furthermore, exercise normalized the expression of ANF, skeletal α-actin, and the α/β-MHC ratio in HT8 rats, augmented the expression of SERCA2a (NC8, 0.93±0.15 vs. NT8, 1.49±0.14; HC8, 0.83±0.13 vs. HT8, 1.32±0.14; P<0.05) and PLBser16 (NC8, 0.89±0.18 vs. NT8, 1.23±0.17; HC8, 0.77±0.17 vs. HT8, 1.32±0.16; P<0.05), and reduced PLBt/SERCA2a (NC8, 1.21±0.19 vs. NT8, 0.50±0.21; HC8, 1.38±0.17 vs. HT8, 0.66±0.21; P<0.05). However, all these adaptations returned to control values within 4weeks of detraining in both SHR and normotensive control animals. In conclusion, low-intensity endurance training induces positive benefits to left ventricular myocyte mechanical and molecular properties, which are reversed within 4weeks of detraining. Copyright © 2013 Elsevier Ltd. All rights reserved.

Macrophage migration inhibitory factor plays a permissive role in the maintenance of cardiac contractile function under starvation through regulation of autophagy.

PubMed

Xu, Xihui; Pacheco, Benjamin D; Leng, Lin; Bucala, Richard; Ren, Jun

2013-08-01

The cytokine macrophage migration inhibitory factor (MIF) protects the heart through AMPK activation. Autophagy, a conserved pathway for bulk degradation of intracellular proteins and organelles, helps preserve and recycle energy and nutrients for cells to survive under starvation. This study was designed to examine the role of MIF in cardiac homeostasis and autophagy regulation following an acute starvation challenge. Wild-type (WT) and MIF knockout mice were starved for 48 h. Echocardiographic data revealed little effect of starvation on cardiac geometry, contractile and intracellular Ca²⁺ properties. MIF deficiency unmasked an increase in left ventricular end-systolic diameter, a drop in fractional shortening associated with cardiomyocyte contractile and intracellular Ca²⁺ anomalies following starvation. Interestingly, the unfavourable effect of MIF deficiency was associated with interruption of starvation-induced autophagy. Furthermore, restoration of autophagy using rapamycin partially protected against starvation-induced cardiomyocyte contractile defects. In our in vitro model of starvation, neonatal mouse cardiomyocytes from WT and MIF-/- mice and H9C2 cells were treated with serum free-glucose free DMEM for 2 h. MIF depletion dramatically attenuated starvation-induced autophagic vacuole formation in neonatal mouse cardiomyocytes and exacerbated starvation-induced cell death in H9C2 cells. In summary, these results indicate that MIF plays a permissive role in the maintenance of cardiac contractile function under starvation by regulation of autophagy.

Smooth muscle-protein translocation and tissue function.

PubMed

Eddinger, Thomas J

2014-09-01

Smooth muscle (SM) tissue is a complex organization of multiple cell types and is regulated by numerous signaling molecules (neurotransmitters, hormones, cytokines, etc.). SM contractile function can be regulated via expression and distribution of the contractile and cytoskeletal proteins, and activation of any of the second messenger pathways that regulate them. Spatial-temporal changes in the contractile, cytoskeletal or regulatory components of SM cells (SMCs) have been proposed to alter SM contractile activity. Ca(2+) sensitization/desensitization can occur as a result of changes at any of these levels, and specific pathways have been identified at all of these levels. Understanding when and how proteins can translocate within the cytoplasm, or to-and-from the plasmalemma and the cytoplasm to alter contractile activity is critical. Numerous studies have reported translocation of proteins associated with the adherens junction and G protein-coupled receptor activation pathways in isolated SMC systems. Specific examples of translocation of vinculin to and from the adherens junction and protein kinase C (PKC) and 17 kDa PKC-potentiated inhibitor of myosin light chain phosphatase (CPI-17) to and from the plasmalemma in isolated SMC systems but not in intact SM tissues are discussed. Using both isolated SMC systems and SM tissues in parallel to pursue these studies will advance our understanding of both the role and mechanism of these pathways as well as their possible significance for Ca(2+) sensitization in intact SM tissues and organ systems. © 2014 Wiley Periodicals, Inc.

Electrophysiological effects of calcitonin gene-related peptide in bull-frog and guinea-pig atrial myocytes.

PubMed Central

Ono, K; Giles, W R

1991-01-01

1. Electrophysiological effects of calcitonin gene-related peptide (CGRP) on action potentials and corresponding transmembrane currents in single myocytes from bull-frog and guinea-pig atria were studied using a whole-cell voltage-clamp method. 2. CGRP at relatively low concentrations increased the height of the action potential plateau in a dose-dependent manner in both bull-frog and guinea-pig myocytes. In addition, in bull-frog cells CGRP accelerated the early phase of repolarization, thus shortening the overall duration of the action potential. In contrast, in guinea-pig myocytes CGRP prolonged the action potential duration at all concentrations that were studied. 3. Voltage-clamp measurements demonstrated that CGRP increased transmembrane calcium current (ICa) in guinea-pig myocytes without a significant change in its voltage dependence. The ED50 value for this effect on ICa was 1.28 +/- 0.55 X 10(-8) M (n = 4). The time course of the inactivation of ICa was not affected by CGRP. 4. CGRP increased the delayed rectifier K+ current (IK) at relatively low concentrations in bull-frog atria, whereas relatively high concentrations were needed to increase IK in guinea-pig myocytes. This effect was observed even after complete inhibition of ICa. 5. CGRP had no significant effect on the inwardly rectifying background K+ current, IK1, even at very high concentrations. 6. Comparison of the time course of ICa augmentation in bull-frog and guinea-pig myocytes revealed an important difference in the effect of CGRP in these two types of cells. CGRP at maximal concentrations increased ICa transiently in bull-frog myocytes, whereas this response was sustained in guinea-pig myocytes. Isoprenaline (Iso) induced sustained increase in ICa in both species. When ICa was fully activated by Iso, CGRP at high concentrations strongly inhibited ICa in the bull-frog, whereas it had little effect on ICa in guinea-pig myocytes. 7. Intracellular application of GTP gamma S (guanosine 5'-O-(3-thiotriphosphate) 10(-4) M) greatly potentiated the CGRP effect on ICa; in contrast, GDP beta S (guanosine 5'-O-(2-thiodiphosphate), 2 x 10(-3) M) partially inhibited the CGRP-induced augmentation of ICa. Taken together, these results indicate that the stimulation of ICa by CGRP is mediated by a GTP-binding protein. 8. The observed dose-dependent changes in ICa and IK in bull-frog and guinea-pig myocytes can explain the different patterns of CGRP-induced changes in action potential shape in these two myocyte preparations. PMID:1905755

Aim44p regulates phosphorylation of Hof1p to promote contractile ring closure during cytokinesis in budding yeast

PubMed Central

Wolken, Dana M. Alessi; McInnes, Joseph; Pon, Liza A.

2014-01-01

Whereas actomyosin and septin ring organization and function in cytokinesis are thoroughly described, little is known regarding the mechanisms by which the actomyosin ring interacts with septins and associated proteins to coordinate cell division. Here we show that the protein product of YPL158C, Aim44p, undergoes septin-dependent recruitment to the site of cell division. Aim44p colocalizes with Myo1p, the type II myosin of the contractile ring, throughout most of the cell cycle. The Aim44p ring does not contract when the actomyosin ring closes. Instead, it forms a double ring that associates with septin rings on mother and daughter cells after cell separation. Deletion of AIM44 results in defects in contractile ring closure. Aim44p coimmunoprecipitates with Hof1p, a conserved F-BAR protein that binds both septins and type II myosins and promotes contractile ring closure. Deletion of AIM44 results in a delay in Hof1p phosphorylation and altered Hof1p localization. Finally, overexpression of Dbf2p, a kinase that phosphorylates Hof1p and is required for relocalization of Hof1p from septin rings to the contractile ring and for Hof1p-triggered contractile ring closure, rescues the cytokinesis defect observed in aim44∆ cells. Our studies reveal a novel role for Aim44p in regulating contractile ring closure through effects on Hof1p. PMID:24451263

Long Term Ablation of Protein Kinase A (PKA)-mediated Cardiac Troponin I Phosphorylation Leads to Excitation-Contraction Uncoupling and Diastolic Dysfunction in a Knock-in Mouse Model of Hypertrophic Cardiomyopathy*

PubMed Central

Dweck, David; Sanchez-Gonzalez, Marcos A.; Chang, Audrey N.; Dulce, Raul A.; Badger, Crystal-Dawn; Koutnik, Andrew P.; Ruiz, Edda L.; Griffin, Brittany; Liang, Jingsheng; Kabbaj, Mohamed; Fincham, Frank D.; Hare, Joshua M.; Overton, J. Michael; Pinto, Jose R.

2014-01-01

The cardiac troponin I (cTnI) R21C (cTnI-R21C) mutation has been linked to hypertrophic cardiomyopathy and renders cTnI incapable of phosphorylation by PKA in vivo. Echocardiographic imaging of homozygous knock-in mice expressing the cTnI-R21C mutation shows that they develop hypertrophy after 12 months of age and have abnormal diastolic function that is characterized by longer filling times and impaired relaxation. Electrocardiographic analyses show that older R21C mice have elevated heart rates and reduced cardiovagal tone. Cardiac myocytes isolated from older R21C mice demonstrate that in the presence of isoproterenol, significant delays in Ca2+ decay and sarcomere relaxation occur that are not present at 6 months of age. Although isoproterenol and stepwise increases in stimulation frequency accelerate Ca2+-transient and sarcomere shortening kinetics in R21C myocytes from older mice, they are unable to attain the corresponding WT values. When R21C myocytes from older mice are treated with isoproterenol, evidence of excitation-contraction uncoupling is indicated by an elevation in diastolic calcium that is frequency-dissociated and not coupled to shorter diastolic sarcomere lengths. Myocytes from older mice have smaller Ca2+ transient amplitudes (2.3-fold) that are associated with reductions (2.9-fold) in sarcoplasmic reticulum Ca2+ content. This abnormal Ca2+ handling within the cell may be attributed to a reduction (2.4-fold) in calsequestrin expression in conjunction with an up-regulation (1.5-fold) of Na+-Ca2+ exchanger. Incubation of permeabilized cardiac fibers from R21C mice with PKA confirmed that the mutation prevents facilitation of mechanical relaxation. Altogether, these results indicate that the inability to enhance myofilament relaxation through cTnI phosphorylation predisposes the heart to abnormal diastolic function, reduced accessibility of cardiac reserves, dysautonomia, and hypertrophy. PMID:24973218

Free Fatty Acid Effects on the Atrial Myocardium: Membrane Ionic Currents Are Remodeled by the Disruption of T-Tubular Architecture

PubMed Central

O’Connell, Ryan P.; Musa, Hassan; Gomez, Mario San Martin; Avula, Uma Mahesh; Herron, Todd J.; Kalifa, Jerome; Anumonwo, Justus M. B.

2015-01-01

Background Epicardial adiposity and plasma levels of free fatty acids (FFAs) are elevated in atrial fibrillation, heart failure and obesity, with potentially detrimental effects on myocardial function. As major components of epicardial fat, FFAs may be abnormally regulated, with a potential to detrimentally modulate electro-mechanical function. The cellular mechanisms underlying such effects of FFAs are unknown. Objective To determine the mechanisms underlying electrophysiological effects of palmitic (PA), stearic (SA) and oleic (OA) FFAs on sheep atrial myocytes. Methods We used electrophysiological techniques, numerical simulations, biochemistry and optical imaging to examine the effects of acutely (≤ 15 min), short-term (4–6 hour) or 24-hour application of individual FFAs (10 μM) on isolated ovine left atrial myocytes (LAMs). Results Acute and short-term incubation in FFAs resulted in no differences in passive or active properties of isolated left atrial myocytes (LAMs). 24-hour application had differential effects depending on the FFA. PA did not affect cellular passive properties but shortened (p<0.05) action potential duration at 30% repolarization (APD30). APD50 and APD80 were unchanged. SA had no effect on resting membrane potential but reduced membrane capacitance by 15% (p<0.05), and abbreviated APD at all values measured (p≤0.001). OA did not significantly affect passive or active properties of LAMs. Measurement of the major voltage-gated ion channels in SA treated LAMs showed a ~60% reduction (p<0.01) of the L-type calcium current (ICa-L) and ~30% reduction (p<0.05) in the transient outward potassium current (ITO). A human atrial cell model recapitulated SA effects on APD. Optical imaging showed that SA incubated for 24 hours altered t-tubular structure in isolated cells (p<0.0001). Conclusions SA disrupts t-tubular architecture and remodels properties of membrane ionic currents in sheep atrial myocytes, with potential implications in arrhythmogenesis. PMID:26274906

Free Fatty Acid Effects on the Atrial Myocardium: Membrane Ionic Currents Are Remodeled by the Disruption of T-Tubular Architecture.

PubMed

O'Connell, Ryan P; Musa, Hassan; Gomez, Mario San Martin; Avula, Uma Mahesh; Herron, Todd J; Kalifa, Jerome; Anumonwo, Justus M B

2015-01-01

Epicardial adiposity and plasma levels of free fatty acids (FFAs) are elevated in atrial fibrillation, heart failure and obesity, with potentially detrimental effects on myocardial function. As major components of epicardial fat, FFAs may be abnormally regulated, with a potential to detrimentally modulate electro-mechanical function. The cellular mechanisms underlying such effects of FFAs are unknown. To determine the mechanisms underlying electrophysiological effects of palmitic (PA), stearic (SA) and oleic (OA) FFAs on sheep atrial myocytes. We used electrophysiological techniques, numerical simulations, biochemistry and optical imaging to examine the effects of acutely (≤ 15 min), short-term (4-6 hour) or 24-hour application of individual FFAs (10 μM) on isolated ovine left atrial myocytes (LAMs). Acute and short-term incubation in FFAs resulted in no differences in passive or active properties of isolated left atrial myocytes (LAMs). 24-hour application had differential effects depending on the FFA. PA did not affect cellular passive properties but shortened (p<0.05) action potential duration at 30% repolarization (APD30). APD50 and APD80 were unchanged. SA had no effect on resting membrane potential but reduced membrane capacitance by 15% (p<0.05), and abbreviated APD at all values measured (p≤0.001). OA did not significantly affect passive or active properties of LAMs. Measurement of the major voltage-gated ion channels in SA treated LAMs showed a ~60% reduction (p<0.01) of the L-type calcium current (ICa-L) and ~30% reduction (p<0.05) in the transient outward potassium current (ITO). A human atrial cell model recapitulated SA effects on APD. Optical imaging showed that SA incubated for 24 hours altered t-tubular structure in isolated cells (p<0.0001). SA disrupts t-tubular architecture and remodels properties of membrane ionic currents in sheep atrial myocytes, with potential implications in arrhythmogenesis.

TRPV2 is critical for the maintenance of cardiac structure and function in mice

PubMed Central

Katanosaka, Yuki; Iwasaki, Keiichiro; Ujihara, Yoshihiro; Takatsu, Satomi; Nishitsuji, Koki; Kanagawa, Motoi; Sudo, Atsushi; Toda, Tatsushi; Katanosaka, Kimiaki; Mohri, Satoshi; Naruse, Keiji

2014-01-01

The heart has a dynamic compensatory mechanism for haemodynamic stress. However, the molecular details of how mechanical forces are transduced in the heart are unclear. Here we show that the transient receptor potential, vanilloid family type 2 (TRPV2) cation channel is critical for the maintenance of cardiac structure and function. Within 4 days of eliminating TRPV2 from hearts of the adult mice, cardiac function declines severely, with disorganization of the intercalated discs that support mechanical coupling with neighbouring myocytes and myocardial conduction defects. After 9 days, cell shortening and Ca2+ handling by single myocytes are impaired in TRPV2-deficient hearts. TRPV2-deficient neonatal cardiomyocytes form no intercalated discs and show no extracellular Ca2+-dependent intracellular Ca2+ increase and insulin-like growth factor (IGF-1) secretion in response to stretch stimulation. We further demonstrate that IGF-1 receptor/PI3K/Akt pathway signalling is significantly downregulated in TRPV2-deficient hearts, and that IGF-1 administration partially prevents chamber dilation and impairment in cardiac pump function in these hearts. Our results improve our understanding of the molecular processes underlying the maintenance of cardiac structure and function. PMID:24874017

TRPV2 is critical for the maintenance of cardiac structure and function in mice.

PubMed

Katanosaka, Yuki; Iwasaki, Keiichiro; Ujihara, Yoshihiro; Takatsu, Satomi; Nishitsuji, Koki; Kanagawa, Motoi; Sudo, Atsushi; Toda, Tatsushi; Katanosaka, Kimiaki; Mohri, Satoshi; Naruse, Keiji

2014-05-29

The heart has a dynamic compensatory mechanism for haemodynamic stress. However, the molecular details of how mechanical forces are transduced in the heart are unclear. Here we show that the transient receptor potential, vanilloid family type 2 (TRPV2) cation channel is critical for the maintenance of cardiac structure and function. Within 4 days of eliminating TRPV2 from hearts of the adult mice, cardiac function declines severely, with disorganization of the intercalated discs that support mechanical coupling with neighbouring myocytes and myocardial conduction defects. After 9 days, cell shortening and Ca(2+) handling by single myocytes are impaired in TRPV2-deficient hearts. TRPV2-deficient neonatal cardiomyocytes form no intercalated discs and show no extracellular Ca(2+)-dependent intracellular Ca(2+) increase and insulin-like growth factor (IGF-1) secretion in response to stretch stimulation. We further demonstrate that IGF-1 receptor/PI3K/Akt pathway signalling is significantly downregulated in TRPV2-deficient hearts, and that IGF-1 administration partially prevents chamber dilation and impairment in cardiac pump function in these hearts. Our results improve our understanding of the molecular processes underlying the maintenance of cardiac structure and function.

In a non-human primate model, aging disrupts the neural control of intestinal smooth muscle contractility in a region-specific manner.

PubMed

Tran, L; Greenwood-Van Meerveld, B

2014-03-01

Incidences of gastrointestinal (GI) motility disorders increase with age. However, there is a paucity of knowledge about the aging mechanisms leading to GI dysmotility. Motility in the GI tract is a function of smooth muscle contractility, which is modulated in part by the enteric nervous system (ENS). Evidence suggests that aging impairs the ENS, thus we tested the hypothesis that senescence in the GI tract precipitates abnormalities in smooth muscle and neurally mediated contractility in a region-specific manner. Jejunal and colonic circular muscle strips were isolated from young (4-10 years) and old (18+ years) baboons. Myogenic responses were investigated using potassium chloride (KCl) and carbachol (CCh). Neurally mediated contractile responses were evoked by electrical field stimulation (EFS) and were recorded in the absence and presence of atropine (1 μM) or NG-Nitro-l-arginine methyl ester (l-NAME; 100 μM). The myogenic responses to KCl in the jejunum and colon were unaffected by age. In the colon, but not the jejunum, CCh-induced contractile responses were reduced in aged animals. Compared to young baboons, there was enhanced EFS-induced contractility of old baboon jejunal smooth muscle in contrast to the reduced contractility in the colon. The effect of atropine on the EFS response was lower in aged colonic tissue, suggesting reduced participation of acetylcholine. In aged jejunal tissue, higher contractile responses to EFS were found to be due to reduced nitregic inhibition. These findings provide key evidence for the importance of intestinal smooth muscle and ENS senescence in age-associated GI motility disorders. © 2014 The Authors. Neurogastroenterology & Motility published by John Wiley & Sons Ltd.

Enhanced cell volume regulation: a key protective mechanism of ischemic preconditioning in rabbit ventricular myocytes.

PubMed

Diaz, Roberto J; Armstrong, Stephen C; Batthish, Michelle; Backx, Peter H; Ganote, Charles E; Wilson, Gregory J

2003-01-01

Accumulation of osmotically active metabolites, which create an osmotic gradient estimated at ~60 mOsM, and cell swelling are prominent features of ischemic myocardial cell death. This study tests the hypothesis that reduction of ischemic swelling by enhanced cell volume regulation is a key mechanism in the delay of ischemic myocardial cell death by ischemic preconditioning (IPC). Experimental protocols address whether: (i) IPC triggers a cell volume regulation mechanism that reduces cardiomyocyte swelling during subsequent index ischemia; (ii) this reduction in ischemic cell swelling is sufficient in magnitude to account for the IPC protection; (iii) the molecular mechanism that mediates IPC also mediates cell volume regulation. Two experimental models with rabbit ventricular myocytes were studied: freshly isolated pelleted myocytes and 48-h cultured myocytes. Myocytes were preconditioned either by distinct short simulated ischemia (SI)/simulated reperfusion protocols (IPC), or by subjecting myocytes to a pharmacological preconditioning (PPC) protocol (1 microM calyculin A, or 1 microM N(6)-2-(4-aminophenyl)ethyladenosine (APNEA), prior to subjecting them to either different durations of long SI or 30 min hypo-osmotic stress. Cell death (percent blue square myocytes) was monitored by trypan blue staining. Cell swelling was determined by either the bromododecane cell flotation assay (qualitative) or video/confocal microscopy (quantitative). Simulated ischemia induced myocyte swelling in both the models. In pelleted myocytes, IPC or PPC with either calyculin A or APNEA produced a marked reduction of ischemic cell swelling as determined by the cell floatation assay. In cultured myocytes, IPC substantially reduced ischemic cell swelling (P < 0.001). This IPC effect on ischemic cell swelling was related to an IPC and PPC (with APNEA) mediated triggering of cell volume regulatory decrease (RVD). IPC and APNEA also significantly (P < 0.001) reduced hypo-osmotic cell swelling. This IPC and APNEA effect was blocked by either adenosine receptor, PKC or Cl(-) channel inhibition. The osmolar equivalent for IPC protection approximated 50-60 mOsM, an osmotic gradient similar to the estimated ischemic osmotic load for preconditioned and non-preconditioned myocytes. The results suggest that cell volume regulation is a key mechanism that accounts for most of the IPC protection in cardiomyocytes.

Contractile properties of rat skeletal muscles following storage at 4 degrees C.

PubMed

van der Heijden, E P; Kroese, A B; Stremel, R W; Bär, P R; Kon, M; Werker, P M

1999-07-01

The purpose of this study was to assess the potential of preservation solutions for protecting skeletal muscle function during storage at 4 degrees C. The soleus and the cutaneus trunci (CT) from the rat were stored for 2, 8 or 16 h at 4 degrees C in University of Wisconsin solution (UW), HTK-Bretschneider solution (HTK) or Krebs-Henseleit solution (KH). After storage, muscles were stimulated electrically to analyse the isometric contractile properties, such as the maximum tetanic tension (P(0)). Histological analysis was also performed. In separate experiments, the effect of the diffusion distance on muscle preservation was studied by bisecting the soleus. After 8 h of storage in UW or HTK, the contractile properties of the CT were similar to those of the control, whereas those of the soleus were reduced (P(0) values of 16% and 69% of control in UW and HTK respectively). At 16 h, the contractile properties of the CT (P(O) 28%) were again better preserved than those of the soleus (P(0) 9%). Muscle function deteriorated most after storage in KH (P(0) at 16 h: soleus, 3%; CT, 17%). The bisected soleus was equally well preserved as the CT (P(O) of bisected soleus at 8 h in UW and HTK: 86%). The functional data corresponded well with the histological data, which showed increasing muscle fibre derangement with increasing storage time. For both muscles and all solutions, the threshold stimulus current increased with increasing storage time (control, 0.1 mA; 16 h, 1.2 mA) and was strongly correlated with the deterioration in contractile properties. It is concluded that, at 4 degrees C, muscle is preserved better in UW and HTK (intracellular-like solutions) than in KH (extracellular-like solution). The soleus and CT were best protected in HTK. The diffusion distance is a critical factor for successful preservation of muscle function at 4 degrees C. The reduced function after 16 h of storage at 4 degrees C was caused by hypercontraction and necrosis of about 25% of the muscle fibres, and by deterioration of the electrical component of excitation-contraction coupling of the remaining fibres.

Preservation of cardiac function by prolonged action potentials in mice deficient of KChIP2.

PubMed

Grubb, Søren; Aistrup, Gary L; Koivumäki, Jussi T; Speerschneider, Tobias; Gottlieb, Lisa A; Mutsaers, Nancy A M; Olesen, Søren-Peter; Calloe, Kirstine; Thomsen, Morten B

2015-08-01

Inherited ion channelopathies and electrical remodeling in heart disease alter the cardiac action potential with important consequences for excitation-contraction coupling. Potassium channel-interacting protein 2 (KChIP2) is reduced in heart failure and interacts under physiological conditions with both Kv4 to conduct the fast-recovering transient outward K(+) current (Ito,f) and with CaV1.2 to mediate the inward L-type Ca(2+) current (ICa,L). Anesthetized KChIP2(-/-) mice have normal cardiac contraction despite the lower ICa,L, and we hypothesized that the delayed repolarization could contribute to the preservation of contractile function. Detailed analysis of current kinetics shows that only ICa,L density is reduced, and immunoblots demonstrate unaltered CaV1.2 and CaVβ₂ protein levels. Computer modeling suggests that delayed repolarization would prolong the period of Ca(2+) entry into the cell, thereby augmenting Ca(2+)-induced Ca(2+) release. Ca(2+) transients in disaggregated KChIP2(-/-) cardiomyocytes are indeed comparable to wild-type transients, corroborating the preserved contractile function and suggesting that the compensatory mechanism lies in the Ca(2+)-induced Ca(2+) release event. We next functionally probed dyad structure, ryanodine receptor Ca(2+) sensitivity, and sarcoplasmic reticulum Ca(2+) load and found that increased temporal synchronicity of the Ca(2+) release in KChIP2(-/-) cardiomyocytes may reflect improved dyad structure aiding the compensatory mechanisms in preserving cardiac contractile force. Thus the bimodal effect of KChIP2 on Ito,f and ICa,L constitutes an important regulatory effect of KChIP2 on cardiac contractility, and we conclude that delayed repolarization and improved dyad structure function together to preserve cardiac contraction in KChIP2(-/-) mice. Copyright © 2015 the American Physiological Society.

A threshold of GATA4 and GATA6 expression is required for cardiovascular development

PubMed Central

Xin, Mei; Davis, Christopher A.; Molkentin, Jeffery D.; Lien, Ching-Ling; Duncan, Stephen A.; Richardson, James A.; Olson, Eric N.

2006-01-01

The zinc-finger transcription factors GATA4 and GATA6 play critical roles in embryonic development. Mouse embryos lacking GATA4 die at embryonic day (E) 8.5 because of failure of ventral foregut closure and cardiac bifida, whereas GATA6 is essential for development of the visceral endoderm. Although mice that are heterozygous for either a GATA4 or GATA6 null allele are normal, we show that compound heterozygosity of GATA4 and GATA6 results in embryonic lethality by E13.5 accompanied by a spectrum of cardiovascular defects, including thin-walled myocardium, ventricular and aortopulmonary septal defects, and abnormal smooth muscle development. Myocardial hypoplasia in GATA4/GATA6 double heterozygous mutant embryos is associated with reduced proliferation of cardiomyocytes, diminished expression of the myogenic transcription factor MEF2C (myocyte enhancer factor 2C), and down-regulation of β-myosin heavy chain expression, a key determinant of cardiac contractility. These findings reveal a threshold of GATA4 and GATA6 activity that is required for gene expression in the developing cardiovascular system and underscore the potential of recessive mutations to perturb the delicate regulation of cardiovascular development. PMID:16847256

ROS-activated Ca/calmodulin kinase IIδ is required for late INa augmentation leading to cellular Na and Ca overload

PubMed Central

Wagner, Stefan; Ruff, Hanna M.; Weber, Sarah L.; Bellmann, Sarah; Sowa, Thomas; Schulte, Timo; Grandi, Eleonora; Bers, Donald M.; Backs, Johannes; Belardinelli, Luiz; Maier, Lars S.

2011-01-01

Rationale In heart failure (HF), CaMKII expression and reactive oxygen species (ROS) are increased. Both ROS and CaMKII can increase late INa leading to intracellular Na accumulation and arrhythmias. It has been shown that ROS can activate CaMKII via oxidation. Objective We tested whether CaMKIIδ is required for ROS-dependent late INa regulation and if ROS-induced Ca released from the sarcoplasmic reticulum (SR) is involved. Methods and Results 40 µmol/L H2O2 significantly increased CaMKII oxidation and autophosphorylation in permeabilized rabbit cardiomyocytes. Without free [Ca]i (5 mmol/L BAPTA/1 mmol/L Br2-BAPTA) or after SR depletion (caffeine 10 mmol/L, thapsigargin 5 µmol/L) the H2O2-dependent CaMKII oxidation and autophosphorylation was abolished. H2O2 significantly increased SR Ca spark frequency (confocal microscopy) but reduced SR Ca load. In wildtype (WT) mouse myocytes, H2O2 increased late INa (whole cell patch-clamp). This increase was abolished in CaMKIIδ−/− myocytes. H2O2-induced [Na]i and [Ca]i accumulation (SBFI and Indo-1 epifluorescence) was significantly slowed in CaMKIIδ−/− myocytes (vs. WT). CaMKIIδ−/− myocytes developed significantly less H2O2-induced arrhythmias, and were more resistant to hypercontracture. Opposite results (increased late INa, [Na]i and [Ca]i accumulation) were obtained by overexpression of CaMKIIδ in rabbit myocytes (adenoviral gene transfer) reversible with CaMKII inhibition (10 µmol/L KN93 or 0.1 µmol/L AIP). Conclusion Free [Ca]i and a functional SR are required for ROS activation of CaMKII. ROS-activated CaMKIIδ enhances late INa, which may lead to cellular Na and Ca overload. This may be of relevance in HF, where enhanced ROS production meets increased CaMKII expression. PMID:21252154

Ser1928 phosphorylation by PKA stimulates the L-type Ca2+ channel CaV1.2 and vasoconstriction during acute hyperglycemia and diabetes.

PubMed

Nystoriak, Matthew A; Nieves-Cintrón, Madeline; Patriarchi, Tommaso; Buonarati, Olivia R; Prada, Maria Paz; Morotti, Stefano; Grandi, Eleonora; Fernandes, Julia Dos Santos; Forbush, Katherine; Hofmann, Franz; Sasse, Kent C; Scott, John D; Ward, Sean M; Hell, Johannes W; Navedo, Manuel F

2017-01-24

Hypercontractility of arterial myocytes and enhanced vascular tone during diabetes are, in part, attributed to the effects of increased glucose (hyperglycemia) on L-type Ca V 1.2 channels. In murine arterial myocytes, kinase-dependent mechanisms mediate the increase in Ca V 1.2 activity in response to increased extracellular glucose. We identified a subpopulation of the Ca V 1.2 channel pore-forming subunit (α1 C ) within nanometer proximity of protein kinase A (PKA) at the sarcolemma of murine and human arterial myocytes. This arrangement depended upon scaffolding of PKA by an A-kinase anchoring protein 150 (AKAP150) in mice. Glucose-mediated increases in Ca V 1.2 channel activity were associated with PKA activity, leading to α1 C phosphorylation at Ser 1928 Compared to arteries from low-fat diet (LFD)-fed mice and nondiabetic patients, arteries from high-fat diet (HFD)-fed mice and from diabetic patients had increased Ser 1928 phosphorylation and Ca V 1.2 activity. Arterial myocytes and arteries from mice lacking AKAP150 or expressing mutant AKAP150 unable to bind PKA did not exhibit increased Ser 1928 phosphorylation and Ca V 1.2 current density in response to increased glucose or to HFD. Consistent with a functional role for Ser 1928 phosphorylation, arterial myocytes and arteries from knockin mice expressing a Ca V 1.2 with Ser 1928 mutated to alanine (S1928A) lacked glucose-mediated increases in Ca V 1.2 activity and vasoconstriction. Furthermore, the HFD-induced increases in Ca V 1.2 current density and myogenic tone were prevented in S1928A knockin mice. These findings reveal an essential role for α1 C phosphorylation at Ser 1928 in stimulating Ca V 1.2 channel activity and vasoconstriction by AKAP-targeted PKA upon exposure to increased glucose and in diabetes. Copyright © 2017, American Association for the Advancement of Science.

MicroRNA-761 regulates mitochondrial biogenesis in mouse skeletal muscle in response to exercise

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xu, Yanli; Zhao, Chaoxian; Sun, Xuewen

MicroRNAs (miRNAs) have been suggested to play critical roles in skeletal muscle in response to exercise. Previous study has shown that miR-761 was involved in a novel model regulating the mitochondrial network. However, its role in mitochondrial biogenesis remains poorly understood. Therefore, the current study was aimed to examine the effect of miR-761 on mitochondrial biogenesis in skeletal muscle. Real-time quantitative PCR analysis demonstrated that aberrantly expressed miR-761 is involved in exercise activity and miR-761 is decreased by exercise training compared with the sedentary control mice. miR-761 suppresses mitochondrial biogenesis of C{sub 2}C{sub 12} myocytes by targeting the 3′-UTR ofmore » peroxisome proliferator-activated receptor gamma (PPARγ) coactivator-1 (PGC-1α). Overexpression of miR-761 was capable of inhibiting the protein expression levels of PGC-1α. Moreover, miR-761 overexpression suppressed the p38 MAPK signaling pathway and down-regulated the expression of phosphorylated MAPK-activated protein kinase-2 (P-MK2), a downstream kinase of p38 MAPK. The phosphorylation of activating transcription factors 2 (ATF2) that plays a functional role in linking the activation of the p38 MAPK pathway to enhanced transcription of the PGC-1α was also inhibited by the overexpression of miR-761. These findings revealed a novel regulation mechanism for miR-761 in skeletal myocytes, and contributed to a better understanding of the modulation of skeletal muscle in response to exercise. - Highlights: • Endurance exercise decreases miR-761 expression in skeletal muscle. • MiR-761 suppresses mitochondrial biogenesis in C{sub 2}C{sub 12} myocytes. • MiR-761 directly targeted PGC-1α expression. • MiR-761 suppresses p38 MAPK signaling pathways in C{sub 2}C{sub 12} myocytes. • A novel mechanism for miR-761 in skeletal myocytes is demonstrated.« less

Cellular distribution of calcium current is unaltered during compensated hypertrophy in the spontaneously hypertensive rat.

PubMed

Fowler, Mark R; Orchard, Clive H; Harrison, Simon M

2007-01-01

Changes in cellular calcium (Ca(2+)) handling are thought to underlie the altered contraction that occurs during cardiac hypertrophy and failure. Recent work has highlighted the importance of t-tubules in the control of intracellular Ca(2+). The present study was performed to investigate whether changes in the distribution of I (Ca) between the surface and t-tubule membranes might contribute to the altered Ca(2+) handling observed during compensated hypertrophy in the spontaneously hypertensive rat (SHR). Experiments were performed on ventricular myocytes isolated from 5-month-old SHR and normotensive Wistar-Kyoto (WKY) control rats. Osmotic shock using formamide was used to disrupt the t-tubular system and the whole-cell patch clamp technique used to monitor I (Ca) in the presence and absence of t-tubules. Membrane capacitance and I (Ca) were greater in control SHR than WKY myocytes; following detubulation, cell capacitance and I (Ca) both decreased and were no longer significantly different in the two cell types. The density of I (Ca) was not significantly different in control SHR and WKY cells or in detubulated myocytes from the two species. These data suggest that the distribution of I (Ca) is unchanged in SHR myocytes compared to WKY controls; I (Ca) density in the t-tubules was 1.2-fold greater than in the sarcolemma in both strains. These data also imply that the increase in surface area in SHR myocytes is due principally to an increase in t-tubular area, which is accompanied by an approximately equivalent increase in I (Ca), so that the density of I (Ca) at the cell surface and in the t-tubules remains the same. These changes would be expected to retain cell function and synchronicity of Ca(2+) release in the SHR at this stage of compensated hypertrophy.

Ser1928 phosphorylation by PKA stimulates the L-type Ca2+ channel CaV1.2 and vasoconstriction during acute hyperglycemia and diabetes

PubMed Central

Nystoriak, Matthew A.; Nieves-Cintrón, Madeline; Patriarchi, Tommaso; Buonarati, Olivia R.; Prada, Maria Paz; Morotti, Stefano; Grandi, Eleonora; Fernandes, Julia Dos Santos; Forbush, Katherine; Hofmann, Franz; Sasse, Kent C.; Scott, John D.; Ward, Sean M.; Hell, Johannes W.; Navedo, Manuel F.

2017-01-01

Hypercontractility of arterial myocytes and enhanced vascular tone during diabetes are, in part, attributed to the effects of increased glucose (hyperglycemia) on L-type CaV1.2 channels. In murine arterial myocytes, kinase-dependent mechanisms mediate the increase in CaV1.2 activity in response to increased extracellular glucose. We identified a subpopulation of the CaV1.2 channel pore-forming subunit (α1C) within nanometer proximity of protein kinase A (PKA) at the sarcolemma of murine and human arterial myocytes. This arrangement depended upon scaffolding of PKA by an A-kinase anchoring protein 150 (AKAP150) in mice. Glucose-mediated increases in CaV1.2 channel activity were associated with PKA activity, leading to α1C phosphorylation at Ser1928. Compared to arteries from low-fat diet (LFD)–fed mice and nondiabetic patients, arteries from high-fat diet (HFD)–fed mice and from diabetic patients had increased Ser1928 phosphorylation and CaV1.2 activity. Arterial myocytes and arteries from mice lacking AKAP150 or expressing mutant AKAP150 unable to bind PKA did not exhibit increased Ser1928 phosphorylation and CaV1.2 current density in response to increased glucose or to HFD. Consistent with a functional role for Ser1928 phosphorylation, arterial myocytes and arteries from knockin mice expressing a CaV1.2 with Ser1928 mutated to alanine (S1928A) lacked glucose-mediated increases in CaV1.2 activity and vasoconstriction. Furthermore, the HFD-induced increases in CaV1.2 current density and myogenic tone were prevented in S1928A knockin mice. These findings reveal an essential role for α1C phosphorylation at Ser1928 in stimulating CaV1.2 channel activity and vasoconstriction by AKAP-targeted PKA upon exposure to increased glucose and in diabetes. PMID:28119464

Down-regulation of CaV1.2 channels during hypertension: how fewer CaV1.2 channels allow more Ca2+ into hypertensive arterial smooth muscle

PubMed Central

Tajada, Sendoa; Cidad, Pilar; Colinas, Olaia; Santana, L Fernando; López-López, José R; Pérez-García, M Teresa

2013-01-01

Hypertension is a clinical syndrome characterized by increased arterial tone. Although the mechanisms are varied, the generally accepted view is that increased CaV1.2 channel function is a common feature of this pathological condition. Here, we investigated the mechanisms underlying vascular dysfunction in a mouse model of genetic hypertension. Contrary to expectation, we found that whole-cell CaV1.2 currents (ICa) were lower in hypertensive (BPH line) than normotensive (BPN line) myocytes. However, local CaV1.2 sparklet activity was higher in BPH cells, suggesting that the relatively low ICa in these cells was produced by a few hyperactive CaV1.2 channels. Furthermore, our data suggest that while the lower expression of the pore-forming α1c subunit of CaV1.2 currents underlies the lower ICa in BPH myocytes, the increased sparklet activity was due to a different composition in the auxiliary subunits of the CaV1.2 complexes. ICa currents in BPN cells were produced by channels composed of α1c/α2δ/β3 subunits, while in BPH myocytes currents were probably generated by the opening of channels formed by α1c/α2δ/β2 subunits. In addition, Ca2+ sparks evoked large conductance, Ca2+-activated K+ (BK) currents of lower magnitude in BPH than in BPN myocytes, because BK channels were less sensitive to Ca2+. Our data are consistent with a model in which a decrease in the global number of CaV1.2 currents coexist with the existence of a subpopulation of highly active channels that dominate the resting Ca2+ influx. The decrease in BK channel activity makes the hyperpolarizing brake ineffective and leads BPH myocytes to a more contracted resting state. PMID:24167226

Use of a Doppler pulmonary artery catheter for continuous measurement of right ventricular pump function and contractility during single lung transplantation.

PubMed

Heerdt, P M; Pond, C G; Kussman, M K; Triantafillou, A N

1993-01-01

Despite numerous technologic advances in intraoperative monitoring, the only methods routinely available for assessment of right ventricular function in lung transplant recipients are continuous measurement of right heart pressures and intermittent thermodilution determination of cardiac output and ejection fraction. Additional data may now be obtained with transesophageal echocardiography, although this technology is expensive and not widely available and requires diverting attention from a potentially unstable patient for data acquisition and analysis. Recently, a Doppler pulmonary artery catheter was introduced that measures beat-to-beat pulmonary artery blood flow-velocity, cross sectional area, and volume flow. Because of data indicating that acceleration of blood in the pulmonary artery (measured as the first derivative of either the velocity or flow waveform) is a sensitive indicator of right ventricular contractility, we have used waveforms obtained with the catheter for assessment of right ventricular pump function (stroke volume and peak pulmonary artery flow rate) and contractility in heart surgery patients. We report here our experience with this method in two patients undergoing left single lung transplantation.

Effect of Substrate Mechanics on Cardiomyocyte Maturation and Growth

PubMed Central

Tallawi, Marwa; Rai, Ranjana; Boccaccini, Aldo. R.

2015-01-01

Cardiac tissue engineering constructs are a promising therapeutic treatment for myocardial infarction, which is one of the leading causes of death. In order to further advance the development and regeneration of engineered cardiac tissues using biomaterial platforms, it is important to have a complete overview of the effects that substrates have on cardiomyocyte (CM) morphology and function. This article summarizes recent studies that investigate the effect of mechanical cues on the CM differentiation, maturation, and growth. In these studies, CMs derived from embryos, neonates, and mesenchymal stem cells were seeded on different substrates of various elastic modulus. Measuring the contractile function by force production, work output, and calcium handling, it was seen that cell behavior on substrates was optimized when the substrate stiffness mimicked that of the native tissue. The contractile function reflected changes in the sarcomeric protein confirmation and organization that promoted the contractile ability. The analysis of the literature also revealed that, in addition to matrix stiffness, mechanical stimulation, such as stretching the substrate during cell seeding, also played an important role during cell maturation and tissue development. PMID:25148904

Myocardial changes in acute Trypanosoma cruzi infection. Ultrastructural evidence of immune damage and the role of microangiopathy.

PubMed Central

Andrade, Z. A.; Andrade, S. G.; Correa, R.; Sadigursky, M.; Ferrans, V. J.

1994-01-01

Histological and ultrastructural studies of the hearts of dogs sacrificed 18 to 26 days after intraperitoneal inoculation with 4 x 10(5) blood forms of the 12 SF strain of Trypanosoma cruzi/kg of body weight disclosed myocarditis characterized by parasitic invasion of some myocytes, damage and necrosis of nonparasitized myocytes, and interstitial infiltration by mononuclear cells. Nonparasitized myocytes showed alterations ranging from mild edema to severe myocytolysis. These changes often were accompanied by contacts of myocytes with lymphocytes (both granular and agranular) and macrophages. These contacts were characterized by focal loss of the myocyte basement membrane and close approximation of the plasma membranes of the two cells. Contacts between lymphocytes and capillary endothelial cells were also frequent. Platelet aggregates and fibrin microthrombi were observed in some capillaries. Our findings suggest that immune effector cells play a major role in the pathogenesis of the myocyte damage and the microangiopathy in acute Chagas' disease. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 PMID:8203476

Sirtuin 1 protects the aging heart from contractile dysfunction mediated through the inhibition of endoplasmic reticulum stress-mediated apoptosis in cardiac-specific Sirtuin 1 knockout mouse model.

PubMed

Hsu, Yu-Juei; Hsu, Shih-Che; Hsu, Chiao-Po; Chen, Yen-Hui; Chang, Yung-Lung; Sadoshima, Junichi; Huang, Shih-Ming; Tsai, Chien-Sung; Lin, Chih-Yuan

2017-02-01

The longevity regulator Sirtuin 1 is an NAD + -dependent histone deacetylase that regulates endoplasmic reticulum stress and influences cardiomyocyte apoptosis during cardiac contractile dysfunction induced by aging. The mechanism underlying Sirtuin 1 function in cardiac contractile dysfunction related to aging has not been completely elucidated. We evaluated cardiac contractile function, endoplasmic reticulum stress, apoptosis, and oxidative stress in 6- and 12month-old cardiac-specific Sirtuin 1 knockout (Sirt1 -/- ) and control (Sirt1 f/f ) mice using western blotting and immunohistochemistry. Mice were injected with a protein disulphide isomerase inhibitor. For in vitro analysis, cultured H9c2 cardiomyocytes were exposed to either a Sirtuin 1 inhibitor or activator, with or without a mitochondrial inhibitor, to evaluate the effects of Sirtuin 1 on endoplasmic reticulum stress, nitric oxide synthase expression, and apoptosis. The effects of protein disulphide isomerase inhibition on oxidative stress and ER stress-related apoptosis were also investigated. Compared with 6-month-old Sirt1 f/f mice, marked impaired contractility was observed in 12-month-old Sirt1 -/- mice. These findings were consistent with increased endoplasmic reticulum stress and apoptosis in the myocardium. Measures of oxidative stress and nitric oxide synthase expression were significantly higher in Sirt1 -/- mice compared with those in Sirt1 f/f mice at 6months. In vitro experiments revealed increased endoplasmic reticulum stress-mediated apoptosis in H9c2 cardiomyocytes treated with a Sirtuin 1 inhibitor; the effects were ameliorated by a Sirtuin 1 activator. Moreover, consistent with the in vitro findings, impaired cardiac contractility was demonstrated in Sirt1 -/- mice injected with a protein disulphide isomerase inhibitor. The present study demonstrates that the aging heart is characterized by contractile dysfunction associated with increased oxidative stress and endoplasmic reticulum stress and Sirtuin 1 might have the ability to protect the aging hearts from the inhibition of endoplasmic reticulum-mediated apoptosis. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Postprandial lymphatic pump function after a high-fat meal: a characterization of contractility, flow, and viscosity

PubMed Central

Kassis, Timothy; Yarlagadda, Sri Charan; Kohan, Alison B.; Tso, Patrick; Breedveld, Victor

2016-01-01

Dietary lipids are transported from the intestine through contractile lymphatics. Chronic lipid loads can adversely affect lymphatic function. However, the acute lymphatic pump response in the mesentery to a postprandial lipid meal has gone unexplored. In this study, we used the rat mesenteric collecting vessel as an in vivo model to quantify the effect of lipoproteins on vessel function. Lipid load was continuously monitored by using the intensity of a fluorescent fatty-acid analog, which we infused along with a fat emulsion through a duodenal cannula. The vessel contractility was simultaneously quantified. We demonstrated for the first time that collecting lymphatic vessels respond to an acute lipid load by reducing pump function. High lipid levels decreased contraction frequency and amplitude. We also showed a strong tonic response through a reduction in the end-diastolic and systolic diameters. We further characterized the changes in flow rate and viscosity and showed that both increase postprandially. In addition, shear-mediated Ca2+ signaling in lymphatic endothelial cells differed when cultured with lipoproteins. Together these results show that the in vivo response could be both shear and lipid mediated and provide the first evidence that high postprandial lipid has an immediate negative effect on lymphatic function even in the acute setting. PMID:26968208

Mechanical unloading of the failing human heart fails to activate the protein kinase B/Akt/glycogen synthase kinase-3beta survival pathway.

PubMed

Razeghi, Peter; Bruckner, Brian A; Sharma, Saumya; Youker, Keith A; Frazier, O H; Taegtmeyer, Heinrich

2003-01-01

Left ventricular assist device (LVAD) support of the failing human heart improves myocyte function and increases cell survival. One potential mechanism underlying this phenomenon is activation of the protein kinase B (PKB)/Akt/glycogen synthase kinase-3beta (GSK-3beta) survival pathway. Left ventricular tissue was obtained both at the time of implantation and explantation of the LVAD (n = 11). Six patients were diagnosed with idiopathic dilated cardiomyopathy, 4 patients with ischemic cardiomyopathy and 1 patient with peripartum cardiomyopathy. The mean duration of LVAD support was 205 +/- 35 days. Myocyte diameter and phosphorylation of ERK were used as indices for reverse remodeling. Transcript levels of genes required for the activation of PKB/Akt (insulin-like growth factor-1, insulin receptor substrate-1) were measured by quantitative RT-PCR. In addition, we measured the relative activity of PKB/Akt and GSK-3beta, and assayed for molecular and histological indices of PKB/Akt activation (cyclooxygenase mRNA levels and glycogen levels). Myocyte diameter and phosphorylation of ERK decreased with LVAD support. In contrast, none of the components of the PKB/Akt/GSK-3beta pathway changed significantly with mechanical unloading. The PKB/Akt/GSK-3beta pathway is not activated during LVAD support. Other signaling pathways must be responsible for the improvement of cellular function and cell survival during LVAD support. Copyright 2003 S. Karger AG, Basel

Mitofusins and the mitochondrial permeability transition: the potential downside of mitochondrial fusion

PubMed Central

Papanicolaou, Kyriakos N.; Phillippo, Matthew M.

2012-01-01

Mitofusins (Mfn-1 and Mfn-2) are transmembrane proteins that bind and hydrolyze guanosine 5′-triphosphate to bring about the merging of adjacent mitochondrial membranes. This event is necessary for mitochondrial fusion, a biological process that is critical for organelle function. The broad effects of mitochondrial fusion on cell bioenergetics have been extensively studied, whereas the local effects of mitofusin activity on the structure and integrity of the fusing mitochondrial membranes have received relatively little attention. From the study of fusogenic proteins, theoretical models, and simulations, it has been noted that the fusion of biological membranes is associated with local perturbations on the integrity of the membrane that present in the form of lipidic holes which open on the opposing bilayers. These lipidic holes represent obligate intermediates that make the fusion process thermodynamically more favorable and at the same time induce leakage to the fusing membranes. In this perspectives article we present the relevant evidence selected from a spectrum of membrane fusion/leakage models and attempt to couple this information with observations conducted with cardiac myocytes or mitochondria deficient in Mfn-1 and Mfn-2. More specifically, we argue in favor of a situation whereby mitochondrial fusion in cardiac myocytes is coupled with outer mitochondrial membrane destabilization that is opportunistically employed during the process of mitochondrial permeability transition. We hope that these insights will initiate research on this new hypothesis of mitochondrial permeability transition regulation, a poorly understood mitochondrial function with significant consequences on myocyte survival. PMID:22636681

IMPACT OF CALCIUM-CHANNEL BLOCKERS ON RIGHT HEART FUNCTION IN A CONTROLLED MODEL OF CHRONIC PULMONARY HYPERTENSION

PubMed Central

Zierer, Andreas; Voeller, Rochus K.; Melby, Spencer J.; Steendijk, Paul; Moon, Marc R.

2009-01-01

Purpose Patients with chronic pulmonary hypertension (CPH) who demonstrate a pulmonary vasodilation following calcium channel blocker (CCB) administration are defined as “responders”. In contrast, “non-responders” are patients who do not show such a pulmonary vasodilation with CCB therapy. The purpose of this investigation was to study the effects of CCB therapy on right heart mechanics in experimental CCB responders versus CCB non-responders. Methods In 12 dogs, right atrial (RA) and ventricular (RV) pressure and volume (conductance catheters) were simultaneously recorded after 3 months of progressive pulmonary artery (PA) banding. Diltiazem was given at 10 mg/hr with the PA constricted (simulated CCB non-responder). Responders were then created by releasing the PA band to unload the ventricle. RA and RV contractility and diastolic stiffness (slope of end-systolic and end-diastolic pressure-volume relations) were calculated and RA reservoir and conduit function were quantified as RA inflow with the tricuspid valve closed versus open, respectively. Results With CCB, RA contractility (p<0.03) and cardiac output (p<0.004) were compromised in simulated non-responders while RA stroke work was pharmacologically depressed in the setting of an unchanged afterload. After simulating a responder by controlled PA band release, the RA became less distensible, causing a shift from reservoir to conduit function (p<0.001) towards physiologic baseline conditions and a recovery in the hyperdynamic compensatory response in both chambers (p<0.007) as evidenced in a declined RA and RV contractility with an improved cardiac output as compared to CPH and simulated non-responders. RA and RV diastolic function in both groups was not affected by CCB. Conclusions CCB did not impact RV function in simulated non-responders, but significantly impaired RA contractility and cardiac output. In simulated responders, afterload fell substantially, thereby allowing the RA and RV to recover from their pathological hyperdynamic contractile response to CPH. This affect was able to outweigh the intrinsic negative effects of CCB therapy on systolic RA function. Current data suggest that the RA in CPH is much more sensitive to CCB therapy than the RV and delineate for the first time why CCB therapy in CPH has been empirically restricted to documented responders. PMID:19237986

Role of endothelin-1 and big endothelin-1 in modulating coronary vascular tone, contractile function and severity of ischemia in rat hearts.

PubMed

Grover, G J; Sleph, P G; Fox, M; Trippodo, N C

1992-12-01

The effect of endothelin-1 (ET-1) and big ET-1 on coronary flow and contractile function was determined in isolated nonischemic and ischemic rat hearts. Both ET-1 (IC50 = 12 pMol) and big ET-1 (IC50 = 2 nMol) reduced coronary flow in a concentration-dependent manner, although ET-1 was > 100-fold more potent. Both compounds decreased contractility, an effect which was lost when coronary flow was held constant, indicating that ET-1 and big ET-1 decrease contractility secondary to reducing coronary flow. Mechanical reduction in coronary flow to levels equivalent to those seen for ET-1 or big ET-1 caused similar reductions in contractility. Both 30 pMol ET-1 and 10 nMol big ET-1 pretreatment significantly reduced the time to contracture in globally ischemic rat hearts, suggesting a proischemic effect. Phosphoramidon (100 microM, endothelin-converting enzyme inhibitor) and BQ-123 (0.3 microM, ETA receptor antagonist) abolished the preischemic increase in coronary perfusion pressure induced by big ET-1 as well as its proischemic effect, whereas only BQ-123 abolished the cardiac effect of ET-1. Neither phosphoramidon nor BQ-123 had an effect on severity of ischemia when given alone. Phosphoramidon was also given i.v. to rats subjected to coronary occlusion and reperfusion and was found to significantly reduce infarct size 24 hr postischemia. Thus, in isolated rat hearts, big ET-1 appears to be converted to ET-1 and is a potent coronary constrictor.(ABSTRACT TRUNCATED AT 250 WORDS)

Cardiac-Specific Overexpression of Catalase Attenuates Lipopolysaccharide-Induced Myocardial Contractile Dysfunction: Role of Autophagy

PubMed Central

Turdi, Subat; Han, Xuefeng; Huff, Anna F.; Roe, Nathan D.; Hu, Nan; Gao, Feng; Ren, Jun

2012-01-01

Lipopolysaccharide (LPS) from Gram-negative bacteria is a major initiator of sepsis, leading to cardiovascular collapse. Accumulating evidence has indicated a role of reactive oxygen species (ROS) in cardiovascular complication in sepsis. This study was designed to examine the effect of cardiac-specific overexpression of catalase in LPS-induced cardiac contractile dysfunction and the underlying mechanism(s) with a focus on autophagy. Catalase transgenic and wild-type FVB mice were challenged with LPS (6 mg/kg) and cardiac function was evaluated. Levels of oxidative stress, autophagy, apoptosis and protein damage were examined using fluorescence microscopy, Western blot, TUNEL assay, caspase-3 activity and carbonyl formation. Kaplan-Meier curve was constructed for survival following LPS treatment. Our results revealed a lower mortality in catalase mice compared with FVB mice following LPS challenge. LPS injection led to depressed cardiac contractile capacity as evidenced by echocardiography and cardiomyocyte contractile function, the effect of which was ablated by catalase overexpression. LPS treatment induced elevated TNF-α level, autophagy, apoptosis (TUNEL, caspase-3 activation, cleaved caspase-3), production of ROS and O2−, and protein carbonyl formation, the effects of which were significantly attenuated by catalase overexpression. Electron microscopy revealed focal myocardial damage characterized by mitochondrial injury following LPS treatment, which was less severe in catalase mice. Interestingly, LPS-induced cardiomyocyte contractile dysfunction was prevented by antioxidant NAC and the autophagy inhibitor 3-methyladenine. Taken together, our data revealed that catalase protects against LPS-induced cardiac dysfunction and mortality, which may be associated with inhibition of oxidative stress and autophagy. PMID:22902401

Effects of the Tibetan herbal formula Padma Lax on visceral nociception and contractility of longitudinal smooth muscle in a rat model.

PubMed

Gschossmann, J M; Krayer, M; Flogerzi, B; Balsiger, B M

2010-09-01

The high prevalence of functional bowel disorders among the general population contrasts with the limited number of pharmacological treatment options for this condition. This has led to an interest for alternative therapeutic approaches. Padma Lax is an herbal laxative on the basis of Tibetan formulas. Our aim is to examine the effect of Padma Lax on visceral nociception in vivo and (B) on contractile activity of longitudinal smooth muscle of the lower gut in vitro and ex vivo. (A) Visceral sensory function in response to colorectal distension was assessed by abdominal wall electromyography in male Wistar rats pretreated with Padma Lax. (B) Effects of Padma Lax on contractility of gut smooth muscles were studied both in vitro with superfusion of the agent and ex vivo following oral administration of the preparation. Activities were measured as area under the curve. (A) For visceral sensitivity, no differences were observed between the Padma Lax and the control group. (B) Proximal colon muscle strips of the Padma Lax pretreated group showed significantly lower spontaneous contractility ex vivo than controls. Cholinergic procontractile stimulation was reduced in Padma Lax pretreated group and in colon strips of naive rats when Padma Lax was superfused in vitro (all P < 0.05). Cholinergic mechanisms appear to be important in the modulation of rat proximal colon contractility of orally and directly applied Padma Lax. These findings help elucidate a potential mechanism of action of this herbal remedy which has undergone clinical testing in patients with constipation predominant irritable bowel syndrome.

Decreased intracellular [Ca2+ ] coincides with reduced expression of Dhprα1s, RyR1, and diaphragmatic dysfunction in a rat model of sepsis.

PubMed

Wang, Meng-Meng; Hao, Li-Ying; Guo, Feng; Zhong, Bin; Zhong, Xiao-Mei; Yuan, Jing; Hao, Yi-Fei; Zhao, Shuang; Sun, Xue-Fei; Lei, Ming; Jiao, Guang-Yu

2017-12-01

Sepsis can cause decreased diaphragmatic contractility. Intracellular calcium as a second messenger is central to diaphragmatic contractility. However, changes in intracellular calcium concentration ([Ca 2+ ]) and the distribution and co-localization of relevant calcium channels [dihydropyridine receptors, (DHPRα1s) and ryanodine receptors (RyR1)] remain unclear during sepsis. In this study we investigated the effect of changed intracellular [Ca 2+ ] and expression and distribution of DHPRα1s and RyR1 on diaphragm function during sepsis. We measured diaphragm contractility and isolated diaphragm muscle cells in a rat model of sepsis. The distribution and co-localization of DHPRα1s and RyR1 were determined using immunohistochemistry and immunofluorescence, whereas intracellular [Ca 2+ ] was measured by confocal microscopy and fluorescence spectrophotometry. Septic rat diaphragm contractility, expression of DHPRα1s and RyR1, and intracellular [Ca 2+ ] were significantly decreased in the rat sepsis model compared with controls. Decreased intracellular [Ca 2+ ] coincides with diaphragmatic contractility and decreased expression of DHPRα1s and RyR1 in sepsis. Muscle Nerve 56: 1128-1136, 2017. © 2017 Wiley Periodicals, Inc.

TRPM4 Is a Novel Component of the Adhesome Required for Focal Adhesion Disassembly, Migration and Contractility

PubMed Central

Cáceres, Mónica; Ortiz, Liliana; Recabarren, Tatiana; Romero, Anibal; Colombo, Alicia; Leiva-Salcedo, Elías; Varela, Diego; Rivas, José; Silva, Ian; Morales, Diego; Campusano, Camilo; Almarza, Oscar; Simon, Felipe; Toledo, Hector; Park, Kang-Sik; Trimmer, James S.; Cerda, Oscar

2015-01-01

Cellular migration and contractility are fundamental processes that are regulated by a variety of concerted mechanisms such as cytoskeleton rearrangements, focal adhesion turnover, and Ca2+ oscillations. TRPM4 is a Ca2+-activated non-selective cationic channel (Ca2+-NSCC) that conducts monovalent but not divalent cations. Here, we used a mass spectrometry-based proteomics approach to identify putative TRPM4-associated proteins. Interestingly, the largest group of these proteins has actin cytoskeleton-related functions, and among these nine are specifically annotated as focal adhesion-related proteins. Consistent with these results, we found that TRPM4 localizes to focal adhesions in cells from different cellular lineages. We show that suppression of TRPM4 in MEFs impacts turnover of focal adhesions, serum-induced Ca2+ influx, focal adhesion kinase (FAK) and Rac activities, and results in reduced cellular spreading, migration and contractile behavior. Finally, we demonstrate that the inhibition of TRPM4 activity alters cellular contractility in vivo, affecting cutaneous wound healing. Together, these findings provide the first evidence, to our knowledge, for a TRP channel specifically localized to focal adhesions, where it performs a central role in modulating cellular migration and contractility. PMID:26110647

Toward an Integrative Computational Model of the Guinea Pig Cardiac Myocyte

PubMed Central

Gauthier, Laura Doyle; Greenstein, Joseph L.; Winslow, Raimond L.

2012-01-01

The local control theory of excitation-contraction (EC) coupling asserts that regulation of calcium (Ca2+) release occurs at the nanodomain level, where openings of single L-type Ca2+ channels (LCCs) trigger openings of small clusters of ryanodine receptors (RyRs) co-localized within the dyad. A consequence of local control is that the whole-cell Ca2+ transient is a smooth continuous function of influx of Ca2+ through LCCs. While this so-called graded release property has been known for some time, its functional importance to the integrated behavior of the cardiac ventricular myocyte has not been fully appreciated. We previously formulated a biophysically based model, in which LCCs and RyRs interact via a coarse-grained representation of the dyadic space. The model captures key features of local control using a low-dimensional system of ordinary differential equations. Voltage-dependent gain and graded Ca2+ release are emergent properties of this model by virtue of the fact that model formulation is closely based on the sub-cellular basis of local control. In this current work, we have incorporated this graded release model into a prior model of guinea pig ventricular myocyte electrophysiology, metabolism, and isometric force production. The resulting integrative model predicts the experimentally observed causal relationship between action potential (AP) shape and timing of Ca2+ and force transients, a relationship that is not explained by models lacking the graded release property. Model results suggest that even relatively subtle changes in AP morphology that may result, for example, from remodeling of membrane transporter expression in disease or spatial variation in cell properties, may have major impact on the temporal waveform of Ca2+ transients, thus influencing tissue level electromechanical function. PMID:22783206

Microdomain-Specific Modulation of L-Type Calcium Channels Leads to Triggered Ventricular Arrhythmia in Heart Failure

PubMed Central

Sanchez-Alonso, Jose L.; Bhargava, Anamika; O’Hara, Thomas; Glukhov, Alexey V.; Schobesberger, Sophie; Bhogal, Navneet; Sikkel, Markus B.; Mansfield, Catherine; Korchev, Yuri E.; Lyon, Alexander R.; Punjabi, Prakash P.; Nikolaev, Viacheslav O.; Trayanova, Natalia A.

2016-01-01

Rationale: Disruption in subcellular targeting of Ca2+ signaling complexes secondary to changes in cardiac myocyte structure may contribute to the pathophysiology of a variety of cardiac diseases, including heart failure (HF) and certain arrhythmias. Objective: To explore microdomain-targeted remodeling of ventricular L-type Ca2+ channels (LTCCs) in HF. Methods and Results: Super-resolution scanning patch-clamp, confocal and fluorescence microscopy were used to explore the distribution of single LTCCs in different membrane microdomains of nonfailing and failing human and rat ventricular myocytes. Disruption of membrane structure in both species led to the redistribution of functional LTCCs from their canonical location in transversal tubules (T-tubules) to the non-native crest of the sarcolemma, where their open probability was dramatically increased (0.034±0.011 versus 0.154±0.027, P<0.001). High open probability was linked to enhance calcium–calmodulin kinase II–mediated phosphorylation in non-native microdomains and resulted in an elevated ICa,L window current, which contributed to the development of early afterdepolarizations. A novel model of LTCC function in HF was developed; after its validation with experimental data, the model was used to ascertain how HF-induced T-tubule loss led to altered LTCC function and early afterdepolarizations. The HF myocyte model was then implemented in a 3-dimensional left ventricle model, demonstrating that such early afterdepolarizations can propagate and initiate reentrant arrhythmias. Conclusions: Microdomain-targeted remodeling of LTCC properties is an important event in pathways that may contribute to ventricular arrhythmogenesis in the settings of HF-associated remodeling. This extends beyond the classical concept of electric remodeling in HF and adds a new dimension to cardiovascular disease. PMID:27572487

Toward an integrative computational model of the Guinea pig cardiac myocyte.

PubMed

Gauthier, Laura Doyle; Greenstein, Joseph L; Winslow, Raimond L

2012-01-01

The local control theory of excitation-contraction (EC) coupling asserts that regulation of calcium (Ca(2+)) release occurs at the nanodomain level, where openings of single L-type Ca(2+) channels (LCCs) trigger openings of small clusters of ryanodine receptors (RyRs) co-localized within the dyad. A consequence of local control is that the whole-cell Ca(2+) transient is a smooth continuous function of influx of Ca(2+) through LCCs. While this so-called graded release property has been known for some time, its functional importance to the integrated behavior of the cardiac ventricular myocyte has not been fully appreciated. We previously formulated a biophysically based model, in which LCCs and RyRs interact via a coarse-grained representation of the dyadic space. The model captures key features of local control using a low-dimensional system of ordinary differential equations. Voltage-dependent gain and graded Ca(2+) release are emergent properties of this model by virtue of the fact that model formulation is closely based on the sub-cellular basis of local control. In this current work, we have incorporated this graded release model into a prior model of guinea pig ventricular myocyte electrophysiology, metabolism, and isometric force production. The resulting integrative model predicts the experimentally observed causal relationship between action potential (AP) shape and timing of Ca(2+) and force transients, a relationship that is not explained by models lacking the graded release property. Model results suggest that even relatively subtle changes in AP morphology that may result, for example, from remodeling of membrane transporter expression in disease or spatial variation in cell properties, may have major impact on the temporal waveform of Ca(2+) transients, thus influencing tissue level electromechanical function.

An Alpha-1A Adrenergic Receptor Agonist Prevents Acute Doxorubicin Cardiomyopathy in Male Mice.

PubMed

Montgomery, Megan D; Chan, Trevor; Swigart, Philip M; Myagmar, Bat-Erdene; Dash, Rajesh; Simpson, Paul C

2017-01-01

Alpha-1 adrenergic receptors mediate adaptive effects in the heart and cardiac myocytes, and a myocyte survival pathway involving the alpha-1A receptor subtype and ERK activation exists in vitro. However, data in vivo are limited. Here we tested A61603 (N-[5-(4,5-dihydro-1H-imidazol-2-yl)-2-hydroxy-5,6,7,8-tetrahydronaphthalen-1-yl]methanesulfonamide), a selective imidazoline agonist for the alpha-1A. A61603 was the most potent alpha-1-agonist in activating ERK in neonatal rat ventricular myocytes. A61603 activated ERK in adult mouse ventricular myocytes and protected the cells from death caused by the anthracycline doxorubicin. A low dose of A61603 (10 ng/kg/d) activated ERK in the mouse heart in vivo, but did not change blood pressure. In male mice, concurrent subcutaneous A61603 infusion at 10 ng/kg/d for 7 days after a single intraperitoneal dose of doxorubicin (25 mg/kg) increased survival, improved cardiac function, heart rate, and cardiac output by echocardiography, and reduced cardiac cell necrosis and apoptosis and myocardial fibrosis. All protective effects were lost in alpha-1A-knockout mice. In female mice, doxorubicin at doses higher than in males (35-40 mg/kg) caused less cardiac toxicity than in males. We conclude that the alpha-1A-selective agonist A61603, via the alpha-1A adrenergic receptor, prevents doxorubicin cardiomyopathy in male mice, supporting the theory that alpha-1A adrenergic receptor agonists have potential as novel heart failure therapies.

Non-canonical Wnt signaling enhances differentiation of Sca1+/c-kit+ adipose-derived murine stromal vascular cells into spontaneously beating cardiac myocytes.

PubMed

Palpant, Nathan J; Yasuda, So-ichiro; MacDougald, Ormond; Metzger, Joseph M

2007-09-01

Recent reports have described a stem cell population termed stromal vascular cells (SVCs) derived from the stromal vascular fraction of adipose tissue, which are capable of intrinsic differentiation into spontaneously beating cardiomyocytes in vitro. The objective of this study was to further define the cardiac lineage differentiation potential of SVCs in vitro and to establish methods for enriching SVC-derived beating cardiac myocytes. SVCs were isolated from the stromal vascular fraction of murine adipose tissue. Cells were cultured in methylcellulose-based murine stem cell media. Analysis of SVC-derived beating myocytes included Western blot and calcium imaging. Enrichment of acutely isolated SVCs was carried out using antibody-tagged magnetic nanoparticles, and pharmacologic manipulation of Wnt and cytokine signaling. Under initial media conditions, spontaneously beating SVCs expressed both cardiac developmental and adult protein isoforms. Functionally, this specialized population can spontaneously contract and pace under field stimulation and shows the presence of coordinated calcium transients. Importantly, this study provides evidence for two independent mechanisms of enriching the cardiac differentiation of SVCs. First, this study shows that differentiation of SVCs into cardiac myocytes is augmented by non-canonical Wnt agonists, canonical Wnt antagonists, and cytokines. Second, SVCs capable of cardiac lineage differentiation can be enriched by selection for stem cell-specific membrane markers Sca1 and c-kit. Adipose-derived SVCs are a unique population of stem cells that show evidence of cardiac lineage development making them a potential source for stem cell-based cardiac regeneration studies.

A cardiac pathway of cyclic GMP-independent signaling of guanylyl cyclase A, the receptor for atrial natriuretic peptide

PubMed Central

Klaiber, Michael; Dankworth, Beatrice; Kruse, Martin; Hartmann, Michael; Nikolaev, Viacheslav O.; Yang, Ruey-Bing; Völker, Katharina; Gaßner, Birgit; Oberwinkler, Heike; Feil, Robert; Freichel, Marc; Groschner, Klaus; Skryabin, Boris V.; Frantz, Stefan; Birnbaumer, Lutz; Pongs, Olaf; Kuhn, Michaela

2011-01-01

Cardiac atrial natriuretic peptide (ANP) regulates arterial blood pressure, moderates cardiomyocyte growth, and stimulates angiogenesis and metabolism. ANP binds to the transmembrane guanylyl cyclase (GC) receptor, GC-A, to exert its diverse functions. This process involves a cGMP-dependent signaling pathway preventing pathological [Ca2+]i increases in myocytes. In chronic cardiac hypertrophy, however, ANP levels are markedly increased and GC-A/cGMP responses to ANP are blunted due to receptor desensitization. Here we show that, in this situation, ANP binding to GC-A stimulates a unique cGMP-independent signaling pathway in cardiac myocytes, resulting in pathologically elevated intracellular Ca2+ levels. This pathway involves the activation of Ca2+‐permeable transient receptor potential canonical 3/6 (TRPC3/C6) cation channels by GC-A, which forms a stable complex with TRPC3/C6 channels. Our results indicate that the resulting cation influx activates voltage-dependent L-type Ca2+ channels and ultimately increases myocyte Ca2+i levels. These observations reveal a dual role of the ANP/GC-A–signaling pathway in the regulation of cardiac myocyte Ca2+i homeostasis. Under physiological conditions, activation of a cGMP-dependent pathway moderates the Ca2+i-enhancing action of hypertrophic factors such as angiotensin II. By contrast, a cGMP-independent pathway predominates under pathophysiological conditions when GC-A is desensitized by high ANP levels. The concomitant rise in [Ca2+]i might increase the propensity to cardiac hypertrophy and arrhythmias. PMID:22027011

Non-canonical Wnt Signaling Enhances differentiation of Sca1+/c-kit+ Adipose-derived Murine Stromal Vascular Cells into Spontaneously Beating Cardiac Myocytes

PubMed Central

Palpant, Nathan J.; Yasuda, So-ichiro; MacDougald, Ormond; Metzger, Joseph M.

2007-01-01

Recent reports have described a stem cell population termed stromal vascular cells (SVCs) derived from the stromal vascular fraction of adipose tissue, which are capable of intrinsic differentiation into spontaneously beating cardiomyocytes in vitro. The objective of this study was to further define the cardiac lineage differentiation potential of SVCs in vitro and to derive methods for enriching SVC-derived beating cardiac myocytes. SVCs were isolated from the stromal vascular fraction of murine adipose tissue. Cells were cultured in methylcellulose-based murine stem cell media. Analysis of SVC-derived beating myocytes included Western blot, and calcium imaging. Enrichment of acutely isolated SVCs was carried out using antibody tagged magnetic nanoparticles, and pharmacologic manipulation of Wnt and cytokine signaling. Under initial media conditions, spontaneously beating SVCs expressed both cardiac developmental and adult protein isoforms. Functionally, this specialized population can spontaneously contract and pace under field stimulation, and shows the presence of coordinated calcium transients. Importantly, this study provides evidence for two independent mechanisms of enriching the cardiac differentiation of SVCs. First, this study shows that differentiation of SVCs into cardiac myocytes is augmented by non-canonical Wnt agonists, canonical Wnt antagonists, and cytokines. Second, SVCs capable of cardiac lineage differentiation can be enriched by selection for stem cell-specific membrane markers Sca1 and c-kit. Adipose-derived SVCs are a unique population of stem cells that show evidence of cardiac lineage development making them a potential source for stem cell-based cardiac regeneration studies. PMID:17706246

Modulation of KCNQ1 alternative splicing regulates cardiac IKs and action potential repolarization.

PubMed

Lee, Hsiang-Chun; Rudy, Yoram; Po-Yuan, Phd; Sheu, Sheng-Hsiung; Chang, Jan-Gowth; Cui, Jianmin

2013-08-01

Slow delayed-rectifier potassium current (IKs) channels, made of the pore-forming KCNQ1 and auxiliary KCNE1 subunits, play a key role in determining action potential duration (APD) in cardiac myocytes. The consequences of drug-induced KCNQ1 splice alteration remain unknown. To study the modulation of KCNQ1 alternative splicing by amiloride and the consequent changes in IKs and action potentials (APs) in ventricular myocytes. Canine endocardial, midmyocardial, and epicardial ventricular myocytes were isolated. Levels of KCNQ1a and KCNQ1b as well as a series of splicing factors were quantified by using the reverse transcriptase-polymerase chain reaction and Western blot. The effect of amiloride-induced changes in the KCNQ1b/total KCNQ1 ratio on AP was measured by using whole-cell patch clamp with and without isoproterenol. With 50 μmol/L of amiloride for 6 hours, KCNQ1a at transcriptional and translational levels increased in midmyocardial myocytes but decreased in endo- and epicardial myocytes. Likewise, changes in splicing factors in midmyocardial were opposite to that in endo- and epicardial myocytes. In midmyocardial myocytes amiloride shortened APD and decreased isoproterenol-induced early afterdepolarizations significantly. The same amiloride-induced effects were demonstrated by using human ventricular myocyte model for AP simulations under beta-adrenergic stimulation. Moreover, amiloride reduced the transmural dispersion of repolarization in pseudo-electrocardiogram. Amiloride regulates IKs and APs with transmural differences and reduces arrhythmogenicity through the modulation of KCNQ1 splicing. We suggested that the modulation of KCNQ1 splicing may help prevent arrhythmia. Copyright © 2013 Heart Rhythm Society. Published by Elsevier Inc. All rights reserved.

Mitochondrial dysfunction and sarcopenia of aging: from signaling pathways to clinical trials

PubMed Central

Marzetti, Emanuele; Calvani, Riccardo; Cesari, Matteo; Buford, Thomas W.; Lorenzi, Maria; Behnke, Bradley J.; Leeuwenburgh, Christiaan

2013-01-01

Sarcopenia, the age-related loss of muscle mass and function, imposes a dramatic burden on individuals and society. The development of preventive and therapeutic strategies against sarcopenia is therefore perceived as an urgent need by health professionals and has instigated intensive research on the pathophysiology of this syndrome. The pathogenesis of sarcopenia is multifaceted and encompasses lifestyle habits, systemic factors (e.g., chronic inflammation and hormonal alterations), local environment perturbations (e.g., vascular dysfunction), and intramuscular specific processes. In this scenario, derangements in skeletal myocyte mitochondrial function are recognized as major factors contributing to the age-dependent muscle degeneration. In this review, we summarize prominent findings and controversial issues on the contribution of specific mitochondrial processes – including oxidative stress, quality control mechanisms and apoptotic signaling – on the development of sarcopenia. Extramuscular alterations accompanying the aging process with a potential impact on myocyte mitochondrial function are also discussed. We conclude with presenting methodological and safety considerations for the design of clinical trials targeting mitochondrial dysfunction to treat sarcopenia. Special emphasis is placed on the importance of monitoring the effects of an intervention on muscle mitochondrial function and identifying the optimal target population for the trial. PMID:23845738

Simultaneous determination of dynamic cardiac metabolism and function using PET/MRI.

PubMed

Barton, Gregory P; Vildberg, Lauren; Goss, Kara; Aggarwal, Niti; Eldridge, Marlowe; McMillan, Alan B

2018-05-01

Cardiac metabolic changes in heart disease precede overt contractile dysfunction. However, metabolism and function are not typically assessed together in clinical practice. The purpose of this study was to develop a cardiac positron emission tomography/magnetic resonance (PET/MR) stress test to assess the dynamic relationship between contractile function and metabolism in a preclinical model. Following an overnight fast, healthy pigs (45-50 kg) were anesthetized and mechanically ventilated. 18 F-fluorodeoxyglucose ( 18 F-FDG) solution was administered intravenously at a constant rate of 0.01 mL/s for 60 minutes. A cardiac PET/MR stress test was performed using normoxic gas (F I O 2  = .209) and hypoxic gas (F I O 2  = .12). Simultaneous cardiac imaging was performed on an integrated 3T PET/MR scanner. Hypoxic stress induced a significant increase in heart rate, cardiac output, left ventricular (LV) ejection fraction (EF), and peak torsion. There was a significant decline in arterial SpO 2 , LV end-diastolic and end-systolic volumes in hypoxia. Increased LV systolic function was coupled with an increase in myocardial FDG uptake (Ki) during hypoxic stress. PET/MR with continuous FDG infusion captures dynamic changes in both cardiac metabolism and contractile function. This technique warrants evaluation in human cardiac disease for assessment of subtle functional and metabolic abnormalities.

Characterization of human septic sera induced gene expression modulation in human myocytes

PubMed Central

Hussein, Shaimaa; Michael, Paul; Brabant, Danielle; Omri, Abdelwahab; Narain, Ravin; Passi, Kalpdrum; Ramana, Chilakamarti V.; Parrillo, Joseph E.; Kumar, Anand; Parissenti, Amadeo; Kumar, Aseem

2009-01-01

To gain a better understanding of the gene expression changes that occurs during sepsis, we have performed a cDNA microarray study utilizing a tissue culture model that mimics human sepsis. This study utilized an in vitro model of cultured human fetal cardiac myocytes treated with 10% sera from septic patients or 10% sera from healthy volunteers. A 1700 cDNA expression microarray was used to compare the transcription profile from human cardiac myocytes treated with septic sera vs normal sera. Septic sera treatment of myocytes resulted in the down-regulation of 178 genes and the up-regulation of 4 genes. Our data indicate that septic sera induced cell cycle, metabolic, transcription factor and apoptotic gene expression changes in human myocytes. Identification and characterization of gene expression changes that occur during sepsis may lead to the development of novel therapeutics and diagnostics. PMID:19684886

BK Channel-Mediated Relaxation of Urinary Bladder Smooth Muscle: A Novel Paradigm for Phosphodiesterase Type 4 Regulation of Bladder Function

PubMed Central

Xin, Wenkuan; Li, Ning; Cheng, Qiuping

2014-01-01

Elevation of intracellular cAMP and activation of protein kinase A (PKA) lead to activation of large conductance voltage- and Ca2+-activated K+ (BK) channels, thus attenuation of detrusor smooth muscle (DSM) contractility. In this study, we investigated the mechanism by which pharmacological inhibition of cAMP-specific phosphodiesterase 4 (PDE4) with rolipram or Ro-20-1724 (C15H22N2O3) suppresses guinea pig DSM excitability and contractility. We used high-speed line-scanning confocal microscopy, ratiometric fluorescence Ca2+ imaging, and perforated whole-cell patch-clamp techniques on freshly isolated DSM cells, along with isometric tension recordings of DSM isolated strips. Rolipram caused an increase in the frequency of Ca2+ sparks and the spontaneous transient BK currents (TBKCs), hyperpolarized the cell membrane potential (MP), and decreased the intracellular Ca2+ levels. Blocking BK channels with paxilline reversed the hyperpolarizing effect of rolipram and depolarized the MP back to the control levels. In the presence of H-89 [N-[2-[[3-(4-bromophenyl)-2-propenyl]amino]ethyl]-5-isoquinolinesulfonamide dihydrochloride], a PKA inhibitor, rolipram did not cause MP hyperpolarization. Rolipram or Ro-20-1724 reduced DSM spontaneous and carbachol-induced phasic contraction amplitude, muscle force, duration, and frequency, and electrical field stimulation-induced contraction amplitude, muscle force, and tone. Paxilline recovered DSM contractility, which was suppressed by pretreatment with PDE4 inhibitors. Rolipram had reduced inhibitory effects on DSM contractility in DSM strips pretreated with paxilline. This study revealed a novel cellular mechanism whereby pharmacological inhibition of PDE4 leads to suppression of guinea pig DSM contractility by increasing the frequency of Ca2+ sparks and the functionally coupled TBKCs, consequently hyperpolarizing DSM cell MP. Collectively, this decreases the global intracellular Ca2+ levels and DSM contractility in a BK channel-dependent manner. PMID:24459245

Identification of Contractile Vacuole Proteins in Trypanosoma cruzi

PubMed Central

Park, Miyoung; Martins, Vicente P.; Atwood, James; Moles, Kristen; Collins, Dalis; Rohloff, Peter; Tarleton, Rick; Moreno, Silvia N. J.; Orlando, Ron; Docampo, Roberto

2011-01-01

Contractile vacuole complexes are critical components of cell volume regulation and have been shown to have other functional roles in several free-living protists. However, very little is known about the functions of the contractile vacuole complex of the parasite Trypanosoma cruzi, the etiologic agent of Chagas disease, other than a role in osmoregulation. Identification of the protein composition of these organelles is important for understanding their physiological roles. We applied a combined proteomic and bioinfomatic approach to identify proteins localized to the contractile vacuole. Proteomic analysis of a T. cruzi fraction enriched for contractile vacuoles and analyzed by one-dimensional gel electrophoresis and LC-MS/MS resulted in the addition of 109 newly detected proteins to the group of expressed proteins of epimastigotes. We also identified different peptides that map to at least 39 members of the dispersed gene family 1 (DGF-1) providing evidence that many members of this family are simultaneously expressed in epimastigotes. Of the proteins present in the fraction we selected several homologues with known localizations in contractile vacuoles of other organisms and others that we expected to be present in these vacuoles on the basis of their potential roles. We determined the localization of each by expression as GFP-fusion proteins or with specific antibodies. Six of these putative proteins (Rab11, Rab32, AP180, ATPase subunit B, VAMP1, and phosphate transporter) predominantly localized to the vacuole bladder. TcSNARE2.1, TcSNARE2.2, and calmodulin localized to the spongiome. Calmodulin was also cytosolic. Our results demonstrate the utility of combining subcellular fractionation, proteomic analysis, and bioinformatic approaches for localization of organellar proteins that are difficult to detect with whole cell methodologies. The CV localization of the proteins investigated revealed potential novel roles of these organelles in phosphate metabolism and provided information on the potential participation of adaptor protein complexes in their biogenesis. PMID:21437209

Contribution of Rho kinase to the early phase of the calcium-contraction coupling in airway smooth muscle.

PubMed

Mbikou, Prisca; Fajmut, Ales; Brumen, Milan; Roux, Etienne

2011-02-01

We investigated theoretically and experimentally the role of Rho kinase (RhoK) in Ca(2+)-contraction coupling in rat airways. Isometric contraction was measured on tracheal, extrapulmonary and intrapulmonary bronchial rings. Intracellular [Ca(2+)] was recorded in freshly isolated tracheal myocytes. Stimulation by carbachol (0.3 and 10 μm) and 50 mm external KCl induced a short-time, Hill-shaped contraction obtained within 90 s, followed by a sustained or an additional delayed contraction. Responses of [Ca(2+)](i) to acetylcholine consisted in a fast peak followed by a plateau and, in 42% of the cells, superimposed Ca(2+) oscillations. The RhoK inhibitor Y27632 (10 μm) did not alter the [Ca(2+)](i) response. Whatever the agonist, Y27632 did not modify the basal tension but decreased the amplitude of the short-duration response, without altering the additional delayed contraction. The Myosin Light Chain Phosphatase (MLCP) inhibitor calyculin A increased the basal tension and abolished the effect of RhoK. KN93 (Ca(2+)-calmodulin-dependent protein kinase II inhibitor) and DIDS (inhibitor of Ca(2+)-activated Cl(-) channels) had no influence on the RhoK effect. We built a theoretical model of Ca(2+)-dependent active/inactive RhoK ratio and subsequent RhoK-dependent MLCP inactivation, which was further coupled with a four-state model of the contractile apparatus and Ca(2+)-dependent MLCK activation. The model explains the time course of the short-duration contraction and the role of RhoK by Ca(2+)-dependent activation of MLCK and RhoK, which inactivates MLCP. Oscillatory and non-oscillatory [Ca(2+)](i) responses result in a non-oscillatory contraction, the amplitude of which is encoded by the plateau value and oscillation frequency. In conclusion, Ca(2+)-dependent but CaMK II-independent RhoK activation contributes to the early phase of the contractile response via MLCP inhibition.

MioLab, a rat cardiac contractile force simulator: Applications to teaching cardiac cell physiology and biophysics.

PubMed

da Silva, Robson Rodrigues; Bissaco, Marcia Aparecida Silva; Goroso, Daniel Gustavo

2015-12-01

Understanding the basic concepts of physiology and biophysics of cardiac cells can be improved by virtual experiments that illustrate the complex excitation-contraction coupling process in cardiac cells. The aim of this study is to propose a rat cardiac myocyte simulator, with which calcium dynamics in excitation-contraction coupling of an isolated cell can be observed. This model has been used in the course "Mathematical Modeling and Simulation of Biological Systems". In this paper we present the didactic utility of the simulator MioLab(®). The simulator enables virtual experiments that can help studying inhibitors and activators in the sarcoplasmic reticulum sodium-calcium exchanger, thus corroborating a better understanding of the effects of medications, which are used to treat arrhythmias, on these compartments. The graphical interfaces were developed not only to facilitate the use of the simulator, but also to promote a constructive learning on the subject, since there are animations and videos for each stage of the simulation. The effectiveness of the simulator was tested by a group of graduate students. Some examples of simulations were presented in order to describe the overall structure of the simulator. Part of these virtual experiments became an activity for Biomedical Engineering graduate students, who evaluated the simulator based on its didactic quality. As a result, students answered a questionnaire on the usability and functionality of the simulator as a teaching tool. All students performed the proposed activities and classified the simulator as an optimal or good learning tool. In their written questions, students indicated as negative characteristics some problems with visualizing graphs; as positive characteristics, they indicated the simulator's didactic function, especially tutorials and videos on the topic of this study. The results show that the simulator complements the study of the physiology and biophysics of the cardiac cell. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Protective effects of vitamin E against myocardial ischemia/reperfusion injury in rats.

PubMed

Saleh, Nermine K; Saleh, Hanan A

2010-02-01

To clarify the cardioprotective effects of a short course of vitamin E treatment (vit E) as compared with a nitric oxide donor, nitroglycerin (GTN) against ischemia-reperfusion induced heart injury in rats. This randomized control study was conducted in the Physiology Department, Faculty of Medicine, Ain Shams University, Cairo, Egypt from 1st June to 31 August 2009. This work was undertaken on 28 female Wistar rats weighing 150- 200 gm. Rats were allocated into 4 groups; control group (non-treated), GTN-treated group (rats received GTN intraperitoneally 25 minutes before sacrifice, in a dose of 120 ug/kg body weight), vit E-treated group (rat received vit E by oral tubal feeding 16-20 hours before sacrifice, in a dose of 250 mg/rat), and vit E and GTN-treated group (rats received vit E and GTN as in both GTN-treated group and vit E -treated group). After sacrifice, the hearts were excised and perfused in a Langendorff preparation and subjected to 30 minutes global ischemia and reperfused for 30 minutes. Following reperfusion, heart tissues were used for assessment of malondialdehyde (MDA) and nicotinamide adenine dinucleotide (NAD)+, and for histological examination. Vitamin E treatment resulted in an enhanced post-ischemic recovery of systolic function in vit E-treated groups (vit E-treated group, and vit E and GTN-treated group) compared to the control group. Post-ischemic recovery of coronary flow was enhanced in the vit E-treated group compared to the GTN-treated group. Post ischemic tissue degeneration indicators: MDA, and NAD+ indicated a cardioprotective effect of vit E. Histological study revealed marked improvement of myocytes and mitochondrial structure in the vit E-treated group as compared with the control group. Preconditioning with vit E treatment afforded substantial recovery of post-ischemic contractile, and vascular functions compared to GTN treatment, the mechanism might involve less opening of mitochondrial permeability transition during postischemic reperfusion.

Identification of ML204, a Novel Potent Antagonist That Selectively Modulates Native TRPC4/C5 Ion Channels*

PubMed Central

Miller, Melissa; Shi, Jie; Zhu, Yingmin; Kustov, Maksym; Tian, Jin-bin; Stevens, Amy; Wu, Meng; Xu, Jia; Long, Shunyou; Yang, Pu; Zholos, Alexander V.; Salovich, James M.; Weaver, C. David; Hopkins, Corey R.; Lindsley, Craig W.; McManus, Owen; Li, Min; Zhu, Michael X.

2011-01-01

Transient receptor potential canonical (TRPC) channels are Ca2+-permeable nonselective cation channels implicated in diverse physiological functions, including smooth muscle contractility and synaptic transmission. However, lack of potent selective pharmacological inhibitors for TRPC channels has limited delineation of the roles of these channels in physiological systems. Here we report the identification and characterization of ML204 as a novel, potent, and selective TRPC4 channel inhibitor. A high throughput fluorescent screen of 305,000 compounds of the Molecular Libraries Small Molecule Repository was performed for inhibitors that blocked intracellular Ca2+ rise in response to stimulation of mouse TRPC4β by μ-opioid receptors. ML204 inhibited TRPC4β-mediated intracellular Ca2+ rise with an IC50 value of 0.96 μm and exhibited 19-fold selectivity against muscarinic receptor-coupled TRPC6 channel activation. In whole-cell patch clamp recordings, ML204 blocked TRPC4β currents activated through either μ-opioid receptor stimulation or intracellular dialysis of guanosine 5′-3-O-(thio)triphosphate (GTPγS), suggesting a direct interaction of ML204 with TRPC4 channels rather than any interference with the signal transduction pathways. Selectivity studies showed no appreciable block by 10–20 μm ML204 of TRPV1, TRPV3, TRPA1, and TRPM8, as well as KCNQ2 and native voltage-gated sodium, potassium, and calcium channels in mouse dorsal root ganglion neurons. In isolated guinea pig ileal myocytes, ML204 blocked muscarinic cation currents activated by bath application of carbachol or intracellular infusion of GTPγS, demonstrating its effectiveness on native TRPC4 currents. Therefore, ML204 represents an excellent novel tool for investigation of TRPC4 channel function and may facilitate the development of therapeutics targeted to TRPC4. PMID:21795696

Pharmacological inhibition of DNA methylation attenuates pressure overload-induced cardiac hypertrophy in rats.

PubMed

Stenzig, Justus; Schneeberger, Yvonne; Löser, Alexandra; Peters, Barbara S; Schaefer, Andreas; Zhao, Rong-Rong; Ng, Shi Ling; Höppner, Grit; Geertz, Birgit; Hirt, Marc N; Tan, Wilson; Wong, Eleanor; Reichenspurner, Hermann; Foo, Roger S-Y; Eschenhagen, Thomas

2018-07-01

Heart failure is associated with altered gene expression and DNA methylation. De novo DNA methylation is associated with gene silencing, but its role in cardiac pathology remains incompletely understood. We hypothesized that inhibition of DNA methyltransferases (DNMT) might prevent the deregulation of gene expression and the deterioration of cardiac function under pressure overload (PO). To test this hypothesis, we evaluated a DNMT inhibitor in PO in rats and analysed DNA methylation in cardiomyocytes. Young male Wistar rats were subjected to PO by transverse aortic constriction (TAC) or to sham surgery. Rats from both groups received solvent or 12.5 mg/kg body weight of the non-nucleosidic DNMT inhibitor RG108, initiated on the day of the intervention. After 4 weeks, we analysed cardiac function by MRI, fibrosis with Sirius Red staining, gene expression by RNA sequencing and qPCR, and DNA methylation by reduced representation bisulphite sequencing (RRBS). RG108 attenuated the ~70% increase in heart weight/body weight ratio of TAC over sham to 47% over sham, partially rescued reduced contractility, diminished the fibrotic response and the downregulation of a set of genes including Atp2a2 (SERCA2a) and Adrb1 (beta1-adrenoceptor). RG108 was associated with significantly lower global DNA methylation in cardiomyocytes by ~2%. The differentially methylated pathways were "cardiac hypertrophy", "cell death" and "xenobiotic metabolism signalling". Among these, "cardiac hypertrophy" was associated with significant methylation differences in the group comparison sham vs. TAC, but not significant between sham+RG108 and TAC+RG108 treatment, suggesting that RG108 partially prevented differential methylation. However, when comparing TAC and TAC+RG108, the pathway cardiac hypertrophy was not significantly differentially methylated. DNMT inhibitor treatment is associated with attenuation of cardiac hypertrophy and moderate changes in cardiomyocyte DNA methylation. The potential mechanistic link between these two effects and the role of non-myocytes need further clarification. Copyright © 2018 Elsevier Ltd. All rights reserved.

Soluble CD14 inhibits contractile function and insulin action in primary adult rat cardiomyocytes.

PubMed

Overhagen, Sabrina; Blumensatt, Marcel; Fahlbusch, Pia; Herzfeld de Wiza, Daniella; Müller, Heidi; Maxhera, Bujar; Akhyari, Payam; Ouwens, D Margriet

2017-02-01

Epicardial adipose tissue (EAT) from patients with type 2 diabetes (T2D) is characterized by monocyte infiltrations and displays an elevated release of the monocyte marker soluble cluster of differentiation 14 (sCD14) versus EAT from patients without T2D. We propose that an increased abundance of sCD14 in EAT from patients with T2D may impair the function and insulin sensitivity of the adjacent cardiomyocytes. To examine this, primary adult rat cardiomyocytes were incubated with increasing concentrations of sCD14 in the presence and absence of the co-receptor lipopolysaccharide (LPS), and analyzed for effects on determinants of contractile function, activation of inflammation signalling and insulin action. Exposing cardiomyocytes to sCD14 increased the phosphorylation of the stress kinases p38 and extracellular-signal regulated kinase (ERK). In contrast, insulin-mediated phosphorylation of Akt on Thr308 and Ser473 was inhibited. Furthermore, sCD14 impaired sarcomere shortening and cytosolic Ca 2+ -fluxes. All responses were concentration-dependent and became significant at 1ng/ml sCD14. LPS, either alone or in complex with sCD14, did not affect contractile function or the activation of stress kinases and insulin signalling pathways. Similar data on protein phosphorylation were obtained when exposing human umbilical vein endothelial cells to sCD14. Finally, pharmacological inhibition of p38 reversed the detrimental effects of sCD14 on contractile function, but not on sCD14-induced insulin resistance. Collectively, these data show that sCD14 impairs the function and insulin sensitivity of cardiomyocytes, suggesting that an enhanced sCD14 release from EAT in patients with T2D may contribute to the pathogenesis of diabetes-related cardiometabolic complications. Copyright © 2016 Elsevier B.V. All rights reserved.

Adenosine triphosphate as a molecular mediator of the vascular response to injury.

PubMed

Guth, Christy M; Luo, Weifung; Jolayemi, Olukemi; Chadalavada, Kalyan S; Komalavilas, Padmini; Cheung-Flynn, Joyce; Brophy, Colleen M

2017-08-01

Human saphenous veins used for arterial bypass undergo stretch injury at the time of harvest and preimplant preparation. Vascular injury promotes intimal hyperplasia, the leading cause of graft failure, but the molecular events leading to this response are largely unknown. This study investigated adenosine triphosphate (ATP) as a potential molecular mediator in the vascular response to stretch injury, and the downstream effects of the purinergic receptor, P2X7R, and p38 MAPK activation. A subfailure stretch rat aorta model was used to determine the effect of stretch injury on release of ATP and vasomotor responses. Stretch-injured tissues were treated with apyrase, the P2X7R antagonist, A438079, or the p38 MAPK inhibitor, SB203580, and subsequent contractile forces were measured using a muscle bath. An exogenous ATP (eATP) injury model was developed and the experiment repeated. Change in p38 MAPK phosphorylation after stretch and eATP tissue injury was determined using Western blotting. Noninjured tissue was incubated in the p38 MAPK activator, anisomycin, and subsequent contractile function and p38 MAPK phosphorylation were analyzed. Stretch injury was associated with release of ATP. Contractile function was decreased in tissue subjected to subfailure stretch, eATP, and anisomycin. Contractile function was restored by apyrase, P2X7R antagonism, and p38-MAPK inhibition. Stretch, eATP, and anisomycin-injured tissue demonstrated increased phosphorylation of p38 MAPK. Taken together, these data suggest that the vascular response to stretch injury is associated with release of ATP and activation of the P2X7R/P38 MAPK pathway, resulting in contractile dysfunction. Modulation of this pathway in vein grafts after harvest and before implantation may reduce the vascular response to injury. Copyright © 2017 Elsevier Inc. All rights reserved.

Effects of temperature on power output and contraction kinetics in the locomotor muscle of the regionally endothermic common thresher shark (Alopias vulpinus).

PubMed

Donley, Jeanine M; Sepulveda, Chugey A; Aalbers, Scott A; McGillivray, David G; Syme, Douglas A; Bernal, Diego

2012-10-01

The common thresher shark (Alopias vulpinus) is a pelagic species with medially positioned red aerobic swimming musculature (RM) and regional RM endothermy. This study tested whether the contractile characteristics of the RM are functionally similar along the length of the body and assessed how the contractile properties of the common thresher shark compare with those of other sharks. Contractile properties of the RM were examined at 8, 16 and 24 °C from anterior and posterior axial positions (0.4 and 0.6 fork length, respectively) using the work loop technique. Experiments were performed to determine whether the contractile properties of the RM are similar along the body of the common thresher shark and to document the effects of temperature on muscle power. Axial differences in contractile properties of RM were found to be small or absent. Isometric twitch kinetics of RM were ~fivefold slower than those of white muscle, with RM twitch durations of about 1 s at 24 °C and exceeding 5 s at 8 °C, a Q(10) of nearly 2.5. Power increased approximately tenfold with the 16 °C increase in temperature, while the cycle frequency for maximal power only increased from about 0.5-1.0 Hz over this temperature range. These data support the hypothesis that the RM is functionally similar along the body of the common thresher shark and corroborate previous findings from shark species both with and without medial RM. While twitch kinetics suggest the endothermic RM is not unusually temperature sensitive, measures of power suggest that the RM is not well suited to function at cool temperatures. The cycle frequency at which power is maximized appeared relatively insensitive to temperature in RM, which may reflect the relatively cooler temperature of the thresher RM compared to that observed in lamnid sharks as well as the relatively slow RM phenotype in these large fish.

2-Deoxyadenosine triphosphate restores the contractile function of cardiac myofibril from adult dogs with naturally occurring dilated cardiomyopathy

PubMed Central

Cheng, Yuanhua; Hogarth, Kaley A.; O'Sullivan, M. Lynne; Regnier, Michael

2015-01-01

Dilated cardiomyopathy (DCM) is a major type of heart failure resulting from loss of systolic function. Naturally occurring canine DCM is a widely accepted experimental paradigm for studying human DCM. 2-Deoxyadenosine triphosphate (dATP) can be used by myosin and is a superior energy substrate over ATP for cross-bridge formation and increased systolic function. The objective of this study was to evaluate the beneficial effect of dATP on contractile function of cardiac myofibrils from dogs with naturally occurring DCM. We measured actomyosin NTPase activity and contraction/relaxation properties of isolated myofibrils from nonfailing (NF) and DCM canine hearts. NTPase assays indicated replacement of ATP with dATP significantly increased myofilament activity in both NF and DCM samples. dATP significantly improved maximal tension of DCM myofibrils to the NF sample level. dATP also restored Ca2+ sensitivity of tension that was reduced in DCM samples. Similarly, dATP increased the kinetics of contractile activation (kACT), with no impact on the rate of cross-bridge tension redevelopment (kTR). Thus, the activation kinetics (kACT/kTR) that were reduced in DCM samples were restored for dATP to NF sample levels. dATP had little effect on relaxation. The rate of early slow-phase relaxation was slightly reduced with dATP, but its duration was not, nor was the fast-phase relaxation or times to 50 and 90% relaxation. Our findings suggest that myosin utilization of dATP improves cardiac myofibril contractile properties of naturally occurring DCM canine samples, restoring them to NF levels, without compromising relaxation. This suggests elevation of cardiac dATP is a promising approach for the treatment of DCM. PMID:26497964

2-Deoxyadenosine triphosphate restores the contractile function of cardiac myofibril from adult dogs with naturally occurring dilated cardiomyopathy.

PubMed

Cheng, Yuanhua; Hogarth, Kaley A; O'Sullivan, M Lynne; Regnier, Michael; Pyle, W Glen

2016-01-01

Dilated cardiomyopathy (DCM) is a major type of heart failure resulting from loss of systolic function. Naturally occurring canine DCM is a widely accepted experimental paradigm for studying human DCM. 2-Deoxyadenosine triphosphate (dATP) can be used by myosin and is a superior energy substrate over ATP for cross-bridge formation and increased systolic function. The objective of this study was to evaluate the beneficial effect of dATP on contractile function of cardiac myofibrils from dogs with naturally occurring DCM. We measured actomyosin NTPase activity and contraction/relaxation properties of isolated myofibrils from nonfailing (NF) and DCM canine hearts. NTPase assays indicated replacement of ATP with dATP significantly increased myofilament activity in both NF and DCM samples. dATP significantly improved maximal tension of DCM myofibrils to the NF sample level. dATP also restored Ca(2+) sensitivity of tension that was reduced in DCM samples. Similarly, dATP increased the kinetics of contractile activation (kACT), with no impact on the rate of cross-bridge tension redevelopment (kTR). Thus, the activation kinetics (kACT/kTR) that were reduced in DCM samples were restored for dATP to NF sample levels. dATP had little effect on relaxation. The rate of early slow-phase relaxation was slightly reduced with dATP, but its duration was not, nor was the fast-phase relaxation or times to 50 and 90% relaxation. Our findings suggest that myosin utilization of dATP improves cardiac myofibril contractile properties of naturally occurring DCM canine samples, restoring them to NF levels, without compromising relaxation. This suggests elevation of cardiac dATP is a promising approach for the treatment of DCM. Copyright © 2016 the American Physiological Society.

Fiber-type-specific sensitivities and phenotypic adaptations to dietary fat overload differentially impact fast- versus slow-twitch muscle contractile function in C57BL/6J mice.

PubMed

Ciapaite, Jolita; van den Berg, Sjoerd A; Houten, Sander M; Nicolay, Klaas; van Dijk, Ko Willems; Jeneson, Jeroen A

2015-02-01

High-fat diets (HFDs) have been shown to interfere with skeletal muscle energy metabolism and cause peripheral insulin resistance. However, understanding of HFD impact on skeletal muscle primary function, i.e., contractile performance, is limited. Male C57BL/6J mice were fed HFD containing lard (HFL) or palm oil (HFP), or low-fat diet (LFD) for 5weeks. Fast-twitch (FT) extensor digitorum longus (EDL) and slow-twitch (ST) soleus muscles were characterized with respect to contractile function and selected biochemical features. In FT EDL muscle, a 30%-50% increase in fatty acid (FA) content and doubling of long-chain acylcarnitine (C14-C18) content in response to HFL and HFP feeding were accompanied by increase in protein levels of peroxisome proliferator-activated receptor-γ coactivator-1α, mitochondrial oxidative phosphorylation complexes and acyl-CoA dehydrogenases involved in mitochondrial FA β-oxidation. Peak force of FT EDL twitch and tetanic contractions was unaltered, but the relaxation time (RT) of twitch contractions was 30% slower compared to LFD controls. The latter was caused by accumulation of lipid intermediates rather than changes in the expression levels of proteins involved in calcium handling. In ST soleus muscle, no evidence for lipid overload was found in any HFD group. However, particularly in HFP group, the peak force of twitch and tetanic contractions was reduced, but RT was faster than LFD controls. The latter was associated with a fast-to-slow shift in troponin T isoform expression. Taken together, these data highlight fiber-type-specific sensitivities and phenotypic adaptations to dietary lipid overload that differentially impact fast- versus slow-twitch skeletal muscle contractile function. Copyright © 2015 Elsevier Inc. All rights reserved.

Proteome- and transcriptome-driven reconstruction of the human myocyte metabolic network and its use for identification of markers for diabetes.

PubMed

Väremo, Leif; Scheele, Camilla; Broholm, Christa; Mardinoglu, Adil; Kampf, Caroline; Asplund, Anna; Nookaew, Intawat; Uhlén, Mathias; Pedersen, Bente Klarlund; Nielsen, Jens

2015-05-12

Skeletal myocytes are metabolically active and susceptible to insulin resistance and are thus implicated in type 2 diabetes (T2D). This complex disease involves systemic metabolic changes, and their elucidation at the systems level requires genome-wide data and biological networks. Genome-scale metabolic models (GEMs) provide a network context for the integration of high-throughput data. We generated myocyte-specific RNA-sequencing data and investigated their correlation with proteome data. These data were then used to reconstruct a comprehensive myocyte GEM. Next, we performed a meta-analysis of six studies comparing muscle transcription in T2D versus healthy subjects. Transcriptional changes were mapped on the myocyte GEM, revealing extensive transcriptional regulation in T2D, particularly around pyruvate oxidation, branched-chain amino acid catabolism, and tetrahydrofolate metabolism, connected through the downregulated dihydrolipoamide dehydrogenase. Strikingly, the gene signature underlying this metabolic regulation successfully classifies the disease state of individual samples, suggesting that regulation of these pathways is a ubiquitous feature of myocytes in response to T2D. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Ginseng Is Useful to Enhance Cardiac Contractility in Animals

PubMed Central

Cherng, Yih-Giun; Chen, Li-Jen; Niu, Ho-Shan; Chang, Chen Kuei; Niu, Chiang-Shan

2014-01-01

Ginseng has been shown to be effective on cardiac dysfunction. Recent evidence has highlighted the mediation of peroxisome proliferator-activated receptors (PPARs) in cardiac function. Thus, we are interested to investigate the role of PPARδ in ginseng-induced modification of cardiac contractility. The isolated hearts in Langendorff apparatus and hemodynamic analysis in catheterized rats were applied to measure the actions of ginseng ex vivo and in vivo. In normal rats, ginseng enhanced cardiac contractility and hemodynamic dP/dt max significantly. Both actions were diminished by GSK0660 at a dose enough to block PPARδ. However, ginseng failed to modify heart rate at the same dose, although it did produce a mild increase in blood pressure. Data of intracellular calcium level and Western blotting analysis showed that both the PPARδ expression and troponin I phosphorylation were raised by ginseng in neonatal rat cardiomyocyte. Thus, we suggest that ginseng could enhance cardiac contractility through increased PPARδ expression in cardiac cells. PMID:24689053