The Small Muscle-Specific Protein Csl Modifies Cell Shape and Promotes Myocyte Fusion in an Insulin-like Growth Factor 1–Dependent Manner

PubMed Central

Palmer, Steve; Groves, Nicola; Schindeler, Aaron; Yeoh, Thomas; Biben, Christine; Wang, Cheng-Chun; Sparrow, Duncan B.; Barnett, Louise; Jenkins, Nancy A.; Copeland, Neal G.; Koentgen, Frank; Mohun, Tim; Harvey, Richard P.

2001-01-01

We have isolated a murine cDNA encoding a 9-kD protein, Chisel (Csl), in a screen for transcriptional targets of the cardiac homeodomain factor Nkx2-5. Csl transcripts were detected in atria and ventricles of the heart and in all skeletal muscles and smooth muscles of the stomach and pulmonary veins. Csl protein was distributed throughout the cytoplasm in fetal muscles, although costameric and M-line localization to the muscle cytoskeleton became obvious after further maturation. Targeted disruption of Csl showed no overt muscle phenotype. However, ectopic expression in C2C12 myoblasts induced formation of lamellipodia in which Csl protein became tethered to membrane ruffles. Migration of these cells was retarded in a monolayer wound repair assay. Csl-expressing myoblasts differentiated and fused normally, although in the presence of insulin-like growth factor (IGF)-1 they showed dramatically enhanced fusion, leading to formation of large dysmorphogenic “myosacs.” The activities of transcription factors nuclear factor of activated T cells (NFAT) and myocyte enhancer–binding factor (MEF)2, were also enhanced in an IGF-1 signaling–dependent manner. The dynamic cytoskeletal localization of Csl and its dominant effects on cell shape and behavior and transcription factor activity suggest that Csl plays a role in the regulatory network through which muscle cells coordinate their structural and functional states during growth, adaptation, and repair. PMID:11381084

Myocyte enhancer factor 2A promotes proliferation and its inhibition attenuates myogenic differentiation via myozenin 2 in bovine skeletal muscle myoblast

PubMed Central

Wang, Ya-Ning; Yang, Wu-Cai; Li, Pei-Wei; Wang, Hong-Bao; Zhang, Ying-Ying

2018-01-01

Myocyte enhancer factor 2A (MEF2A) is widely distributed in various tissues or organs and plays crucial roles in multiple biological processes. To examine the potential effects of MEF2A on skeletal muscle myoblast, the functional role of MFE2A in myoblast proliferation and differentiation was investigated. In this study, we found that the mRNA expression level of Mef2a was dramatically increased during the myogenesis of bovine skeletal muscle primary myoblast. Overexpression of MEF2A significantly promoted myoblast proliferation, while knockdown of MEF2A inhibited the proliferation and differentiation of myoblast. RT-PCR and western blot analysis revealed that this positive effect of MEF2A on the proliferation of myoblast was carried out by triggering cell cycle progression by activating CDK2 protein expression. Besides, MEF2A was found to be an important transcription factor that bound to the myozenin 2 (MyoZ2) proximal promoter and performed upstream of MyoZ2 during myoblast differentiation. This study provides the first experimental evidence that MEF2A is a positive regulator in skeletal muscle myoblast proliferation and suggests that MEF2A regulates myoblast differentiation via regulating MyoZ2. PMID:29698438

Insulin antagonises pigment epithelium-derived factor (PEDF)-induced modulation of lineage commitment of myocytes and heterotrophic ossification.

PubMed

Carnagarin, Revathy; Elahy, Mina; Dharmarajan, Arun M; Dass, Crispin R

2017-12-16

Extensive bone defects arising as a result of trauma, infection and tumour resection and other bone pathologies necessitates the identification of effective strategies in the form of tissue engineering, gene therapy and osteoinductive agents to enhance the bone repair process. PEDF is a multifunctional glycoprotein which plays an important role in regulating osteoblastic differentiation and bone formation. PEDF treatment of mice and human skeletal myocytes at physiological concentration inhibited myogenic differentiation and activated Erk1/2 MAPK- dependent osteogenic transdifferentiation of myocytes. In mice, insulin, a promoter of bone regeneration, attenuated PEDF-induced expression of osteogenic markers such as osteocalcin, alkaline phosphatase and mineralisation for bone formation in the muscle and surrounding adipose tissue. These results provide new insights into the molecular aspects of the antagonising effect of insulin on PEDF-dependent modulation of the differentiation commitment of musculoskeletal environment into osteogenesis, and suggest that PEDF may be developed as an effective clinical therapy for bone regeneration as its heterotopic ossification can be controlled via co-administration of insulin. Copyright © 2017 Elsevier B.V. All rights reserved.

Ultrasonic destruction of albumin microbubbles enhances gene transfection and expression in cardiac myocytes.

PubMed

Wang, Guo-zhong; Liu, Jing-hua; Lü, Shu-zheng; Lü, Yun; Guo, Cheng-jun; Zhao, Dong-hui; Fang, Dong-ping; He, Dong-fang; Zhou, Yuan; Ge, Chang-jiang

2011-05-01

It has been proven that ultrasonic destruction of microbubbles can enhance gene transfection efficiency into the noncardiac cells, but there are few reports about cardiac myocytes. Moreover, the exact mechanisms are not yet clear; whether the characteristic of microbubbles can affect the gene transfection efficiency or not is still controversial. This study was designed to investigate whether the ultrasound destruction of gene-loaded microbubbles could enhance the plasmids carried reporter gene transfection in primary cultured myocardial cell, and evaluate the effects of microbubbles characteristics on the transgene expression in cardiac myocytes. The β-galactosidase plasmids attached to the two types of microbubbles, air-contained sonicated dextrose albumin (ASDA) and perfluoropropane-exposed sonicated dextrose albumin (PESDA) were prepared. The gene transfection into cardiac myocytes was performed in vitro by naked plasmids, ultrasound exposure, ultrasonic destruction of gene-loaded microbubbles and calcium phosphate precipitation, and then the gene expression and cell viability were analyzed. The ultrasonic destruction of gene-loaded microbubbles enhanced gene expression in cardiac myocytes compared with naked plasmid transfection ((51.95 ± 2.41) U/g or (29.28 ± 3.65) U/g vs. (0.84 ± 0.21) U/g, P < 0.01), and ultrasonic destruction PESDA resulted in more significant gene expression than ASDA ((51.95 ± 2.41) U/g vs. (29.28 ± 3.65) U/g, P < 0.05). Ultrasonic destruction of microbubbles during calcium phosphate precipitation gene transfection enhanced β-galactosidase activity nearly 8-fold compared with calcium phosphate precipitation gene transfection alone ((111.35 ± 11.21) U/g protein vs. (14.13 ± 2.58) U/g protein, P < 0.01). Even 6 hours after calcium phosphate precipitation gene transfection, ultrasound-mediated microbubbles destruction resulted in more intense gene expression ((35.63 ± 7.65) U/g vs. (14.13 ± 2.58) U/g, P < 0.05). Ultrasonic destruction of microbubbles might be a promising method for the delivery of non-viral DNA into cardiac myocytes, and the gene tranfection is related to the characteristics of microbubbles.

Regulation of muscle GLUT-4 transcription by AMP-activated protein kinase.

PubMed

Zheng, D; MacLean, P S; Pohnert, S C; Knight, J B; Olson, A L; Winder, W W; Dohm, G L

2001-09-01

Skeletal muscle GLUT-4 transcription in response to treatment with 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR), a known activator of AMP-activated protein kinase (AMPK), was studied in rats and mice. The increase in GLUT-4 mRNA levels in response to a single subcutaneous injection of AICAR, peaked at 13 h in white and red quadriceps muscles but not in the soleus muscle. The mRNA level of chloramphenicol acyltransferase reporter gene which is driven by 1,154 or 895 bp of the human GLUT-4 proximal promoter was increased in AICAR-treated transgenic mice, demonstrating the transcriptional upregulation of the GLUT-4 gene by AICAR. However, this induction of transcription was not apparent with 730 bp of the promoter. In addition, nuclear extracts from AICAR-treated mice bound to the consensus sequence of myocyte enhancer factor-2 (from -473 to -464) to a greater extent than from saline-injected mice. Thus AMP-activated protein kinase activation by AICAR increases GLUT-4 transcription by a mechanism that requires response elements within 895 bp of human GLUT-4 proximal promoter and that may be cooperatively mediated by myocyte enhancer factor-2.

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.

Purinergic modulation of adult guinea pig cardiomyocytes in long term cultures and co-cultures with extracardiac or intrinsic cardiac neurones.

PubMed

Horackova, M; Huang, M H; Armour, J A

1994-05-01

To determine the capacity of ATP to modify cardiomyocytes directly or indirectly via peripheral autonomic neurones, the effects of various purinergic agents were studied on long term cultures of adult guinea pig ventricular myocytes and their co-cultures with extracardiac (stellate ganglion) or intrinsic cardiac neurones. Ventricular myocytes and cardiac neurones were enzymatically dissociated and plated together or alone (myocytes only). Myocyte cultures were used for experiments after three to six weeks. The electrical and contractile properties of cultured myocytes and myocyte-neuronal networks were investigated. The spontaneous beating frequency of ventricular myocytes co-cultured with stellate ganglion neurones increased by approximately 140% (p < 0.001) following superfusion with 10(-5) M ATP. This effect was not modified significantly by tetrodotoxin or by beta adrenoceptor blockade (10(-5) M timolol), but was eliminated following application of the P2 antagonist suramin (10(-5) M). Basal spontaneous contractile rate was reduced by approximately 86% (p < 0.001) in the presence of suramin, indicating the existence of tonically active purinergic synaptic mechanisms in stellate ganglion neurone-myocyte cocultures. Suramin did not significantly affect non-innervated myocyte cultures. ATP increased myocyte contractile rate in intrinsic cardiac neurone-myocyte co-cultures by approximately 40% (p < 0.01) under control conditions, but when beta adrenergic receptors of tetrodotoxin sensitive neural responses were blocked, ATP induced greater augmentation (> 100%). In contrast, ATP induced much smaller effects in non-innervated myocyte cultures (approximately 26%, p < 0.01). Analogues of AT) showed the following order of potency: ATP > UTP > MSATP > beta gamma ATP > alpha beta ATP. Adenosine (10(-4) M) attenuated the beating frequency of myocytes in both types of co-culture, while not significantly affecting non-innervated myocyte cultures. The experimental model used in this study showed that extrinsic and intrinsic cardiac neurones which possess P2 receptors can greatly enhance cardiac myocyte contractile rate when activated by ATP. Since adenosine reduced contractile rate in both types of co-cultures while not affecting non-innervated myocytes, it is concluded that some of these neurones possess P1 receptors.

Acute administration of tumour necrosis factor-α induces spontaneous calcium release via the reactive oxygen species pathway in atrial myocytes.

PubMed

Zuo, Song; Li, Lin-Ling; Ruan, Yan-Fei; Jiang, Le; Li, Xin; Li, Song-Nan; Wen, Song-Nan; Bai, Rong; Liu, Nian; Du, Xin; Dong, Jian-Zeng; Ma, Chang-Sheng

2017-10-17

The arrhythmogenic mechanisms of atrial fibrillation (AF) that are induced by acute inflammation, such as postoperative AF, are not well understood. We investigated the acute effects of tumour necrosis factor-α (TNF-α) that mimic acute inflammation on Ca2+ handling in isolated atrial myocytes and its underlying mechanisms. Cytosol Ca2+ handling and mitochondrial reactive oxygen species (ROS) production were studied in freshly isolated atrial myocytes of wild-type mice that were exposed to TNF-α (0.05 ng/mL) for 2 h by Ionoptix and confocal microscopy. The acute effects of TNF-α on Ca2+ handling were decreased amplitudes and prolonged decay times of Ca2+ transients in isolated atrial myocytes. A significant reduction in the sarcoplasmic reticulum (SR) Ca2+ content was detected in TNF-α treated cells, which was associated with increased spontaneous Ca2+ release events. In particular, physiological concentrations of TNF-α dramatically promoted the frequency of spontaneous Ca2+ waves and Ca2+ sparks, while the spark mass presented with reduced amplitudes and prolonged durations. The underlying mechanisms of pro-arrhythmic effects of TNF-α were further investigated. Acute exposure to TNF-α rapidly promoted mitochondrial ROS production that was correlated with the acute effect of TNF-α on Ca2+ handling, and enhanced the oxidation of calcium/calmodulin-dependent protein kinase II (CaMKII) and the phosphorylation of RyR2. However, the performance of ROS inhibitor, DL-Dithiothreitol (DTT), reversed Ca2+ handling disorders induced by TNF-α. Tumour necrosis factor-α rapidly increases spontaneous Ca2+ release and promotes atrial arrhythmogenesis via the ROS pathway, which suggests that antioxidant therapy is a promising strategy for acute inflammation related AF. Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2017. For permissions, please email: journals.permissions@oup.com.

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

Crosstalk between monocytes and myometrial smooth muscle in culture generates synergistic pro-inflammatory cytokine production and enhances myocyte contraction, with effects opposed by progesterone.

PubMed

Rajagopal, S P; Hutchinson, J L; Dorward, D A; Rossi, A G; Norman, J E

2015-08-01

Both term and preterm parturition are characterized by an influx of macrophages and neutrophils into the myometrium and cervix, with co-incident increased peripheral blood monocyte activation. Infection and inflammation are strongly implicated in the pathology of preterm labour (PTL), with progesterone considered a promising candidate for its prevention or treatment. In this study, we investigated the effect of monocytes on myometrial smooth muscle cell inflammatory cytokine production both alone and in response to LPS, a TLR4 agonist used to trigger PTL in vivo. We also investigated the effect of monocytes on myocyte contraction. Monocytes, isolated from peripheral blood samples from term pregnant women, were cultured alone, or co-cultured with PHM1-41 myometrial smooth muscle cells, for 24 h. In a third set of experiments, PHM1-41 myocytes were cultured for 24 h in isolation. Cytokine secretion was determined by ELISA or multiplex assays. Co-culture of monocytes and myocytes led to synergistic secretion of pro-inflammatory cytokines and chemokines including IL-6, IL-8 and MCP-1, with the secretion being further enhanced by LPS (100 ng/ml). The synergistic secretion of IL-6 and IL-8 from co-cultures was mediated in part by direct cell-cell contact, and by TNF. Conditioned media from co-cultures stimulated contraction of PHM1-41 myocytes, and the effect was inhibited by progesterone. Both progesterone and IL-10 inhibited LPS-stimulated IL-6 and IL-8 secretion from co-cultures, while progesterone also inhibited chemokine secretion. These data suggest that monocytes infiltrating the myometrium at labour participate in crosstalk that potentiates pro-inflammatory cytokine secretion, an effect that is enhanced by LPS, and can augment myocyte contraction. These effects are all partially inhibited by progesterone. © The Author 2015. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology.

Connective tissue growth factor induces cardiac hypertrophy through Akt signaling

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

Hayata, Nozomi; Fujio, Yasushi; Yamamoto, Yasuhiro

2008-05-30

In the process of cardiac remodeling, connective tissue growth factor (CTGF/CCN2) is secreted from cardiac myocytes. Though CTGF is well known to promote fibroblast proliferation, its pathophysiological effects in cardiac myocytes remain to be elucidated. In this study, we examined the biological effects of CTGF in rat neonatal cardiomyocytes. Cardiac myocytes stimulated with full length CTGF and its C-terminal region peptide showed the increase in cell surface area. Similar to hypertrophic ligands for G-protein coupled receptors, such as endothelin-1, CTGF activated amino acid uptake; however, CTGF-induced hypertrophy is not associated with the increased expression of skeletal actin or BNP, analyzedmore » by Northern-blotting. CTGF treatment activated ERK1/2, p38 MAPK, JNK and Akt. The inhibition of Akt by transducing dominant-negative Akt abrogated CTGF-mediated increase in cell size, while the inhibition of MAP kinases did not affect the cardiac hypertrophy. These findings indicate that CTGF is a novel hypertrophic factor in cardiac myocytes.« less

Sphingosine mediates the immediate negative inotropic effects of tumor necrosis factor-alpha in the adult mammalian cardiac myocyte.

PubMed

Oral, H; Dorn, G W; Mann, D L

1997-02-21

To determine whether activation of the neutral sphingomyelinase pathway was responsible for the immediate (<30 min) negative inotropic effects of tumor necrosis factor-alpha (TNF-alpha), we examined sphingosine levels in diluent and TNF-alpha-stimulated cardiac myocytes. TNF-alpha stimulation of adult feline cardiac myocytes provoked a rapid (<15 min) increase in the hydrolysis of [14C]sphingomyelin in cell-free extracts, as well as an increase in ceramide mass, consistent with cytokine-induced activation of the neutral sphingomyelinase pathway. High performance liquid chromatographic analysis of lipid extracts from TNF-alpha-stimulated cardiac myocytes showed that TNF-alpha stimulation produced a rapid (<30 min) increase in free sphingosine levels. Moreover, exogenous D-sphingosine mimicked the effects of TNF-alpha on intracellular calcium homeostasis, as well as the negative inotropic effects of TNF-alpha in isolated contracting myocytes; time course studies showed that exogenous D-sphingosine produced abnormalities in cell shortening that were maximal at 5 min. Finally, blocking sphingosine production using an inhibitor of ceramidase, n-oleoylethanolamine, completely abrogated the negative inotropic effects of TNF-alpha in isolated contracting cardiac myocytes. Additional studies employing biologically active ceramide analogs and sphingosine 1-phosphate suggested that neither the immediate precursor of sphingosine nor the immediate metabolite of sphingosine, respectively, were likely to be responsible for the immediate negative inotropic effects of TNF-alpha. Thus, these studies suggest that sphingosine mediates the immediate negative inotropic effects of TNF-alpha in isolated cardiac myocytes.

Distinct Effects of Abelson Kinase Mutations on Myocytes and Neurons in Dissociated Drosophila Embryonic Cultures: Mimicking of High Temperature

PubMed Central

Liu, Lijuan; Wu, Chun-Fang

2014-01-01

Abelson tyrosine kinase (Abl) is known to regulate axon guidance, muscle development, and cell-cell interaction in vivo. The Drosophila primary culture system offers advantages in exploring the cellular mechanisms mediated by Abl with utilizing various experimental manipulations. Here we demonstrate that single-embryo cultures exhibit stage-dependent characteristics of cellular differentiation and developmental progression in neurons and myocytes, as well as nerve-muscle contacts. In particular, muscle development critically depends on the stage of dissociated embryos. In wild-type (WT) cultures derived from embryos before stage 12, muscle cells remained within cell clusters and were rarely detected. Interestingly, abundant myocytes were spotted in Abl mutant cultures, exhibiting enhanced myocyte movement and fusion, as well as neuron-muscle contacts even in cultures dissociated from younger, stage 10 embryos. Notably, Abl myocytes frequently displayed well-expanded lamellipodia. Conversely, Abl neurons were characterized with fewer large veil-like lamellipodia, but instead had increased numbers of filopodia and darker nodes along neurites. These distinct phenotypes were equally evident in both homo- and hetero-zygous cultures (Abl/Abl vs. Abl/+) of different alleles (Abl1 and Abl4) indicating dominant mutational effects. Strikingly, in WT cultures derived from stage 10 embryos, high temperature (HT) incubation promoted muscle migration and fusion, partially mimicking the advanced muscle development typical of Abl cultures. However, HT enhanced neuronal growth with increased numbers of enlarged lamellipodia, distinct from the characteristic Abl neuronal morphology. Intriguingly, HT incubation also promoted Abl lamellipodia expansion, with a much greater effect on nerve cells than muscle. Our results suggest that Abl is an essential regulator for myocyte and neuron development and that high-temperature incubation partially mimics the faster muscle development typical of Abl cultures. Despite the extensive alterations by Abl mutations, we observed myocyte fusion events and nerve-muscle contact formation between WT and Abl cells in mixed WT and Abl cultures derived from labeled embryos. PMID:24466097

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.

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.

Neuropeptide Y rapidly enhances [Ca2+]i transients and Ca2+ sparks in adult rat ventricular myocytes through Y1 receptor and PLC activation.

PubMed

Heredia, María del Puy; Delgado, Carmen; Pereira, Laetitia; Perrier, Romain; Richard, Sylvain; Vassort, Guy; Bénitah, Jean-Pierre; Gómez, Ana María

2005-01-01

Neuropeptide Y (NPY) is the most abundant peptide in the mammalian heart, but its cardiac actions are not fully understood. Here we investigate the effect of NPY in intracellular Ca2+ release, using isolated rat cardiac myocytes and confocal microscopy. Cardiac myocytes were field-stimulated at 1 Hz. The evoked [Ca2+]i transient was of higher amplitude and of faster decay in the presence of 100 nM NPY. Cell contraction was also increased by NPY. We analyzed the occurrence of Ca2+ sparks and their characteristics after NPY application. NPY significantly increased Ca2+ sparks frequency in quiescent cells. The Ca2+ spark amplitude was enhanced by NPY but the other characteristics of Ca2+ sparks were not significantly altered. Because cardiac myocytes express both Y1 and Y2 NPY receptors, we repeated the experiments in the presence of the receptor blockers, BIBP3226 and BIIE0246. We found that Y1 NPY receptor blockade completely inhibited NPY effects on [Ca2+]i transient. PTX-sensitive G-proteins and/or phospholypase C (PLC) have been invoked to mediate NPY effects in other cell types. We tested these two hypotheses. In PTX-treated myocytes NPY was still effective, which suggests that the observed NPY actions are not mediated by PTX-sensitive G-proteins. In contrast, the increase in [Ca2+]i transient by NPY was completely inhibited by the PLC inhibitor U73122. In conclusion, we find that NPY has a positive inotropic effect in isolated rat cardiac myocytes, which involves increase in Ca2+ release after activation of Y1 NPY receptor and subsequent stimulation of PLC.

JS-K, a GST-activated nitric oxide donor prodrug, enhances chemo-sensitivity in renal carcinoma cells and prevents cardiac myocytes toxicity induced by Doxorubicin.

PubMed

Qiu, Mingning; Ke, Longzhi; Zhang, Sai; Zeng, Xin; Fang, Zesong; Liu, Jianjun

2017-08-01

Doxorubicin, a highly effective and widely used anthracycline antibiotic in multiple chemotherapy regimens, has been limited by its cardiotoxicity. The aim of this study is to investigate the effect of nitric oxide donor prodrug JS-K on proliferation and apoptosis in renal carcinoma cells and cardiac myocytes toxicity induced by Doxorubicin and to explore possible p53-related mechanism in renal carcinoma cells. The effect of JS-K on anti-cancer activity of Doxorubicin was investigated in renal carcinoma cells via detecting cell proliferation, cytotoxicity, cell death and apoptosis and expressions of apoptotic-related proteins. Effect of p53 on the combination of JS-K and Doxorubicin was determined using p53 inhibitor Pifithrin-α and p53 activator III. Furthermore, the effect of JS-K on cardiac myocytes toxicity of Doxorubicin was investigated in H9c2 (2-1) cardiac myocytes via measuring cell growth, cell death and apoptosis, expressions of proteins involved in apoptosis and intracellular reactive oxygen species. We demonstrated that JS-K could increase Doxorubicin-induced renal carcinoma cell growth suppression and apoptosis and could increase expressions of proteins that are involved in apoptosis. Additionally, Pifithrin-α reversed the promoting effect of JS-K on Doxorubicin-induced renal carcinoma cell apoptosis; conversely, the p53 activator III exacerbated the promoting effect of JS-K on Doxorubicin-induced renal carcinoma cell apoptosis. Furthermore, JS-K protected H9c2 (2-1) cardiac myocytes against Doxorubicin-induced toxicity and decreased Doxorubicin-induced reactive oxygen species production. JS-K enhances the anti-cancer activity of Doxorubicin in renal carcinoma cells by upregulating p53 expression and prevents cardiac myocytes toxicity of Doxorubicin by decreasing oxidative stress.

Protein kinase C enhances the swelling-induced chloride current in human atrial myocytes.

PubMed

Li, Ye-Tao; Du, Xin-Ling

2016-06-01

Swelling-activated chloride currents (ICl.swell) are thought to play a role in several physiologic and pathophysiologic processes and thus represent a target for therapeutic approaches. However, the mechanism of ICl.swell regulation remains unclear. In this study, we used the whole-cell patch-clamp technique to examine the role of protein kinase C (PKC) in the regulation of ICl.swell in human atrial myocytes. Atrial myocytes were isolated from the right atrial appendages of patients undergoing coronary artery bypass and enzymatically dissociated. ICl.swell was evoked in hypotonic solution and recorded using the whole-cell patch-clamp technique. The PKC agonist phorbol dibutyrate (PDBu) enhanced ICl.swell in a concentration-dependent manner, which was reversed in isotonic solution and by a chloride current inhibitor, 9-anthracenecarboxylicacid. Furthermore, the PKC inhibitor bis-indolylmaleimide attenuated the effect and 4α-PDBu, an inactive PDBu analog, had no effect on ICl.swell. These results, obtained using the whole-cell patch-clamp technique, demonstrate the ability of PKC to activate ICl,swell in human atrial myocytes. This observation was consistent with a previous study using a single-channel patch-clamp technique, but differed from some findings in other species.

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.

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

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.

Physiological activation of Akt by PHLPP1 deletion protects against pathological hypertrophy.

PubMed

Moc, Courtney; Taylor, Amy E; Chesini, Gino P; Zambrano, Cristina M; Barlow, Melissa S; Zhang, Xiaoxue; Gustafsson, Åsa B; Purcell, Nicole H

2015-02-01

To examine the role of physiological Akt signalling in pathological hypertrophy through analysis of PHLPP1 (PH domain leucine-rich repeat protein phosphatase) knock-out (KO) mice. To investigate the in vivo requirement for 'physiological' control of Akt activation in cardiac growth, we examined the effect of deleting the Akt phosphatase, PHLPP, on the induction of cardiac hypertrophy. Basal Akt phosphorylation increased nearly two-fold in the cardiomyocytes from PHLPP1 KO mice and physiological hypertrophy induced by swimming exercise was accentuated as assessed by increased heart size and myocyte cell area. In contrast, the development of pathophysiological hypertrophy induced by pressure overload and assessed by increases in heart size, myocyte cell area, and hypertrophic gene expression was attenuated. This attenuation coincided with decreased fibrosis and cell death in the KO mice. Cast moulding revealed increased capillary density basally in the KO hearts, which was further elevated relative to wild-type mouse hearts in response to pressure overload. In vitro studies with isolated myocytes in co-culture also demonstrated that PHLPP1 deletion in cardiomyocytes can enhance endothelial tube formation. Expression of the pro-angiogenic factor VEGF was also elevated basally and accentuated in response to transverse aortic constriction in hearts from KO mice. Our data suggest that enhancing Akt activity by inhibiting its PHLPP1-mediated dephosphorylation promotes processes associated with physiological hypertrophy that may be beneficial in attenuating the development of pathological hypertrophy. Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2014. For permissions please email: journals.permissions@oup.com.

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

Interleukin 1 and Tumor Necrosis Factor Inhibit Cardiac Myocyte β -adrenergic Responsiveness

NASA Astrophysics Data System (ADS)

Gulick, Tod; Chung, Mina K.; Pieper, Stephen J.; Lange, Louis G.; Schreiner, George F.

1989-09-01

Reversible congestive heart failure can accompany cardiac allograft rejection and inflammatory myocarditis, conditions associated with an immune cell infiltrate of the myocardium. To determine whether immune cell secretory products alter cardiac muscle metabolism without cytotoxicity, we cultured cardiac myocytes in the presence of culture supernatants from activated immune cells. We observed that these culture supernatants inhibit β -adrenergic agonist-mediated increases in cultured cardiac myocyte contractility and intracellular cAMP accumulation. The myocyte contractile response to increased extracellular Ca2+ concentration is unaltered by prior exposure to these culture supernatants, as is the increase in myocyte intracellular cAMP concentration in response to stimulation with forskolin, a direct adenyl cyclase activator. Inhibition occurs in the absence of alteration in β -adrenergic receptor density or ligand binding affinity. Suppressive activity is attributable to the macrophage-derived cytokines interleukin 1 and tumor necrosis factor. Thus, these observations describe a role for defined cytokines in regulating the hormonal responsiveness and function of contractile cells. The effects of interleukin 1 and tumor necrosis factor on intracellular cAMP accumulation may be a model for immune modulation of other cellular functions dependent upon cyclic nucleotide metabolism. The uncoupling of agonist-occupied receptors from adenyl cyclase suggests that β -receptor or guanine nucleotide binding protein function is altered by the direct or indirect action of cytokines on cardiac muscle cells.

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

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

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

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.

Effects of insulin-like growth factor-I, insulin, and leucine on protein turnover and pathways that regulate ubiquitin ligase expression in rainbow trout primary myocytes

USDA-ARS?s Scientific Manuscript database

The effects of insulin-like growth factor-I (IGF-I), insulin, and leucine on protein turnover and pathways that regulate proteolytic gene expression and protein polyubiquitination were investigated in primary cultures of four day old rainbow trout myocytes. Supplementing media with 100 nM IGF-I inc...

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

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

IGF-1 induces skeletal myocyte hypertrophy through calcineurin in association with GATA-2 and NF-ATc1

NASA Technical Reports Server (NTRS)

Musaro, A.; McCullagh, K. J.; Naya, F. J.; Olson, E. N.; Rosenthal, N.

1999-01-01

Localized synthesis of insulin-like growth factors (IGFs) has been broadly implicated in skeletal muscle growth, hypertrophy and regeneration. Virally delivered IGF-1 genes induce local skeletal muscle hypertrophy and attenuate age-related skeletal muscle atrophy, restoring and improving muscle mass and strength in mice. Here we show that the molecular pathways underlying the hypertrophic action of IGF-1 in skeletal muscle are similar to those responsible for cardiac hypertrophy. Transfected IGF-1 gene expression in postmitotic skeletal myocytes activates calcineurin-mediated calcium signalling by inducing calcineurin transcripts and nuclear localization of calcineurin protein. Expression of activated calcineurin mimics the effects of IGF-1, whereas expression of a dominant-negative calcineurin mutant or addition of cyclosporin, a calcineurin inhibitor, represses myocyte differentiation and hypertrophy. Either IGF-1 or activated calcineurin induces expression of the transcription factor GATA-2, which accumulates in a subset of myocyte nuclei, where it associates with calcineurin and a specific dephosphorylated isoform of the transcription factor NF-ATc1. Thus, IGF-1 induces calcineurin-mediated signalling and activation of GATA-2, a marker of skeletal muscle hypertrophy, which cooperates with selected NF-ATc isoforms to activate gene expression programs.

Caveolin-3 Overexpression Attenuates Cardiac Hypertrophy via Inhibition of T-type Ca2+ Current Modulated by Protein Kinase Cα in Cardiomyocytes*

PubMed Central

Markandeya, Yogananda S.; Phelan, Laura J.; Woon, Marites T.; Keefe, Alexis M.; Reynolds, Courtney R.; August, Benjamin K.; Hacker, Timothy A.; Roth, David M.; Patel, Hemal H.; Balijepalli, Ravi C.

2015-01-01

Pathological cardiac hypertrophy is characterized by subcellular remodeling of the ventricular myocyte with a reduction in the scaffolding protein caveolin-3 (Cav-3), altered Ca2+ cycling, increased protein kinase C expression, and hyperactivation of calcineurin/nuclear factor of activated T cell (NFAT) signaling. However, the precise role of Cav-3 in the regulation of local Ca2+ signaling in pathological cardiac hypertrophy is unclear. We used cardiac-specific Cav-3-overexpressing mice and in vivo and in vitro cardiac hypertrophy models to determine the essential requirement for Cav-3 expression in protection against pharmacologically and pressure overload-induced cardiac hypertrophy. Transverse aortic constriction and angiotensin-II (Ang-II) infusion in wild type (WT) mice resulted in cardiac hypertrophy characterized by significant reduction in fractional shortening, ejection fraction, and a reduced expression of Cav-3. In addition, association of PKCα and angiotensin-II receptor, type 1, with Cav-3 was disrupted in the hypertrophic ventricular myocytes. Whole cell patch clamp analysis demonstrated increased expression of T-type Ca2+ current (ICa, T) in hypertrophic ventricular myocytes. In contrast, the Cav-3-overexpressing mice demonstrated protection from transverse aortic constriction or Ang-II-induced pathological hypertrophy with inhibition of ICa, T and intact Cav-3-associated macromolecular signaling complexes. siRNA-mediated knockdown of Cav-3 in the neonatal cardiomyocytes resulted in enhanced Ang-II stimulation of ICa, T mediated by PKCα, which caused nuclear translocation of NFAT. Overexpression of Cav-3 in neonatal myocytes prevented a PKCα-mediated increase in ICa, T and nuclear translocation of NFAT. In conclusion, we show that stable Cav-3 expression is essential for protecting the signaling mechanisms in pharmacologically and pressure overload-induced cardiac hypertrophy. PMID:26170457

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.

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).

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.

Inhibition of autophagy by berberine enhances the survival of H9C2 myocytes following hypoxia.

PubMed

Jia, Zhuyin; Lin, Lu; Huang, Shanjun; Zhu, Zhouyang; Huang, Weijian; Huang, Zhouqing

2017-08-01

Hypoxia may induce apoptosis and autophagy to promote cardiomyocyte injury. The present study investigated the effect of berberine, a natural extract of Rhizoma Coptidis, on hypoxia‑induced autophagy and apoptosis in the H9c2 rat myocardial cell line. Expression levels of apoptosis and autophagy markers were upregulated in H9c2 myocytes during hypoxia and cell viability was reduced. However, berberine significantly reduced hypoxia‑induced autophagy in H9c2 myocytes, as demonstrated by the ratio of microtubule‑associated proteins 1A/1B light chain 3 I/II and the expression levels of B‑cell lymphoma 2 (Bcl‑2)/adenovirus E1B 19 kDa protein‑interacting protein 3, and promoted cell viability. In addition, expression levels of the Bcl‑2 anti‑apoptotic protein were significantly downregulated, and expression levels of pro‑apoptotic proteins Bcl‑2‑associated X protein and cleaved caspase‑3 were upregulated during hypoxia injury in cardiac myocytes. This was reversed by treatment with berberine or the autophagy inhibitor 3‑methyladenine, whereas the autophagy agonist rapamycin had the opposite effects, suggesting that berberine reduces myocyte cell death via inhibition of autophagy and apoptosis during hypoxia. In addition, Compound C, a 5' adenosine monophosphate‑activated protein kinase (AMPK) inhibitor, reduced apoptosis and autophagy in hypoxic myocytes, suggesting that the activation of the AMPK signaling pathway may be involved in this process. These findings suggested that berberine protects cells from hypoxia‑induced apoptosis via inhibition of autophagy and suppression of AMPK activation. Therefore, berberine may be a potential therapeutic agent for the treatment of patients with cardiac myocyte injury and ischemia.

Profound regulation of Na/K pump activity by transient elevations of cytoplasmic calcium in murine cardiac myocytes

PubMed Central

Lu, Fang-Min; Deisl, Christine; Hilgemann, Donald W

2016-01-01

Small changes of Na/K pump activity regulate internal Ca release in cardiac myocytes via Na/Ca exchange. We now show conversely that transient elevations of cytoplasmic Ca strongly regulate cardiac Na/K pumps. When cytoplasmic Na is submaximal, Na/K pump currents decay rapidly during extracellular K application and multiple results suggest that an inactivation mechanism is involved. Brief activation of Ca influx by reverse Na/Ca exchange enhances pump currents and attenuates current decay, while repeated Ca elevations suppress pump currents. Pump current enhancement reverses over 3 min, and results are similar in myocytes lacking the regulatory protein, phospholemman. Classical signaling mechanisms, including Ca-activated protein kinases and reactive oxygen, are evidently not involved. Electrogenic signals mediated by intramembrane movement of hydrophobic ions, such as hexyltriphenylphosphonium (C6TPP), increase and decrease in parallel with pump currents. Thus, transient Ca elevation and Na/K pump inactivation cause opposing sarcolemma changes that may affect diverse membrane processes. DOI: http://dx.doi.org/10.7554/eLife.19267.001 PMID:27627745

T-tubule Disruption Promotes Calcium Alternans in Failing Ventricular Myocytes: Mechanistic Insights from Computational Modeling

PubMed Central

Nivala, Michael; Song, Zhen; Weiss, James N.; Qu, Zhilin

2015-01-01

In heart failure (HF), T-tubule (TT) disruption contributes to dyssynchronous calcium (Ca) release and impaired contraction, but its role in arrhythmogenesis remains unclear. In this study, we investigate the mechanisms of TT disruption and other HF remodeling factors on Ca alternans in ventricular myocytes using computer modeling. A ventricular myocyte model with detailed spatiotemporal Ca cycling modeled by a coupled Ca release unit (CRU) network was used, in which the L-type Ca channels and the ryanodine receptor (RyR) channels were simulated by random Markov transitions. TT disruption, which removes the L-type Ca channels from the associated CRUs, results in “orphaned” RyR clusters and thus provides increased opportunity for spark-induced Ca sparks to occur. This effect combined with other HF remodeling factors promoted alternans by two distinct mechanisms: 1) for normal sarco-endoplasmic reticulum Ca ATPase (SERCA) activity, alternans was caused by both CRU refractoriness and coupling. The increased opportunity for spark-induced sparks by TT disruption combined with the enhanced CRU coupling by Ca elevation in the presence or absence of increased RyR leakiness facilitated spark synchronization on alternate beats to promote Ca alternans; 2) for down-regulated SERCA, alternans was caused by the sarcoplasmic reticulum (SR) Ca load-dependent mechanism, independent of CRU refractoriness. TT disruption and increased RyR leakiness shifted and steepened the SR Ca release-load relationship, which combines with down-regulated SERCA to promote Ca alternans. In conclusion, the mechanisms of Ca alternans for normal and down-regulated SERCA are different, and TT disruption promotes Ca alternans by both mechanisms, which may contribute to alternans at different stages of HF. PMID:25450613

T-tubule disruption promotes calcium alternans in failing ventricular myocytes: mechanistic insights from computational modeling.

PubMed

Nivala, Michael; Song, Zhen; Weiss, James N; Qu, Zhilin

2015-02-01

In heart failure (HF), T-tubule (TT) disruption contributes to dyssynchronous calcium (Ca) release and impaired contraction, but its role in arrhythmogenesis remains unclear. In this study, we investigate the effects of TT disruption and other HF remodeling factors on Ca alternans in ventricular myocytes using computer modeling. A ventricular myocyte model with detailed spatiotemporal Ca cycling modeled by a coupled Ca release unit (CRU) network was used, in which the L-type Ca channels and the ryanodine receptor (RyR) channels were simulated by random Markov transitions. TT disruption, which removes the L-type Ca channels from the associated CRUs, results in "orphaned" RyR clusters and thus provides increased opportunity for spark-induced Ca sparks to occur. This effect combined with other HF remodeling factors promoted alternans by two distinct mechanisms: 1) for normal sarco-endoplasmic reticulum Ca ATPase (SERCA) activity, alternans was caused by both CRU refractoriness and coupling. The increased opportunity for spark-induced sparks by TT disruption combined with the enhanced CRU coupling by Ca elevation in the presence or absence of increased RyR leakiness facilitated spark synchronization on alternate beats to promote Ca alternans; 2) for down-regulated SERCA, alternans was caused by the sarcoplasmic reticulum (SR) Ca load-dependent mechanism, independent of CRU refractoriness. TT disruption and increased RyR leakiness shifted and steepened the SR Ca release-load relationship, which combines with down-regulated SERCA to promote Ca alternans. In conclusion, the mechanisms of Ca alternans for normal and down-regulated SERCA are different, and TT disruption promotes Ca alternans by both mechanisms, which may contribute to alternans at different stages of HF. Copyright © 2014 Elsevier Ltd. All rights reserved.

Mangiferin Enhanced Autophagy via Inhibiting mTORC1 Pathway to Prevent High Glucose-Induced Cardiomyocyte Injury.

PubMed

Hou, Jun; Zheng, Dezhi; Xiao, Wenjing; Li, Dandan; Ma, Jie; Hu, Yonghe

2018-01-01

Mangiferin functions as a perfect anti-oxidative compound in the diabetic heart, however, the exact mechanism remains to be elucidated. Here, we show the cardioprotective effect of mangiferin under high glucose-induced cardiotoxic condition mainly contributed to enhanced autophagy via suppressing mTORC1 downstream signal transduction. Primary neonatal rat cardiomyocytes were cultured to detect myocytes injury, autophagy, and related signal transduction under different doses of glucose and mangiferin treatment. High glucose (30 mM) reduced autophagic flux, and increased myocyte apoptosis and death compared with normal glucose (5.5 mM) as determined by variation of autophagy markers LC3-II, p62, parkin, GFP-LC3, or mRFP-LC3 fluorescence puncta, cell viability, cleaved caspase 3, cleaved PARP apoptosis indices, reactive oxygen species (ROS), MAO, and PI death indices. Conversely, mangiferin inhibited hyperglycemia associated oxidative stress by reducing ROS, MAO, cleaved caspase 3, and cleaved PARP generation, reestablishing cell viability, mitochondrial membrane potential, and enhancing autophagic flux, thereby preventing myocytes from high glucose-induced toxicity. Furthermore, cardioprotection with mangiferin was potentially related to the decreased mTOR phosphorylation and suppression of mTORC1 downstream signaling pathway. These data indicated the valuable effects of mangiferin on regulation of cardiac autophagy and pointed to the promising utilization for hyperglycemia control.

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.

Oxidative Stress-Responsive Apoptosis Inducing Protein (ORAIP) Plays a Critical Role in High Glucose-Induced Apoptosis in Rat Cardiac Myocytes and Murine Pancreatic β-Cells.

PubMed

Yao, Takako; Fujimura, Tsutomu; Murayama, Kimie; Okumura, Ko; Seko, Yoshinori

2017-10-18

We previously identified a novel apoptosis-inducing humoral factor in the conditioned medium of hypoxic/reoxygenated-cardiac myocytes. We named this novel post-translationally-modified secreted-form of eukaryotic translation initiation factor 5A Oxidative stress-Responsive Apoptosis-Inducing Protein (ORAIP). We confirmed that myocardial ischemia/reperfusion markedly increased plasma ORAIP levels and rat myocardial ischemia/reperfusion injury was clearly suppressed by neutralizing anti-ORAIP monoclonal antibodies (mAbs) in vivo. In this study, to investigate the mechanism of cell injury of cardiac myocytes and pancreatic β-cells involved in diabetes mellitus (DM), we analyzed plasma ORAIP levels in DM model rats and the role of ORAIP in high glucose-induced apoptosis of cardiac myocytes in vitro. We also examined whether recombinant-ORAIP induces apoptosis in pancreatic β-cells. Plasma ORAIP levels in DM rats during diabetic phase were about 18 times elevated as compared with non-diabetic phase. High glucose induced massive apoptosis in cardiac myocytes (66.2 ± 2.2%), which was 78% suppressed by neutralizing anti-ORAIP mAb in vitro. Furthermore, recombinant-ORAIP clearly induced apoptosis in pancreatic β-cells in vitro. These findings strongly suggested that ORAIP plays a pivotal role in hyperglycemia-induced myocardial injury and pancreatic β-cell injury in DM. ORAIP will be a biomarker and a critical therapeutic target for cardiac injury and progression of DM itself.

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

The mammalian target of rapamycin signaling pathway regulates myocyte enhancer factor-2C phosphorylation levels through integrin-linked kinase in goat skeletal muscle satellite cells.

PubMed

Wu, Haiqing; Ren, Yu; Pan, Wei; Dong, Zhenguo; Cang, Ming; Liu, Dongjun

2015-11-01

Mammalian target of rapamycin (mTOR) signaling pathway plays a key role in muscle development and is involved in multiple intracellular signaling pathways. Myocyte enhancer factor-2 (MEF2) regulates muscle cell proliferation and differentiation. However, how the mTOR signaling pathway regulates MEF2 activity remains unclear. We isolated goat skeletal muscle satellite cells (gSSCs) as model cells to explore mTOR signaling pathway regulation of MEF2C. We inhibited mTOR activity in gSSCs with PP242 and found that MEF2C phosphorylation was decreased and that muscle creatine kinase (MCK) expression was suppressed. Subsequently, we detected integrin-linked kinase (ILK) using MEF2C coimmunoprecipitation; ILK and MEF2C were colocalized in the gSSCs. We found that inhibiting mTOR activity increased ILK phosphorylation levels and that inhibiting ILK activity with Cpd 22 and knocking down ILK with small interfering RNA increased MEF2C phosphorylation and MCK expression. In the presence of Cpd 22, mTOR activity inhibition did not affect MEF2C phosphorylation. Moreover, ILK dephosphorylated MEF2C in vitro. These results suggest that the mTOR signaling pathway regulates MEF2C positively and regulates ILK negatively and that ILK regulates MEF2C negatively. It appears that the mTOR signaling pathway regulates MEF2C through ILK, further regulating the expression of muscle-related genes in gSSCs. © 2015 International Federation for Cell Biology.

Enhanced Functional Genomic Screening Identifies Novel Mediators of Dual Leucine Zipper Kinase-Dependent Injury Signaling in Neurons.

PubMed

Welsbie, Derek S; Mitchell, Katherine L; Jaskula-Ranga, Vinod; Sluch, Valentin M; Yang, Zhiyong; Kim, Jessica; Buehler, Eugen; Patel, Amit; Martin, Scott E; Zhang, Ping-Wu; Ge, Yan; Duan, Yukan; Fuller, John; Kim, Byung-Jin; Hamed, Eman; Chamling, Xitiz; Lei, Lei; Fraser, Iain D C; Ronai, Ze'ev A; Berlinicke, Cynthia A; Zack, Donald J

2017-06-21

Dual leucine zipper kinase (DLK) has been implicated in cell death signaling secondary to axonal damage in retinal ganglion cells (RGCs) and other neurons. To better understand the pathway through which DLK acts, we developed enhanced functional genomic screens in primary RGCs, including use of arrayed, whole-genome, small interfering RNA libraries. Explaining why DLK inhibition is only partially protective, we identify leucine zipper kinase (LZK) as cooperating with DLK to activate downstream signaling and cell death in RGCs, including in a mouse model of optic nerve injury, and show that the same pathway is active in human stem cell-derived RGCs. Moreover, we identify four transcription factors, JUN, activating transcription factor 2 (ATF2), myocyte-specific enhancer factor 2A (MEF2A), and SRY-Box 11 (SOX11), as being the major downstream mediators through which DLK/LZK activation leads to RGC cell death. Increased understanding of the DLK pathway has implications for understanding and treating neurodegenerative diseases. Copyright © 2017 Elsevier Inc. All rights reserved.

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

The IGF-1 Receptor Identifies a Pool of Human Cardiac Stem Cells with Superior Therapeutic Potential for Myocardial Regeneration

PubMed Central

D’Amario, Domenico; Cabral-Da-Silva, Mauricio; Zheng, Hanqiao; Fiorini, Claudia; Goichberg, Polina; Steadman, Elisabeth; Ferreira-Martins, João; Sanada, Fumihiro; Piccoli, Marco; Cappetta, Donato; D’Alessandro, David A.; Michler, Robert E.; Hosoda, Toru; Anastasia, Luigi; Rota, Marcello; Leri, Annarosa; Anversa, Piero; Kajstura, Jan

2012-01-01

Rationale Age and coronary artery disease may negatively affect the function of human cardiac stem cells (hCSCs) and their potential therapeutic efficacy for autologous cell transplantation in the failing heart. Objective Insulin-like growth factor 1 (IGF-1) and 2 (IGF-2), and angiotensin II (Ang II) and their receptors, IGF-1R, IGF-2R and AT1R, were characterized in c-kit-positive-hCSCs to establish whether these systems would allow us to separate hCSC classes with different growth reserve in the aging and diseased myocardium. Methods and Results C-kit-positive-hCSCs were collected from myocardial samples obtained from 24 patients, 48 to 86 years of age, undergoing elective cardiac surgery for coronary artery disease. The expression of IGF-1R in hCSCs recognized a young cell phenotype defined by long telomeres, high telomerase activity, enhanced cell proliferation and attenuated apoptosis. In addition to IGF-1, IGF-1R-positive-hCSCs secreted IGF-2 that promoted myocyte differentiation. Conversely, the presence of IGF-2R and AT1R, in the absence of IGF-1R, identified senescent hCSCs with impaired growth reserve and increased susceptibility to apoptosis. The ability of IGF-1R-positive-hCSCs to regenerate infarcted myocardium was then compared with that of unselected c-kit-positive-hCSCs. IGF-1R-positive-hCSCs improved cardiomyogenesis and vasculogenesis. Pretreatment of IGF-1R-positive-hCSCs with IGF-2 resulted in the formation of more mature myocytes and superior recovery of ventricular structure. Conclusions hCSCs expressing only IGF-1R synthesize both IGF-1 and IGF-2, which are potent modulators of stem cell replication, commitment to the myocyte lineage and myocyte differentiation, pointing to this hCSC subset as the ideal candidate cell for the management of human heart failure. PMID:21546606

STAT3 balances myocyte hypertrophy vis-à-vis autophagy in response to Angiotensin II by modulating the AMPKα/mTOR axis.

PubMed

Chen, Lei; Zhao, Lin; Samanta, Anweshan; Mahmoudi, Seyed Morteza; Buehler, Tanner; Cantilena, Amy; Vincent, Robert J; Girgis, Magdy; Breeden, Joshua; Asante, Samuel; Xuan, Yu-Ting; Dawn, Buddhadeb

2017-01-01

Signal transducers and activators of transcription 3 (STAT3) is known to participate in various cardiovascular signal transduction pathways, including those responsible for cardiac hypertrophy and cytoprotection. However, the role of STAT3 signaling in cardiomyocyte autophagy remains unclear. We tested the hypothesis that Angiotensin II (Ang II)-induced cardiomyocyte hypertrophy is effected, at least in part, through STAT3-mediated inhibition of cellular autophagy. In H9c2 cells, Ang II treatment resulted in STAT3 activation and cellular hypertrophy in a dose-dependent manner. Ang II enhanced autophagy, albeit without impacting AMPKα/mTOR signaling or cellular ADP/ATP ratio. Pharmacologic inhibition of STAT3 with WP1066 suppressed Ang II-induced myocyte hypertrophy and mRNA expression of hypertrophy-related genes ANP and β-MHC. These molecular events were recapitulated in cells with STAT3 knockdown. Genetic or pharmacologic inhibition of STAT3 significantly increased myocyte ADP/ATP ratio and enhanced autophagy through AMPKα/mTOR signaling. Pharmacologic activation and inhibition of AMPKα attenuated and exaggerated, respectively, the effects of Ang II on ANP and β-MHC gene expression, while concomitant inhibition of STAT3 accentuated the inhibition of hypertrophy. Together, these data indicate that novel nongenomic effects of STAT3 influence myocyte energy status and modulate AMPKα/mTOR signaling and autophagy to balance the transcriptional hypertrophic response to Ang II stimulation. These findings may have significant relevance for various cardiovascular pathological processes mediated by Ang II signaling.

STAT3 balances myocyte hypertrophy vis-à-vis autophagy in response to Angiotensin II by modulating the AMPKα/mTOR axis

PubMed Central

Samanta, Anweshan; Mahmoudi, Seyed Morteza; Buehler, Tanner; Cantilena, Amy; Vincent, Robert J.; Girgis, Magdy; Breeden, Joshua; Asante, Samuel; Xuan, Yu-Ting

2017-01-01

Signal transducers and activators of transcription 3 (STAT3) is known to participate in various cardiovascular signal transduction pathways, including those responsible for cardiac hypertrophy and cytoprotection. However, the role of STAT3 signaling in cardiomyocyte autophagy remains unclear. We tested the hypothesis that Angiotensin II (Ang II)-induced cardiomyocyte hypertrophy is effected, at least in part, through STAT3-mediated inhibition of cellular autophagy. In H9c2 cells, Ang II treatment resulted in STAT3 activation and cellular hypertrophy in a dose-dependent manner. Ang II enhanced autophagy, albeit without impacting AMPKα/mTOR signaling or cellular ADP/ATP ratio. Pharmacologic inhibition of STAT3 with WP1066 suppressed Ang II-induced myocyte hypertrophy and mRNA expression of hypertrophy-related genes ANP and β-MHC. These molecular events were recapitulated in cells with STAT3 knockdown. Genetic or pharmacologic inhibition of STAT3 significantly increased myocyte ADP/ATP ratio and enhanced autophagy through AMPKα/mTOR signaling. Pharmacologic activation and inhibition of AMPKα attenuated and exaggerated, respectively, the effects of Ang II on ANP and β-MHC gene expression, while concomitant inhibition of STAT3 accentuated the inhibition of hypertrophy. Together, these data indicate that novel nongenomic effects of STAT3 influence myocyte energy status and modulate AMPKα/mTOR signaling and autophagy to balance the transcriptional hypertrophic response to Ang II stimulation. These findings may have significant relevance for various cardiovascular pathological processes mediated by Ang II signaling. PMID:28686615

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

Myocyte enhancer factor (MEF)-2 plays essential roles in T-cell transformation associated with HTLV-1 infection by stabilizing complex between Tax and CREB.

PubMed

Jain, Pooja; Lavorgna, Alfonso; Sehgal, Mohit; Gao, Linlin; Ginwala, Rashida; Sagar, Divya; Harhaj, Edward W; Khan, Zafar K

2015-02-27

The exact molecular mechanisms regarding HTLV-1 Tax-mediated viral gene expression and CD4 T-cell transformation have yet to be fully delineated. Herein, utilizing virus-infected primary CD4+ T cells and the virus-producing cell line, MT-2, we describe the involvement and regulation of Myocyte enhancer factor-2 (specifically MEF-2A) during the course of HTLV-1 infection and associated disease syndrome. Inhibition of MEF-2 expression by shRNA and its activity by HDAC9 led to reduced viral replication and T-cell transformation in correlation with a heightened expression of MEF-2 in ATL patients. Mechanistically, MEF-2 was recruited to the viral promoter (LTR, long terminal repeat) in the context of chromatin, and constituted Tax/CREB transcriptional complex via direct binding to the HTLV-1 LTR. Furthermore, an increase in MEF-2 expression was observed upon infection in an extent similar to CREB (known Tax-interacting transcription factor), and HATs (p300, CBP, and p/CAF). Confocal imaging confirmed MEF-2 co-localization with Tax and these proteins were also shown to interact by co-immunoprecipitation. MEF-2 stabilization of Tax/CREB complex was confirmed by a novel promoter-binding assay that highlighted the involvement of NFAT (nuclear factor of activated T cells) in this process via Tax-mediated activation of calcineurin (a calcium-dependent serine-threonine phosphatase). MEF-2-integrated signaling pathways (PI3K/Akt, NF-κB, MAPK, JAK/STAT, and TGF-β) were also activated during HTLV-1 infection of primary CD4+ T cells, possibly regulating MEF-2 activity. We demonstrate the involvement of MEF-2 in Tax-mediated LTR activation, viral replication, and T-cell transformation in correlation with its heightened expression in ATL patients through direct binding to DNA within the HTLV-1 LTR.

Novel mechanisms for caspase inhibition protecting cardiac function with chronic pressure overload

PubMed Central

Vatner, Stephen F.; Yan, Lin; Gao, Shumin; Yoon, Seunghun; Lee, Grace Jung Ah; Xie, Lai-Hua; Kitsis, Richard N.; Vatner, Dorothy E.

2013-01-01

Myocyte apoptosis is considered a major mechanism in the pathogenesis of heart failure. Accordingly, manipulations that inhibit apoptosis are assumed to preserve cardiac function by maintaining myocyte numbers. We tested this assumption by examining the effects of caspase inhibition (CI) on cardiac structure and function in C57BL/6 mouse with pressure overload model induced by transverse aortic constriction (TAC). CI preserved left ventricular (LV) function following TAC compared with the vehicle. TAC increased apoptosis in non-myocytes more than in myocytes and these increases were blunted more in non-myocytes by CI. Total myocyte number, however, did not differ significantly among control and TAC groups and there was no correlation between myocyte number and apoptosis, but there was a strong correlation between myocyte number and an index of myocyte proliferation, Ki67-positive myocytes. Despite comparable pressure gradients, LV hypertrophy was less in the CI group, likely attributable to decreased wall stress. Since changes in myocyte numbers did not account for protection from TAC, several other CI-mediated mechanisms were identified including: (a) lessening of TAC-induced fibrosis, (b) augmentation of isolated myocyte contractility, and (c) increased angiogenesis and Ki67-positive myocytes, which were due almost entirely to the non-myocyte apoptosis, but not myocyte apoptosis, with CI. CI maintained LV function following TAC not by protecting against myocyte loss, but rather by augmenting myocyte contractile function, myocyte proliferation, and angiogenesis resulting in reduced LV wall stress, hypertrophy, and fibrosis. PMID:23277091

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

Roles of mitochondrial fragmentation and reactive oxygen species in mitochondrial dysfunction and myocardial insulin resistance

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

Watanabe, Tomoyuki; Saotome, Masao, E-mail: msaotome@hama-med.ac.jp; Nobuhara, Mamoru

Purpose: Evidence suggests an association between aberrant mitochondrial dynamics and cardiac diseases. Because myocardial metabolic deficiency caused by insulin resistance plays a crucial role in heart disease, we investigated the role of dynamin-related protein-1 (DRP1; a mitochondrial fission protein) in the pathogenesis of myocardial insulin resistance. Methods and Results: DRP1-expressing H9c2 myocytes, which had fragmented mitochondria with mitochondrial membrane potential (ΔΨ{sub m}) depolarization, exhibited attenuated insulin signaling and 2-deoxy-D-glucose (2-DG) uptake, indicating insulin resistance. Treatment of the DRP1-expressing myocytes with Mn(III)tetrakis(1-methyl-4-pyridyl)porphyrin pentachloride (TMPyP) significantly improved insulin resistance and mitochondrial dysfunction. When myocytes were exposed to hydrogen peroxide (H{sub 2}O{sub 2}),more » they increased DRP1 expression and mitochondrial fragmentation, resulting in ΔΨ{sub m} depolarization and insulin resistance. When DRP1 was suppressed by siRNA, H{sub 2}O{sub 2}-induced mitochondrial dysfunction and insulin resistance were restored. Our results suggest that a mutual enhancement between DRP1 and reactive oxygen species could induce mitochondrial dysfunction and myocardial insulin resistance. In palmitate-induced insulin-resistant myocytes, neither DRP1-suppression nor TMPyP restored the ΔΨ{sub m} depolarization and impaired 2-DG uptake, however they improved insulin signaling. Conclusions: A mutual enhancement between DRP1 and ROS could promote mitochondrial dysfunction and inhibition of insulin signal transduction. However, other mechanisms, including lipid metabolite-induced mitochondrial dysfunction, may be involved in palmitate-induced insulin resistance. - Highlights: • DRP1 promotes mitochondrial fragmentation and insulin-resistance. • A mutual enhancement between DRP1 and ROS ipromotes insulin-resistance. • Palmitate increases DRP1 expression and induces insulin-resistance. • Inhibition of DRP or ROS failed to improve palmitate-induced insulin-resistance. • Mitochondrial dysfunction by lipid metabolites would induce insulin-resistance.« less

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.

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.

Phospholamban, a predicted candidate for early cardiac problem detection using signal processing techniques.

PubMed

Hejase de Trad, C

2005-01-01

Heart failure has been identified as a serious international problem, in particular for aging groups, posing both an increasing number of patients on waiting lists in countries susceptible with Medicare systems and increasing financial burdens. It may be imperative to develop a marker that can identify such problems at an early stage. It is believed that certain proteins have crucial roles in early detection of cardiovascular disease, the number one killer in United Arab Emirates. This might be accomplished by recognition of unusual features in protein candidates. Phospholamban (PLB) is a 52 amino acid phosphoprotein which regulates the calcium pump of cardiac sarcoplasmic reticulum (SR). During muscle contraction, PLB inhibits the Ca⁺⁺ pump. During muscle relaxation, it can be phosphorylated, removing the inhibition and allowing Ca⁺⁺ to be pumped back into SR. With the calcium pump disrupted, the heart muscle is probably weakened, resulting in congestive heart failure. Interleukin 6 (IL-6) is considered as a better predictor of heart attack in elderly people. It could serve as an early warning sign since its level increases early in the inflammatory process. Also, it has been established that myocyte enhancer factor 2A (MEF2A) plays a vital role in the development of cardiovascular problems like atherosclerosis and restenosis after angioplasty inflammation. In this paper, the resonance recognition method (RRM) has been employed to determine the characteristic frequencies of the above-mentioned proteins. It has been found that phospholamban and IL-6 share the same characteristic frequency, 0.3320 plusmn 0.0002 suggesting their common probable contribution to heart failure. Myocyte enhancer factor 2A does not share the same characteristic frequency. Hence, phospholamban is suggested as a highly probable early marker for cardiac problem detection.

Myocyte enhancer factor 2D provides a cross-talk between chronic inflammation and lung cancer.

PubMed

Zhu, Hai-Xing; Shi, Lin; Zhang, Yong; Zhu, Yi-Chun; Bai, Chun-Xue; Wang, Xiang-Dong; Zhou, Jie-Bai

2017-03-24

Lung cancer is the leading cause of cancer-related morbidity and mortality worldwide. Patients with chronic respiratory diseases, such as chronic obstructive pulmonary disease (COPD), are exposed to a higher risk of developing lung cancer. Chronic inflammation may play an important role in the lung carcinogenesis among those patients. The present study aimed at identifying candidate biomarker predicting lung cancer risk among patients with chronic respiratory diseases. We applied clinical bioinformatics tools to analyze different gene profile datasets with a special focus on screening the potential biomarker during chronic inflammation-lung cancer transition. Then we adopted an in vitro model based on LPS-challenged A549 cells to validate the biomarker through RNA-sequencing, quantitative real time polymerase chain reaction, and western blot analysis. Bioinformatics analyses of the 16 enrolled GSE datasets from Gene Expression Omnibus online database showed myocyte enhancer factor 2D (MEF2D) level significantly increased in COPD patients coexisting non-small-cell lung carcinoma (NSCLC). Inflammation challenge increased MEF2D expression in NSCLC cell line A549, associated with the severity of inflammation. Extracellular signal-regulated protein kinase inhibition could reverse the up-regulation of MEF2D in inflammation-activated A549. MEF2D played a critical role in NSCLC cell bio-behaviors, including proliferation, differentiation, and movement. Inflammatory conditions led to increased MEF2D expression, which might further contribute to the development of lung cancer through influencing cancer microenvironment and cell bio-behaviors. MEF2D might be a potential biomarker during chronic inflammation-lung cancer transition, predicting the risk of lung cancer among patients with chronic respiratory diseases.

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.

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.

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

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

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

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

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.

The myogenic repressor gene Holes in muscles is a direct transcriptional target of Twist and Tinman in the Drosophila embryonic mesoderm

PubMed Central

Elwell, Jennifer A.; Lovato, TyAnna L.; Adams, Melanie M.; Baca, Erica M.; Lee, Thai; Cripps, Richard M.

2015-01-01

Understanding the regulatory circuitry controlling myogenesis is critical to understanding developmental mechanisms and developmentally-derived diseases. We analyzed the transcriptional regulation of a Drosophila myogenic repressor gene, Holes in muscles (Him). Previously, Him was shown to inhibit Myocyte enhancer factor-2 (MEF2) activity, and is expressed in myoblasts but not differentiating myotubes. We demonstrate that different phases of Him embryonic expression arise through the actions of different enhancers, and we characterize the enhancer required for its early mesoderm expression. This Him early mesoderm enhancer contains two conserved binding sites for the basic helix-loop-helix regulator Twist, and one binding site for the NK homeodomain protein Tinman. The sites for both proteins are required for enhancer activity in early embryos. Twist and Tinman activate the enhancer in tissue culture assays, and ectopic expression of either factor is sufficient to direct ectopic expression of a Him-lacZ reporter, or of the endogenous Him gene. Moreover, sustained expression of twist expression in the mesoderm up-regulates mesodermal Him expression in late embryos. Our findings provide a model to define mechanistically how Twist can both promotes myogenesis through direct activation of Mef2, and can place a brake on myogenesis, through direct activation of Him. PMID:25704510

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.

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

Adipocyte-myocyte crosstalk in skeletal muscle insulin resistance; is there a role for thyroid hormone?

PubMed

Havekes, Bas; Sauerwein, Hans P

2010-11-01

To review original research studies and reviews that present data on adipocyte-myocyte crosstalk in the development of skeletal muscle insulin resistance with a specific focus on thyroid hormone. Adipose tissue communicates with skeletal muscle not only through free fatty acids but also through secretion of various products called adipokines. Adipokines came out as governors of insulin sensitivity and are deregulated in obesity. In addition to well known leptin, adiponectin, interleukin-6 and tumor necrosis factor-alpha, newer adipokines like retinol-binding protein 4 have been associated with insulin resistance. There is mounting evidence that not only adipose tissue but also skeletal muscle produces and secretes biologically active proteins or 'myokines' that facilitate metabolic crosstalk between organ systems. In recent years, increased expression of myostatin, a secreted anabolic inhibitor of muscle growth and development, has been associated with obesity and insulin resistance. Both hypothyroidism and hyperthyroidism affect insulin sensitivity in multiple ways that might overlap adipocyte-myocyte crosstalk. Recent studies have provided new insights in effects of processing of the parent hormone T4 to the active T3 at the level of the skeletal muscle. Adipocyte-myocyte crosstalk is an important modulator in the development of skeletal muscle insulin resistance. Thyroid disorders are very common and may have detrimental effects on skeletal muscle insulin resistance, potentially by interacting with adipocyte-myocyte crosstalk.

[Virus-like inclusions in the myocytes of the skeletal muscle in lateral amyotrophic sclerosis].

PubMed

Musaeva, L S; Sakharova, A V; Zavalishin, I A

2004-01-01

Microscopic examination of musculus gastrocnemius biopsies was made in four cases of sporadic lateral amyotrophic sclerosis (LAS). The validity of the clinical diagnosis was confirmed by detected neurotrophic atrophy of the muscular fibers typical for LAS. Electron microscopic study revealed virus-like inclusions 200-450 nm in size in sarcoplasm of myocytes of all the patients. The inclusions consist of lined cells of hexagonal shape at the distance of 37-41 nm from each other. The inclusions resemble enteroviruses but are not identical to them both by size and structure of their elements. There were also specific ultrastructural changes of myocytes corresponding to viral infection. The above virus-like inclusions should be considered as specific structures formed as a result of metabolic shifts caused by productive action on the cell of infective or unknown factor.

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.

Cardiac Hypertrophy Is Inhibited by a Local Pool of cAMP Regulated by Phosphodiesterase 2.

PubMed

Zoccarato, Anna; Surdo, Nicoletta C; Aronsen, Jan M; Fields, Laura A; Mancuso, Luisa; Dodoni, Giuliano; Stangherlin, Alessandra; Livie, Craig; Jiang, He; Sin, Yuan Yan; Gesellchen, Frank; Terrin, Anna; Baillie, George S; Nicklin, Stuart A; Graham, Delyth; Szabo-Fresnais, Nicolas; Krall, Judith; Vandeput, Fabrice; Movsesian, Matthew; Furlan, Leonardo; Corsetti, Veronica; Hamilton, Graham; Lefkimmiatis, Konstantinos; Sjaastad, Ivar; Zaccolo, Manuela

2015-09-25

Chronic elevation of 3'-5'-cyclic adenosine monophosphate (cAMP) levels has been associated with cardiac remodeling and cardiac hypertrophy. However, enhancement of particular aspects of cAMP/protein kinase A signaling seems to be beneficial for the failing heart. cAMP is a pleiotropic second messenger with the ability to generate multiple functional outcomes in response to different extracellular stimuli with strict fidelity, a feature that relies on the spatial segregation of the cAMP pathway components in signaling microdomains. How individual cAMP microdomains affect cardiac pathophysiology remains largely to be established. The cAMP-degrading enzymes phosphodiesterases (PDEs) play a key role in shaping local changes in cAMP. Here we investigated the effect of specific inhibition of selected PDEs on cardiac myocyte hypertrophic growth. Using pharmacological and genetic manipulation of PDE activity, we found that the rise in cAMP resulting from inhibition of PDE3 and PDE4 induces hypertrophy, whereas increasing cAMP levels via PDE2 inhibition is antihypertrophic. By real-time imaging of cAMP levels in intact myocytes and selective displacement of protein kinase A isoforms, we demonstrate that the antihypertrophic effect of PDE2 inhibition involves the generation of a local pool of cAMP and activation of a protein kinase A type II subset, leading to phosphorylation of the nuclear factor of activated T cells. Different cAMP pools have opposing effects on cardiac myocyte cell size. PDE2 emerges as a novel key regulator of cardiac hypertrophy in vitro and in vivo, and its inhibition may have therapeutic applications. © 2015 American Heart Association, Inc.

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

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.

[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.

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.

Global Intracoronary Infusion of Allogeneic Cardiosphere-Derived Cells Improves Ventricular Function and Stimulates Endogenous Myocyte Regeneration throughout the Heart in Swine with Hibernating Myocardium

PubMed Central

Suzuki, Gen; Weil, Brian R.; Leiker, Merced M.; Ribbeck, Amanda E.; Young, Rebeccah F.; Cimato, Thomas R.; Canty, John M.

2014-01-01

Background Cardiosphere-derived cells (CDCs) improve ventricular function and reduce fibrotic volume when administered via an infarct-related artery using the “stop-flow” technique. Unfortunately, myocyte loss and dysfunction occur globally in many patients with ischemic and non-ischemic cardiomyopathy, necessitating an approach to distribute CDCs throughout the entire heart. We therefore determined whether global intracoronary infusion of CDCs under continuous flow improves contractile function and stimulates new myocyte formation. Methods and Results Swine with hibernating myocardium from a chronic LAD occlusion were studied 3-months after instrumentation (n = 25). CDCs isolated from myocardial biopsies were infused into each major coronary artery (∼33×106 icCDCs). Global icCDC infusion was safe and while ∼3% of injected CDCs were retained, they did not affect ventricular function or myocyte proliferation in normal animals. In contrast, four-weeks after icCDCs were administered to animals with hibernating myocardium, %LADWT increased from 23±6 to 51±5% (p<0.01). In diseased hearts, myocyte proliferation (phospho-histone-H3) increased in hibernating and remote regions with a concomitant increase in myocyte nuclear density. These effects were accompanied by reductions in myocyte diameter consistent with new myocyte formation. Only rare myocytes arose from sex-mismatched donor CDCs. Conclusions Global icCDC infusion under continuous flow is feasible and improves contractile function, regresses myocyte cellular hypertrophy and increases myocyte proliferation in diseased but not normal hearts. New myocytes arising via differentiation of injected cells are rare, implicating stimulation of endogenous myocyte regeneration as the primary mechanism of repair. PMID:25402428

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.

Na/K pump inactivation, subsarcolemmal Na measurements, and cytoplasmic ion turnover kinetics contradict restricted Na spaces in murine cardiac myocytes

PubMed Central

Lu, Fang-Min

2017-01-01

Decades ago, it was proposed that Na transport in cardiac myocytes is modulated by large changes in cytoplasmic Na concentration within restricted subsarcolemmal spaces. Here, we probe this hypothesis for Na/K pumps by generating constitutive transsarcolemmal Na flux with the Na channel opener veratridine in whole-cell patch-clamp recordings. Using 25 mM Na in the patch pipette, pump currents decay strongly during continuous activation by extracellular K (τ, ∼2 s). In contradiction to depletion hypotheses, the decay becomes stronger when pump currents are decreased by hyperpolarization. Na channel currents are nearly unchanged by pump activity in these conditions, and conversely, continuous Na currents up to 0.5 nA in magnitude have negligible effects on pump currents. These outcomes are even more pronounced using 50 mM Li as a cytoplasmic Na congener. Thus, the Na/K pump current decay reflects mostly an inactivation mechanism that immobilizes Na/K pump charge movements, not cytoplasmic Na depletion. When channel currents are increased beyond 1 nA, models with unrestricted subsarcolemmal diffusion accurately predict current decay (τ ∼15 s) and reversal potential shifts observed for Na, Li, and K currents through Na channels opened by veratridine, as well as for Na, K, Cs, Li, and Cl currents recorded in nystatin-permeabilized myocytes. Ion concentrations in the pipette tip (i.e., access conductance) track without appreciable delay the current changes caused by sarcolemmal ion flux. Importantly, cytoplasmic mixing volumes, calculated from current decay kinetics, increase and decrease as expected with osmolarity changes (τ >30 s). Na/K pump current run-down over 20 min reflects a failure of pumps to recover from inactivation. Simulations reveal that pump inactivation coupled with Na-activated recovery enhances the rapidity and effectivity of Na homeostasis in cardiac myocytes. In conclusion, an autoregulatory mechanism enhances cardiac Na/K pump activity when cytoplasmic Na rises and suppresses pump activity when cytoplasmic Na declines. PMID:28606910

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.

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.

Increased arterial smooth muscle Ca2+ signaling, vasoconstriction, and myogenic reactivity in Milan hypertensive rats

PubMed Central

Linde, Cristina I.; Karashima, Eiji; Raina, Hema; Zulian, Alessandra; Wier, Withrow G.; Hamlyn, John M.; Ferrari, Patrizia; Blaustein, Mordecai P.

2012-01-01

The Milan hypertensive strain (MHS) rats are a genetic model of hypertension with adducin gene polymorphisms linked to enhanced renal tubular Na+ reabsorption. Recently we demonstrated that Ca2+ signaling is augmented in freshly isolated mesenteric artery myocytes from MHS rats. This is associated with greatly enhanced expression of Na+/Ca2+ exchanger-1 (NCX1), C-type transient receptor potential (TRPC6) protein, and sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA2) compared with arteries from Milan normotensive strain (MNS) rats. Here, we test the hypothesis that the enhanced Ca2+ signaling in MHS arterial smooth muscle is directly reflected in augmented vasoconstriction [myogenic and phenylephrine (PE)-evoked responses] in isolated mesenteric small arteries. Systolic blood pressure was higher in MHS (145 ± 1 mmHg) than in MNS (112 ± 1 mmHg; P < 0.001; n = 16 each) rats. Pressurized mesenteric resistance arteries from MHS rats had significantly augmented myogenic tone and reactivity and enhanced constriction to low-dose (1–100 nM) PE. Isolated MHS arterial myocytes exhibited approximately twofold increased peak Ca2+ signals in response to 5 μM PE or ATP in the absence and presence of extracellular Ca2+. These augmented responses are consistent with increased vasoconstrictor-evoked sarcoplasmic reticulum (SR) Ca2+ release and increased Ca2+ entry, respectively. The increased SR Ca2+ release correlates with a doubling of inositol 1,4,5-trisphosphate receptor type 1 and tripling of SERCA2 expression. Pressurized MHS arteries also exhibited a ∼70% increase in 100 nM ouabain-induced vasoconstriction compared with MNS arteries. These functional alterations reveal that, in a genetic model of hypertension linked to renal dysfunction, multiple mechanisms within the arterial myocytes contribute to enhanced Ca2+ signaling and myogenic and vasoconstrictor-induced arterial constriction. MHS rats have elevated plasma levels of endogenous ouabain, which may initiate the protein upregulation and enhanced Ca2+ signaling. These molecular and functional changes provide a mechanism for the increased peripheral vascular resistance (whole body autoregulation) that underlies the sustained hypertension. PMID:22140038

The myogenic repressor gene Holes in muscles is a direct transcriptional target of Twist and Tinman in the Drosophila embryonic mesoderm.

PubMed

Elwell, Jennifer A; Lovato, TyAnna L; Adams, Melanie M; Baca, Erica M; Lee, Thai; Cripps, Richard M

2015-04-15

Understanding the regulatory circuitry controlling myogenesis is critical to understanding developmental mechanisms and developmentally-derived diseases. We analyzed the transcriptional regulation of a Drosophila myogenic repressor gene, Holes in muscles (Him). Previously, Him was shown to inhibit Myocyte enhancer factor-2 (MEF2) activity, and is expressed in myoblasts but not differentiating myotubes. We demonstrate that different phases of Him embryonic expression arises through the actions of different enhancers, and we characterize the enhancer required for its early mesoderm expression. This Him early mesoderm enhancer contains two conserved binding sites for the basic helix-loop-helix regulator Twist, and one binding site for the NK homeodomain protein Tinman. The sites for both proteins are required for enhancer activity in early embryos. Twist and Tinman activate the enhancer in tissue culture assays, and ectopic expression of either factor is sufficient to direct ectopic expression of a Him-lacZ reporter, or of the endogenous Him gene. Moreover, sustained expression of twist in the mesoderm up-regulates mesodermal Him expression in late embryos. Our findings provide a model to define mechanistically how Twist can both promotes myogenesis through direct activation of Mef2, and can place a brake on myogenesis, through direct activation of Him. Copyright © 2015 Elsevier Inc. All rights reserved.

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

Improving cardiac conduction with a skeletal muscle sodium channel by gene and cell therapy

PubMed Central

Lu, Jia; Wang, Hong-Zhan; Jia, Zhiheng; Zuckerman, Joan; Lu, Zhongju; Guo, Yuanjian; Boink, Gerard J.J.; Brink, Peter R.; Robinson, Richard B.; Entcheva, Emilia; Cohen, Ira S.

2012-01-01

The voltage-gated Na+ channel is a critical determinant of the action potential upstroke. Increasing Na+ conductance may speed action potential propagation. Here we propose use of the skeletal muscle Na+ channel SkM1 as a more favorable gene than the cardiac isoform SCN5A to enhance conduction velocity in depolarized cardiac tissue. We used cells which electrically coupled with cardiac myocytes as a delivery platform to introduce the Na+ channels. HEK293 cells were stably transfected with SkM1 or SCN5A. SkM1 had a more depolarized (18mV shift) inactivation curve than SCN5A. We also found that SkM1 recovered faster from inactivation than SCN5A. When coupled with SkM1 expressing cells, cultured myocytes showed an increase in the dV/dtmax of the action potential. Expression of SCN5A had no such effect. In an in vitro cardiac syncytium, coculture of neonatal cardiac myocytes with SkM1 expressing but not SCN5A expressing cells significantly increased the conduction velocity under both normal and depolarized conditions. In an in vitro re-entry model induced by high frequency stimulation, expression of SkM1 also enhanced angular velocity of the induced re-entry. These results suggest that cells carrying a Na+ channel with a more depolarized inactivation curve can improve cardiac excitability and conduction in depolarized tissues. PMID:22526298

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

Nuclear accumulation of myocyte muscle LIM protein is regulated by heme oxygenase 1 and correlates with cardiac function in the transition to failure

PubMed Central

Paudyal, Anju; Dewan, Sukriti; Ikie, Cindy; Whalley, Benjamin J; de Tombe, Pieter P.

2016-01-01

Key points The present study investigated the mechanism associated with impaired cardiac mechanosensing that leads to heart failure by examining the factors regulating muscle LIM protein subcellular distribution in myocytes.In myocytes, muscle LIM protein subcellular distribution is regulated by cell contractility rather than passive stretch via heme oxygenase‐1 and histone deacetylase signalling. The result of the present study provide new insights into mechanotransduction in cardiac myocytes.Myocyte mechanosensitivity, as indicated by the muscle LIM protein ratio, is also correlated with cardiac function in the transition to failure in a guinea‐pig model of disease. This shows that the loss mechanosensitivity plays an important role during the transition to failure in the heart.The present study provides the first indication that mechanosensing could be modified pharmacologically during the transition to heart failure. Abstract Impaired mechanosensing leads to heart failure and a decreased ratio of cytoplasmic to nuclear CSRP3/muscle LIM protein (MLP ratio) is associated with a loss of mechanosensitivity. In the present study, we tested whether passive or active stress/strain was important in modulating the MLP ratio and determined whether this correlated with heart function during the transition to failure. We exposed cultured neonatal rat myocytes to a 10% cyclic mechanical stretch at 1 Hz, or electrically paced myocytes at 6.8 V (1 Hz) for 48 h. The MLP ratio decreased by 50% (P < 0.05, n = 4) only in response to electrical pacing, suggesting impaired mechanosensitivity. Inhibition of contractility with 10 μm blebbistatin resulted in an ∼3‐fold increase in the MLP ratio (n = 8, P < 0.05), indicating that myocyte contractility regulates nuclear MLP. Inhibition of histone deacetylase (HDAC) signalling with trichostatin A increased nuclear MLP following passive stretch, suggesting that HDACs block MLP nuclear accumulation. Inhibition of heme oxygenase1 (HO‐1) activity with protoporphyrin IX zinc(II) blocked MLP nuclear accumulation. To examine how mechanosensitivity changes during the transition to heart failure, we studied a guinea‐pig model of angiotensin II infusion (400 ng kg–1 min–1) over 12 weeks. Using subcellular fractionation, we showed that the MLP ratio increased by 88% (n = 4, P < 0.01) during compensated hypertrophy but decreased significantly during heart failure (P < 0.001, n = 4). The MLP ratio correlated significantly with the E/A ratio (r = 0.71, P < 0.01, n = 12), a clinical measure of diastolic function. These data indicate for the first time that myocyte mechanosensitivity as indicated by the MLP ratio is regulated primarily by myocyte contractility via HO‐1 and HDAC signalling. PMID:26847743

Vinpocetine Attenuates Pathological Cardiac Remodeling by Inhibiting Cardiac Hypertrophy and Fibrosis

PubMed Central

Wu, Mei-ping; Zhang, Yi-shuai; Xu, Xiangbin; Zhou, Qian

2017-01-01

Purpose Pathological cardiac remodeling, characterized by cardiac hypertrophy and fibrosis, is a pathological feature of many cardiac disorders that leads to heart failure and cardiac arrest. Vinpocetine, a derivative of the alkaloid vincamine, has been used for enhancing cerebral blood flow to treat cognitive impairment. However, its role in pathological cardiac remodeling remains unknown. The aim of this study is to examine the effect of vinpocetine on pathological cardiac remodeling induced by chronic stimulation with angiotensin II (Ang II). Methods Mice received Ang II infusion via osmotic pumps in the presence of vehicle or vinpocetine. Cardiac hypertrophy and fibrosis were assessed by morphological, histological, and biochemical analyses. Mechanistic studies were carried out in vitro with isolated mouse adult cardiac myocytes and fibroblasts. Results We showed that chronic Ang II infusion caused cardiac hypertrophy and fibrosis, which were all significantly attenuated by systemic administration of vinpocetine. In isolated adult mouse cardiomyocytes, vinpocetine suppressed Ang II-stimulated myocyte hypertrophic growth. In cultured cardiac fibroblasts, vinpocetine suppressed TGFβ-induced fibroblast activation and matrix gene expression, consistent with its effect in attenuating cardiac fibrosis. The effects of vinpocetine on cardiac myocyte hypertrophy and fibroblast activation are likely mediated by targeting cyclic nucleotide phosphodiesterase 1 (PDE1). Conclusions Our results reveal a novel protective effect of vinpocetine in attenuating pathological cardiac remodeling through suppressing cardiac myocyte hypertrophic growth and fibroblast activation and fibrotic gene expression. These studies may also shed light on developing novel therapeutic agents for antagonizing pathological cardiac remodeling. PMID:28321644

Vinpocetine Attenuates Pathological Cardiac Remodeling by Inhibiting Cardiac Hypertrophy and Fibrosis.

PubMed

Wu, Mei-Ping; Zhang, Yi-Shuai; Xu, Xiangbin; Zhou, Qian; Li, Jian-Dong; Yan, Chen

2017-04-01

Pathological cardiac remodeling, characterized by cardiac hypertrophy and fibrosis, is a pathological feature of many cardiac disorders that leads to heart failure and cardiac arrest. Vinpocetine, a derivative of the alkaloid vincamine, has been used for enhancing cerebral blood flow to treat cognitive impairment. However, its role in pathological cardiac remodeling remains unknown. The aim of this study is to examine the effect of vinpocetine on pathological cardiac remodeling induced by chronic stimulation with angiotensin II (Ang II). Mice received Ang II infusion via osmotic pumps in the presence of vehicle or vinpocetine. Cardiac hypertrophy and fibrosis were assessed by morphological, histological, and biochemical analyses. Mechanistic studies were carried out in vitro with isolated mouse adult cardiac myocytes and fibroblasts. We showed that chronic Ang II infusion caused cardiac hypertrophy and fibrosis, which were all significantly attenuated by systemic administration of vinpocetine. In isolated adult mouse cardiomyocytes, vinpocetine suppressed Ang II-stimulated myocyte hypertrophic growth. In cultured cardiac fibroblasts, vinpocetine suppressed TGFβ-induced fibroblast activation and matrix gene expression, consistent with its effect in attenuating cardiac fibrosis. The effects of vinpocetine on cardiac myocyte hypertrophy and fibroblast activation are likely mediated by targeting cyclic nucleotide phosphodiesterase 1 (PDE1). Our results reveal a novel protective effect of vinpocetine in attenuating pathological cardiac remodeling through suppressing cardiac myocyte hypertrophic growth and fibroblast activation and fibrotic gene expression. These studies may also shed light on developing novel therapeutic agents for antagonizing pathological cardiac remodeling.

A mathematical model of action potential heterogeneity in adult rat left ventricular myocytes.

PubMed Central

Pandit, S V; Clark, R B; Giles, W R; Demir, S S

2001-01-01

Mathematical models were developed to reconstruct the action potentials (AP) recorded in epicardial and endocardial myocytes isolated from the adult rat left ventricle. The main goal was to obtain additional insight into the ionic mechanisms responsible for the transmural AP heterogeneity. The simulation results support the hypothesis that the smaller density and the slower reactivation kinetics of the Ca(2+)-independent transient outward K(+) current (I(t)) in the endocardial myocytes can account for the longer action potential duration (APD), and more prominent rate dependence in that cell type. The larger density of the Na(+) current (I(Na)) in the endocardial myocytes results in a faster upstroke (dV/dt(max)). This, in addition to the smaller magnitude of I(t), is responsible for the larger peak overshoot of the simulated endocardial AP. The prolonged APD in the endocardial cell also leads to an enhanced amplitude of the sustained K(+) current (I(ss)), and a larger influx of Ca(2+) ions via the L-type Ca(2+) current (I(CaL)). The latter results in an increased sarcoplasmic reticulum (SR) load, which is mainly responsible for the higher peak systolic value of the Ca(2+) transient [Ca(2+)](i), and the resultant increase in the Na(+)-Ca(2+) exchanger (I(NaCa)) activity, associated with the simulated endocardial AP. In combination, these calculations provide novel, quantitative insights into the repolarization process and its naturally occurring transmural variations in the rat left ventricle. PMID:11720973

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.

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.

Promoter Variant-Dependent mRNA Expression of the MEF2A in Longissimus Dorsi Muscle in Cattle

PubMed Central

Starzyński, Rafał Radosław; Wicińska, Krystyna; Flisikowski, Krzysztof

2012-01-01

The myocyte enhancer factor 2A (MEF2A) gene encodes a member of the myocyte enhancer factor 2 (MEF2) protein family that is involved in vertebrate skeletal, cardiac, and smooth muscle development and differentiation during myogenesis. According to recent studies, MEF2 genes might be major regulators of postnatal skeletal muscle growth; thus, they are considered to be important, novel candidates for muscle development and body growth in farm animals. The aim of the present study was to search for polymorphisms in the bovine MEF2A gene and analyze their effect on the MEF2A mRNA expression level in the longissimus dorsi muscle of Polish Holstein-Fresian cattle. In total, 4094 bp of the whole coding sequence and the promoter region of MEF2A were re-sequenced in 30 animals, resulting in the detection of 6 novel variants as well as one previously reported SNP. Three linked mutations in the promoter region (-780T/G, g.-768T/G, and g.-222A/G) and only two genotypes were identified in two Polish breeds (TTA/TTA and TTA/GGG). Three SNPs in the coding region [g.1599G/A (421aa), g.1626G/A (429aa), and g.1641G/A (434aa)] appeared to be silent substitutions and segregated as two intragene haplotypes: GGG and AAA. Expression analysis showed that the mutations in the promoter region are highly associated with the MEF2A mRNA level in the longissimus dorsi muscle of bulls carrying two different genotypes. The higher MEF2A mRNA level was estimated in the muscle of bulls carrying the TTA/TTA (p<0.01) genotype as compared with those with TTA/GGG. The results obtained suggest that the nucleotide sequence mutation in MEF2A might be useful marker for body growth traits in cattle. PMID:22320864

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.

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.

An Optimized and Simplified System of Mouse Embryonic Stem Cell Cardiac Differentiation for the Assessment of Differentiation Modifiers

PubMed Central

Hartman, Matthew E.; Librande, Jason R.; Medvedev, Ivan O.; Ahmad, Rabiah N.; Moussavi-Harami, Farid; Gupta, Pritha P.; Chien, Wei-Ming; Chin, Michael T.

2014-01-01

Generating cardiomyocytes from embryonic stem cells is an important technique for understanding cardiovascular development, the origins of cardiovascular diseases and also for providing potential reagents for cardiac repair. Numerous methods have been published but often are technically challenging, complex, and are not easily adapted to assessment of specific gene contributions to cardiac myocyte differentiation. Here we report the development of an optimized protocol to induce the differentiation of mouse embryonic stem cells to cardiac myocytes that is simplified and easily adapted for genetic studies. Specifically, we made four critical findings that distinguish our protocol: 1) mouse embryonic stem cells cultured in media containing CHIR99021 and PD0325901 to maintain pluripotency will efficiently form embryoid bodies containing precardiac mesoderm when cultured in these factors at a reduced dosage, 2) low serum conditions promote cardiomyocyte differentiation and can be used in place of commercially prepared StemPro nutrient supplement, 3) the Wnt inhibitor Dkk-1 is dispensable for efficient cardiac differentiation and 4) tracking differentiation efficiency may be done with surface expression of PDGFRα alone. In addition, cardiac mesodermal precursors generated by this system can undergo lentiviral infection to manipulate the expression of specific target molecules to assess effects on cardiac myocyte differentiation and maturation. Using this approach, we assessed the effects of CHF1/Hey2 on cardiac myocyte differentiation, using both gain and loss of function. Overexpression of CHF1/Hey2 at the cardiac mesoderm stage had no apparent effect on cardiac differentiation, while knockdown of CHF1/Hey2 resulted in increased expression of atrial natriuretic factor and connexin 43, suggesting an alteration in the phenotype of the cardiomyocytes. In summary we have generated a detailed and simplified protocol for generating cardiomyocytes from mES cells that is optimized for investigating factors that affect cardiac differentiation. PMID:24667642

β-Adrenergic Stimulation Induces Histone Deacetylase 5 (HDAC5) Nuclear Accumulation in Cardiomyocytes by B55α-PP2A-Mediated Dephosphorylation.

PubMed

Weeks, Kate L; Ranieri, Antonella; Karaś, Agnieszka; Bernardo, Bianca C; Ashcroft, Alexandra S; Molenaar, Chris; McMullen, Julie R; Avkiran, Metin

2017-03-25

Class IIa histone deacetylase (HDAC) isoforms such as HDAC5 are critical signal-responsive repressors of maladaptive cardiomyocyte hypertrophy, through nuclear interactions with transcription factors including myocyte enhancer factor-2. β-Adrenoceptor (β-AR) stimulation, a signal of fundamental importance in regulating cardiac function, has been proposed to induce both phosphorylation-independent nuclear export and phosphorylation-dependent nuclear accumulation of cardiomyocyte HDAC5. The relative importance of phosphorylation at Ser259/Ser498 versus Ser279 in HDAC5 regulation is also controversial. We aimed to determine the impact of β-AR stimulation on the phosphorylation, localization, and function of cardiomyocyte HDAC5 and delineate underlying molecular mechanisms. A novel 3-dimensional confocal microscopy method that objectively quantifies the whole-cell nuclear/cytoplasmic distribution of green fluorescent protein tagged HDAC5 revealed the β-AR agonist isoproterenol to induce β 1 -AR-mediated and protein kinase A-dependent HDAC5 nuclear accumulation in adult rat cardiomyocytes, which was accompanied by dephosphorylation at Ser259/279/498. Mutation of Ser259/Ser498 to Ala promoted HDAC5 nuclear accumulation and myocyte enhancer factor-2 inhibition, whereas Ser279 ablation had no such effect and did not block isoproterenol-induced nuclear accumulation. Inhibition of the Ser/Thr phosphatase PP2A blocked isoproterenol-induced HDAC5 dephosphorylation. Co-immunoprecipitation revealed a specific interaction of HDAC5 with the PP2A targeting subunit B55α, as well as catalytic and scaffolding subunits, which increased >3-fold with isoproterenol. Knockdown of B55α in neonatal cardiomyocytes attenuated isoproterenol-induced HDAC5 dephosphorylation. β-AR stimulation induces HDAC5 nuclear accumulation in cardiomyocytes by a mechanism that is protein kinase A-dependent but requires B55α-PP2A-mediated dephosphorylation of Ser259/Ser498 rather than protein kinase A-mediated phosphorylation of Ser279. © 2017 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley Blackwell.

FoxO1 regulates myocardial glucose oxidation rates via transcriptional control of pyruvate dehydrogenase kinase 4 expression.

PubMed

Gopal, Keshav; Saleme, Bruno; Al Batran, Rami; Aburasayn, Hanin; Eshreif, Amina; Ho, Kim L; Ma, Wayne K; Almutairi, Malak; Eaton, Farah; Gandhi, Manoj; Park, Edwards A; Sutendra, Gopinath; Ussher, John R

2017-09-01

Pyruvate dehydrogenase (PDH) is the rate-limiting enzyme for glucose oxidation and a critical regulator of metabolic flexibility during the fasting to feeding transition. PDH is regulated via both PDH kinases (PDHK) and PDH phosphatases, which phosphorylate/inactivate and dephosphorylate/activate PDH, respectively. Our goal was to determine whether the transcription factor forkhead box O1 (FoxO1) regulates PDH activity and glucose oxidation in the heart via increasing the expression of Pdk4 , the gene encoding PDHK4. To address this question, we differentiated H9c2 myoblasts into cardiac myocytes and modulated FoxO1 activity, after which Pdk4 /PDHK4 expression and PDH phosphorylation/activity were assessed. We assessed binding of FoxO1 to the Pdk4 promoter in cardiac myocytes in conjunction with measuring the role of FoxO1 on glucose oxidation in the isolated working heart. Both pharmacological (1 µM AS1842856) and genetic (siRNA mediated) inhibition of FoxO1 decreased Pdk4 /PDHK4 expression and subsequent PDH phosphorylation in H9c2 cardiac myocytes, whereas 10 µM dexamethasone-induced Pdk4 /PDHK4 expression was abolished via pretreatment with 1 µM AS1842856. Furthermore, transfection of H9c2 cardiac myocytes with a vector expressing FoxO1 increased luciferase activity driven by a Pdk4 promoter construct containing the FoxO1 DNA-binding element region, but not in a Pdk4 promoter construct lacking this region. Finally, AS1842856 treatment in fasted mice enhanced glucose oxidation rates during aerobic isolated working heart perfusions. Taken together, FoxO1 directly regulates Pdk4 transcription in the heart, thereby controlling PDH activity and subsequent glucose oxidation rates. NEW & NOTEWORTHY Although studies have shown an association between FoxO1 activity and pyruvate dehydrogenase kinase 4 expression, our study demonstrated that pyruvate dehydrogenase kinase 4 is a direct transcriptional target of FoxO1 (but not FoxO3/FoxO4) in the heart. Furthermore, we report here, for the first time, that FoxO1 inhibition increases glucose oxidation in the isolated working mouse heart. Copyright © 2017 the American Physiological Society.

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.

Inhibitory Effect of Vascular Endothelial Growth Factor on the Slowly Activating Delayed Rectifier Potassium Current in Guinea Pig Ventricular Myocytes.

PubMed

Lin, Zhenhao; Xing, Wenlu; Gao, Chuanyu; Wang, Xianpei; Qi, Datun; Dai, Guoyou; Zhao, Wen; Yan, Ganxin

2018-01-26

Vascular endothelial growth factor (VEGF) exerts a number of beneficial effects on ischemic myocardium via its angiogenic properties. However, little is known about whether VEGF has a direct effect on the electrical properties of cardiomyocytes. In the present study, we investigated the effects of different concentrations of VEGF on delayed rectifier potassium currents (I K ) in guinea pig ventricular myocytes and their effects on action potential (AP) parameters. I K and AP were recorded by the whole-cell patch clamp method in ventricular myocytes. Cells were superfused with control solution or solution containing VEGF at different concentrations for 10 minutes before recording. Some ventricular myocytes were pretreated with a phosphatidylinositol 3-kinase inhibitor for 1 hour before the addition of VEGF. We found that VEGF inhibited the slowly activating delayed rectifier potassium current (I K s ) in a concentration-dependent manner (18.13±1.04 versus 12.73±0.34, n=5, P =0.001; 12.73±0.34 versus 9.05±1.20, n=5, P =0.036) and prolonged AP duration (894.5±36.92 versus 746.3±33.71, n=5, P =0.021). Wortmannin, a phosphatidylinositol 3-kinase inhibitor, eliminated these VEGF-induced effects. VEGF had no significant effect on the rapidly activating delayed rectifier potassium current (I K r ), resting membrane potential, AP amplitude, or maximal velocity of depolarization. VEGF inhibited I K s in a concentration-dependent manner through a phosphatidylinositol 3-kinase-mediated signaling pathway, leading to AP prolongation. The results indicate a promising therapeutic potential of VEGF in prevention of ventricular tachyarrhythmias under conditions of high sympathetic activity and ischemia. © 2018 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley.

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

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.

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

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.

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.

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

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.

Human colonic myocytes are involved in postischemic inflammation through ADAM17-dependent TNFα production

PubMed Central

Jarry, Anne; Bach-Ngohou, Kalyane; Masson, Damien; Dejoie, Thomas; Lehur, Paul-Antoine; Mosnier, Jean-François; Denis, Marc G; Laboisse, Christian L

2005-01-01

The aim of this study was to identify human colonic resident cells able to initiate an inflammatory response in postischemic injury. Postischemic colonic injury, a condition relevant to various clinical settings, involves an inflammatory cascade in intestinal tissues through the recruitment of circulating inflammatory cells. However, there is no information on the nature of resident cells of the different intestinal layers able to initiate a postischemic inflammatory response. It is however an important issue in the context of a pharmacological approach of the early phase of intestinal ischemia. We reasoned that maintaining the different colonic layers as explant cultures in an oxygenated medium immediately after colonic resection, that is, after an ischemic period, would allow one to identify the resident cells able to initiate an inflammatory cascade, without interference of recruited inflammatory/immune cells. To this end, we designed an explant culture system that operationally defines three compartments in surgical specimens of the human colon, based on the microdissected layers, that is, mucosa, submucosa (containing muscularis mucosae) and muscularis propria. To validate the results obtained in explant cultures in the clinical setting of ischemic colitis, eight cases of sigmoid volvulus were examined. Only the myocytes-containing explants produced tumor necrosis factor alpha (TNFα), via an ADAM17 (a disintegrin and metalloproteinase-17)-dependent pathway, as shown by the abrogation of TNFα production by the inhibitor Tapi-2. Immunofluorescence studies identified nonvascular and vascular myocytes as resident cells coexpressing TNFα and ADAM17, both in our postischemic explant system and in surgical specimens from ischemic colitis patients. Finally, time-course experiments on explanted tissues showed that TNFα production by myocytes was an early event triggered by a postischemic oxidative stress involving nuclear factor kappa B (NF-κB). In conclusion, this study identifies human intestinal myocytes as resident cells able to initiate an inflammatory reaction through TNFα production in postischemic conditions, and delineates two points of control in TNFα production, NF-κB and ADAM17, which can be targeted by pharmacological manipulation. PMID:16273118

PDSM, a motif for phosphorylation-dependent SUMO modification

PubMed Central

Hietakangas, Ville; Anckar, Julius; Blomster, Henri A.; Fujimoto, Mitsuaki; Palvimo, Jorma J.; Nakai, Akira; Sistonen, Lea

2006-01-01

SUMO (small ubiquitin-like modifier) modification regulates many cellular processes, including transcription. Although sumoylation often occurs on specific lysines within the consensus tetrapeptide ΨKxE, other modifications, such as phosphorylation, may regulate the sumoylation of a substrate. We have discovered PDSM (phosphorylation-dependent sumoylation motif), composed of a SUMO consensus site and an adjacent proline-directed phosphorylation site (ΨKxExxSP). The highly conserved motif regulates phosphorylation-dependent sumoylation of multiple substrates, such as heat-shock factors (HSFs), GATA-1, and myocyte enhancer factor 2. In fact, the majority of the PDSM-containing proteins are transcriptional regulators. Within the HSF family, PDSM is conserved between two functionally distinct members, HSF1 and HSF4b, whose transactivation capacities are repressed through the phosphorylation-dependent sumoylation. As the first recurrent sumoylation determinant beyond the consensus tetrapeptide, the PDSM provides a valuable tool in predicting new SUMO substrates. PMID:16371476

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.

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

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

Neuronal Na+ Channels Are Integral Components of Pro-arrhythmic Na+/Ca2+ Signaling Nanodomain That Promotes Cardiac Arrhythmias During β-adrenergic Stimulation

PubMed Central

Radwański, Przemysław B.; Ho, Hsiang-Ting; Veeraraghavan, Rengasayee; Brunello, Lucia; Liu, Bin; Belevych, Andriy E.; Unudurthi, Sathya D.; Makara, Michael A.; Priori, Silvia G.; Volpe, Pompeo; Armoundas, Antonis A.; Dillmann, Wolfgang H.; Knollmann, Bjorn C.; Mohler, Peter J.; Hund, Thomas J.; Györke, Sándor

2016-01-01

Background Cardiac arrhythmias are a leading cause of death in the US. Vast majority of these arrhythmias including catecholaminergic polymorphic ventricular tachycardia (CPVT) are associated with increased levels of circulating catecholamines and involve abnormal impulse formation secondary to aberrant Ca2+ and Na+ handling. However, the mechanistic link between β-AR stimulation and the subcellular/molecular arrhythmogenic trigger(s) remains elusive. Methods and Results We performed functional and structural studies to assess Ca2+ and Na+ signaling in ventricular myocyte as well as surface electrocardiograms in mouse models of cardiac calsequestrin (CASQ2)-associated CPVT. We demonstrate that a subpopulation of Na+ channels (neuronal Na+ channels; nNav) that colocalize with RyR2 and Na+/Ca2+ exchanger (NCX) are a part of the β-AR-mediated arrhythmogenic process. Specifically, augmented Na+ entry via nNav in the settings of genetic defects within the RyR2 complex and enhanced sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA)-mediated SR Ca2+ refill is both an essential and a necessary factor for the arrhythmogenesis. Furthermore, we show that augmentation of Na+ entry involves β-AR-mediated activation of CAMKII subsequently leading to nNav augmentation. Importantly, selective pharmacological inhibition as well as silencing of Nav1.6 inhibit myocyte arrhythmic potential and prevent arrhythmias in vivo. Conclusion These data suggest that the arrhythmogenic alteration in Na+/Ca2+ handling evidenced ruing β-AR stimulation results, at least in part, from enhanced Na+ influx through nNav. Therefore, selective inhibition of these channels and Nav1.6 in particular can serve as a potential antiarrhythmic therapy. PMID:27747307

A mathematical model of the electrophysiological alterations in rat ventricular myocytes in type-I diabetes.

PubMed

Pandit, Sandeep V; Giles, Wayne R; Demir, Semahat S

2003-02-01

Our mathematical model of the rat ventricular myocyte (Pandit et al., 2001) was utilized to explore the ionic mechanism(s) that underlie the altered electrophysiological characteristics associated with the short-term model of streptozotocin-induced, type-I diabetes. The simulations show that the observed reductions in the Ca(2+)-independent transient outward K(+) current (I(t)) and the steady-state outward K(+) current (I(ss)), along with slowed inactivation of the L-type Ca(2+) current (I(CaL)), can result in the prolongation of the action potential duration, a well-known experimental finding. In addition, the model demonstrates that the slowed reactivation kinetics of I(t) in diabetic myocytes can account for the more pronounced rate-dependent action potential duration prolongation in diabetes, and that a decrease in the electrogenic Na(+)-K(+) pump current (I(NaK)) results in a small depolarization in the resting membrane potential (V(rest)). This depolarization reduces the availability of the Na(+) channels (I(Na)), thereby resulting in a slower upstroke (dV/dt(max)) of the diabetic action potential. Additional simulations suggest that a reduction in the magnitude of I(CaL), in combination with impaired sarcoplasmic reticulum uptake can lead to a decreased sarcoplasmic reticulum Ca(2+) load. These factors contribute to characteristic abnormal [Ca(2+)](i) homeostasis (reduced peak systolic value and rate of decay) in myocytes from diabetic animals. In combination, these simulation results provide novel information and integrative insights concerning plausible ionic mechanisms for the observed changes in cardiac repolarization and excitation-contraction coupling in rat ventricular myocytes in the setting of streptozotocin-induced, type-I diabetes.

Intracellular tortuosity underlies slow cAMP diffusion in adult ventricular myocytes.

PubMed

Richards, Mark; Lomas, Oliver; Jalink, Kees; Ford, Kerrie L; Vaughan-Jones, Richard D; Lefkimmiatis, Konstantinos; Swietach, Pawel

2016-06-01

3',5'-Cyclic adenosine monophosphate (cAMP) signals in the heart are often confined to concentration microdomains shaped by cAMP diffusion and enzymatic degradation. While the importance of phosphodiesterases (degradative enzymes) in sculpting cAMP microdomains is well established in cardiomyocytes, less is known about cAMP diffusivity (DcAMP) and factors affecting it. Many earlier studies have reported fast diffusivity, which argues against sharply defined microdomains. [cAMP] dynamics in the cytoplasm of adult rat ventricular myocytes were imaged using a fourth generation genetically encoded FRET-based sensor. The [cAMP]-response to the addition and removal of isoproterenol (β-adrenoceptor agonist) quantified the rates of cAMP synthesis and degradation. To obtain a read out of DcAMP, a stable [cAMP] gradient was generated using a microfluidic device which delivered agonist to one half of the myocyte only. After accounting for phosphodiesterase activity, DcAMP was calculated to be 32 µm(2)/s; an order of magnitude lower than in water. Diffusivity was independent of the amount of cAMP produced. Saturating cAMP-binding sites with the analogue 6-Bnz-cAMP did not accelerate DcAMP, arguing against a role of buffering in restricting cAMP mobility. cAMP diffused at a comparable rate to chemically unrelated but similar sized molecules, arguing for a common physical cause of restricted diffusivity. Lower mitochondrial density and order in neonatal cardiac myocytes allowed for faster diffusion, demonstrating the importance of mitochondria as physical barriers to cAMP mobility. In adult cardiac myocytes, tortuosity due to physical barriers, notably mitochondria, restricts cAMP diffusion to levels that are more compatible with microdomain signalling. © The Author 2016. Published by Oxford University Press on behalf of the European Society of Cardiology.

Supplementation with IL-6 and Muscle Cell Culture Conditioned Media Enhances Myogenic Differentiation of Adipose Tissue-Derived Stem Cells through STAT3 Activation.

PubMed

Seo, Eunhui; Kang, Hwansu; Lim, Oh-Kyung; Jun, Hee-Sook

2018-05-24

Mature skeletal muscle cells cannot be expanded in culture systems. Therefore, it is difficult to construct an in vitro model for muscle diseases. To establish an efficient protocol for myogenic differentiation of human adipose tissue-derived stem cells (hADSCs), we investigated whether addition of IL-6 and/or myocyte-conditioned media (CM) to conventional differentiation media can shorten the differentiation period. hADSCs were differentiated to myocytes using the conventional protocol or modified with the addition of 25 pg/mL IL-6 and/or C2C12 CM (25% v / v ). The expression of MyoD and myogenine mRNA was significantly higher at 5⁻6 days after differentiation using the modified protocol than with the conventional protocol. mRNA and protein expression of myosin heavy chain, a marker of myotubes, was significantly upregulated at 28 and 42 days of differentiation using the modified protocol, and the level achieved after a 4-week differentiation period was similar to that achieved at 6 weeks using the conventional protocol. The expression of p-STAT3 was significantly increased when the modified protocol was used. Similarly, addition of colivelin, a STAT3 activator, instead of IL-6 and C2C12 CM, promoted the myogenic differentiation of ADSCs. The modified protocol improved differentiation efficiency and reduced the time required for differentiation of myocytes. It might be helpful to save cost and time when preparing myocytes for cell therapies and drug discovery.

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

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.

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.

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.

Metabolic remodeling of human skeletal myocytes by cocultured adipocytes depends on the lipolytic state of the system.

PubMed

Kovalik, Jean-Paul; Slentz, Dorothy; Stevens, Robert D; Kraus, William E; Houmard, Joseph A; Nicoll, James B; Lea-Currie, Y Renee; Everingham, Karen; Kien, C Lawrence; Buehrer, Benjamin M; Muoio, Deborah M

2011-07-01

Adipocyte infiltration of the musculoskeletal system is well recognized as a hallmark of aging, obesity, and type 2 diabetes. Intermuscular adipocytes might serve as a benign storage site for surplus lipid or play a role in disrupting energy homeostasis as a result of dysregulated lipolysis or secretion of proinflammatory cytokines. This investigation sought to understand the net impact of local adipocytes on skeletal myocyte metabolism. Interactions between these two tissues were modeled using a coculture system composed of primary human adipocytes and human skeletal myotubes derived from lean or obese donors. Metabolic analysis of myocytes was performed after coculture with lipolytically silent or activated adipocytes and included transcript and metabolite profiling along with assessment of substrate selection and insulin action. Cocultured adipocytes increased myotube mRNA expression of genes involved in oxidative metabolism, regardless of the donor and degree of lipolytic activity. Adipocytes in the basal state sequestered free fatty acids, thereby forcing neighboring myotubes to rely more heavily on glucose fuel. Under this condition, insulin action was enhanced in myotubes from lean but not obese donors. In contrast, when exposed to lipolytically active adipocytes, cocultured myotubes shifted substrate use in favor of fatty acids, which was accompanied by intracellular accumulation of triacylglycerol and even-chain acylcarnitines, decreased glucose oxidation, and modest attenuation of insulin signaling. The effects of cocultured adipocytes on myocyte substrate selection and insulin action depended on the metabolic state of the system. These findings are relevant to understanding the metabolic consequences of intermuscular adipogenesis. © 2011 by the American Diabetes Association.

Metabolic Remodeling of Human Skeletal Myocytes by Cocultured Adipocytes Depends on the Lipolytic State of the System

PubMed Central

Kovalik, Jean-Paul; Slentz, Dorothy; Stevens, Robert D.; Kraus, William E.; Houmard, Joseph A.; Nicoll, James B.; Lea-Currie, Y. Renee; Everingham, Karen; Kien, C. Lawrence; Buehrer, Benjamin M.; Muoio, Deborah M.

2011-01-01

OBJECTIVE Adipocyte infiltration of the musculoskeletal system is well recognized as a hallmark of aging, obesity, and type 2 diabetes. Intermuscular adipocytes might serve as a benign storage site for surplus lipid or play a role in disrupting energy homeostasis as a result of dysregulated lipolysis or secretion of proinflammatory cytokines. This investigation sought to understand the net impact of local adipocytes on skeletal myocyte metabolism. RESEARCH DESIGN AND METHODS Interactions between these two tissues were modeled using a coculture system composed of primary human adipocytes and human skeletal myotubes derived from lean or obese donors. Metabolic analysis of myocytes was performed after coculture with lipolytically silent or activated adipocytes and included transcript and metabolite profiling along with assessment of substrate selection and insulin action. RESULTS Cocultured adipocytes increased myotube mRNA expression of genes involved in oxidative metabolism, regardless of the donor and degree of lipolytic activity. Adipocytes in the basal state sequestered free fatty acids, thereby forcing neighboring myotubes to rely more heavily on glucose fuel. Under this condition, insulin action was enhanced in myotubes from lean but not obese donors. In contrast, when exposed to lipolytically active adipocytes, cocultured myotubes shifted substrate use in favor of fatty acids, which was accompanied by intracellular accumulation of triacylglycerol and even-chain acylcarnitines, decreased glucose oxidation, and modest attenuation of insulin signaling. CONCLUSIONS The effects of cocultured adipocytes on myocyte substrate selection and insulin action depended on the metabolic state of the system. These findings are relevant to understanding the metabolic consequences of intermuscular adipogenesis. PMID:21602515

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.

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

Impaired Na⁺-dependent regulation of acetylcholine-activated inward-rectifier K⁺ current modulates action potential rate dependence in patients with chronic atrial fibrillation.

PubMed

Voigt, Niels; Heijman, Jordi; Trausch, Anne; Mintert-Jancke, Elisa; Pott, Lutz; Ravens, Ursula; Dobrev, Dobromir

2013-08-01

Shortened action-potential duration (APD) and blunted APD rate adaptation are hallmarks of chronic atrial fibrillation (cAF). Basal and muscarinic (M)-receptor-activated inward-rectifier K(+) currents (IK1 and IK,ACh, respectively) contribute to regulation of human atrial APD and are subject to cAF-dependent remodeling. Intracellular Na(+) ([Na(+)]i) enhances IK,ACh in experimental models but the effect of [Na(+)]i-dependent regulation of inward-rectifier K(+) currents on APD in human atrial myocytes is currently unknown. Here, we report a [Na(+)]i-dependent inhibition of outward IK1 in atrial myocytes from sinus rhythm (SR) or cAF patients. In contrast, IK,ACh activated by carbachol, a non-selective M-receptor agonist, increased with elevation of [Na(+)]i in SR. This [Na(+)]i-dependent IK,ACh regulation was absent in cAF. Including [Na(+)]i dependence of IK1 and IK,ACh in a recent computational model of the human atrial myocyte revealed that [Na(+)]i accumulation at fast rates inhibits IK1 and blunts physiological APD rate dependence in both groups. [Na(+)]i-dependent IK,ACh augmentation at fast rates increased APD rate dependence in SR, but not in cAF. These results identify impaired Na(+)-sensitivity of IK,ACh as one potential mechanism contributing to the blunted APD rate dependence in patients with cAF. This article is part of a Special Issue entitled "Na(+) Regulation in Cardiac Myocytes". Copyright © 2013 The Authors. Published by Elsevier Ltd.. All rights reserved.

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.

Recovery of methamphetamine associated cardiomyopathy predicted by late gadolinium enhanced cardiovascular magnetic resonance.

PubMed

Lopez, Javier E; Yeo, Khung; Caputo, Gary; Buonocore, Michael; Schaefer, Saul

2009-11-11

Methamphetamine is known to cause a cardiomyopathy which may be reversible with appropriate medical therapy and cessation of use. Late gadolinium enhancement cardiovascular magnetic resonance (CMR) has been shown to identify fibrosis in ischemic and non-ischemic cardiomyopathies. We present a case of severe methamphetamine-associated cardiomyopathy in which cardiac function recovered after 6 months. Evaluation by CMR using late gadolinium enhancement was notable for an absence of enhancement, suggesting an absence of irreversible myocyte injury and a good prognosis. CMR may be useful to predict recovery in toxin-associated non-ischemic cardiomyopathies.

Recovery of methamphetamine associated cardiomyopathy predicted by late gadolinium enhanced cardiovascular magnetic resonance

PubMed Central

2009-01-01

Methamphetamine is known to cause a cardiomyopathy which may be reversible with appropriate medical therapy and cessation of use. Late gadolinium enhancement cardiovascular magnetic resonance (CMR) has been shown to identify fibrosis in ischemic and non-ischemic cardiomyopathies. We present a case of severe methamphetamine-associated cardiomyopathy in which cardiac function recovered after 6 months. Evaluation by CMR using late gadolinium enhancement was notable for an absence of enhancement, suggesting an absence of irreversible myocyte injury and a good prognosis. CMR may be useful to predict recovery in toxin-associated non-ischemic cardiomyopathies. PMID:19906310

Myosin light chain 2-based selection of human iPSC-derived early ventricular cardiac myocytes.

PubMed

Bizy, Alexandra; Guerrero-Serna, Guadalupe; Hu, Bin; Ponce-Balbuena, Daniela; Willis, B Cicero; Zarzoso, Manuel; Ramirez, Rafael J; Sener, Michelle F; Mundada, Lakshmi V; Klos, Matthew; Devaney, Eric J; Vikstrom, Karen L; Herron, Todd J; Jalife, José

2013-11-01

Applications of human induced pluripotent stem cell derived-cardiac myocytes (hiPSC-CMs) would be strengthened by the ability to generate specific cardiac myocyte (CM) lineages. However, purification of lineage-specific hiPSC-CMs is limited by the lack of cell marking techniques. Here, we have developed an iPSC-CM marking system using recombinant adenoviral reporter constructs with atrial- or ventricular-specific myosin light chain-2 (MLC-2) promoters. MLC-2a and MLC-2v selected hiPSC-CMs were purified by fluorescence-activated cell sorting and their biochemical and electrophysiological phenotypes analyzed. We demonstrate that the phenotype of both populations remained stable in culture and they expressed the expected sarcomeric proteins, gap junction proteins and chamber-specific transcription factors. Compared to MLC-2a cells, MLC-2v selected CMs had larger action potential amplitudes and durations. In addition, by immunofluorescence, we showed that MLC-2 isoform expression can be used to enrich hiPSC-CM consistent with early atrial and ventricular myocyte lineages. However, only the ventricular myosin light chain-2 promoter was able to purify a highly homogeneous population of iPSC-CMs. Using this approach, it is now possible to develop ventricular-specific disease models using iPSC-CMs while atrial-specific iPSC-CM cultures may require additional chamber-specific markers. © 2013.

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.

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.

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.

Histone deacetylase degradation andMEF2 activation promote the formation of slow-twitch myofibers

PubMed Central

Potthoff, Matthew J.; Wu, Hai; Arnold, Michael A.; Shelton, John M.; Backs, Johannes; McAnally, John; Richardson, James A.; Bassel-Duby, Rhonda; Olson, Eric N.

2007-01-01

Skeletal muscle is composed of heterogeneous myofibers with distinctive rates of contraction, metabolic properties, and susceptibility to fatigue. We show that class II histone deacetylase (HDAC) proteins, which function as transcriptional repressors of the myocyte enhancer factor 2 (MEF2) transcription factor, fail to accumulate in the soleus, a slow muscle, compared with fast muscles (e.g., white vastus lateralis). Accordingly, pharmacological blockade of proteasome function specifically increases expression of class II HDAC proteins in the soleus in vivo. Using gain- and loss-of-function approaches in mice, we discovered that class II HDAC proteins suppress the formation of slow twitch, oxidative myofibers through the repression of MEF2 activity. Conversely, expression of a hyperactive form of MEF2 in skeletal muscle of transgenic mice promotes the formation of slow fibers and enhances running endurance, enabling mice to run almost twice the distance of WT littermates. Thus, the selective degradation of class II HDACs in slow skeletal muscle provides a mechanism for enhancing physical performance and resistance to fatigue by augmenting the transcriptional activity of MEF2. These findings provide what we believe are new insights into the molecular basis of skeletal muscle function and have important implications for possible therapeutic interventions into muscular diseases. PMID:17786239

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

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.

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

Pan-histone deacetylase inhibitors regulate signaling pathways involved in proliferative and pro-inflammatory mechanisms in H9c2 cells

PubMed Central

2012-01-01

Background We have shown previously that pan-HDAC inhibitors (HDACIs) m-carboxycinnamic acid bis-hydroxamide (CBHA) and trichostatin A (TSA) attenuated cardiac hypertrophy in BALB/c mice by inducing hyper-acetylation of cardiac chromatin that was accompanied by suppression of pro-inflammatory gene networks. However, it was not feasible to determine the precise contribution of the myocytes- and non-myocytes to HDACI-induced gene expression in the intact heart. Therefore, the current study was undertaken with a primary goal of elucidating temporal changes in the transcriptomes of cardiac myocytes exposed to CBHA and TSA. Results We incubated H9c2 cardiac myocytes in growth medium containing either of the two HDACIs for 6h and 24h and analyzed changes in gene expression using Illumina microarrays. H9c2 cells exposed to TSA for 6h and 24h led to differential expression of 468 and 231 genes, respectively. In contrast, cardiac myocytes incubated with CBHA for 6h and 24h elicited differential expression of 768 and 999 genes, respectively. We analyzed CBHA- and TSA-induced differentially expressed genes by Ingenuity Pathway (IPA), Kyoto Encyclopedia of Genes and Genomes (KEGG) and Core_TF programs and discovered that CBHA and TSA impinged on several common gene networks. Thus, both HDACIs induced a repertoire of signaling kinases (PTEN-PI3K-AKT and MAPK) and transcription factors (Myc, p53, NFkB and HNF4A) representing canonical TGFβ, TNF-α, IFNγ and IL-6 specific networks. An overrepresentation of E2F, AP2, EGR1 and SP1 specific motifs was also found in the promoters of the differentially expressed genes. Apparently, TSA elicited predominantly TGFβ- and TNF-α-intensive gene networks regardless of the duration of treatment. In contrast, CBHA elicited TNF-α and IFNγ specific networks at 6 h, followed by elicitation of IL-6 and IFNγ-centered gene networks at 24h. Conclusions Our data show that both CBHA and TSA induced similar, but not identical, time-dependent, gene networks in H9c2 cardiac myocytes. Initially, both HDACIs impinged on numerous genes associated with adipokine signaling, intracellular metabolism and energetics, and cell cycle. A continued exposure to either CBHA or TSA led to the emergence of a number of apoptosis- and inflammation-specific gene networks that were apparently suppressed by both HDACIs. Based on these data we posit that the anti-inflammatory and anti-proliferative actions of HDACIs are myocyte-intrinsic. These findings advance our understanding of the mechanisms of actions of HDACIs on cardiac myocytes and reveal potential signaling pathways that may be targeted therapeutically. PMID:23249388

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.

Ca2+ transients in cardiac myocytes measured with high and low affinity Ca2+ indicators.

PubMed Central

Berlin, J R; Konishi, M

1993-01-01

Intracellular calcium ion ([Ca2+]i) transients were measured in voltage-clamped rat cardiac myocytes with fura-2 or furaptra to quantitate rapid changes in [Ca2+]i. Patch electrode solutions contained the K+ salt of fura-2 (50 microM) or furaptra (300 microM). With identical experimental conditions, peak amplitude of stimulated [Ca2+]i transients in furaptra-loaded myocytes was 4- to 6-fold greater than that in fura-2-loaded cells. To determine the reason for this discrepancy, intracellular fura-2 Ca2+ buffering, kinetics of Ca2+ binding, and optical properties were examined. Decreasing cellular fura-2 concentration by lowering electrode fura-2 concentration 5-fold, decreased the difference between the amplitudes of [Ca2+]i transients in fura-2 and furaptra-loaded myocytes by twofold. Thus, fura-2 buffers [Ca2+]i under these conditions; however, Ca2+ buffering is not the only factor that explains the different amplitudes of the [Ca2+]i transients measured with these indicators. From the temporal comparison of the [Ca2+]i transients measured with fura-2 and furaptra, the apparent reverse rate constant for Ca2+ binding of fura-2 was at least 65s-1, much faster than previously reported in skeletal muscle fibers. These binding kinetics do not explain the difference in the size of the [Ca2+]i transients reported by fura-2 and furaptra. Parameters for fura-2 calibration, Rmin, Rmax, and beta, were obtained in salt solutions (in vitro) and in myocytes exposed to the Ca2+ ionophore, 4-Br A23187, in EGTA-buffered solutions (in situ). Calibration of fura-2 fluorescence signals with these in situ parameters yielded [Ca2+]i transients whose peak amplitude was 50-100% larger than those calculated with in vitro parameters. Thus, in vitro calibration of fura-2 fluorescence significantly underestimates the amplitude of the [Ca2+]i transient. These data suggest that the difference in amplitude of [Ca2+]i transients in fura-2 and furaptra-loaded myocytes is due, in part, to Ca2+ buffering by fura-2 and use of in vitro calibration parameters. PMID:8274651

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

Phospholipase Cϵ Scaffolds to Muscle-specific A Kinase Anchoring Protein (mAKAPβ) and Integrates Multiple Hypertrophic Stimuli in Cardiac Myocytes*

PubMed Central

Zhang, Lianghui; Malik, Sundeep; Kelley, Grant G.; Kapiloff, Michael S.; Smrcka, Alan V.

2011-01-01

To define a role for phospholipase Cϵ (PLCϵ) signaling in cardiac myocyte hypertrophic growth, PLCϵ protein was depleted from neonatal rat ventricular myocytes (NRVMs) using siRNA. NRVMs with PLCϵ depletion were stimulated with endothelin (ET-1), norepinephrine, insulin-like growth factor-1 (IGF-1), or isoproterenol and assessed for development of hypertrophy. PLCϵ depletion dramatically reduced hypertrophic growth and gene expression induced by all agonists tested. PLCϵ catalytic activity was required for hypertrophy development, yet PLCϵ depletion did not reduce global agonist-stimulated inositol phosphate production, suggesting a requirement for localized PLC activity. PLCϵ was found to be scaffolded to a muscle-specific A kinase anchoring protein (mAKAPβ) in heart and NRVMs, and mAKAPβ localizes to the nuclear envelope in NRVMs. PLCϵ-mAKAP interaction domains were defined and overexpressed to disrupt endogenous mAKAPβ-PLCϵ complexes in NRVMs, resulting in significantly reduced ET-1-dependent NRVM hypertrophy. We propose that PLCϵ integrates multiple upstream signaling pathways to generate local signals at the nucleus that regulate hypertrophy. PMID:21550986

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.

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

Distance constraints on activation of TRPV4 channels by AKAP150-bound PKCα in arterial myocytes

PubMed Central

Moreno, Claudia M.; O’Dwyer, Samantha; Woods, Sean

2017-01-01

TRPV4 (transient receptor potential vanilloid 4) channels are Ca2+-permeable channels that play a key role in regulating vascular tone. In arterial myocytes, opening of TRPV4 channels creates local increases in Ca2+ influx, detectable optically as “TRPV4 sparklets.” TRPV4 sparklet activity can be enhanced by the action of the vasoconstrictor angiotensin II (AngII). This modulation depends on the activation of subcellular signaling domains that comprise protein kinase C α (PKCα) bound to the anchoring protein AKAP150. Here, we used super-resolution nanoscopy, patch-clamp electrophysiology, Ca2+ imaging, and mathematical modeling approaches to test the hypothesis that AKAP150-dependent modulation of TRPV4 channels is critically dependent on the distance between these two proteins in the sarcolemma of arterial myocytes. Our data show that the distance between AKAP150 and TRPV4 channel clusters varies with sex and arterial bed. Consistent with our hypothesis, we further find that basal and AngII-induced TRPV4 channel activity decays exponentially as the distance between TRPV4 and AKAP150 increases. Our data suggest a maximum radius of action of ∼200 nm for local modulation of TRPV4 channels by AKAP150-associated PKCα. PMID:28507079

Effects of astragaloside IV on action potentials and ionic currents in guinea-pig ventricular myocytes.

PubMed

Zhao, Meimi; Zhao, Jinsheng; He, Guilin; Sun, Xuefei; Huang, Xueshi; Hao, Liying

2013-01-01

Astragaloside IV (AS-IV) is one of the main active constituents of Astragalus membranaceus, which has various actions on the cardiovascular system. However, its electrophysiological mechanisms are not clear. In the present study, we investigated the effects of AS-IV on action potentials and membrane currents using the whole-cell patch clamp technique in isolated guinea-pig ventricular myocytes. AS-IV prolonged the action potential duration (APD) at all three tested concentrations. The peak effect was achieved with 1×10(-6) M, at which concentration AS-IV significantly prolonged the APD at 95% repolarization from 313.1±38.9 to 785.3±83.7 ms. AS-IV at 1×10(-6) M also enhanced the inward rectifier K(+) currents (I(K1)) and inhibited the delayed rectifier K(+) currents (I(K)). AS-IV (1×10(-6) M) strongly depressed the peak of voltage-dependent Ca(2+) channel current (I(CaL)) from -607.3±37.5 to -321.1±38.3 pA. However, AS-IV was not found to affect the Na(+) currents. Taken together, AS-IV prolonged APD of guinea-pig ventricular myocytes, which might be explained by its inhibition of I(K). AS-IV also influences Ca(2+) signaling through suppressing ICaL.

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

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

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.

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

Substantial protection against MPTP-associated Parkinson's neurotoxicity in vitro and in vivo by anti-cancer agent SU4312 via activation of MEF2D and inhibition of MAO-B.

PubMed

Guo, Baojian; Hu, Shengquan; Zheng, Chengyou; Wang, Hongyu; Luo, Fangcheng; Li, Haitao; Cui, Wei; Yang, Xifei; Cui, Guozhen; Mak, Shinghung; Choi, Tony Chung-Lit; Ma, Edmond Dik-Lung; Wang, Yuqiang; Lee, Simon Ming Yuen; Zhang, Zaijun; Han, Yifan

2017-11-01

We have previously demonstrated the unexpected neuroprotection of the anti-cancer agent SU4312 in cellular models associated with Parkinson's disease (PD). However, the precise mechanisms underlying its neuroprotection are still unknown, and the effects of SU4312 on rodent models of PD have not been characterized. In the current study, we found that the protection of SU4312 against 1-methyl-4-phenylpyridinium ion (MPP + )-induced neurotoxicity in PC12 cells was achieved through the activation of transcription factor myocyte enhancer factor 2D (MEF2D), as evidenced by the fact that SU4312 stimulated myocyte enhancer factor 2 (MEF2) transcriptional activity and prevented the inhibition of MEF2D protein expression caused by MPP + , and that short hairpin RNA (ShRNA)-mediated knockdown of MEF2D significantly abolished the neuroprotection of SU4312. Additionally, Western blotting analysis revealed that SU4312 potentiated pro-survival PI3-K/Akt pathway to down-regulate MEF2D inhibitor glycogen synthase kinase-3beta (GSK3β). Furthermore, using the in vivo PD model of C57BL/6 mice insulted with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), we found that intragastrical administration of SU4312 (0.2 and 1 mg/kg) greatly ameliorated Parkinsonian motor defects, and restored protein levels of MEF2D, phosphorylated-Ser473-Akt and phosphorylated-Ser9-GSK3β. Meanwhile, SU4312 effectively reversed the decrease in protein expression of tyrosine hydroxylase in substantia nigra pars compacta dopaminergic neurons, inhibited oxidative stress, maintained mitochondrial biogenesis and partially prevented the depletion of dopamine and its metabolites. Very encouragingly, SU4312 was able to selectively inhibit monoamine oxidase-B (MAO-B) activity both in vitro and in vivo, with an IC 50 value of 0.2 μM. These findings suggest that SU4312 provides therapeutic benefits in cellular and animal models of PD, possibly through multiple mechanisms including enhancement of MEF2D through the activation of PI3-K/Akt pathway, maintenance of mitochondrial biogenesis and inhibition of MAO-B activity. SU4312 thus may be an effective drug candidate for the prevention or even modification of the pathological processes of PD. Copyright © 2017 Elsevier Ltd. All rights reserved.

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.

Overexpression of Striated Muscle Activator of Rho Signaling (STARS) Increases C2C12 Skeletal Muscle Cell Differentiation.

PubMed

Wallace, Marita A; Della Gatta, Paul A; Ahmad Mir, Bilal; Kowalski, Greg M; Kloehn, Joachim; McConville, Malcom J; Russell, Aaron P; Lamon, Séverine

2016-01-01

Skeletal muscle growth and regeneration depend on the activation of satellite cells, which leads to myocyte proliferation, differentiation and fusion with existing muscle fibers. Skeletal muscle cell proliferation and differentiation are tightly coordinated by a continuum of molecular signaling pathways. The striated muscle activator of Rho signaling (STARS) is an actin binding protein that regulates the transcription of genes involved in muscle cell growth, structure and function via the stimulation of actin polymerization and activation of serum-response factor (SRF) signaling. STARS mediates cell proliferation in smooth and cardiac muscle models; however, whether STARS overexpression enhances cell proliferation and differentiation has not been investigated in skeletal muscle cells. We demonstrate for the first time that STARS overexpression enhances differentiation but not proliferation in C2C12 mouse skeletal muscle cells. Increased differentiation was associated with an increase in the gene levels of the myogenic differentiation markers Ckm, Ckmt2 and Myh4, the differentiation factor Igf2 and the myogenic regulatory factors (MRFs) Myf5 and Myf6. Exposing C2C12 cells to CCG-1423, a pharmacological inhibitor of SRF preventing the nuclear translocation of its co-factor MRTF-A, had no effect on myotube differentiation rate, suggesting that STARS regulates differentiation via a MRTF-A independent mechanism. These findings position STARS as an important regulator of skeletal muscle growth and regeneration.

Cardioprotective benefits of adenosine triphosphate-sensitive potassium channel opener diazoxide are lost with administration after the onset of stress in mouse and human myocytes.

PubMed

Janjua, M Burhan; Makepeace, Carol M; Anastacio, Melissa M; Schuessler, Richard B; Nichols, Colin G; Lawton, Jennifer S

2014-10-01

Adenosine triphosphate-sensitive (KATP) potassium channel opener diazoxide (DZX) maintains myocyte volume and contractility during stress via an unknown mechanism when administered at the onset of stress. This study was performed to investigate the cardioprotective potential of DZX when added after the onset of the stresses of hyperkalemic cardioplegia, metabolic inhibition, and hypo-osmotic stress. Isolated mouse ventricular and human atrial myocytes were exposed to control Tyrode's solution (TYR) for 10 to 20 minutes, test solution for 30 minutes (hypothermic hyperkalemic cardioplegia [CPG], CPG + 100uM diazoxide [CPG+DZX], metabolic inhibition [MI], MI+DZX, mild hypo-osmotic stress [0.9T], or 0.9T + DZX), with DZX added after 10 or 20 minutes of stress, followed by 20 minutes of re-exposure to TYR (±DZX). Myocyte volume (human + mouse) and contractility (mouse) were compared. Mouse and human myocytes demonstrated significant swelling during exposure to CPG, MI, and hypo-osmotic stress that was not prevented by DZX when administered either at 10 or 20 minutes after the onset of stress. Contractility after the stress of CPG in mouse myocytes significantly declined when DZX was administered 20 minutes after the onset of stress (p < 0.05 vs TYR). Contractility after hypo-osmotic stress in mouse myocytes was not altered by the addition of DZX. To maintain myocyte volume homeostasis and contractility during stress (hyperkalemic cardioplegia, metabolic inhibition, and hypo-osmotic stress), KATP channel opener diazoxide requires administration at the onset of stress in this isolated myocyte model. These data have potential implications for any future clinical application of diazoxide. Copyright © 2014 American College of Surgeons. Published by Elsevier Inc. All rights reserved.

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

Vitamin D treatment attenuates cardiac FGF23/FGFR4 signaling and hypertrophy in uremic rats.

PubMed

Leifheit-Nestler, Maren; Grabner, Alexander; Hermann, Laura; Richter, Beatrice; Schmitz, Karin; Fischer, Dagmar-Christiane; Yanucil, Christopher; Faul, Christian; Haffner, Dieter

2017-09-01

Vitamin D deficiency and excess of circulating fibroblast growth factor 23 (FGF23) contribute to cardiovascular mortality in patients with chronic kidney disease (CKD). FGF23 activates FGF receptor 4 and (FGFR4) calcineurin/nuclear factor of activated T cells (NFAT) signaling in cardiac myocytes, thereby causing left ventricular hypertrophy (LVH). Here, we determined if 1,25-dihydroxyvitamin D (calcitriol) inhibits FGF23-induced cardiac signaling and LVH. 5/6 nephrectomized (5/6 Nx) rats were treated with different doses of calcitriol for 4 or 10 weeks and cardiac expression of FGF23/FGFR4 and activation of calcineurin/NFAT as well as LVH were analyzed. FGFR4 activation and hypertrophic cell growth were studied in cultured cardiac myocytes that were co-treated with FGF23 and calcitriol. In 5/6Nx rats with LVH, we detected elevated FGF23 expression in bone and myocardium, increased cardiac expression of FGFR4 and elevated cardiac activation of calcineurin/NFAT signaling. Cardiac expression levels of FGF23 and FGFR4 significantly correlated with the presence of LVH in uremic rats. Treatment with calcitriol reduced LVH as well as cardiac FGFR4 expression and calcineurin/NFAT activation. Bone and cardiac FGF23 expression were further stimulated by calcitriol in a dose-dependent manner, but levels of intact cardiac FGF23 protein were suppressed by high-dose calcitriol. In cultured cardiac myocytes, co-treatment with calcitriol blocked FGF23-induced activation of FGFR4 and hypertrophic cell growth. Our data suggest that in CKD, cardioprotective effects of calcitriol stem from its inhibitory actions on the cardiac FGF23/FGFR4 system, and based on their counterbalancing effects on cardiac myocytes, high FGF23 and low calcitriol synergistically contribute to cardiac hypertrophy. © The Author 2017. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved.

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

J chain and myocyte enhancer factor 2B are useful in differentiating classical Hodgkin lymphoma from nodular lymphocyte predominant Hodgkin lymphoma and primary mediastinal large B-cell lymphoma.

PubMed

Moore, Erika M; Swerdlow, Steven H; Gibson, Sarah E

2017-10-01

Although most classical Hodgkin lymphomas (CHLs) are easily distinguished from nodular lymphocyte predominant Hodgkin lymphoma (NLPHL) and primary mediastinal large B-cell lymphoma (PMBL), cases with significant CD20 expression cause diagnostic confusion. Although the absence of OCT-2 and BOB.1 are useful in these circumstances, a variable proportion of CHLs are positive for these antigens. We investigated the utility of J chain and myocyte enhancer factor 2B (MEF2B) in the diagnosis of CHL; NLPHL; PMBL; T-cell/histiocyte-rich large B-cell lymphoma (TCRLBL); and B-cell lymphoma, unclassifiable, with features intermediate between diffuse large B-cell lymphoma and CHL, compared with OCT-2 and BOB.1. J chain and MEF2B highlighted lymphocyte predominant (LP) cells in 20/20 (100%) NLPHLs and were negative in 43/43 (100%) CHLs. Fourteen of 15 (93%) PMBLs and 4/4 (100%) TCRLBLs were MEF2B positive, whereas 67% of PMBLs and 50% of TCRLBLs were J chain positive. Three of 3 B-cell lymphomas, unclassifiable, with features intermediate between diffuse large B-cell lymphoma and CHL, were negative for J chain and MEF2B. J chain and MEF2B were 100% sensitive and specific for NLPHL versus CHL. MEF2B was 100% sensitive and 98% specific for PMBL versus CHL. Whereas loss of OCT-2 and/or BOB.1 expression had a sensitivity of only 86% and specificity of 100% for CHL versus NLPHL, PMBL, and TCRLBL, lack of both J chain and MEF2B expression was 100% sensitive and 97% specific. J chain and MEF2B are highly sensitive and specific markers of NLPHL versus CHL; are particularly useful in highlighting LP cells; and, with rare exception, are of greater utility than OCT-2 and BOB.1 in differentiating CHL from NLPHL and other large B-cell lymphomas. Copyright © 2017 Elsevier Inc. All rights reserved.

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

The transcription factor MEF2C mediates cardiomyocyte hypertrophy induced by IGF-1 signaling

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

Munoz, Juan Pablo; Collao, Andres; Chiong, Mario

2009-10-09

Myocyte enhancer factor 2C (MEF2C) plays an important role in cardiovascular development and is a key transcription factor for cardiac hypertrophy. Here, we describe MEF2C regulation by insulin-like growth factor-1 (IGF-1) and its role in IGF-1-induced cardiac hypertrophy. We found that IGF-1 addition to cultured rat cardiomyocytes activated MEF2C, as evidenced by its increased nuclear localization and DNA binding activity. IGF-1 stimulated MEF2 dependent-gene transcription in a time-dependent manner, as indicated by increased MEF2 promoter-driven reporter gene activity; IGF-1 also induced p38-MAPK phosphorylation, while an inhibitor of p38-MAPK decreased both effects. Additionally, inhibitors of phosphatidylinositol 3-kinase and calcineurin prevented IGF-1-inducedmore » MEF2 transcriptional activity. Via MEF2C-dependent signaling, IGF-1 also stimulated transcription of atrial natriuretic factor and skeletal {alpha}-actin but not of fos-lux reporter genes. These novel data suggest that MEF2C activation by IGF-1 mediates the pro-hypertrophic effects of IGF-1 on cardiac gene expression.« less

Spine growth in the anterior cingulate cortex is necessary for the consolidation of contextual fear memory

PubMed Central

Vetere, Gisella; Restivo, Leonardo; Cole, Christina J.; Ross, P. Joel; Ammassari-Teule, Martine; Josselyn, Sheena A.; Frankland, Paul W.

2011-01-01

Remodeling of cortical connectivity is thought to allow initially hippocampus-dependent memories to be expressed independently of the hippocampus at remote time points. Consistent with this, consolidation of a contextual fear memory is associated with dendritic spine growth in neurons of the anterior cingulate cortex (aCC). To directly test whether such cortical structural remodeling is necessary for memory consolidation, we disrupted spine growth in the aCC at different times following contextual fear conditioning in mice. We took advantage of previous studies showing that the transcription factor myocyte enhancer factor 2 (MEF2) negatively regulates spinogenesis both in vitro and in vivo. We found that increasing MEF2-dependent transcription in the aCC during a critical posttraining window (but not at later time points) blocked both the consolidation-associated dendritic spine growth and subsequent memory expression. Together, these data strengthen the causal link between cortical structural remodeling and memory consolidation and, further, identify MEF2 as a key regulator of these processes. PMID:21531906

Cardiomyocytes undergo apoptosis in human immunodeficiency virus cardiomyopathy through mitochondrion- and death receptor-controlled pathways

PubMed Central

Twu, Cheryl; Liu, Nancy Q.; Popik, Waldemar; Bukrinsky, Michael; Sayre, James; Roberts, Jaclyn; Rania, Shammas; Bramhandam, Vishnu; Roos, Kenneth P.; MacLellan, W. Robb; Fiala, Milan

2002-01-01

We investigated 18 AIDS hearts (5 with and 13 without cardiomyopathy) by using immunocytochemistry and computerized image analysis regarding the roles of HIV-1 proteins and tumor necrosis factor ligands in HIV cardiomyopathy (HIVCM). HIVCM and cardiomyocyte apoptosis were significantly related to each other and to the expression by inflammatory cells of gp120 and tumor necrosis factor-α. In HIVCM heart, active caspase 9, a component of the mitochondrion-controlled apoptotic pathway, and the elements of the death receptor-mediated pathway, tumor necrosis factor-α and Fas ligand, were expressed strongly on macrophages and weakly on cardiomyocytes. HIVCM showed significantly greater macrophage infiltration and cardiomyocyte apoptosis rate compared with non-HIVCM. HIV-1 entered cultured neonatal rat ventricular myocytes by macropinocytosis but did not replicate. HIV-1- or gp120-induced apoptosis of rat myocytes through a mitochondrion-controlled pathway, which was inhibited by heparin, AOP-RANTES, or pertussis toxin, suggesting that cardiomyocyte apoptosis is induced by signaling through chemokine receptors. In conclusion, in patients with HIVCM, cardiomyocytes die through both mitochondrion- and death receptor-controlled apoptotic pathways. PMID:12379743

Cardiomyocytes undergo apoptosis in human immunodeficiency virus cardiomyopathy through mitochondrion- and death receptor-controlled pathways.

PubMed

Twu, Cheryl; Liu, Nancy Q; Popik, Waldemar; Bukrinsky, Michael; Sayre, James; Roberts, Jaclyn; Rania, Shammas; Bramhandam, Vishnu; Roos, Kenneth P; MacLellan, W Robb; Fiala, Milan

2002-10-29

We investigated 18 AIDS hearts (5 with and 13 without cardiomyopathy) by using immunocytochemistry and computerized image analysis regarding the roles of HIV-1 proteins and tumor necrosis factor ligands in HIV cardiomyopathy (HIVCM). HIVCM and cardiomyocyte apoptosis were significantly related to each other and to the expression by inflammatory cells of gp120 and tumor necrosis factor-alpha. In HIVCM heart, active caspase 9, a component of the mitochondrion-controlled apoptotic pathway, and the elements of the death receptor-mediated pathway, tumor necrosis factor-alpha and Fas ligand, were expressed strongly on macrophages and weakly on cardiomyocytes. HIVCM showed significantly greater macrophage infiltration and cardiomyocyte apoptosis rate compared with non-HIVCM. HIV-1 entered cultured neonatal rat ventricular myocytes by macropinocytosis but did not replicate. HIV-1- or gp120-induced apoptosis of rat myocytes through a mitochondrion-controlled pathway, which was inhibited by heparin, AOP-RANTES, or pertussis toxin, suggesting that cardiomyocyte apoptosis is induced by signaling through chemokine receptors. In conclusion, in patients with HIVCM, cardiomyocytes die through both mitochondrion- and death receptor-controlled apoptotic pathways.

Spire, an actin nucleation factor, regulates cell division during Drosophila heart development.

PubMed

Xu, Peng; Johnson, Tamara L; Stoller-Conrad, Jessica R; Schulz, Robert A

2012-01-01

The Drosophila dorsal vessel is a beneficial model system for studying the regulation of early heart development. Spire (Spir), an actin-nucleation factor, regulates actin dynamics in many developmental processes, such as cell shape determination, intracellular transport, and locomotion. Through protein expression pattern analysis, we demonstrate that the absence of spir function affects cell division in Myocyte enhancer factor 2-, Tinman (Tin)-, Even-skipped- and Seven up (Svp)-positive heart cells. In addition, genetic interaction analysis shows that spir functionally interacts with Dorsocross, tin, and pannier to properly specify the cardiac fate. Furthermore, through visualization of double heterozygous embryos, we determines that spir cooperates with CycA for heart cell specification and division. Finally, when comparing the spir mutant phenotype with that of a CycA mutant, the results suggest that most Svp-positive progenitors in spir mutant embryos cannot undergo full cell division at cell cycle 15, and that Tin-positive progenitors are arrested at cell cycle 16 as double-nucleated cells. We conclude that Spir plays a crucial role in controlling dorsal vessel formation and has a function in cell division during heart tube morphogenesis.

Targeting p35/Cdk5 signalling via CIP-peptide promotes angiogenesis in hypoxia.

PubMed

Bosutti, Alessandra; Qi, Jie; Pennucci, Roberta; Bolton, David; Matou, Sabine; Ali, Kamela; Tsai, Li-Huei; Krupinski, Jerzy; Petcu, Eugene B; Montaner, Joan; Al Baradie, Raid; Caccuri, Francesca; Caruso, Arnaldo; Alessandri, Giulio; Kumar, Shant; Rodriguez, Cristina; Martinez-Gonzalez, Jose; Slevin, Mark

2013-01-01

Cyclin-dependent kinase-5 (Cdk5) is over-expressed in both neurons and microvessels in hypoxic regions of stroke tissue and has a significant pathological role following hyper-phosphorylation leading to calpain-induced cell death. Here, we have identified a critical role of Cdk5 in cytoskeleton/focal dynamics, wherein its activator, p35, redistributes along actin microfilaments of spreading cells co-localising with p(Tyr15)Cdk5, talin/integrin beta-1 at the lamellipodia in polarising cells. Cdk5 inhibition (roscovitine) resulted in actin-cytoskeleton disorganisation, prevention of protein co-localization and inhibition of movement. Cells expressing Cdk5 (D144N) kinase mutant, were unable to spread, migrate and form tube-like structures or sprouts, while Cdk5 wild-type over-expression showed enhanced motility and angiogenesis in vitro, which was maintained during hypoxia. Gene microarray studies demonstrated myocyte enhancer factor (MEF2C) as a substrate for Cdk5-mediated angiogenesis in vitro. MEF2C showed nuclear co-immunoprecipitation with Cdk5 and almost complete inhibition of differentiation and sprout formation following siRNA knock-down. In hypoxia, insertion of Cdk5/p25-inhibitory peptide (CIP) vector preserved and enhanced in vitro angiogenesis. These results demonstrate the existence of critical and complementary signalling pathways through Cdk5 and p35, and through which coordination is a required factor for successful angiogenesis in sustained hypoxic condition.

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.

Calcium-calmodulin-dependent protein kinase mediates the intracellular signalling pathways of cardiac apoptosis in mice with impaired glucose tolerance.

PubMed

Federico, Marilen; Portiansky, Enrique L; Sommese, Leandro; Alvarado, Francisco J; Blanco, Paula G; Zanuzzi, Carolina N; Dedman, John; Kaetzel, Marcia; Wehrens, Xander H T; Mattiazzi, Alicia; Palomeque, Julieta

2017-06-15

Spontaneous sarcoplasmic reticulum (SR) Ca 2+ release events increased in fructose-rich diet mouse (FRD) myocytes vs. control diet (CD) mice, in the absence of significant changes in SR Ca 2+ load. In HEK293 cells, hyperglycaemia significantly enhanced [ 3 H]ryanodine binding and Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) phosphorylation of RyR2-S2814 residue vs. normoglycaemia. These increases were prevented by CaMKII inhibition. FRD significantly augmented cardiac apoptosis in WT vs. CD-WT mice, which was prevented by co-treatment with the reactive oxygen species scavenger Tempol. Oxidative stress was also increased in FRD-SR-autocamide inhibitory peptide (AIP) mice, expressing the SR-targeted CaMKII inhibitor AIP, without any significant enhancement of apoptosis vs. CD-SR-AIP mice. FRD produced mitochondrial swelling and membrane depolarization in FRD-WT mice but not in FRD-S2814A mice, in which the CaMKII site on ryanodine receptor 2 was ablated. FRD decreased mitochondrial area, mean Feret diameter and the mean distance between SR and the outer mitochondrial membrane vs. CD hearts. This remodelling was prevented in AC3I mice, with cardiac-targeted CaMKII inhibition. The impact of cardiac apoptosis in pre-diabetic stages of diabetic cardiomyopathy is unknown. We show that myocytes from fructose-rich diet (FRD) animals exhibit arrhythmias produced by exacerbated Ca 2+ /calmodulin-protein kinase (CaMKII) activity, ryanodine receptor 2 (RyR2) phosphorylation and sarcoplasmic reticulum (SR) Ca 2+ leak. We tested the hypothesis that this mechanism also underlies cardiac apoptosis in pre-diabetes. We generated a pre-diabetic model in FRD mice. FRD mice showed an increase in oxidative stress, hypertrophy and systolic dysfunction. FRD myocytes exhibited enhanced SR Ca 2+ spontaneous events in the absence of SR Ca 2+ load alterations vs. control-diet (CD) myocytes. In HEK293 cells, hyperglycaemia significantly enhanced [ 3 H]ryanodine binding and CaMKII phosphorylation of RyR2-S2814 residue vs. normoglycaemia. CaMKII inhibition prevented hyperglycaemia-induced alterations. FRD also evoked cardiac apoptosis in WT mice vs. CD-WT mice. Co-treatment with the reactive oxygen species scavenger Tempol prevented FRD-induced apoptosis in WT mice. In contrast, FRD enhanced oxidative stress but not apoptosis in FRD-SR-AIP mice, in which a CaMKII inhibitor is targeted to the SR. FRD produced mitochondrial membrane depolarization in WT mice but not in S2814A mice, in which the CaMKII phosphorylation site on RyR2 was ablated. Furthermore, FRD decreased mitochondrial area, mean Feret diameter and mean SR-mitochondrial distance vs. CD-WT hearts. This remodelling was prevented in AC3I mice, with cardiac-targeted CaMKII inhibition. CaMKII phosphorylation of RyR2, SR Ca 2+ leak and mitochondrial membrane depolarization are critically involved in the apoptotic pathway of the pre-diabetic heart. The FRD-induced decrease in SR-mitochondrial distance is likely to additionally favour Ca 2+ transit between the two organelles. © 2017 The Authors. The Journal of Physiology © 2017 The Physiological Society.

Matrix elasticity regulates the optimal cardiac myocyte shape for contractility

PubMed Central

McCain, Megan L.; Yuan, Hongyan; Pasqualini, Francesco S.; Campbell, Patrick H.

2014-01-01

Concentric hypertrophy is characterized by ventricular wall thickening, fibrosis, and decreased myocyte length-to-width aspect ratio. Ventricular thickening is considered compensatory because it reduces wall stress, but the functional consequences of cell shape remodeling in this pathological setting are unknown. We hypothesized that decreases in myocyte aspect ratio allow myocytes to maximize contractility when the extracellular matrix becomes stiffer due to conditions such as fibrosis. To test this, we engineered neonatal rat ventricular myocytes into rectangles mimicking the 2-D profiles of healthy and hypertrophied myocytes on hydrogels with moderate (13 kPa) and high (90 kPa) elastic moduli. Actin alignment was unaffected by matrix elasticity, but sarcomere content was typically higher on stiff gels. Microtubule polymerization was higher on stiff gels, implying increased intracellular elastic modulus. On moderate gels, myocytes with moderate aspect ratios (∼7:1) generated the most peak systolic work compared with other cell shapes. However, on stiffer gels, low aspect ratios (∼2:1) generated the most peak systolic work. To compare the relative contributions of intracellular vs. extracellular elasticity to contractility, we developed an analytical model and used our experimental data to fit unknown parameters. Our model predicted that matrix elasticity dominates over intracellular elasticity, suggesting that the extracellular matrix may potentially be a more effective therapeutic target than microtubules. Our data and model suggest that myocytes with lower aspect ratios have a functional advantage when the elasticity of the extracellular matrix decreases due to conditions such as fibrosis, highlighting the role of the extracellular matrix in cardiac disease. PMID:24682394

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

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.

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.

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

Delayed innocent bystander cell death following hypoxia in Caenorhabditis elegans

PubMed Central

Sun, C-L; Kim, E; Crowder, C M

2014-01-01

After hypoxia, cells may die immediately or have a protracted course, living or dying depending on an incompletely understood set of cell autonomous and nonautonomous factors. In stroke, for example, some neurons are thought to die from direct hypoxic injury by cell autonomous primary mechanisms, whereas other so called innocent bystander neurons die from factors released from the primarily injured cells. A major limitation in identifying these factors is the inability of current in vivo models to selectively target a set of cells for hypoxic injury so that the primarily injured cells and the innocent bystanders are clearly delineated. In order to develop such a model, we generated transgenic Caenorhabditis elegans strains where 2–3% of somatic cells were made selectively sensitive to hypoxia. This was accomplished by cell type-specific wild-type rescue in either pharyngeal myocytes or GABAergic neurons of a hypoxia resistance-producing translation factor mutation. Surprisingly, hypoxic targeting of these relatively small subsets of non-essential cells produced widespread innocent bystander cell injury, behavioral dysfunction and eventual organismal death. The hypoxic injury phenotypes of the myocyte or neuron sensitized strains were virtually identical. Using this model, we show that the C. elegans insulin receptor/FOXO transcription factor pathway improves survival when activated only after hypoxic injury and blocks innocent bystander death. PMID:24317200

Delayed innocent bystander cell death following hypoxia in Caenorhabditis elegans.

PubMed

Sun, C-L; Kim, E; Crowder, C M

2014-04-01

After hypoxia, cells may die immediately or have a protracted course, living or dying depending on an incompletely understood set of cell autonomous and nonautonomous factors. In stroke, for example, some neurons are thought to die from direct hypoxic injury by cell autonomous primary mechanisms, whereas other so called innocent bystander neurons die from factors released from the primarily injured cells. A major limitation in identifying these factors is the inability of current in vivo models to selectively target a set of cells for hypoxic injury so that the primarily injured cells and the innocent bystanders are clearly delineated. In order to develop such a model, we generated transgenic Caenorhabditis elegans strains where 2-3% of somatic cells were made selectively sensitive to hypoxia. This was accomplished by cell type-specific wild-type rescue in either pharyngeal myocytes or GABAergic neurons of a hypoxia resistance-producing translation factor mutation. Surprisingly, hypoxic targeting of these relatively small subsets of non-essential cells produced widespread innocent bystander cell injury, behavioral dysfunction and eventual organismal death. The hypoxic injury phenotypes of the myocyte or neuron sensitized strains were virtually identical. Using this model, we show that the C. elegans insulin receptor/FOXO transcription factor pathway improves survival when activated only after hypoxic injury and blocks innocent bystander death.

Intrinsic-mediated caspase activation is essential for cardiomyocyte hypertrophy

PubMed Central

Putinski, Charis; Abdul-Ghani, Mohammad; Stiles, Rebecca; Brunette, Steve; Dick, Sarah A.; Fernando, Pasan; Megeney, Lynn A.

2013-01-01

Cardiomyocyte hypertrophy is the cellular response that mediates pathologic enlargement of the heart. This maladaptation is also characterized by cell behaviors that are typically associated with apoptosis, including cytoskeletal reorganization and disassembly, altered nuclear morphology, and enhanced protein synthesis/translation. Here, we investigated the requirement of apoptotic caspase pathways in mediating cardiomyocyte hypertrophy. Cardiomyocytes treated with hypertrophy agonists displayed rapid and transient activation of the intrinsic-mediated cell death pathway, characterized by elevated levels of caspase 9, followed by caspase 3 protease activity. Disruption of the intrinsic cell death pathway at multiple junctures led to a significant inhibition of cardiomyocyte hypertrophy during agonist stimulation, with a corresponding reduction in the expression of known hypertrophic markers (atrial natriuretic peptide) and transcription factor activity [myocyte enhancer factor-2, nuclear factor kappa B (NF-κB)]. Similarly, in vivo attenuation of caspase activity via adenoviral expression of the biologic effector caspase inhibitor p35 blunted cardiomyocyte hypertrophy in response to agonist stimulation. Treatment of cardiomyocytes with procaspase 3 activating compound 1, a small-molecule activator of caspase 3, resulted in a robust induction of the hypertrophy response in the absence of any agonist stimulation. These results suggest that caspase-dependent signaling is necessary and sufficient to promote cardiomyocyte hypertrophy. These results also confirm that cell death signal pathways behave as active remodeling agents in cardiomyocytes, independent of inducing an apoptosis response. PMID:24101493

Contractile reserve and intracellular calcium regulation in mouse myocytes from normal and hypertrophied failing hearts

NASA Technical Reports Server (NTRS)

Ito, K.; Yan, X.; Tajima, M.; Su, Z.; Barry, W. H.; Lorell, B. H.; Schneider, M. (Principal Investigator)

2000-01-01

Mouse myocyte contractility and the changes induced by pressure overload are not fully understood. We studied contractile reserve in isolated left ventricular myocytes from mice with ascending aortic stenosis (AS) during compensatory hypertrophy (4-week AS) and the later stage of early failure (7-week AS) and from control mice. Myocyte contraction and [Ca(2+)](i) transients with fluo-3 were measured simultaneously. At baseline (0.5 Hz, 1.5 mmol/L [Ca(2+)](o), 25 degrees C), the amplitude of myocyte shortening and peak-systolic [Ca(2+)](i) in 7-week AS were not different from those of controls, whereas contraction, relaxation, and the decline of [Ca(2+)](i) transients were slower. In response to the challenge of high [Ca(2+)](o), fractional cell shortening was severely depressed with reduced peak-systolic [Ca(2+)](i) in 7-week AS compared with controls. In response to rapid pacing stimulation, cell shortening and peak-systolic [Ca(2+)](i) increased in controls, but this response was depressed in 7-week AS. In contrast, the responses to both challenge with high [Ca(2+)](o) and rapid pacing in 4-week AS were similar to those of controls. Although protein levels of Na(+)-Ca(2+) exchanger were increased in both 4-week and 7-week AS, the ratio of SR Ca(2+)-ATPase to phospholamban protein levels was depressed in 7-week AS compared with controls but not in 4-week AS. This was associated with an impaired capacity to increase sarcoplasmic reticulum Ca(2+) load during high work states in 7-week AS myocytes. In hypertrophied failing mouse myocytes, depressed contractile reserve is related to an impaired augmentation of systolic [Ca(2+)](i) and SR Ca(2+) load and simulates findings in human failing myocytes.

Contractile reserve and calcium regulation are depressed in myocytes from chronically unloaded hearts

NASA Technical Reports Server (NTRS)

Ito, Kenta; Nakayama, Masaharu; Hasan, Faisal; Yan, Xinhua; Schneider, Michael D.; Lorell, Beverly H.

2003-01-01

BACKGROUND: Chronic cardiac unloading of the normal heart results in the reduction of left ventricular (LV) mass, but effects on myocyte contractile function are not known. METHODS AND RESULTS: Cardiac unloading and reduction in LV mass were induced by heterotopic heart transplantation to the abdominal aorta in isogenic rats. Contractility and [Ca(2+)](i) regulation in LV myocytes were studied at both 2 and 5 weeks after transplantation. Native in situ hearts from recipient animals were used as the controls for all experiments. Contractile function indices in myocytes from 2-week unloaded and native (control) hearts were similar under baseline conditions (0.5 Hz, 1.2 mmol/L [Ca(2+)](o), and 36 degrees C) and in response to stimulation with high [Ca(2+)](o) (range 2.5 to 4.0 mmol/L). In myocytes from 5-week unloaded hearts, there were no differences in fractional cell shortening and peak-systolic [Ca(2+)](i) at baseline; however, time to 50% relengthening and time to 50% decline in [Ca(2+)](i) were prolonged compared with controls. Severe defects in fractional cell shortening and peak-systolic [Ca(2+)](i) were elicited in myocytes from 5-week unloaded hearts in response to high [Ca(2+)](o). However, there were no differences in the contractile response to isoproterenol between myocytes from unloaded and native hearts. In 5-week unloaded hearts, but not in 2-week unloaded hearts, LV protein levels of phospholamban were increased (345% of native heart values). Protein levels of sarcoplasmic reticulum Ca(2+) ATPase and the Na(+)/Ca(2+) exchanger were not changed. CONCLUSIONS: Chronic unloading of the normal heart caused a time-dependent depression of myocyte contractile function, suggesting the potential for impaired performance in states associated with prolonged cardiac atrophy.

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

Cloning and characterization of largemouth bass ( Micropterus salmoides) myostatin encoding gene and its promoter

NASA Astrophysics Data System (ADS)

Li, Shengjie; Bai, Junjie; Wang, Lin

2008-08-01

Myostatin or GDF-8, a member of the transforming growth factor-β (TGF-β) superfamily, has been demonstrated to be a negative regulator of skeletal muscle mass in mammals. In the present study, we obtained a 5.64 kb sequence of myostatin encoding gene and its promoter from largemouth bass ( Micropterus salmoides). The myostatin encoding gene consisted of three exons (488 bp, 371 bp and 1779 bp, respectively) and two introns (390 bp and 855 bp, respectively). The intron-exon boundaries were conservative in comparison with those of mammalian myostatin encoding genes, whereas the size of introns was smaller than that of mammals. Sequence analysis of 1.569 kb of the largemouth bass myostatin gene promoter region revealed that it contained two TATA boxes, one CAAT box and nine putative E-boxes. Putative muscle growth response elements for myocyte enhancer factor 2 (MEF2), serum response factor (SRF), activator protein 1 (AP1), etc., and muscle-specific Mt binding site (MTBF) were also detected. Some of the transcription factor binding sites were conserved among five teleost species. This information will be useful for studying the transcriptional regulation of myostatin in fish.

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.

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.

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.

Akt1/protein kinase B enhances transcriptional reprogramming of fibroblasts to functional cardiomyocytes

PubMed Central

Zhou, Huanyu; Dickson, Matthew E.; Kim, Min Soo; Bassel-Duby, Rhonda; Olson, Eric N.

2015-01-01

Conversion of fibroblasts to functional cardiomyocytes represents a potential approach for restoring cardiac function after myocardial injury, but the technique thus far has been slow and inefficient. To improve the efficiency of reprogramming fibroblasts to cardiac-like myocytes (iCMs) by cardiac transcription factors [Gata4, Hand2, Mef2c, and Tbx5 (GHMT)], we screened 192 protein kinases and discovered that Akt/protein kinase B dramatically accelerates and amplifies this process in three different types of fibroblasts (mouse embryo, adult cardiac, and tail tip). Approximately 50% of reprogrammed mouse embryo fibroblasts displayed spontaneous beating after 3 wk of induction by Akt plus GHMT. Furthermore, addition of Akt1 to GHMT evoked a more mature cardiac phenotype for iCMs, as seen by enhanced polynucleation, cellular hypertrophy, gene expression, and metabolic reprogramming. Insulin-like growth factor 1 (IGF1) and phosphoinositol 3-kinase (PI3K) acted upstream of Akt whereas the mitochondrial target of rapamycin complex 1 (mTORC1) and forkhead box o3 (Foxo3a) acted downstream of Akt to influence fibroblast-to-cardiomyocyte reprogramming. These findings provide insights into the molecular basis of cardiac reprogramming and represent an important step toward further application of this technique. PMID:26354121

Ketamine produces antidepressant-like effects through phosphorylation-dependent nuclear export of histone deacetylase 5 (HDAC5) in rats

PubMed Central

Choi, Miyeon; Lee, Seung Hoon; Wang, Sung Eun; Ko, Seung Yeon; Song, Mihee; Choi, June-Seek; Duman, Ronald S.; Son, Hyeon

2015-01-01

Ketamine produces rapid antidepressant-like effects in animal assays for depression, although the molecular mechanisms underlying these behavioral actions remain incomplete. Here, we demonstrate that ketamine rapidly stimulates histone deacetylase 5 (HDAC5) phosphorylation and nuclear export in rat hippocampal neurons through calcium/calmodulin kinase II- and protein kinase D-dependent pathways. Consequently, ketamine enhanced the transcriptional activity of myocyte enhancer factor 2 (MEF2), which leads to regulation of MEF2 target genes. Transfection of a HDAC5 phosphorylation-defective mutant (Ser259/Ser498 replaced by Ala259/Ala498, HDAC5-S/A), resulted in resistance to ketamine-induced nuclear export, suppression of ketamine-mediated MEF2 transcriptional activity, and decreased expression of MEF2 target genes. Behaviorally, viral-mediated hippocampal knockdown of HDAC5 blocked or occluded the antidepressant effects of ketamine both in unstressed and stressed animals. Taken together, our results reveal a novel role of HDAC5 in the actions of ketamine and suggest that HDAC5 could be a potential mechanism contributing to the therapeutic actions of ketamine. PMID:26647181

Genomic analyses identify recurrent MEF2D fusions in acute lymphoblastic leukaemia

PubMed Central

Gu, Zhaohui; Churchman, Michelle; Roberts, Kathryn; Li, Yongjin; Liu, Yu; Harvey, Richard C.; McCastlain, Kelly; Reshmi, Shalini C.; Payne-Turner, Debbie; Iacobucci, Ilaria; Shao, Ying; Chen, I-Ming; Valentine, Marcus; Pei, Deqing; Mungall, Karen L.; Mungall, Andrew J.; Ma, Yussanne; Moore, Richard; Marra, Marco; Stonerock, Eileen; Gastier-Foster, Julie M.; Devidas, Meenakshi; Dai, Yunfeng; Wood, Brent; Borowitz, Michael; Larsen, Eric E.; Maloney, Kelly; Mattano Jr, Leonard A.; Angiolillo, Anne; Salzer, Wanda L.; Burke, Michael J.; Gianni, Francesca; Spinelli, Orietta; Radich, Jerald P.; Minden, Mark D.; Moorman, Anthony V.; Patel, Bella; Fielding, Adele K.; Rowe, Jacob M.; Luger, Selina M.; Bhatia, Ravi; Aldoss, Ibrahim; Forman, Stephen J.; Kohlschmidt, Jessica; Mrózek, Krzysztof; Marcucci, Guido; Bloomfield, Clara D.; Stock, Wendy; Kornblau, Steven; Kantarjian, Hagop M.; Konopleva, Marina; Paietta, Elisabeth; Willman, Cheryl L.; L. Loh, Mignon; P. Hunger, Stephen; Mullighan, Charles G.

2016-01-01

Chromosomal rearrangements are initiating events in acute lymphoblastic leukaemia (ALL). Here using RNA sequencing of 560 ALL cases, we identify rearrangements between MEF2D (myocyte enhancer factor 2D) and five genes (BCL9, CSF1R, DAZAP1, HNRNPUL1 and SS18) in 22 B progenitor ALL (B-ALL) cases with a distinct gene expression profile, the most common of which is MEF2D-BCL9. Examination of an extended cohort of 1,164 B-ALL cases identified 30 cases with MEF2D rearrangements, which include an additional fusion partner, FOXJ2; thus, MEF2D-rearranged cases comprise 5.3% of cases lacking recurring alterations. MEF2D-rearranged ALL is characterized by a distinct immunophenotype, DNA copy number alterations at the rearrangement sites, older diagnosis age and poor outcome. The rearrangements result in enhanced MEF2D transcriptional activity, lymphoid transformation, activation of HDAC9 expression and sensitive to histone deacetylase inhibitor treatment. Thus, MEF2D-rearranged ALL represents a distinct form of high-risk leukaemia, for which new therapeutic approaches should be considered. PMID:27824051

Adiponectin downregulation is associated with volume overload-induced myocyte dysfunction in rats

PubMed Central

Wang, Li-li; Miller, Dori; Wanders, Desiree; Nanayakkara, Gayani; Amin, Rajesh; Judd, Robert; Morrison, Edward E; Zhong, Ju-ming

2016-01-01

Aim: Adiponectin has been reported to exert protective effects during pathological ventricular remodeling, but the role of adiponectin in volume overload-induced heart failure remains unclear. In this study we investigated the effect of adiponectin on cardiac myocyte contractile dysfunction following volume overload in rats. Methods: Volume overload was surgically induced in rats by infrarenal aorta-vena cava fistula. The rats were intravenously administered adenoviral adiponectin at 2-, 6- and 9-weeks following fistula. The protein expression of adiponectin, adiponectin receptors (AdipoR1/R2 and T-cadherin) and AMPK activity were measured using Western blot analyses. Isolated ventricular myocytes were prepared at 12 weeks post-fistula to examine the contractile performance of myocytes and intracellular Ca2+ transient. Results: A-V fistula resulted in significant reductions in serum and myocardial adiponectin levels, myocardial adiponectin receptor (AdipoR1/R2 and T-cadherin) levels, as well as myocardial AMPK activity. Consistent with these changes, the isolated myocytes exhibited significant depression in cell shortening and intracellular Ca2+ transient. Administration of adenoviral adiponectin significantly increased serum adiponectin levels and prevented myocyte contractile dysfunction in fistula rats. Furthermore, pretreatment of isolated myocytes with recombinant adiponectin (2.5 μg/mL) significantly improved their contractile performance in fistula rats, but had no effects in control or adenoviral adiponectin-administered rats. Conclusion: These results demonstrate a positive correlation between adiponectin downregulation and volume overload-induced ventricular remodeling. Adiponectin plays a protective role in volume overload-induced heart failure. PMID:26616727

Pokemon promotes the invasiveness of hepatocellular carcinoma by enhancing MEF2D transcription.

PubMed

Kong, Jing; Liu, Xiaoping; Li, Xiangqian; Wu, Jinsheng; Wu, Ning; Chen, Jun; Fang, Fang

2016-05-01

Pokemon, a master oncogene crucial for the tumorigenicity and progression of a variety of cancers, has been demonstrated to enhance the proliferation and survival of hepatocellular carcinoma (HCC). However, the contribution of Pokemon to the invasiveness of HCC has not yet been studied. In this study, we employed HCC cells to investigate the role of Pokemon in the invasion of HCC with multidisciplinary approaches. Pokemon overexpression was found to be closely associated with invasion and intrahepatic metastasis of HCC in clinical specimens. Suppression of Pokemon attenuated the invasion of HCC cells by in vitro transwell and wound-healing assays. Myocyte enhancer factor 2D (MEF2D), an oncogene that can promote the invasiveness of HCC, was found to be underexpressed during Pokemon silencing in HCC cells. Restoration of MEF2D abolished the effect of Pokemon downregulation on the migration of HCC cells. Further experiments verified that Pokemon binds two putative recognition sites located within the upstream region of the MEF2D promoter and enhances its transcription. The association between Pokemon and MEF2D was further confirmed in HCC specimens. Animal experiments further confirmed that Pokemon downregulation attenuated the metastasis of HCC cells in mice. Collectively, Pokemon was found to enhance the migration and invasion of HCC by increasing MEF2D expression. Thus, targeting Pokemon and MEF2D may be an effective strategy to suppress the metastasis of HCC.

Glutathionylation-Dependence of Na+-K+-Pump Currents Can Mimic Reduced Subsarcolemmal Na+ Diffusion

PubMed Central

Garcia, Alvaro; Liu, Chia-Chi; Cornelius, Flemming; Clarke, Ronald J.; Rasmussen, Helge H.

2016-01-01

The existence of a subsarcolemmal space with restricted diffusion for Na+ in cardiac myocytes has been inferred from a transient peak electrogenic Na+-K+ pump current beyond steady state on reexposure of myocytes to K+ after a period of exposure to K+-free extracellular solution. The transient peak current is attributed to enhanced electrogenic pumping of Na+ that accumulated in the diffusion-restricted space during pump inhibition in K+-free extracellular solution. However, there are no known physical barriers that account for such restricted Na+ diffusion, and we examined if changes of activity of the Na+-K+ pump itself cause the transient peak current. Reexposure to K+ reproduced a transient current beyond steady state in voltage-clamped ventricular myocytes as reported by others. Persistence of it when the Na+ concentration in patch pipette solutions perfusing the intracellular compartment was high and elimination of it with K+-free pipette solution could not be reconciled with restricted subsarcolemmal Na+ diffusion. The pattern of the transient current early after pump activation was dependent on transmembrane Na+- and K+ concentration gradients suggesting the currents were related to the conformational poise imposed on the pump. We examined if the currents might be accounted for by changes in glutathionylation of the β1 Na+-K+ pump subunit, a reversible oxidative modification that inhibits the pump. Susceptibility of the β1 subunit to glutathionylation depends on the conformational poise of the Na+-K+ pump, and glutathionylation with the pump stabilized in conformations equivalent to those expected to be imposed on voltage-clamped myocytes supported this hypothesis. So did elimination of the transient K+-induced peak Na+-K+ pump current when we included glutaredoxin 1 in patch pipette solutions to reverse glutathionylation. We conclude that transient K+-induced peak Na+-K+ pump current reflects the effect of conformation-dependent β1 pump subunit glutathionylation, not restricted subsarcolemmal diffusion of Na+. PMID:26958887

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

Glutathionylation-Dependence of Na(+)-K(+)-Pump Currents Can Mimic Reduced Subsarcolemmal Na(+) Diffusion.

PubMed

Garcia, Alvaro; Liu, Chia-Chi; Cornelius, Flemming; Clarke, Ronald J; Rasmussen, Helge H

2016-03-08

The existence of a subsarcolemmal space with restricted diffusion for Na(+) in cardiac myocytes has been inferred from a transient peak electrogenic Na(+)-K(+) pump current beyond steady state on reexposure of myocytes to K(+) after a period of exposure to K(+)-free extracellular solution. The transient peak current is attributed to enhanced electrogenic pumping of Na(+) that accumulated in the diffusion-restricted space during pump inhibition in K(+)-free extracellular solution. However, there are no known physical barriers that account for such restricted Na(+) diffusion, and we examined if changes of activity of the Na(+)-K(+) pump itself cause the transient peak current. Reexposure to K(+) reproduced a transient current beyond steady state in voltage-clamped ventricular myocytes as reported by others. Persistence of it when the Na(+) concentration in patch pipette solutions perfusing the intracellular compartment was high and elimination of it with K(+)-free pipette solution could not be reconciled with restricted subsarcolemmal Na(+) diffusion. The pattern of the transient current early after pump activation was dependent on transmembrane Na(+)- and K(+) concentration gradients suggesting the currents were related to the conformational poise imposed on the pump. We examined if the currents might be accounted for by changes in glutathionylation of the β1 Na(+)-K(+) pump subunit, a reversible oxidative modification that inhibits the pump. Susceptibility of the β1 subunit to glutathionylation depends on the conformational poise of the Na(+)-K(+) pump, and glutathionylation with the pump stabilized in conformations equivalent to those expected to be imposed on voltage-clamped myocytes supported this hypothesis. So did elimination of the transient K(+)-induced peak Na(+)-K(+) pump current when we included glutaredoxin 1 in patch pipette solutions to reverse glutathionylation. We conclude that transient K(+)-induced peak Na(+)-K(+) pump current reflects the effect of conformation-dependent β1 pump subunit glutathionylation, not restricted subsarcolemmal diffusion of Na(+). Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Electrophysiological effects of Chinese medicine Shen song Yang xin (SSYX) on Chinese miniature swine heart and isolated guinea pig ventricular myocytes.

PubMed

Feng, Li; Gong, Jing; Jin, Zhen-yi; Li, Ning; Sun, Li-ping; Wu, Yi-ling; Pu, Jie-lin

2009-07-05

Shen song Yang xin (SSYX) is a compound of Chinese medicine with the effect of increasing heart rate (HR). This study aimed to evaluate its electrophysiological properties at heart and cellular levels. The Chinese miniature swines were randomly assigned to two groups, administered with SSYX or placebo for 4 weeks (n = 8 per group). Cardiac electrophysiological study (EPS) was performed before and after drug administration. The guinea pig ventricular myocytes were enzymatically isolated and whole cell voltage-clamp technique was used to evaluate the effect of SSYX on cardiac action potential (AP). SSYX treatment accelerated the HR from (141.8 +/- 36.0) beats per minute to (163.0 +/- 38.0) beats per minute (P = 0.013) without changing the other parameters in surface electrocardiogram. After blockage of the autonomic nervous system with metoprolol and atropin, SSYX had no effect on intrinsic HR (IHR), but decreased corrected sinus node recovery time (CSNRT) and sinus atrium conducting time (SACT). Intra cardiac EPS showed that SSYX significantly decreased the A-H and A-V intervals as well as shortened the atrial (A), atrioventricular node (AVN) and ventricular (V) effective refractory period (ERP). In isolated guinea pig ventricular myocytes, the most obvious effect of SSYX on action potential was a shortening of the action potential duration (APD) without change in shape of action potential. The shortening rates of APD(30), APD(50) and APD(90) were 19.5%, 17.8% and 15.3%, respectively. The resting potential (Em) and the interval between the end of APD(30) and APD(90) did not significantly change. The present study demonstrates that SSYX increases the HR and enhances the conducting capacity of the heart in the condition of the intact autonomic nervous system. SSYX homogenously decreases the ERP of the heart and shortens the APD of the myocytes, suggesting its antiarrhythmic effect without proarrhythmia.

Purkinje cells from RyR2 mutant mice are highly arrhythmogenic but responsive to targeted therapy.

PubMed

Kang, Guoxin; Giovannone, Steven F; Liu, Nian; Liu, Fang-Yu; Zhang, Jie; Priori, Silvia G; Fishman, Glenn I

2010-08-20

The Purkinje fiber network has been proposed as the source of arrhythmogenic Ca(2+) release events in catecholaminergic polymorphic ventricular tachycardia (CPVT), yet evidence supporting this mechanism at the cellular level is lacking. We sought to determine the frequency and severity of spontaneous Ca(2+) release events and the response to the antiarrhythmic agent flecainide in Purkinje cells and ventricular myocytes from RyR2(R4496C/+) CPVT mutant mice and littermate controls. We crossed RyR2(R4496C/+) knock-in mice with the newly described Cntn2-EGFP BAC transgenic mice, which express a fluorescent reporter gene in cells of the cardiac conduction system, including the distal Purkinje fiber network. Isolated ventricular myocytes (EGFP(-)) and Purkinje cells (EGFP(+)) from wild-type hearts and mutant hearts were distinguished by epifluorescence and intracellular Ca(2+) dynamics recorded by microfluorimetry. Both wild-type and RyR2(R4496C/+) mutant Purkinje cells displayed significantly slower kinetics of activation and relaxation compared to ventricular myocytes of the same genotype, and tau(decay) in the mutant Purkinje cells was significantly slower than that observed in wild-type Purkinje cells. Of the 4 groups studied, RyR2(R4496C/+) mutant Purkinje cells were also most likely to develop spontaneous Ca(2+) release events, and the number of events per cell was also significantly greater. Furthermore, with isoproterenol treatment, although all 4 groups showed increases in the frequency of arrhythmogenic Ca(2+(i)) events, the RyR2(R4496C/+) Purkinje cells responded with the most profound abnormalities in intracellular Ca(2+) handling, including a significant increase in the frequency of unstimulated Ca(2+(i)) events and the development of alternans, as well as isolated and sustained runs of triggered beats. Both Purkinje cells and ventricular myocytes from wild-type mice showed suppression of spontaneous Ca(2+) release events with flecainide, whereas in RyR2(R4496C/+) mice, the Purkinje cells were preferentially responsive to drug. In contrast, the RyR2 blocker tetracaine was equally efficacious in mutant Purkinje cells and ventricular myocytes. Purkinje cells display a greater propensity to develop abnormalities in intracellular Ca(2+) handling than ventricular myocytes. This proarrhythmic behavior is enhanced by disease-causing mutations in the RyR2 Ca(2+) release channel and greatly exacerbated by catecholaminergic stimulation, with the development of arrhythmogenic triggered beats. These data support the concept that Purkinje cells are critical contributors to arrhythmic triggers in animal models and humans with CPVT and suggest a broader role for the Purkinje fiber network in the genesis of ventricular arrhythmias.

Can optical recordings of membrane potential be used to screen for drug-induced action potential prolongation in single cardiac myocytes?

PubMed

Hardy, M E L; Lawrence, C L; Standen, N B; Rodrigo, G C

2006-01-01

Potential-sensitive dyes have primarily been used to optically record action potentials (APs) in whole heart tissue. Using these dyes to record drug-induced changes in AP morphology of isolated cardiac myocytes could provide an opportunity to develop medium throughout assays for the pharmaceutical industry. Ideally, this requires that the dye has a consistent and rapid response to membrane potential, is insensitive to movement, and does not itself affect AP morphology. We recorded the AP from isolated adult guinea-pig ventricular myocytes optically using di-8-ANEPPS in a single-excitation dual-emission ratiometric system, either separately in electrically field stimulated myocytes, or simultaneously with an electrical AP recorded with a patch electrode in the whole-cell bridge mode. The ratio of di-8-ANEPPS fluorescence signal was calibrated against membrane potential using a switch-clamp to voltage clamp the myocyte. Our data show that the ratio of the optical signals emitted at 560/620 nm is linearly related to voltage over the voltage range of an AP, producing a change in ratio of 7.5% per 100 mV, is unaffected by cell movement and is identical to the AP recorded simultaneously with a patch electrode. However, the APD90 recorded optically in myocytes loaded with di-8-ANEPPS was significantly longer than in unloaded myocytes recorded with a patch electrode (355.6+/-13.5 vs. 296.2+/-16.2 ms; p<0.01). Despite this effect, the apparent IC50 for cisapride, which prolongs the AP by blocking IKr, was not significantly different whether determined optically or with a patch electrode (91+/-46 vs. 81+/-20 nM). These data show that the optical AP recorded ratiometrically using di-8-ANEPPS from a single ventricular myocyte accurately follows the action potential morphology. This technique can be used to estimate the AP prolonging effects of a compound, although di-8-ANEPPS itself prolongs APD90. Optical dyes require less technical skills and are less invasive than conventional electrophysiological techniques and, when coupled to ventricular myocytes, decreases animal usage and facilitates higher throughput assays.

Retinoic Acid Increases Fatty Acid Oxidation and Irisin Expression in Skeletal Muscle Cells and Impacts Irisin In Vivo.

PubMed

Amengual, Jaume; García-Carrizo, Francisco J; Arreguín, Andrea; Mušinović, Hana; Granados, Nuria; Palou, Andreu; Bonet, M Luisa; Ribot, Joan

2018-01-01

All-trans retinoic acid (ATRA) has protective effects against obesity and metabolic syndrome. We here aimed to gain further insight into the interaction of ATRA with skeletal muscle metabolism and secretory activity as important players in metabolic health. Cultured murine C2C12 myocytes were used to study direct effects of ATRA on cellular fatty acid oxidation (FAO) rate (using radioactively-labelled palmitate), glucose uptake (using radioactively-labelled 2-deoxy-D-glucose), triacylglycerol levels (by an enzymatic method), and the expression of genes related to FAO and glucose utilization (by RT-real time PCR). We also studied selected myokine production (using ELISA and immunohistochemistry) in ATRA-treated myocytes and intact mice. Exposure of C2C12 myocytes to ATRA led to increased fatty acid consumption and decreased cellular triacylglycerol levels without affecting glucose uptake, and induced the expression of the myokine irisin at the mRNA and secreted protein level in a dose-response manner. ATRA stimulatory effects on FAO-related genes and the Fndc5 gene (encoding irisin) were reproduced by agonists of peroxisome proliferator-activated receptor β/δ and retinoid X receptors, but not of retinoic acid receptors, and were partially blocked by an AMP-dependent protein kinase inhibitor. Circulating irisin levels were increased by 5-fold in ATRA-treated mice, linked to increased Fndc5 transcription in liver and adipose tissues, rather than skeletal muscle. Immunohistochemistry analysis of FNDC5 suggested that ATRA treatment enhances the release of FNDC5/irisin from skeletal muscle and the liver and its accumulation in interscapular brown and inguinal white adipose depots. These results provide new mechanistic insights on how ATRA globally stimulates FAO and enhances irisin secretion, thereby contributing to leaning effects and improved metabolic status. © 2018 The Author(s). Published by S. Karger AG, Basel.

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.

Three-dimensional histology: tools and application to quantitative assessment of cell-type distribution in rabbit heart

PubMed Central

Burton, Rebecca A.B.; Lee, Peter; Casero, Ramón; Garny, Alan; Siedlecka, Urszula; Schneider, Jürgen E.; Kohl, Peter; Grau, Vicente

2014-01-01

Aims Cardiac histo-anatomical organization is a major determinant of function. Changes in tissue structure are a relevant factor in normal and disease development, and form targets of therapeutic interventions. The purpose of this study was to test tools aimed to allow quantitative assessment of cell-type distribution from large histology and magnetic resonance imaging- (MRI) based datasets. Methods and results Rabbit heart fixation during cardioplegic arrest and MRI were followed by serial sectioning of the whole heart and light-microscopic imaging of trichrome-stained tissue. Segmentation techniques developed specifically for this project were applied to segment myocardial tissue in the MRI and histology datasets. In addition, histology slices were segmented into myocytes, connective tissue, and undefined. A bounding surface, containing the whole heart, was established for both MRI and histology. Volumes contained in the bounding surface (called ‘anatomical volume’), as well as that identified as containing any of the above tissue categories (called ‘morphological volume’), were calculated. The anatomical volume was 7.8 cm3 in MRI, and this reduced to 4.9 cm3 after histological processing, representing an ‘anatomical’ shrinkage by 37.2%. The morphological volume decreased by 48% between MRI and histology, highlighting the presence of additional tissue-level shrinkage (e.g. an increase in interstitial cleft space). The ratio of pixels classified as containing myocytes to pixels identified as non-myocytes was roughly 6:1 (61.6 vs. 9.8%; the remaining fraction of 28.6% was ‘undefined’). Conclusion Qualitative and quantitative differentiation between myocytes and connective tissue, using state-of-the-art high-resolution serial histology techniques, allows identification of cell-type distribution in whole-heart datasets. Comparison with MRI illustrates a pronounced reduction in anatomical and morphological volumes during histology processing. PMID:25362175

Hysteresis effect implicates calcium cycling as a mechanism of repolarization alternans.

PubMed

Walker, Mariah L; Wan, Xiaoping; Kirsch, Glenn E; Rosenbaum, David S

2003-11-25

T-wave alternans is due to alternation of membrane repolarization at the cellular level and is a risk factor for sudden cardiac death. Recently, a hysteresis effect has been reported in patients whereby T-wave alternans, once induced by rapid heart rate, persists even when heart rate is subsequently slowed. We hypothesized that alternans hysteresis is an intrinsic property of cardiac myocytes, directly related to an underlying mechanism for repolarization alternans that involves intracellular calcium cycling. Stepwise pacing was used to induce alternans in Langendorff-perfused guinea pig hearts from which optical action potentials were recorded simultaneously at 256 ventricular sites with voltage-sensitive dyes and in whole-cell patch-clamped cardiac myocytes treated with or without BAPTA-AM (1,2-bis[2-aminophenoxy]ethane-N,N,N',N'-tetraacetic acid tetrakis [acetoxymethyl ester]). Alternans hysteresis was observed in every isolated heart: threshold heart rate for alternans was 280+/-12 bpm, but during subsequent deceleration of pacing, alternans persisted to significantly slower heart rates (238+/-5 bpm, P<0.05). Optical mapping showed that this effect also applied to the threshold for spatially discordant alternans (313+/-2.2 bpm during acceleration versus 250+/-6.6 bpm during deceleration, P<0.05). Alternans hysteresis was also observed in isolated cardiac myocytes. Moreover, calcium chelation by BAPTA-AM raised the threshold for alternans and inhibited hysteresis in a dose-dependent manner with no effect on baseline action potential duration. Alternans hysteresis is an intrinsic property of cardiac myocytes that can lead to persistence of arrhythmogenic discordant alternans even after heart rate is slowed. These results also support an important underlying role of calcium cycling in the mechanism of alternans.

Vector-averaged gravity alters myocyte and neuron properties in cell culture

NASA Technical Reports Server (NTRS)

Gruener, Raphael; Hoeger, Glenn

1991-01-01

The effect of changes in the gravitational field of developing neurons and myocytes on the development of these cells was investigated using observations of rotated cultures of embryonic spinal neurons and myocytes in a horizontal clinostat, in which rotation produces, from the cells' perspective, a 'vector-free' gravity environment by continous averaging of the vector, thus simulating the microgravity of space. It was found that, at rotation rates between 1 and 50 rpm, cellular and nuclear areas of myocytes become significantly enlarged and the number of presumptive nucleoli increase; in neurons, frequent and large swellings appeared along neuritic shafts. Some of these changes were reversible after the cessation of rotation.

Modeling hypertrophic IP3 transients in the cardiac myocyte.

PubMed

Cooling, Michael; Hunter, Peter; Crampin, Edmund J

2007-11-15

Cardiac hypertrophy is a known risk factor for heart disease, and at the cellular level is caused by a complex interaction of signal transduction pathways. The IP3-calcineurin pathway plays an important role in stimulating the transcription factor NFAT which binds to DNA cooperatively with other hypertrophic transcription factors. Using available kinetic data, we construct a mathematical model of the IP3 signal production system after stimulation by a hypertrophic alpha-adrenergic agonist (endothelin-1) in the mouse atrial cardiac myocyte. We use a global sensitivity analysis to identify key controlling parameters with respect to the resultant IP3 transient, including the phosphorylation of cell-membrane receptors, the ligand strength and binding kinetics to precoupled (with G(alpha)GDP) receptor, and the kinetics associated with precoupling the receptors. We show that the kinetics associated with the receptor system contribute to the behavior of the system to a great extent, with precoupled receptors driving the response to extracellular ligand. Finally, by reparameterizing for a second hypertrophic alpha-adrenergic agonist, angiotensin-II, we show that differences in key receptor kinetic and membrane density parameters are sufficient to explain different observed IP3 transients in essentially the same pathway.

Effects of seasonal acclimatization on action potentials and sarcolemmal K+ currents in roach (Rutilus rutilus) cardiac myocytes.

PubMed

Badr, Ahmed; Hassinen, Minna; El-Sayed, Mohamed F; Vornanen, Matti

2017-03-01

Temperature sensitivity of electrical excitability is a potential limiting factor for high temperature tolerance of ectotherms. The present study examines whether heat resistance of electrical excitability of cardiac myocytes is modified by seasonal thermal acclimatization in roach (Rutilus rutilus), a eurythermal teleost species. To this end, temperature dependencies of ventricular action potentials (APs), and atrial and ventricular K + currents were measured from winter-acclimatized (WiR) and summer-acclimatized (SuR) roach. Under patch-clamp recording conditions, ventricular APs could be triggered over a wide range of temperatures (4-43°C) with prominent changes in resting membrane potential (RMP), AP duration and amplitude. In general, APs of SuR were slightly more tolerant to high temperatures than those of WiR, e.g. the break point temperature (T BP ) of RMP was 37.6±0.4°C in WiR and 41±1°C in SuR (p<0.05). Of the two major cardiac K + currents, the inward rectifier K + current (I K1 ) was particularly heat resistant in both SuR (T BP 39.4±0.4°C) and WiR (T BP 40.0±0.4°C) ventricular myocytes. The delayed rectifier K + current (I Kr ) was not as heat resistant as I K1 . Surprisingly, I Kr of WiR tolerated heat better (T BP 31.9±0.8°C) than I Kr of SuR (T BP 24.1±0.5°C) (p<0.05). I Kr (Erg2) channel transcripts of both atrial and ventricular myocytes were up-regulated in WiR. I K1 (Kir2) channel transcripts were not affected by seasonal acclimatization, although ventricular I K1 current was up-regulated in summer. Collectively, these findings show that thermal tolerance limits of K + currents in isolated myocytes between seasonally acclimatized roach are much less pronounced than the heat sensitivity of ECG variables in intact fish. Copyright © 2016 Elsevier Inc. All rights reserved.

How Does the Ca2+-paradox Injury Induce Contracture in the Heart?—A Combined Study of the Intracellular Ca2+ Dynamics and Cell Structures in Perfused Rat Hearts—

PubMed Central

Mani, Hiroki; Tanaka, Hideo; Adachi, Tetsuya; Ikegawa, Masaya; Dai, Ping; Fujita, Naohisa; Takamatsu, Tetsuro

2015-01-01

The calcium (Ca2+)-paradox injury of the heart, induced by restoration of extracellular Ca2+ after its short-term depletion, is known to provoke cardiomyocyte contracture. However, undetermined is how the Ca2+-paradox provokes such a distinctive presentation of myocytes in the heart. To address this, we imaged sequential intracellular Ca2+ dynamics and concomitant structures of the subepicardial ventricular myocytes in fluo3-loaded, Langendorff-perfused rat hearts produced by the Ca2+ paradox. Under rapid-scanning confocal microscopy, repletion of Ca2+ following its depletion produced high-frequency Ca2+ waves in individual myocytes with asynchronous localized contractions, resulting in contracture within 10 min. Such alterations of myocytes were attenuated by 5-mM NiCl2, but not by verapamil, SEA0400, or combination of ryanodine and thapsigargin, indicating a contribution of non-specific transmembrane Ca2+ influx in the injury. However, saponin-induced membrane permeabilization of Ca2+ showed no apparent contracture despite the emergence of high-frequency Ca2+ waves, indicating an essential role of myocyte-myocyte and myocyte-extracellular matrix (ECM) mechanical connections in the Ca2+ paradox. In immunohistochemistry Ca2+ depletion produced separation of the intercalated disc that expresses cadherin and dissipation of β-dystroglycan located along the sarcolemma. Taken together, along with the trans-sarcolemmal Ca2+ influx, disruption of cell-cell and cell-ECM connections is essential for contracture in the Ca2+-paradox injury. PMID:25861132

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.

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

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.

Anabolic steroids and cardiovascular risk.

PubMed

Angell, Peter; Chester, Neil; Green, Danny; Somauroo, John; Whyte, Greg; George, Keith

2012-02-01

Recent reports from needle exchange programmes and other public health initiatives have suggested growing use of anabolic steroids (AS) in the UK and other countries. Data indicate that AS use is not confined to body-builders or high-level sportsmen. Use has spread to professionals working in emergency services, casual fitness enthusiasts and subelite sportsmen and women. Although the precise health consequences of AS use is largely undefined, AS use represents a growing public health concern. Data regarding the consequences of AS use on cardiovascular health are limited to case studies and a modest number of small cohort studies. Numerous case studies have linked AS use with a variety of cardiovascular disease (CVD) events or endpoints, including myocardial infarction, stroke and death. Large-scale epidemiological studies to support these links are absent. Consequently, the impact of AS use upon known CVD risk factors has been studied in relatively small, case-series studies. Data relating AS use to elevated blood pressure, altered lipid profiles and ECG abnormalities have been reported, but are often limited in scope, and other studies have often produced equivocal outcomes. The use of AS has been linked to the appearance of concentric left ventricular hypertrophy as well as endothelial dysfunction but the data again remains controversial. The mechanisms responsible for the negative effect of AS on cardiovascular health are poorly understood, especially in humans. Possibilities include direct effects on myocytes and endothelial cells, reduced intracellular Ca2+ levels, increased release of apoptogenic factors, as well as increased collagen crosslinks between myocytes. New data relating AS use to cardiovascular health risks are emerging, as novel technologies are developed (especially in non-invasive imaging) that can assess physiological structure and function. Continued efforts to fully document the cardiovascular health consequences of AS use is important to provide a clear, accurate, public health message to the many groups now using AS for performance and image enhancement.

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

First-in-Human Phase 1 Trial of Agarose Beads Containing Murine RENCA Cells in Advanced Solid Tumors.

PubMed

Smith, Barry H; Parikh, Tapan; Andrada, Zoe P; Fahey, Thomas J; Berman, Nathaniel; Wiles, Madeline; Nazarian, Angelica; Thomas, Joanne; Arreglado, Anna; Akahoho, Eugene; Wolf, David J; Levine, Daniel M; Parker, Thomas S; Gazda, Lawrence S; Ocean, Allyson J

2016-01-01

Agarose macrobeads containing mouse renal adenocarcinoma cells (RMBs) release factors, suppressing the growth of cancer cells and prolonging survival in spontaneous or induced tumor animals, mediated, in part, by increased levels of myocyte-enhancing factor (MEF2D) via EGFR-and AKT-signaling pathways. The primary objective of this study was to determine the safety of RMBs in advanced, treatment-resistant metastatic cancers, and then its efficacy (survival), which is the secondary objective. Thirty-one patients underwent up to four intraperitoneal implantations of RMBs (8 or 16 macrobeads/kg) via laparoscopy in this single-arm trial (FDA BB-IND 10091; NCT 00283075). Serial physical examinations, laboratory testing, and PET-CT imaging were performed before and three months after each implant. RMBs were well tolerated at both dose levels (mean 660.9 per implant). AEs were (Grade 1/2) with no treatment-related SAEs. The data support the safety of RMB therapy in advanced-malignancy patients, and the preliminary evidence for their potential efficacy is encouraging. A Phase 2 efficacy trial is ongoing.

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

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.

Larger late sodium current density as well as greater sensitivities to ATX II and ranolazine in rabbit left atrial than left ventricular myocytes.

PubMed

Luo, Antao; Ma, Jihua; Song, Yejia; Qian, Chunping; Wu, Ying; Zhang, Peihua; Wang, Leilei; Fu, Chen; Cao, Zhenzhen; Shryock, John C

2014-02-01

An increase of cardiac late sodium current (INa.L) is arrhythmogenic in atrial and ventricular tissues, but the densities of INa.L and thus the potential relative contributions of this current to sodium ion (Na(+)) influx and arrhythmogenesis in atria and ventricles are unclear. In this study, whole-cell and cell-attached patch-clamp techniques were used to measure INa.L in rabbit left atrial and ventricular myocytes under identical conditions. The density of INa.L was 67% greater in left atrial (0.50 ± 0.09 pA/pF, n = 20) than in left ventricular cells (0.30 ± 0.07 pA/pF, n = 27, P < 0.01) when elicited by step pulses from -120 to -20 mV at a rate of 0.2 Hz. Similar results were obtained using step pulses from -90 to -20 mV. Anemone toxin II (ATX II) increased INa.L with an EC50 value of 14 ± 2 nM and a Hill slope of 1.4 ± 0.1 (n = 9) in atrial myocytes and with an EC50 of 21 ± 5 nM and a Hill slope of 1.2 ± 0.1 (n = 12) in ventricular myocytes. Na(+) channel open probability (but not mean open time) was greater in atrial than in ventricular cells in the absence and presence of ATX II. The INa.L inhibitor ranolazine (3, 6, and 9 μM) reduced INa.L more in atrial than ventricular myocytes in the presence of 40 nM ATX II. In summary, rabbit left atrial myocytes have a greater density of INa.L and higher sensitivities to ATX II and ranolazine than rabbit left ventricular myocytes.

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

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

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

Heat stress responses modulate calcium regulations and electrophysiological characteristics in atrial myocytes.

PubMed

Chen, Yao-Chang; Kao, Yu-Hsun; Huang, Chun-Feng; Cheng, Chen-Chuan; Chen, Yi-Jen; Chen, Shih-Ann

2010-04-01

Heat stress-induced responses change the ionic currents and calcium homeostasis. However, the molecular insights into the heat stress responses on calcium homeostasis remain unclear. The purposes of this study were to examine the mechanisms of heat stress responses on calcium handling and electrophysiological characteristics in atrial myocytes. We used indo-1 fluorimetric ratio technique and whole-cell patch clamp to investigate the intracellular calcium, action potentials, and ionic currents in isolated rabbit single atrial cardiomyocytes with or without (control) exposure to heat stress (43 degrees C, 15 min) 5+/-1 h before experiments. The expressions of sarcoplasmic reticulum ATPase (SERCA2a), and Na(+)-Ca(2+) exchanger (NCX) in the control and heat stress-treated atrial myocytes were evaluated by Western blot and real-time PCR. As compared with control myocytes, the heat stress-treated myocytes had larger sarcoplasmic reticulum calcium content and larger intracellular calcium transient with a shorter decay portion. Heat stress-treated myocytes also had larger L-type calcium currents, transient outward potassium currents, but smaller NCX currents. Heat stress responses increased the protein expressions, SERCA2a, NCX, and heat shock protein. However, heat stress responses did not change the RNA expression of SERCA2a and NCX. In conclusion, heat stress responses change calcium handling through protein but not RNA regulation. Copyright (c) 2009 Elsevier Ltd. All rights reserved.

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.

Myocardial deletion of transcription factor CHF1/Hey2 results in altered myocyte action potential and mild conduction system expansion but does not alter conduction system function or promote spontaneous arrhythmias.

PubMed

Hartman, Matthew E; Liu, Yonggang; Zhu, Wei-Zhong; Chien, Wei-Ming; Weldy, Chad S; Fishman, Glenn I; Laflamme, Michael A; Chin, Michael T

2014-07-01

CHF1/Hey2 is a Notch-responsive basic helix-loop-helix transcription factor involved in cardiac development. Common variants in Hey2 are associated with Brugada syndrome. We hypothesized that absence of CHF1/Hey2 would result in abnormal cellular electrical activity, altered cardiac conduction system (CCS) development, and increased arrhythmogenesis. We isolated neonatal CHF/Hey2-knockout (KO) cardiac myocytes and measured action potentials and ion channel subunit gene expression. We also crossed myocardial-specific CHF1/Hey2-KO mice with cardiac conduction system LacZ reporter mice and stained for conduction system tissue. We also performed ambulatory ECG monitoring for arrhythmias and heart rate variability. Neonatal cardiomyocytes from CHF1/Hey2-KO mice demonstrate a 50% reduction in action potential dV/dT, a 50-75% reduction in SCN5A, KCNJ2, and CACNA1C ion channel subunit gene expression, and an increase in delayed afterdepolarizations from 0/min to 12/min. CHF1/Hey2 cKO CCS-lacZ mice have a ∼3-fold increase in amount of CCS tissue. Ambulatory ECG monitoring showed no difference in cardiac conduction, arrhythmias, or heart rate variability. Wild-type cells or animals were used in all experiments. CHF1/Hey2 may contribute to Brugada syndrome by influencing the expression of SCN5A and formation of the cardiac conduction system, but its absence does not cause baseline conduction defects or arrhythmias in the adult mouse.-Hartman, M. E., Liu, Y., Zhu, W.-Z., Chien, W.-M., Weldy, C. S., Fishman, G. I., Laflamme, M. A., Chin, M. T. Myocardial deletion of transcription factor CHF1/Hey2 results in altered myocyte action potential and mild conduction system expansion but does not alter conduction system function or promote spontaneous arrhythmias. © FASEB.

Vitamin D and the immunomodulation of rotator cuff injury

PubMed Central

Dougherty, Kaitlin A; Dilisio, Matthew F; Agrawal, Devendra K

2016-01-01

Tendon-to-bone healing after rotator cuff repair surgery has a failure rate of 20%–94%. There has been a recent interest to determine the factors that act as determinants between successful and unsuccessful rotator cuff repair. Vitamin D level in patients is one of the factors that have been linked to bone and muscle proliferation and healing, and it may have an effect on tendon-to-bone healing. The purpose of this article is to critically review relevant published research that relates to the effect of vitamin D on rotator cuff tears and subsequent healing. A review of the literature was conducted to identify all studies that investigate the relationship between vitamin D and tendon healing, in addition to its mechanism of action. The data were then analyzed in order to summarize what is currently known about vitamin D, rotator cuff pathology, and tendon-to-bone healing. The activated metabolite of vitamin D, 1α,25-dihydroxyvitamin D3, affects osteoblast proliferation and differentiation. Likewise, vitamin D plays a significant role in the tendon-to-bone healing process by increasing the bone mineral density and strengthening the skeletal muscles. The 1α,25-dihydroxyvitamin D3 binds to vitamin D receptors on myocytes to stimulate growth and proliferation. The form of vitamin D produced by the liver, calcifediol, is a key initiator of the myocyte healing process by moving phosphate into myocytes, which improves function and metabolism. Investigation into the effect of vitamin D on tendons has been sparse, but limited studies have been promising. Matrix metalloproteinases play an active role in remodeling the extracellular matrix (ECM) of tendons, particularly deleterious remodeling of the collagen fibers. Also, the levels of transforming growth factor-β3 positively influence the success of the surgery for rotator cuff repair. In the tendon-to-bone healing process, vitamin D has been shown to successfully influence bone and muscle healing, but more research is needed to delve into the mechanisms of vitamin D as a factor in skeletal tendon health and healing. PMID:27366101

Invited review: mesenchymal progenitor cells in intramuscular connective tissue development.

PubMed

Miao, Z G; Zhang, L P; Fu, X; Yang, Q Y; Zhu, M J; Dodson, M V; Du, M

2016-01-01

The abundance and cross-linking of intramuscular connective tissue contributes to the background toughness of meat, and is thus undesirable. Connective tissue is mainly synthesized by intramuscular fibroblasts. Myocytes, adipocytes and fibroblasts are derived from a common pool of progenitor cells during the early embryonic development. It appears that multipotent mesenchymal stem cells first diverge into either myogenic or non-myogenic lineages; non-myogenic mesenchymal progenitors then develop into the stromal-vascular fraction of skeletal muscle wherein adipocytes, fibroblasts and derived mesenchymal progenitors reside. Because non-myogenic mesenchymal progenitors mainly undergo adipogenic or fibrogenic differentiation during muscle development, strengthening progenitor proliferation enhances the potential for both intramuscular adipogenesis and fibrogenesis, leading to the elevation of both marbling and connective tissue content in the resulting meat product. Furthermore, given the bipotent developmental potential of progenitor cells, enhancing their conversion to adipogenesis reduces fibrogenesis, which likely results in the overall improvement of marbling (more intramuscular adipocytes) and tenderness (less connective tissue) of meat. Fibrogenesis is mainly regulated by the transforming growth factor (TGF) β signaling pathway and its regulatory cascade. In addition, extracellular matrix, a part of the intramuscular connective tissue, provides a niche environment for regulating myogenic differentiation of satellite cells and muscle growth. Despite rapid progress, many questions remain in the role of extracellular matrix on muscle development, and factors determining the early differentiation of myogenic, adipogenic and fibrogenic cells, which warrant further studies.

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.

Pertussis toxin treatment attenuates some effects of insulin in BC3H-1 murine myocytes

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

Luttrell, L.M.; Hewlett, E.L.; Romero, G.

1988-05-05

The effects of pertussis toxin (PT) treatment on insulin-stimulated myristoyl-diacylglycerol (DAG) generation, hexose transport, and thymidine incorporation were studied in differentiated BC3H-1 mycocytes. Insulin treatment caused a biphasic increase in myristoyl-DAG production which was abolished in myocytes treated with PT. There was no effect of PT treatment on basal (nonstimulated) myristoyl-DAG production. Insulin-stimulated hydrolysis of a membrane phosphatidylinositol glycan was blocked by PT treatment. ADP-ribosylation of BC3H-1 plasma membranes with (/sup 32/P)NAD revealed a 40-kDa protein as the major PT substrate in vivo and in vitro. The time course and dose dependence of the effects of PT on diacylglycerol generationmore » correlated with the in vivo ADP-ribosylation of the 40-kDa substrate. Pertussis toxin treatment resulted in a 71% attenuation of insulin-stimulated hexose uptake without effect on either basal or phorbol ester-stimulated uptake. The stimulatory effects of insulin and fetal calf serum on (/sup 3/H)thymidine incorporation into quiescent myocytes were attenuated by 61 and 59%, respectively, when PT was added coincidently with the growth factors. Nonstimulated and EGF-stimulated (/sup 3/H)thymidine incorporation was unaffected by PT treatment. These data suggest that a PT-sensitive G protein is involved in the cellular signaling mechanisms of insulin.« less

Myosin light chain 2-based selection of human iPSC-derived early ventricular cardiac myocytes

PubMed Central

Bizy, Alexandra; Guerrero-Serna, Guadalupe; Hu, Bin; Ponce-Balbuena, Daniela; Willis, B. Cicero; Zarzoso, Manuel; Ramirez, Rafael J.; Sener, Michelle F.; Mundada, Lakshmi V.; Klos, Matthew; Devaney, Eric J.; Vikstrom, Karen L.; Herron, Todd J.; Jalife, José

2014-01-01

Applications of human induced pluripotent stemcell derived-cardiac myocytes (hiPSC-CMs) would be strengthened by the ability to generate specific cardiac myocyte (CM) lineages. However, purification of lineage-specific hiPSC-CMs is limited by the lack of cell marking techniques. Here, we have developed an iPSC-CM marking system using recombinant adenoviral reporter constructs with atrial- or ventricular-specific myosin light chain-2 (MLC-2) promoters. MLC-2a and MLC-2v selected hiPSC-CMs were purified by fluorescence-activated cell sorting and their biochemical and electrophysiological phenotypes analyzed. We demonstrate that the phenotype of both populations remained stable in culture and they expressed the expected sarcomeric proteins, gap junction proteins and chamber-specific transcription factors. Compared to MLC-2a cells, MLC-2v selected CMs had larger action potential amplitudes and durations. In addition, by immunofluorescence, we showed that MLC-2 isoform expression can be used to enrich hiPSC-CM consistent with early atrial and ventricularmyocyte lineages. However, only the ventricular myosin light chain-2 promoter was able to purify a highly homogeneous population of iPSC-CMs. Using this approach, it is now possible to develop ventricular-specific disease models using iPSC-CMs while atrial-specific iPSC-CM cultures may require additional chamber-specific markers. PMID:24095945

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

Long Noncoding RNA MHRT Protects Cardiomyocytes against H2O2-Induced Apoptosis

PubMed Central

Zhang, Jianying; Gao, Caihua; Meng, Meijuan; Tang, Hongxia

2016-01-01

Acute myocardial infarction (AMI) remains a leading cause of morbidity and mortality worldwide. The exploration of new biomarkers with high sensitivity and specificity for early diagnosis of AMI therefore becomes one of the primary task. In the current study, we aim to detect whether there is any heart specific long noncoding RNA (lncRNA) releasing into the circulation during AMI, and explore its function in the neonatal rat cardiac myocytes injury induced by H2O2. Our results revealed that the cardiac-specific lncRNA MHRT (Myosin Heavy Chain Associated RNA Transcripts) was significantly elevated in the blood from AMI patients compared with the healthy control (*p<0.05). Using an in vitro neonatal rat cardiac myocytes injury model, we demonstrated that lncRNA MHRT was upregulated in the cardiac myocytes after treatment with hydrogen peroxide (H2O2) via real-time RT-PCR (qRT-PCR). Furthermore, we knockdowned the MHRT gene by siRNA to confirm its roles in the H2O2-induced cardiac cell apoptosis, and found that knockdown of MHRT led to significant more apoptotic cells than the non-target control (**p<0.01), indicating that the lncRNA MHRT is a protective factor for cardiomyocyte and the plasma concentration of MHRT may serve as a biomarker for myocardial infarction diagnosis in humans AMI. PMID:26759697

5'-AMP-activated protein kinase increases glucose uptake independent of GLUT4 translocation in cardiac myocytes.

PubMed

Lee, Christopher T; Ussher, John R; Mohammad, Askar; Lam, Anna; Lopaschuk, Gary D

2014-04-01

Glucose uptake and glycolysis are increased in the heart during ischemia, and this metabolic alteration constitutes an important contributing factor towards ischemic injury. Therefore, it is important to understand glucose uptake regulation in the ischemic heart. There are primarily 2 glucose transporters controlling glucose uptake into cardiac myocytes: GLUT1 and GLUT4. In the non-ischemic heart, insulin stimulates GLUT4 translocation to the sarcolemmal membrane, while both GLUT1 and GLUT4 translocation can occur following AMPK stimulation. Using a newly developed technique involving [(3)H]2-deoxyglucose, we measured glucose uptake in H9c2 ventricular myoblasts, and demonstrated that while insulin has no detectable effect on glucose uptake, phenformin-induced AMPK activation increases glucose uptake 2.5-fold. Furthermore, insulin treatment produced no discernible effect on either Akt serine 473 phosphorylation or AMPKα threonine 172 phosphorylation, while treatment with phenformin results in an increase in AMPKα threonine 172 phosphorylation, and a decrease in Akt serine 473 phosphorylation. Visualization of a dsRed-GLUT4 fusion construct in H9c2 cells by laser confocal microscopy showed that unlike insulin, AMPK activation did not redistribute GLUT4 to the sarcolemmal membrane, suggesting that AMPK may regulate glucose uptake via another glucose transporter. These studies suggest that AMPK is a major regulator of glucose uptake in cardiac myocytes.

Cardioprotective stress response in the human fetal heart.

PubMed

Coles, John G; Boscarino, Cathy; Takahashi, Mark; Grant, Diane; Chang, Astra; Ritter, Julia; Dai, Xiaojing; Du, Changqing; Musso, Gabriel; Yamabi, Hideaki; Goncalves, Jason; Kumar, Ashu Sunny; Woodgett, James; Lu, Huanzhang; Hannigan, Gregory

2005-05-01

We propose that the fetal heart is highly resilient to hypoxic stress. Our objective was to elucidate the human fetal gene expression profile in response to simulated ischemia and reperfusion to identify molecular targets that account for the innate cardioprotection exhibited by the fetal phenotype. Primary cultures of human fetal cardiac myocytes (gestational age, 15-20 weeks) were exposed to simulated ischemia and reperfusion in vitro by using a simulated ischemic buffer under anoxic conditions. Total RNA from treated and baseline cells were isolated, reverse transcribed, and labeled with Cy3 or Cy5 and hybridized to a human cDNA microarray for expression analysis. This analysis revealed a highly significant (false discovery rate, <3%) suppression of interleukin 6 transcript levels during the reperfusion phase confirmed by means of quantitative polymerase chain reaction (0.25 +/- 0.11-fold). Interleukin 6 signaling during ischemia and reperfusion was assessed at the protein expression level by means of Western measurements of interleukin 6 receptor, the signaling subunit of the interleukin 6 receptor complex (gp130), and signal transducer of activated transcription 3. Posttranslational changes in the protein kinase B signaling pathway were determined on the basis of the phosphorylation status of protein kinase B, mitogen-activated protein kinase, and glycogen synthase kinase 3beta. The effect of suppression of a prohypertrophic kinase, integrin-linked kinase, with short-interfering RNA was determined in an ischemia and reperfusion-stressed neonatal rat cardiac myocyte model. Endogenous secretion of interleukin 6 protein in culture supernatants was measured by enzyme-linked immunosorbent assay. Human fetal cardiac myocytes exhibited a significantly lower rate of apoptosis induction during ischemia and reperfusion and after exposure to staurosporine and recombinant interleukin 6 compared with that observed in neonatal rat cardiac myocytes ( P < .05 for all comparisons, analysis of variance). Exposure to exogenously added recombinant interleukin 6 increased the apoptotic rate in both rat and human fetal cardiac myocytes ( P < .05). Short-interfering RNA-mediated suppression of integrin-linked kinase, a prohypertrophy upstream kinase regulating protein kinase B and glycogen synthase kinase 3beta phosphorylation, was cytoprotective against ischemia and reperfusion-induced apoptosis in neonatal rat cardiac myocytes ( P < .05). Human fetal cardiac myocytes exhibit a uniquely adaptive transcriptional response to ischemia and reperfusion that is associated with an apoptosis-resistant phenotype. The stress-inducible fetal cardiac myocyte gene repertoire is a useful platform for identification of targets relevant to the mitigation of cardiac ischemic injury and highlights a novel avenue involving interleukin 6 modulation for preventing the cardiac myocyte injury associated with ischemia and reperfusion.

Erythropoietin and carbamylated erythropoietin promote histone deacetylase 5 phosphorylation and nuclear export in rat hippocampal neurons

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

Jo, Hye-Ryeong; Kim, Yong-Seok; Department of Biochemistry and Molecular Biology, College of Medicine, Hanyang University, 17 Haengdang-dong, Sungdong-gu, Seoul 133-791

2016-01-29

Erythropoietin (EPO) produces neurotrophic effects in animal model of neurodegeneration. However, clinical use of EPO is limited due to thrombotic risk. Carbamylated EPO (cEPO), devoid of thrombotic risk, has been proposed as a novel neuroprotective and neurotrophic agent although the molecular mechanisms of cEPO remain incomplete. Here, we show a previously unidentified role of histone deacetylase 5 (HDAC5) in the actions of EPO and cEPO. EPO and cEPO regulate the HDAC5 phosphorylation at two critical sites, Ser259 and Ser498 through a protein kinase D (PKD) dependent pathway. In addition, EPO and cEPO rapidly stimulates nuclear export of HDAC5 in ratmore » hippocampal neurons which expressing HDAC5-GFP. Consequently, EPO and cEPO enhanced the myocyte enhancer factor-2 (MEF2) target gene expression. Taken together, our results reveal that EPO and cEPO mediate MEF2 target gene expression via the regulation of HDAC5 phosphorylation at Ser259/498, and suggest that HDAC5 could be a potential mechanism contributing to the therapeutic actions of EPO and cEPO.« less

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.

Integrative Analysis of PRKAG2 Cardiomyopathy iPS and Microtissue Models Identifies AMPK as a Regulator of Metabolism, Survival, and Fibrosis.

PubMed

Hinson, J Travis; Chopra, Anant; Lowe, Andre; Sheng, Calvin C; Gupta, Rajat M; Kuppusamy, Rajarajan; O'Sullivan, John; Rowe, Glenn; Wakimoto, Hiroko; Gorham, Joshua; Burke, Michael A; Zhang, Kehan; Musunuru, Kiran; Gerszten, Robert E; Wu, Sean M; Chen, Christopher S; Seidman, Jonathan G; Seidman, Christine E

2016-12-20

AMP-activated protein kinase (AMPK) is a metabolic enzyme that can be activated by nutrient stress or genetic mutations. Missense mutations in the regulatory subunit, PRKAG2, activate AMPK and cause left ventricular hypertrophy, glycogen accumulation, and ventricular pre-excitation. Using human iPS cell models combined with three-dimensional cardiac microtissues, we show that activating PRKAG2 mutations increase microtissue twitch force by enhancing myocyte survival. Integrating RNA sequencing with metabolomics, PRKAG2 mutations that activate AMPK remodeled global metabolism by regulating RNA transcripts to favor glycogen storage and oxidative metabolism instead of glycolysis. As in patients with PRKAG2 cardiomyopathy, iPS cell and mouse models are protected from cardiac fibrosis, and we define a crosstalk between AMPK and post-transcriptional regulation of TGFβ isoform signaling that has implications in fibrotic forms of cardiomyopathy. Our results establish critical connections among metabolic sensing, myocyte survival, and TGFβ signaling. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

Three-Dimensional Geometric Modeling of Membrane-bound Organelles in Ventricular Myocytes: Bridging the Gap between Microscopic Imaging and Mathematical Simulation

PubMed Central

Yu, Zeyun; Holst, Michael J.; Hayashi, Takeharu; Bajaj, Chandrajit L.; Ellisman, Mark H.; McCammon, J. Andrew; Hoshijima, Masahiko

2009-01-01

A general framework of image-based geometric processing is presented to bridge the gap between three-dimensional (3D) imaging that provides structural details of a biological system and mathematical simulation where high-quality surface or volumetric meshes are required. A 3D density map is processed in the order of image pre-processing (contrast enhancement and anisotropic filtering), feature extraction (boundary segmentation and skeletonization), and high-quality and realistic surface (triangular) and volumetric (tetrahedral) mesh generation. While the tool-chain described is applicable to general types of 3D imaging data, the performance is demonstrated specifically on membrane-bound organelles in ventricular myocytes that are imaged and reconstructed with electron microscopic (EM) tomography and two-photon microscopy (T-PM). Of particular interest in this study are two types of membrane-bound Ca2+-handling organelles, namely, transverse tubules (T-tubules) and junctional sarcoplasmic reticulum (jSR), both of which play an important role in regulating the excitation-contraction (E-C) coupling through dynamic Ca2+ mobilization in cardiomyocytes. PMID:18835449

Three-dimensional geometric modeling of membrane-bound organelles in ventricular myocytes: bridging the gap between microscopic imaging and mathematical simulation.

PubMed

Yu, Zeyun; Holst, Michael J; Hayashi, Takeharu; Bajaj, Chandrajit L; Ellisman, Mark H; McCammon, J Andrew; Hoshijima, Masahiko

2008-12-01

A general framework of image-based geometric processing is presented to bridge the gap between three-dimensional (3D) imaging that provides structural details of a biological system and mathematical simulation where high-quality surface or volumetric meshes are required. A 3D density map is processed in the order of image pre-processing (contrast enhancement and anisotropic filtering), feature extraction (boundary segmentation and skeletonization), and high-quality and realistic surface (triangular) and volumetric (tetrahedral) mesh generation. While the tool-chain described is applicable to general types of 3D imaging data, the performance is demonstrated specifically on membrane-bound organelles in ventricular myocytes that are imaged and reconstructed with electron microscopic (EM) tomography and two-photon microscopy (T-PM). Of particular interest in this study are two types of membrane-bound Ca(2+)-handling organelles, namely, transverse tubules (T-tubules) and junctional sarcoplasmic reticulum (jSR), both of which play an important role in regulating the excitation-contraction (E-C) coupling through dynamic Ca(2+) mobilization in cardiomyocytes.

Evidence for a direct effect of captopril on early steps of insulin action in BC3H-1 myocytes.

PubMed

Moisés, Regina S; Carvalho, Carla R O; Shiota, Debora; Saad, Mario J A

2003-03-01

Captopril, an angiotensin-converting enzyme (ACE) inhibitor, has been reported to improve insulin sensitivity. However, despite extensive investigation, the mechanisms responsible for this effect are not fully understood. Reduction of plasma angiotensin II and inhibition of kininase II have been suggested to contribute to improve insulin sensitivity. Insulin binding was measured at tracer insulin concentration in intact cells with or without captopril treatment. Specific binding, expressed as percent of total insulin added, was not different in control and captopril-treated cells. However, captopril treatment caused an increase in insulin-induced insulin receptor substrate-1 (IRS-1) phosphorylation accompanied by an increased association of IRS-1 with phosphoinositide-3 kinase (PI-3 kinase), despite no change on insulin receptor (IR) autophosphorylation. There was also an increased threonine kinase B (AKT) phosphorylation in captopril-treated cells followed by enhanced basal and insulin-stimulated glucose uptake. These results indicate that captopril treatment has a direct effect on early phosphorylation events induced by insulin in BC3H-1 myocytes. Copyright 2003, Elsevier Science (USA). All rights reserved.

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.

Length dependence of force generation exhibit similarities between rat cardiac myocytes and skeletal muscle fibres.

PubMed

Hanft, Laurin M; McDonald, Kerry S

2010-08-01

According to the Frank-Starling relationship, increased ventricular volume increases cardiac output, which helps match cardiac output to peripheral circulatory demand. The cellular basis for this relationship is in large part the myofilament length-tension relationship. Length-tension relationships in maximally calcium activated preparations are relatively shallow and similar between cardiac myocytes and skeletal muscle fibres. During twitch activations length-tension relationships become steeper in both cardiac and skeletal muscle; however, it remains unclear whether length dependence of tension differs between striated muscle cell types during submaximal activations. The purpose of this study was to compare sarcomere length-tension relationships and the sarcomere length dependence of force development between rat skinned left ventricular cardiac myocytes and fast-twitch and slow-twitch skeletal muscle fibres. Muscle cell preparations were calcium activated to yield 50% maximal force, after which isometric force and rate constants (k(tr)) of force development were measured over a range of sarcomere lengths. Myofilament length-tension relationships were considerably steeper in fast-twitch fibres compared to slow-twitch fibres. Interestingly, cardiac myocyte preparations exhibited two populations of length-tension relationships, one steeper than fast-twitch fibres and the other similar to slow-twitch fibres. Moreover, myocytes with shallow length-tension relationships were converted to steeper length-tension relationships by protein kinase A (PKA)-induced myofilament phosphorylation. Sarcomere length-k(tr) relationships were distinct between all three cell types and exhibited patterns markedly different from Ca(2+) activation-dependent k(tr) relationships. Overall, these findings indicate cardiac myocytes exhibit varied length-tension relationships and sarcomere length appears a dominant modulator of force development rates. Importantly, cardiac myocyte length-tension relationships appear able to switch between slow-twitch-like and fast-twitch-like by PKA-mediated myofibrillar phosphorylation, which implicates a novel means for controlling Frank-Starling relationships.

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

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.

Regulation of inward rectifier potassium current ionic channel remodeling by AT1 -Calcineurin-NFAT signaling pathway in stretch-induced hypertrophic atrial myocytes.

PubMed

He, Jionghong; Xu, Yanan; Yang, Long; Xia, Guiling; Deng, Na; Yang, Yongyao; Tian, Ye; Fu, Zenan; Huang, Yongqi

2018-05-02

Previous studies have shown that the activation of angiotensin II receptor type I (AT 1 ) is attributed to cardiac remodeling stimulated by increased heart load, and that it is followed by the activation of the calcineurin-nuclear factor of activated T-cells (NFAT) signaling pathway. Additionally, AT 1 has been found to be a regulator of cardiocyte ionic channel remodeling, and calcineurin-NFAT signals participate in the regulation of cardiocyte ionic channel expression. A hypothesis therefore follows that stretch stimulation may regulate cardiocyte ionic channel remodeling by activating the AT 1 -calcineurin-NFAT pathway. Here, we investigated the role of the AT 1 -calcineurin-NFAT pathway in the remodeling of inward rectifier potassium (I k1 ) channel, in addition to its role in changing action potential, in stretch-induced hypertrophic atrial myocytes of neonatal rats. Our results showed that increased stretch significantly led to atrial myocytes hypertrophy; it also increased the activity of calcineurin enzymatic activity, which was subsequently attenuated by telmisartan or cyclosporine-A. The level of NFAT 3 protein in nuclear extracts, the mRNA and protein expression of Kir2.1 in whole cell extracts, and the density of I k1 were noticeably increased in stretched samples. Stretch stimulation significantly shortened the action potential duration (APD) of repolarization at the 50% and 90% level. Telmisartan, cyclosporine-A, and 11R-VIVIT attenuated stretch-induced alterations in the levels of NFAT 3 , mRNA and protein expression of Kir2.1, the density of I k1 , and the APD. Our findings suggest that the AT 1 -calcineurin-NFAT signaling pathway played an important role in regulating I k1 channel remodeling and APD change in stretch-induced hypertrophic atrial myocytes of neonatal rats. This article is protected by copyright. All rights reserved.

Electrical coupling of single cardiac rat myocytes to field-effect and bipolar transistors.

PubMed

Kind, Thomas; Issing, Matthias; Arnold, Rüdiger; Müller, Bernt

2002-12-01

A novel bipolar transistor for extracellular recording the electrical activity of biological cells is presented, and the electrical behavior compared with the field-effect transistor (FET). Electrical coupling is examined between single cells separated from the heart of adults rats (cardiac myocytes) and both types of transistors. To initiate a local extracellular voltage, the cells are periodically stimulated by a patch pipette in voltage clamp and current clamp mode. The local extracellular voltage is measured by the planar integrated electronic sensors: the bipolar and the FET. The small signal transistor currents correspond to the local extracellular voltage. The two types of sensor transistors used here were developed and manufactured in the laboratory of our institute. The manufacturing process and the interfaces between myocytes and transistors are described. The recordings are interpreted by way of simulation based on the point-contact model and the single cardiac myocyte model.

Induced Pluripotent Stem Cells 10 Years Later: For Cardiac Applications.

PubMed

Yoshida, Yoshinori; Yamanaka, Shinya

2017-06-09

Induced pluripotent stem cells (iPSCs) are reprogrammed cells that have features similar to embryonic stem cells, such as the capacity of self-renewal and differentiation into many types of cells, including cardiac myocytes. Although initially the reprogramming efficiency was low, several improvements in reprogramming methods have achieved robust and efficient generation of iPSCs without genomic insertion of transgenes. iPSCs display clonal variations in epigenetic and genomic profiles and cellular behavior in differentiation. iPSC-derived cardiac myocytes (iPSC cardiac myocytes) recapitulate phenotypic differences caused by genetic variations, making them attractive human disease models, and are useful for drug discovery and toxicology testing. In addition, iPSC cardiac myocytes can help with patient stratification in regard to drug responsiveness. Furthermore, they can be used as source cells for cardiac regeneration in animal models. Here, we review recent progress in iPSC technology and its applications to cardiac diseases. © 2017 American Heart Association, Inc.

Mature adipocyte-derived dedifferentiated fat cells can transdifferentiate into skeletal myocytes in vitro

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

Kazama, Tomohiko; Fujie, Masaki; Endo, Tuyoshi

2008-12-19

We have previously reported the establishment of preadipocyte cell lines, termed dedifferentiated fat (DFAT) cells, from mature adipocytes of various animals. DFAT cells possess long-term viability and can redifferentiate into adipocytes both in vivo and in vitro. Furthermore, DFAT cells can transdifferentiate into osteoblasts and chondrocytes under appropriate culture conditions. However, it is unclear whether DFAT cells are capable of transdifferentiating into skeletal myocytes, which is common in the mesodermal lineage. Here, we show that DFAT cells can be induced to transdifferentiate into skeletal myocytes in vitro. Myogenic induction of DFAT cells resulted in the expression of MyoD and myogenin,more » followed by cell fusion and formation of multinucleated cells expressing sarcomeric myosin heavy chain. These results indicate that DFAT cells derived from mature adipocytes can transdifferentiate into skeletal myocytes in vitro.« less

[Low extracellular pH increases the persistent sodium current in guinea pig ventricular myocytes].

PubMed

Ma, Ji-Hua; Luo, An-Tao; Wang, Wei-Ping; Zhang, Pei-Hua

2007-04-25

Whole-cell and cell-attached patch-clamp techniques were used to record the changes of persistent sodium current (I(Na.P)) in ventricular myocytes of guinea pig to investigate the effect of low extracellular pH on I(Na.P) and its mechanism. The results showed that low extracellular pH (7.0, 6.8 and 6.5) obviously increased the amplitude of whole-cell I(Na.P) in a [H(+)] concentration-dependent manner. Under the condition of extracellular pH 6.5, I(Na.P) was markedly augmented from control (pH 7.4) value of (0.347+/-0.067) pA/pF to (0.817+/- 0.137) pA/pF (P<0.01, n=6), whereas the reducing agent dithiothreitiol (DTT, 1 mmol/L) reversed the increased IN(Na.P) from (0.817+/-0.137) pA/pF to (0.233+/-0.078) pA/pF (P<0.01 vs pH 6.5, n=6). Decreasing extracellular pH to 6.5 also increased the persistent sodium channel activity in cell-attached patches. The mean open probability and mean open time were increased from control value of 0.021+/-0.007 and (0.899+/-0.074) ms to 0.205+/-0.023 and (1.593+/-0.158) ms, respectively (both P<0.01, n=6), and such enhancement was reversed by application of 1 mmol/L DTT [to 0.019+/-0.005 and (0.868+/-0.190) ms, both P<0.01 vs pH 6.5, n=6]. Furthermore, protein kinase C (PKC) inhibitor bisindolylmaleimide (BIM, 5 micromol/L) reduced the enhanced mean open probability and mean open time at pH 6.5 from 0.214+/-0.024 and (1.634+/-0.137) ms to 0.025+/-0.006 and (0.914+/-0.070) ms, respectively (both P<0.01 vs pH 6.5, n=6). The results demonstrate that low extracellular pH markedly increases I(Na.P) in guinea pig ventricular myocytes, in which activation of PKC may be involved.

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

Synergistic Anti-arrhythmic Effects in Human Atria with Combined Use of Sodium Blockers and Acacetin

PubMed Central

Ni, Haibo; Whittaker, Dominic G.; Wang, Wei; Giles, Wayne R.; Narayan, Sanjiv M.; Zhang, Henggui

2017-01-01

Atrial fibrillation (AF) is the most common cardiac arrhythmia. Developing effective and safe anti-AF drugs remains an unmet challenge. Simultaneous block of both atrial-specific ultra-rapid delayed rectifier potassium (K+) current (IKur) and the Na+ current (INa) has been hypothesized to be anti-AF, without inducing significant QT prolongation and ventricular side effects. However, the antiarrhythmic advantage of simultaneously blocking these two channels vs. individual block in the setting of AF-induced electrical remodeling remains to be documented. Furthermore, many IKur blockers such as acacetin and AVE0118, partially inhibit other K+ currents in the atria. Whether this multi-K+-block produces greater anti-AF effects compared with selective IKur-block has not been fully understood. The aim of this study was to use computer models to (i) assess the impact of multi-K+-block as exhibited by many IKur blokers, and (ii) evaluate the antiarrhythmic effect of blocking IKur and INa, either alone or in combination, on atrial and ventricular electrical excitation and recovery in the setting of AF-induced electrical-remodeling. Contemporary mathematical models of human atrial and ventricular cells were modified to incorporate dose-dependent actions of acacetin (a multichannel blocker primarily inhibiting IKur while less potently blocking Ito, IKr, and IKs). Rate- and atrial-selective inhibition of INa was also incorporated into the models. These single myocyte models were then incorporated into multicellular two-dimensional (2D) and three-dimensional (3D) anatomical models of the human atria. As expected, application of IKur blocker produced pronounced action potential duration (APD) prolongation in atrial myocytes. Furthermore, combined multiple K+-channel block that mimicked the effects of acacetin exhibited synergistic APD prolongations. Synergistically anti-AF effects following inhibition of INa and combined IKur/K+-channels were also observed. The attainable maximal AF-selectivity of INa inhibition was greatly augmented by blocking IKur or multiple K+-currents in the atrial myocytes. This enhanced anti-arrhythmic effects of combined block of Na+- and K+-channels were also seen in 2D and 3D simulations; specially, there was an enhanced efficacy in terminating re-entrant excitation waves, exerting improved antiarrhythmic effects in the human atria as compared to a single-channel block. However, in the human ventricular myocytes and tissue, cellular repolarization and computed QT intervals were modestly affected in the presence of actions of acacetin and INa blockers (either alone or in combination). In conclusion, this study demonstrates synergistic antiarrhythmic benefits of combined block of IKur and INa, as well as those of INa and combined multi K+-current block of acacetin, without significant alterations of ventricular repolarization and QT intervals. This approach may be a valuable strategy for the treatment of AF. PMID:29218016

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

MEF2 responds to multiple calcium-regulated signals in the control of skeletal muscle fiber type

PubMed Central

Wu, Hai; Naya, Francisco J.; McKinsey, Timothy A.; Mercer, Brian; Shelton, John M.; Chin, Eva R.; Simard, Alain R.; Michel, Robin N.; Bassel-Duby, Rhonda; Olson, Eric N.; Williams, R. Sanders

2000-01-01

Different patterns of motor nerve activity drive distinctive programs of gene transcription in skeletal muscles, thereby establishing a high degree of metabolic and physiological specialization among myofiber subtypes. Recently, we proposed that the influence of motor nerve activity on skeletal muscle fiber type is transduced to the relevant genes by calcineurin, which controls the functional activity of NFAT (nuclear family of activated T cell) proteins. Here we demonstrate that calcineurin-dependent gene regulation in skeletal myocytes is mediated also by MEF2 transcription factors, and is integrated with additional calcium-regulated signaling inputs, specifically calmodulin-dependent protein kinase activity. In skeletal muscles of transgenic mice, both NFAT and MEF2 binding sites are necessary for properly regulated function of a slow fiber-specific enhancer, and either forced expression of activated calcineurin or motor nerve stimulation up-regulates a MEF2-dependent reporter gene. These results provide new insights into the molecular mechanisms by which specialized characteristics of skeletal myofiber subtypes are established and maintained. PMID:10790363

Maturation of the myogenic program is induced by postmitotic expression of insulin-like growth factor I.

PubMed

Musarò, A; Rosenthal, N

1999-04-01

The molecular mechanisms underlying myogenic induction by insulin-like growth factor I (IGF-I) are distinct from its proliferative effects on myoblasts. To determine the postmitotic role of IGF-I on muscle cell differentiation, we derived L6E9 muscle cell lines carrying a stably transfected rat IGF-I gene under the control of a myosin light chain (MLC) promoter-enhancer cassette. Expression of MLC-IGF-I exclusively in differentiated L6E9 myotubes, which express the embryonic form of myosin heavy chain (MyHC) and no endogenous IGF-I, resulted in pronounced myotube hypertrophy, accompanied by activation of the neonatal MyHC isoform. The hypertrophic myotubes dramatically increased expression of myogenin, muscle creatine kinase, beta-enolase, and IGF binding protein 5 and activated the myocyte enhancer factor 2C gene which is normally silent in this cell line. MLC-IGF-I induction in differentiated L6E9 cells also increased the expression of a transiently transfected LacZ reporter driven by the myogenin promoter, demonstrating activation of the differentiation program at the transcriptional level. Nuclear reorganization, accumulation of skeletal actin protein, and an increased expression of beta1D integrin were also observed. Inhibition of the phosphatidyl inositol (PI) 3-kinase intermediate in IGF-I-mediated signal transduction confirmed that the PI 3-kinase pathway is required only at early stages for IGF-I-mediated hypertrophy and neonatal MyHC induction in these cells. Expression of IGF-I in postmitotic muscle may therefore play an important role in the maturation of the myogenic program.

Highly efficient gene transfer into adult ventricular myocytes by recombinant adenovirus.

PubMed Central

Kirshenbaum, L A; MacLellan, W R; Mazur, W; French, B A; Schneider, M D

1993-01-01

Molecular dissection of mechanisms that govern the differentiated cardiac phenotype has, for cogent technical reasons, largely been undertaken to date in neonatal ventricular myocytes. To circumvent expected limitations of other methods, the present study was initiated to determine whether replication-deficient adenovirus would enable efficient gene transfer to adult cardiac cells in culture. Adult rat ventricular myocytes were infected, 24 h after plating, with adenovirus type 5 containing a cytomegalovirus immediate-early promoter-driven lacZ reporter gene and were assayed for the presence of beta-galactosidase 48 h after infection. The frequency of lacZ+ rod-shaped myocytes was half-maximal at 4 x 10(5) plaque-forming units (PFU) and approached 90% at 1 x 10(8) PFU. Uninfected cells and cells infected with lacZ- virus remained colorless. Beta-galactosidase activity concurred with the proportion of lacZ+ cells and was contingent on the exogenous lacZ gene. At 10(8) PFU/dish, cell number, morphology, and viability each were comparable to uninfected cells. Thus, adult ventricular myocytes are amenable to efficient gene transfer with recombinant adenovirus. The relative uniformity for gene transfer by adenovirus should facilitate tests to determine the impact of putative regulators upon the endogenous genes and gene products of virally modified adult ventricular muscle cells. Images PMID:8326005

Protein Kinase D-dependent Phosphorylation and Nuclear Export of Histone Deacetylase 5 Mediates Vascular Endothelial Growth Factor-induced Gene Expression and Angiogenesis*S⃞

PubMed Central

Ha, Chang Hoon; Wang, Weiye; Jhun, Bong Sook; Wong, Chelsea; Hausser, Angelika; Pfizenmaier, Klaus; McKinsey, Timothy A.; Olson, Eric N.; Jin, Zheng-Gen

2008-01-01

Vascular endothelial growth factor (VEGF) is essential for normal and pathological angiogenesis. However, the signaling pathways linked to gene regulation in VEGF-induced angiogenesis are not fully understood. Here we demonstrate a critical role of protein kinase D (PKD) and histone deacetylase 5 (HDAC5) in VEGF-induced gene expression and angiogenesis. We found that VEGF stimulated HDAC5 phosphorylation and nuclear export in endothelial cells through a VEGF receptor 2-phospholipase Cγ-protein kinase C-PKD-dependent pathway. We further showed that the PKD-HDAC5 pathway mediated myocyte enhancer factor-2 transcriptional activation and a specific subset of gene expression in response to VEGF, including NR4A1, an orphan nuclear receptor involved in angiogenesis. Specifically, inhibition of PKD by overexpression of the PKD kinase-negative mutant prevents VEGF-induced HDAC5 phosphorylation and nuclear export as well as NR4A1 induction. Moreover, a mutant of HDAC5 specifically deficient in PKD-dependent phosphorylation inhibited VEGF-mediated NR4A1 expression, endothelial cell migration, and in vitro angiogenesis. These findings suggest that the PKD-HDAC5 pathway plays an important role in VEGF regulation of gene transcription and angiogenesis. PMID:18332134

ATM-Dependent Phosphorylation of MEF2D Promotes Neuronal Survival after DNA Damage

PubMed Central

Chan, Shing Fai; Sances, Sam; Brill, Laurence M.; Okamoto, Shu-ichi; Zaidi, Rameez; McKercher, Scott R.; Akhtar, Mohd W.; Nakanishi, Nobuki

2014-01-01

Mutations in the ataxia telangiectasia mutated (ATM) gene, which encodes a kinase critical for the normal DNA damage response, cause the neurodegenerative disorder ataxia-telangiectasia (AT). The substrates of ATM in the brain are poorly understood. Here we demonstrate that ATM phosphorylates and activates the transcription factor myocyte enhancer factor 2D (MEF2D), which plays a critical role in promoting survival of cerebellar granule cells. ATM associates with MEF2D after DNA damage and phosphorylates the transcription factor at four ATM consensus sites. Knockdown of endogenous MEF2D with a short-hairpin RNA (shRNA) increases sensitivity to etoposide-induced DNA damage and neuronal cell death. Interestingly, substitution of endogenous MEF2D with an shRNA-resistant phosphomimetic MEF2D mutant protects cerebellar granule cells from cell death after DNA damage, whereas an shRNA-resistant nonphosphorylatable MEF2D mutant does not. In vivo, cerebella in Mef2d knock-out mice manifest increased susceptibility to DNA damage. Together, our results show that MEF2D is a substrate for phosphorylation by ATM, thus promoting survival in response to DNA damage. Moreover, dysregulation of the ATM–MEF2D pathway may contribute to neurodegeneration in AT. PMID:24672010

N-acetylcysteine reverses cardiac myocyte dysfunction in a rodent model of behavioral stress

PubMed Central

Chen, Fangping; Hadfield, Jessalyn M.; Berzingi, Chalak; Hollander, John M.; Miller, Diane B.; Nichols, Cody E.

2013-01-01

Compelling clinical reports reveal that behavioral stress alone is sufficient to cause reversible myocardial dysfunction in selected individuals. We developed a rodent stress cardiomyopathy model by a combination of prenatal and postnatal behavioral stresses (Stress). We previously reported a decrease in percent fractional shortening by echo, both systolic and diastolic dysfunction by catheter-based hemodynamics, as well as attenuated hemodynamic and inotropic responses to the β-adrenergic agonist, isoproterenol (ISO) in Stress rats compared with matched controls (Kan H, Birkle D, Jain AC, Failinger C, Xie S, Finkel MS. J Appl Physiol 98: 77–82, 2005). We now report enhanced catecholamine responses to behavioral stress, as evidenced by increased circulating plasma levels of norepinephrine (P < 0.01) and epinephrine (P < 0.01) in Stress rats vs. controls. Cardiac myocytes isolated from Stress rats also reveal evidence of oxidative stress, as indicated by decreased ATP, increased GSSG, and decreased GSH-to-GSSG ratio in the presence of increased GSH peroxidase and catalase activities (P < 0.01, for each). We also report blunted inotropic and intracellular Ca2+ concentration responses to extracellular Ca2+ (P < 0.05), as well as altered inotropic responses to the intracellular calcium regulator, caffeine (20 mM; P < 0.01). Treatment of cardiac myocytes with N-acetylcysteine (NAC) (10−3 M) normalized calcium handling in response to ISO and extracellular Ca2+ concentration and inotropic response to caffeine (P < 0.01, for each). NAC also attenuated the blunted inotropic response to ISO and Ca2+ (P < 0.01, for each). Surprisingly, NAC did not reverse the changes in GSH, GSSG, or GSH-to-GSSG ratio. These data support a GSH-independent salutary effect of NAC on intracellular calcium signaling in this rodent model of stress-induced cardiomyopathy. PMID:23722706

Cardiac safety of lacosamide: the non-clinical perspective.

PubMed

Delaunois, A; Colomar, A; Depelchin, B O; Cornet, M

2015-11-01

Lacosamide is indicated for the adjunctive treatment of partial-onset seizures in adult patients. Unlike other sodium channel-blocking antiepileptic drugs, lacosamide selectively enhances sodium channel slow inactivation. Potential effects of lacosamide on cardiac sodium channels and their cardiovascular consequences were comprehensively assessed. This manuscript presents the non-clinical cardiac safety profile of lacosamide. Lacosamide was tested in vitro on sodium and L-type calcium currents from isolated human atrial myocytes and on hERG-mediated potassium currents from stably transfected HEK293 cells. Cardiac action potentials were recorded in guinea pig ventricular myocytes. In vivo, hemodynamic and ECG parameters were evaluated in anesthetized dogs and monkeys receiving acute cumulative intravenous doses of lacosamide. Following intravenous dosing with lacosamide, dose-dependent PR and QRS prolongation and ECG abnormalities (loss of P waves, atrioventricular and intraventricular blocks, junctional premature contractions) were observed in anesthetized dogs and monkeys. In vitro, lacosamide reduced human cardiac sodium currents in a concentration-, voltage- and state-dependent manner. Lacosamide reductions in Vmax in guinea pig myocytes were similar to lamotrigine and carbamazepine. Lacosamide showed no relevant inhibitory effects on hERG and L-type calcium channels and did not prolong QTc in vivo. ECG findings in anesthetized animals correlate well with in vitro sodium channel-related effects and are also consistent with those (PR prolongation, first-degree atrioventricular block) reported in healthy volunteers and patients with epilepsy. Both in vivo and in vitro effects were detected from exposure levels 1.5- to 2-fold above those achieved with the maximum-recommended human lacosamide dose (400 mg/day). © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

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

Control of cytoplasmic and nuclear protein kinase A by phosphodiesterases and phosphatases in cardiac myocytes

PubMed Central

Haj Slimane, Zeineb; Bedioune, Ibrahim; Lechêne, Patrick; Varin, Audrey; Lefebvre, Florence; Mateo, Philippe; Domergue-Dupont, Valérie; Dewenter, Matthias; Richter, Wito; Conti, Marco; El-Armouche, Ali; Zhang, Jin; Fischmeister, Rodolphe; Vandecasteele, Grégoire

2014-01-01

Aims The cAMP-dependent protein kinase (PKA) mediates β-adrenoceptor (β-AR) regulation of cardiac contraction and gene expression. Whereas PKA activity is well characterized in various subcellular compartments of adult cardiomyocytes, its regulation in the nucleus remains largely unknown. The aim of the present study was to compare the modalities of PKA regulation in the cytoplasm and nucleus of cardiomyocytes. Methods and results Cytoplasmic and nuclear cAMP and PKA activity were measured with targeted fluorescence resonance energy transfer probes in adult rat ventricular myocytes. β-AR stimulation with isoprenaline (Iso) led to fast cAMP elevation in both compartments, whereas PKA activity was fast in the cytoplasm but markedly slower in the nucleus. Iso was also more potent and efficient in activating cytoplasmic than nuclear PKA. Similar slow kinetics of nuclear PKA activation was observed upon adenylyl cyclase activation with L-858051 or phosphodiesterase (PDE) inhibition with 3-isobutyl-1-methylxantine. Consistently, pulse stimulation with Iso (15 s) maximally induced PKA and myosin-binding protein C phosphorylation in the cytoplasm, but marginally activated PKA and cAMP response element-binding protein phosphorylation in the nucleus. Inhibition of PDE4 or ablation of the Pde4d gene in mice prolonged cytoplasmic PKA activation and enhanced nuclear PKA responses. In the cytoplasm, phosphatase 1 (PP1) and 2A (PP2A) contributed to the termination of PKA responses, whereas only PP1 played a role in the nucleus. Conclusion Our study reveals a differential integration of cytoplasmic and nuclear PKA responses to β-AR stimulation in cardiac myocytes. This may have important implications in the physiological and pathological hypertrophic response to β-AR stimulation. PMID:24550350

Role of ion channels and subcellular Ca2+ signaling in arachidonic acid-induced dilation of pressurized retinal arterioles.

PubMed

Kur, Joanna; McGahon, Mary K; Fernández, Jose A; Scholfield, C Norman; McGeown, J Graham; Curtis, Tim M

2014-05-02

To investigate the mechanisms responsible for the dilatation of rat retinal arterioles in response to arachidonic acid (AA). Changes in the diameter of isolated, pressurized rat retinal arterioles were measured in the presence of AA alone and following pre-incubation with pharmacologic agents inhibiting Ca(2+) sparks and oscillations and K(+) channels. Subcellular Ca(2+) signals were recorded in arteriolar myocytes using Fluo-4-based confocal imaging. The effects of AA on membrane currents of retinal arteriolar myocytes were studied using whole-cell perforated patch clamp recording. Arachidonic acid dilated pressurized retinal arterioles under conditions of myogenic tone. Eicosatetraynoic acid (ETYA) exerted a similar effect, but unlike AA, its effects were rapidly reversible. Arachidonic acid-induced dilation was associated with an inhibition of subcellular Ca(2+) signals. Interventions known to block Ca(2+) sparks and oscillations in retinal arterioles caused dilatation and inhibited AA-induced vasodilator responses. Arachidonic acid accelerated the rate of inactivation of the A-type Kv current and the voltage dependence of inactivation was shifted to more negative membrane potentials. It also enhanced voltage-activated and spontaneous large-conductance calcium-activated K(+) (BK) currents, but only at positive membrane potentials. Pharmacologic inhibition of A-type Kv and BK currents failed to block AA-induced vasodilator responses. Arachidonic acid suppressed L-type Ca(2+) currents. These results suggest that AA induces retinal arteriolar vasodilation by inhibiting subcellular Ca(2+)-signaling activity in retinal arteriolar myocytes, most likely through a mechanism involving the inhibition of L-type Ca(2+)-channel activity. Arachidonic acid actions on K(+) currents are inconsistent with a model in which K(+) channels contribute to the vasodilator effects of AA.

Mild hypoxia-induced cardiomyocyte hypertrophy via up-regulation of HIF-1α-mediated TRPC signalling

PubMed Central

Chu, Wenfeng; Wan, Lin; Zhao, Dan; Qu, Xuefeng; Cai, Fulai; Huo, Rong; Wang, Ning; Zhu, Jiuxin; Zhang, Chun; Zheng, Fangfang; Cai, Ruijun; Dong, Deli; Lu, Yanjie; Yang, Baofeng

2012-01-01

Hypoxia-inducible factor-1 alpha (HIF-1α) is a central transcriptional regulator of hypoxic response. The present study was designed to investigate the role of HIF-1α in mild hypoxia-induced cardiomyocytes hypertrophy and its underlying mechanism. Mild hypoxia (MH, 10% O2) caused hypertrophy in cultured neonatal rat cardiac myocytes, which was accompanied with increase of HIF-1α mRNA and accumulation of HIF-1α protein in nuclei. Transient receptor potential canonical (TRPC) channels including TRPC3 and TRPC6, except for TRPC1, were increased, and Ca2+-calcineurin signals were also enhanced in a time-dependent manner under MH condition. MH-induced cardiomyocytes hypertrophy, TRPC up-regulation and enhanced Ca2+-calcineurin signals were inhibited by an HIF-1α specific blocker, SC205346 (30 μM), whereas promoted by HIF-1α overexpression. Electrophysiological voltage-clamp demonstrated that DAG analogue, OAG (30 μM), induced TRPC current by as much as 170% in neonatal rat cardiomyocytes overexpressing HIF-1α compared to negative control. These results implicate that HIF-1α plays a key role in development of cardiac hypertrophy in responses to hypoxic stress. Its mechanism is associated with up-regulating TRPC3, TRPC6 expression, activating TRPC current and subsequently leading to enhanced Ca2+-calcineurin signals. PMID:22129453

Short-Term Caloric Restriction Suppresses Cardiac Oxidative Stress and Hypertrophy Caused by Chronic Pressure Overload.

PubMed

Kobara, Miyuki; Furumori-Yukiya, Akiko; Kitamura, Miho; Matsumura, Mihoko; Ohigashi, Makoto; Toba, Hiroe; Nakata, Tetsuo

2015-08-01

Caloric restriction (CR) prevents senescent changes, in which reactive oxygen species (ROS) have a critical role. Left ventricular (LV) hypertrophy is a risk factor for cardiovascular diseases. We examined whether CR alters cardiac redox state and hypertrophy from chronic pressure overload. Male c57BL6 mice were subjected to ascending aortic constriction (AAC) with ad libitum caloric intake (AL + AAC group) or 40% restricted caloric intake (CR + AAC group). CR was initiated 2 weeks before AAC and was continued for 4 weeks. Two weeks after constriction, AAC increased LV wall thickness, impaired transmitral flow velocity, and augmented myocyte hypertrophy and fibrosis, in association with enhancement of BNP and collagen III expressions in the AL + AAC group. In the AL + AAC group, oxidative stress in cardiac tissue and mitochondria were enhanced, and NADPH oxidase activity and mitochondrial ROS production were elevated. These changes were significantly attenuated in the CR + AAC group. Additionally, in antioxidant systems, myocardial glutathione peroxidase and superoxide dismutase activities were enhanced in the CR + AAC group. Chronic pressure overload increased cardiac oxidative damage, in association with cardiac hypertrophy and fibrosis. Short-term CR suppressed oxidative stress and improved cardiac function, suggesting that short-term CR could be a useful strategy to prevent pressure overload-induced cardiac injury. Copyright © 2015 Elsevier Inc. All rights reserved.

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

Inhibition of potassium currents is involved in antiarrhythmic effect of moderate ethanol on atrial fibrillation

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

Yang, Baode; Li, Chenxing

Excessive consumption of alcohol is a well-established risk factor of atrial fibrillation (AF). However, the effects of moderate alcohol drinking remain to be elucidated. This study was designed to determine the effects of moderate ethanol ingestion on atrial fibrillation and the electrophysiological mechanisms. In acetylcholine-induced canine and mouse AF models, the moderate ethanol prevented the generation and persistence of AF through prolonging the latent period of AF and shortening the duration of AF. The action potential duration (APD) was remarkably prolonged under the concentration range of 12.5–50.0 mM ethanol in guinea pig atrial myocytes. Ultra-rapid delayed rectified potassium currents (I{submore » Kv1.5}) were markedly inhibited by 12.5–50.0 mM ethanol in a concentration-dependent manner. Ethanol with 50.0 mM could inhibit rapid delayed rectifier potassium currents (I{sub hERG}). Ethanol under 6.25–50.0 mM did not affect on inward rectifier potassium currents (I{sub Kir2.1}). Collectively, the present study provided an evidence that moderate ethanol intake can prolong the APD of atrial myocytes by inhibition of I{sub Kv1.5} and I{sub hERG}, which contributed to preventing the development and duration of AF. - Highlights: • Moderate ethanol prevented the development of AF in animal models. • Moderate ethanol prolonged APD in guinea pig atrial myocytes. • Moderate ethanol inhibited Kv1.5 currents.« less

Multifunction of Chrysin in Parkinson's Model: Anti-Neuronal Apoptosis, Neuroprotection via Activation of MEF2D, and Inhibition of Monoamine Oxidase-B.

PubMed

Guo, Baojian; Zheng, Chengyou; Cai, Wei; Cheng, Jiehong; Wang, Hongyu; Li, Haitao; Sun, Yewei; Cui, Wei; Wang, Yuqiang; Han, Yifan; Lee, Simon Ming-Yuen; Zhang, Zaijun

2016-07-06

Chrysin, a flavonoid compound existing in several plants, is applied as a dietary supplement because of its beneficial effects on general human health and alleviation of neurological disorders. However, mechanisms underlying neuroprotection of chrysin has not been fully elucidated, and the effects of chrysin on the Parkinson's disease (PD) model in vivo have not been investigated. It is here shown that chrysin protects primary granular neurons against 1-methyl-4-phenylpyridinium ion insult via antiapoptosis by reversing the dysregulated expression of Bcl-2, Bax, and caspase 3. The mechanisms also involved activating transcriptional factor myocyte enhancer factor 2D (MEF2D) via regulation of AKT-GSK3β signaling. In this in vivo model of PD, chrysin rescued the dopaminergic neurons loss and alleviated the decrease in dopamine level induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine in mice. Moreover, chrysin markedly inhibited monoamine oxidase-B activity in vitro and in vivo. In conclusion, chrysin exerts beneficial effects to PD, possibly through multitarget mechanisms including antineuronal apoptosis, activation of the AKT-GSK3β/MEF2D pathway, and inhibition of the MAO-B activity.

Effect of siRNA silencing of inducible co-stimulatory molecule on myocardial cell hypertrophy after cardiac infarction in rats.

PubMed

Wang, W M; Liu, Z; Chen, G

2016-05-20

As the most common cardiac disease, myocardial infarction is followed by hypertrophy of cardiac myocytes and reconstruction of ventricular structure. The up-regulation of a series of factors including metalloproteinases, inflammatory factors, and growth factors after primary infarction lead to the hypertrophy, apoptosis, necrosis, and fibroblast proliferation in cardiac muscle tissues. Recent studies have reported on the potency of small interfering RNA (siRNA) in treating cardiac diseases. We thus investigated the efficacy of inducible co-stimulatory molecule (ICOS)-specific siRNA silencing in myocardial hypertrophy in a cardiac infarction rat model. This cardiac infarction model was prepared by ligating the left anterior descending coronary artery. ICOS-siRNA treatment was administered in parallel with non-sense siRNA. After 18 days, the cross-sectional area of cardiac muscle tissues and the left ventricle weight index were measured, along with ICOS mRNA and protein expression levels, and pathological staining. Compared to those in the control groups, in myocardial infarcted rats, the application of ICOS-siRNA effectively decreased the left ventricle weight index, as well as the surface area of cardiac myocytes. Both mRNA and protein levels of ICOS were also significantly decreased. HE staining was consistent with these results. In conclusion, ICOS-targeted siRNA can effectively silence gene expression of ICOS, and provided satisfactory treatment efficacy for myocardial cell hypertrophy after infarction.

[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.

Stimulation of ICa by basal PKA activity is facilitated by caveolin-3 in cardiac ventricular myocytes.

PubMed

Bryant, Simon; Kimura, Tomomi E; Kong, Cherrie H T; Watson, Judy J; Chase, Anabelle; Suleiman, M Saadeh; James, Andrew F; Orchard, Clive H

2014-03-01

L-type Ca channels (LTCC), which play a key role in cardiac excitation-contraction coupling, are located predominantly at the transverse (t-) tubules in ventricular myocytes. Caveolae and the protein caveolin-3 (Cav-3) are also present at the t-tubules and have been implicated in localizing a number of signaling molecules, including protein kinase A (PKA) and β2-adrenoceptors. The present study investigated whether disruption of Cav-3 binding to its endogenous binding partners influenced LTCC activity. Ventricular myocytes were isolated from male Wistar rats and LTCC current (ICa) recorded using the whole-cell patch-clamp technique. Incubation of myocytes with a membrane-permeable peptide representing the scaffolding domain of Cav-3 (C3SD) reduced basal ICa amplitude in intact, but not detubulated, myocytes, and attenuated the stimulatory effects of the β2-adrenergic agonist zinterol on ICa. The PKA inhibitor H-89 also reduced basal ICa; however, the inhibitory effects of C3SD and H-89 on basal ICa amplitude were not summative. Under control conditions, myocytes stained with antibody against phosphorylated LTCC (pLTCC) displayed a striated pattern, presumably reflecting localization at the t-tubules. Both C3SD and H-89 reduced pLTCC staining at the z-lines but did not affect staining of total LTCC or Cav-3. These data are consistent with the idea that the effects of C3SD and H-89 share a common pathway, which involves PKA and is maximally inhibited by H-89, and suggest that Cav-3 plays an important role in mediating stimulation of ICa at the t-tubules via PKA-induced phosphorylation under basal conditions, and in response to β2-adrenoceptor stimulation. Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.

Left ventricular hypertrophy in ascending aortic stenosis mice: anoikis and the progression to early failure

NASA Technical Reports Server (NTRS)

Ding, B.; Price, R. L.; Goldsmith, E. C.; Borg, T. K.; Yan, X.; Douglas, P. S.; Weinberg, E. O.; Bartunek, J.; Thielen, T.; Didenko, V. V.;

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

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.

Purification of cardiac myocytes from human heart biopsies for gene expression analysis.

PubMed

Kosloski, L M; Bales, I K; Allen, K B; Walker, B L; Borkon, A M; Stuart, R S; Pak, A F; Wacker, M J

2009-09-01

The collection of gene expression data from human heart biopsies is important for understanding the cellular mechanisms of arrhythmias and diseases such as cardiac hypertrophy and heart failure. Many clinical and basic research laboratories conduct gene expression analysis using RNA from whole cardiac biopsies. This allows for the analysis of global changes in gene expression in areas of the heart, while eliminating the need for more complex and technically difficult single-cell isolation procedures (such as flow cytometry, laser capture microdissection, etc.) that require expensive equipment and specialized training. The abundance of fibroblasts and other cell types in whole biopsies, however, can complicate gene expression analysis and the interpretation of results. Therefore, we have designed a technique to quickly and easily purify cardiac myocytes from whole cardiac biopsies for RNA extraction. Human heart tissue samples were collected, and our purification method was compared with the standard nonpurification method. Cell imaging using acridine orange staining of the purified sample demonstrated that >98% of total RNA was contained within identifiable cardiac myocytes. Real-time RT-PCR was performed comparing nonpurified and purified samples for the expression of troponin T (myocyte marker), vimentin (fibroblast marker), and alpha-smooth muscle actin (smooth muscle marker). Troponin T expression was significantly increased, and vimentin and alpha-smooth muscle actin were significantly decreased in the purified sample (n = 8; P < 0.05). Extracted RNA was analyzed during each step of the purification, and no significant degradation occurred. These results demonstrate that this isolation method yields a more purified cardiac myocyte RNA sample suitable for downstream applications, such as real-time RT-PCR, and allows for more accurate gene expression changes in cardiac myocytes from heart biopsies.

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.

Myristoylated peptides potentiate the funny current (If) in sinoatrial myocytes

PubMed Central

Liao, Zhandi; St Clair, Joshua R; Larson, Eric D

2011-01-01

The funny current, If, in sinoatrial myocytes is thought to contribute to the sympathetic fight-or-flight increase in heart rate. If is produced by hyperpolarization-activated cyclic nucleotide sensitive-4 (HCN4) channels, and it is widely believed that sympathetic regulation of If occurs via direct binding of cAMP to HCN4, independent of phosphorylation. However, we have recently shown that Protein Kinase A (PKA) activity is required for sympathetic regulation of If, and that PKA can directly phosphorylate HCN4.1 In the present study, we examined the effects of a myristoylated PKA inhibitory peptide (myr-PKI) on If in mouse sinoatrial myocytes. We found that myr-PKI and another myristoylated peptide potently and specifically potentiated If via a mechanism that did not involve PKA inhibition and that was independent of the peptide sequence, Protein Kinase C or phosphatidylinositol-4,5-bisphosphate. The off-target activation of If by myristoylated peptides limits their usefulness for studies of pacemaker mechanisms in sinoatrial myocytes. PMID:21150293

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

Rapid Responses and Mechanism of Action for Low-Dose Bisphenol S on ex Vivo Rat Hearts and Isolated Myocytes: Evidence of Female-Specific Proarrhythmic Effects

PubMed Central

Gao, Xiaoqian; Ma, Jianyong; Chen, Yamei

2015-01-01

Background Bisphenol S (BPS) has increasingly been used as a substitute for bisphenol A (BPA) in some “BPA-free” consumer goods and in thermal papers. Wide human exposure to BPS has been reported; however, the biological and potential toxic effects of BPS are poorly understood. Objective In this study, we sought to elucidate the sex-specific rapid effect of BPS in rat hearts and its underlying mechanism. Methods We examined the rapid effects of BPS in rat hearts using electrophysiology, confocal and conventional fluorescence imaging, and immunoblotting. Treatment was administered via acute perfusion of excised hearts or isolated cardiac myocytes. Results In female rat hearts acutely exposed to 10–9 M BPS, the heart rate was increased; in the presence of catecholamine-induced stress, the frequency of ventricular arrhythmia events was markedly increased. BPS-exposed hearts showed increased incidence of arrhythmogenic-triggered activities in female ventricular myocytes and altered myocyte Ca2+ handling, particularly spontaneous Ca2+ release from the sarcoplasmic reticulum. The dose responses of BPS actions were inverted U-shaped. The impact of BPS on myocyte Ca2+ handling was mediated by estrogen receptor β signaling and by rapid increases in the phosphorylation of key Ca2+ handling proteins, including ryanodine receptor and phospholamban. The proarrhythmic effects of BPS were female specific; male rat hearts were not affected by BPS at the organ, myocyte, or protein levels. Conclusion Rapid exposure to low-dose BPS showed proarrhythmic impact on female rat hearts; these effects at the organ, cellular, and molecular levels are remarkably similar to those reported for BPA. Evaluation of the bioactivity and safety of BPS and other BPA analogs is necessary before they are used as BPA alternatives in consumer products. Citation Gao X, Ma J, Chen Y, Wang HS. 2015. Rapid responses and mechanism of action for low-dose bisphenol S on ex vivo rat hearts and isolated myocytes: evidence of female-specific proarrhythmic effects. Environ Health Perspect 123:571–578; http://dx.doi.org/10.1289/ehp.1408679 PMID:25723814

β-Hydroxy-β-Methylbutyrate (HMB) Promotes Neurite Outgrowth in Neuro2a Cells.

PubMed

Salto, Rafael; Vílchez, Jose D; Girón, María D; Cabrera, Elena; Campos, Nefertiti; Manzano, Manuel; Rueda, Ricardo; López-Pedrosa, Jose M

2015-01-01

β-Hydroxy-β-methylbutyrate (HMB) has been shown to enhance cell survival, differentiation and protein turnover in muscle, mainly activating phosphoinositide-3-kinase/protein kinase B (PI3K/Akt) and mitogen-activated protein kinases/ extracellular-signal-regulated kinases (MAPK/ERK) signaling pathways. Since these two pathways are related to neuronal survival and differentiation, in this study, we have investigated the neurotrophic effects of HMB in mouse neuroblastoma Neuro2a cells. In Neuro2a cells, HMB promotes differentiation to neurites independent from any effects on proliferation. These effects are mediated by activation of both the PI3K/Akt and the extracellular-signal-regulated kinases (ERK1/2) signaling as demonstrated by the use of specific inhibitors of these two pathways. As myocyte-enhancer factor 2 (MEF2) family of transcription factors are involved in neuronal survival and plasticity, the transcriptional activity and protein levels of MEF2 were also evaluated. HMB promoted MEF2-dependent transcriptional activity mediated by the activation of Akt and ERK1/2 pathways. Furthermore, HMB increases the expression of brain glucose transporters 1 (GLUT1) and 3 (GLUT3), and mTOR phosphorylation, which translates in a higher protein synthesis in Neuro2a cells. Furthermore, Torin1 and rapamycin effects on MEF2 transcriptional activity and HMB-dependent neurite outgrowth support that HMB acts through mTORC2. Together, these findings provide clear evidence to support an important role of HMB in neurite outgrowth.

β-Hydroxy-β-Methylbutyrate (HMB) Promotes Neurite Outgrowth in Neuro2a Cells

PubMed Central

Girón, María D.; Cabrera, Elena; Campos, Nefertiti; Manzano, Manuel; Rueda, Ricardo; López-Pedrosa, Jose M.

2015-01-01

β-Hydroxy-β-methylbutyrate (HMB) has been shown to enhance cell survival, differentiation and protein turnover in muscle, mainly activating phosphoinositide-3-kinase/protein kinase B (PI3K/Akt) and mitogen-activated protein kinases/ extracellular-signal-regulated kinases (MAPK/ERK) signaling pathways. Since these two pathways are related to neuronal survival and differentiation, in this study, we have investigated the neurotrophic effects of HMB in mouse neuroblastoma Neuro2a cells. In Neuro2a cells, HMB promotes differentiation to neurites independent from any effects on proliferation. These effects are mediated by activation of both the PI3K/Akt and the extracellular-signal-regulated kinases (ERK1/2) signaling as demonstrated by the use of specific inhibitors of these two pathways. As myocyte-enhancer factor 2 (MEF2) family of transcription factors are involved in neuronal survival and plasticity, the transcriptional activity and protein levels of MEF2 were also evaluated. HMB promoted MEF2-dependent transcriptional activity mediated by the activation of Akt and ERK1/2 pathways. Furthermore, HMB increases the expression of brain glucose transporters 1 (GLUT1) and 3 (GLUT3), and mTOR phosphorylation, which translates in a higher protein synthesis in Neuro2a cells. Furthermore, Torin1 and rapamycin effects on MEF2 transcriptional activity and HMB-dependent neurite outgrowth support that HMB acts through mTORC2. Together, these findings provide clear evidence to support an important role of HMB in neurite outgrowth. PMID:26267903

Postnatal Loss of Mef2c Results in Dissociation of Effects on Synapse Number and Learning and Memory.

PubMed

Adachi, Megumi; Lin, Pei-Yi; Pranav, Heena; Monteggia, Lisa M

2016-07-15

Myocyte enhancer factor 2 (MEF2) transcription factors play critical roles in diverse cellular processes during central nervous system development. Studies attempting to address the role of MEF2 in brain have largely relied on overexpression of a constitutive MEF2 construct that impairs memory formation or knockdown of MEF2 function that increases spine numbers and enhances memory formation. Genetic deletion of individual MEF2 isoforms in brain during embryogenesis demonstrated that Mef2c loss negatively regulates spine numbers resulting in learning and memory deficits, possibly as a result of its essential role in development. To investigate MEF2C function in brain further, we genetically deleted Mef2c during postnatal development in mice. We characterized these conditional Mef2c knockout mice in an array of behavioral paradigms and examined the impact of postnatal loss of Mef2c on long-term potentiation. We observed increased spine numbers in hippocampus of the conditional Mef2c knockout mice. However, the postnatal loss of Mef2c did not impact learning and memory, long-term potentiation, or social and repetitive behaviors. Our findings demonstrate a critical role for MEF2C in the regulation of spine numbers with a dissociation of learning and memory, synaptic plasticity, and measures of autism-related behaviors in postnatal brain. Copyright © 2016 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.

Physical Inactivity, Obesity, and Type 2 Diabetes: An Evolutionary Perspective

ERIC Educational Resources Information Center

Eaton, S. Boyd; Eaton, Stanley B.

2017-01-01

Physical inactivity (and unhealthy nutrition) has distorted body composition and, in turn, reordered the proportions of myocyte and adipocyte insulin receptors. Insulin acting on adipocyte receptors produces less glucose uptake than does comparable interaction with myocyte receptors. Accordingly, in individuals with disproportionate muscle/fat…

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.

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

Bucindolol, a nonselective beta 1- and beta 2-adrenergic receptor antagonist, decreases beta-adrenergic receptor density in cultured embryonic chick cardiac myocyte membranes.

PubMed

Asano, K; Zisman, L S; Yoshikawa, T; Headley, V; Bristow, M R; Port, J D

2001-06-01

Bucindolol and carvedilol, nonselective beta1- and beta2-adrenergic receptor antagonists, have been widely used in clinical therapeutic trials of congestive heart failure. The aim of the current study was to investigate long-term effects of bucindolol or carvedilol on beta-adrenergic receptor protein and gene expression in cardiac myocytes. Embryonic chick cardiac myocytes were cultured and incubated with bucindolol (1 microM), carvedilol (1 microM), or norepinephrine (1 microM) for 24 h. 125I-iodocyanopindolol binding assays demonstrated that incubation with norepinephrine or bucindolol, but not carvedilol, significantly decreased beta-adrenergic receptor density in crude membranes prepared from the myocytes. Neither bucindolol nor carvedilol significantly stimulated adenylyl cyclase activity in membranes from drug-untreated cells. Unlike by norepinephrine, the receptor density reduction by bucindolol incubation was not accompanied by a change in beta1-adrenergic receptor messenger RNA abundance. A decrease in membrane beta-adrenergic receptor density without a change in cognate messenger RNA abundance was also observed in hamster DDT1 MF2 cell line incubated with bucindolol (1 microM, 24 h). We conclude that incubation with bucindolol, but not carvedilol, results in true reduction of beta-adrenergic receptor density in chick cardiac myocyte membranes by mechanisms that are distinct from those responsible for receptor density reduction by the agonist norepinephrine.

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.

Mesenchymal stem cells: The roles and functions in cutaneous wound healing and tumor growth.

PubMed

Motegi, Sei-Ichiro; Ishikawa, Osamu

2017-05-01

Mesenchymal stem cells (MSCs) are bone marrow-derived non-hematopoietic progenitor cells. MSCs are able to differentiate into various types of cells, including chondrocytes, adipocytes, osteocytes, myocytes, endothelial cells, and keratinocytes. There is increasing evidence that MSCs might be located external to the vasculature, and that perivascular cells in the skin, generally called as "pericytes", might include MSCs. It has been suggested that MSCs localized around blood vessels might migrate into wounds and contribute to the restoration of injured tissues. Many studies have demonstrated that intravenous or intradermal administration of MSCs enhanced cutaneous wound healing, such as acute incisional and excisional wounds, diabetic ulcers, radiation ulcers, and burns in animals and humans. Several mechanisms of the acceleration of wound healing by MSCs have been identified, including the enhancement of angiogenesis by secretion of pro-angiogenic factors and the differentiation into endothelial cells and/or pericytes, M2 macrophages polarization, the recruitment of endogenous stem/progenitor cells, extracellular matrix production and remodeling, and immunosuppressive effects. Since the microenvironments of wounds and/or injured tissues are similar to those of tumors, MSCs also play similar roles in malignant tumors, such as the enhancement of angiogenesis, M2 macrophages polarization, and immunosuppressive effects. In addition, the mechanisms of homing of MSCs might have a commonality in the pathogenesis of wound healing and tumors. Thus, the regulating factors of MSCs, including MFG-E8, could be a therapeutic target and lead to the establishment of new therapeutic approaches for both intractable wound healing and tumors. Copyright © 2016 Japanese Society for Investigative Dermatology. Published by Elsevier B.V. All rights reserved.

EXPOSURE OF CULTURED MYOCYTES TO ZINC RESULTS IN ALTERED BEAT RATE AND INTERCELLULAR COMMUNICATION.

EPA Science Inventory

Exposure of cultured myocytes to zinc results in altered beat rate and intercellular communication

Graff, Donald W, Devlin, Robert B, Brackhan, Joseph A, Muller-Borer, Barbara J, Bowman, Jill S, Cascio, Wayne E.

Exposure to ambient air pollution particulate matter (...

Cardiac and renal function in patients with type 2 diabetes who have chronic kidney disease: potential effects of bardoxolone methyl.

PubMed

McCullough, Peter A; Ali, Sajid

2012-01-01

The intracellular and tissue balance of oxidant and antioxidant forces is a potential therapeutic target for a variety of agents in the treatment of complications due to chronic disease including diabetes mellitus and hypertension. There are a myriad of processes controlled at the level of genes, transcription factors, and protein messages that work to control the normal use of oxidative reactions within cells. Loss of control of these processes may lead to reversible dysfunction in many cell lines including the podocyte, renal tubular cells, and cardiac myocytes. Bardoxolone methyl is a novel nuclear regulator factor (Nrf-2) activator which works to tip the balance of effects towards antioxidation and as an observation made serendipitously, improves renal filtration function in humans after approximately 12 weeks of therapy. The improvement in estimated glomerular filtration can be up to 30% in those with stage 3 and 4 chronic kidney disease. However, experimental evidence suggests there may be a consequence of relative hyperfiltration in diseased kidneys as well as potential adverse effects on skeletal and cardiac myocytes. Only large, prospective randomized trials with carefully collected and adjudicated clinical outcomes will inform the research community on the therapeutic risks and benefits of this important new agent.

Cardiac and renal function in patients with type 2 diabetes who have chronic kidney disease: potential effects of bardoxolone methyl

PubMed Central

McCullough, Peter A; Ali, Sajid

2012-01-01

The intracellular and tissue balance of oxidant and antioxidant forces is a potential therapeutic target for a variety of agents in the treatment of complications due to chronic disease including diabetes mellitus and hypertension. There are a myriad of processes controlled at the level of genes, transcription factors, and protein messages that work to control the normal use of oxidative reactions within cells. Loss of control of these processes may lead to reversible dysfunction in many cell lines including the podocyte, renal tubular cells, and cardiac myocytes. Bardoxolone methyl is a novel nuclear regulator factor (Nrf-2) activator which works to tip the balance of effects towards antioxidation and as an observation made serendipitously, improves renal filtration function in humans after approximately 12 weeks of therapy. The improvement in estimated glomerular filtration can be up to 30% in those with stage 3 and 4 chronic kidney disease. However, experimental evidence suggests there may be a consequence of relative hyperfiltration in diseased kidneys as well as potential adverse effects on skeletal and cardiac myocytes. Only large, prospective randomized trials with carefully collected and adjudicated clinical outcomes will inform the research community on the therapeutic risks and benefits of this important new agent. PMID:22787387

Simulation of the effect of rogue ryanodine receptors on a calcium wave in ventricular myocytes with heart failure.

PubMed

Lu, Luyao; Xia, Ling; Ye, Xuesong; Cheng, Heping

2010-05-26

Calcium homeostasis is considered to be one of the most important factors for the contraction and relaxation of the heart muscle. However, under some pathological conditions, such as heart failure (HF), calcium homeostasis is disordered, and spontaneous waves may occur. In this study, we developed a mathematical model of formation and propagation of a calcium wave based upon a governing system of diffusion-reaction equations presented by Izu et al (2001 Biophys. J. 80 103-20) and integrated non-clustered or 'rogue' ryanodine receptors (rogue RyRs) into a two-dimensional (2D) model of ventricular myocytes isolated from failing hearts in which sarcoplasmic reticulum (SR) Ca(2+) pools are partially unloaded. The model was then used to simulate the effect of rogue RyRs on initiation and propagation of the calcium wave in ventricular myocytes with HF. Our simulation results show that rogue RyRs can amplify the diastolic SR Ca(2+) leak in the form of Ca(2+) quarks, increase the probability of occurrence of spontaneous Ca(2+) waves even with smaller SR Ca(2+) stores, accelerate Ca(2+) wave propagation, and hence lead to delayed afterdepolarizations (DADs) and cardiac arrhythmia in the diseased heart. This investigation suggests that incorporating rogue RyRs in the Ca(2+) wave model under HF conditions provides a new view of Ca(2+) dynamics that could not be mimicked by adjusting traditional parameters involved in Ca(2+) release units and other ion channels, and contributes to understanding the underlying mechanism of HF.

RSK3 is required for concentric myocyte hypertrophy in an activated Raf1 model for Noonan syndrome.

PubMed

Passariello, Catherine L; Martinez, Eliana C; Thakur, Hrishikesh; Cesareo, Maria; Li, Jinliang; Kapiloff, Michael S

2016-04-01

Noonan syndrome (NS) is a congenital disorder resulting from mutations of the Ras-Raf signaling pathway. Hypertrophic cardiomyopathy associated with RAF1 "RASopathy" mutations is a major risk factor for heart failure and death in NS and has been attributed to activation of MEK1/2-ERK1/2 mitogen-activated protein kinases. We recently discovered that type 3 p90 ribosomal S6 kinase (RSK3) is an ERK effector that is required, like ERK1/2, for concentric myocyte hypertrophy in response to pathological stress such as pressure overload. In order to test whether RSK3 also contributes to NS-associated hypertrophic cardiomyopathy, RSK3 knock-out mice were crossed with mice bearing the Raf1(L613V) human NS mutation. We confirmed that Raf1(L613V) knock-in confers a NS-like phenotype, including cardiac hypertrophy. Active RSK3 was increased in Raf1(L613V) mice. Constitutive RSK3 gene deletion prevented the Raf1(L613V)-dependent concentric growth in width of the cardiac myocyte and attenuated cardiac hypertrophy in female mice. These results are consistent with RSK3 being an important mediator of ERK1/2-dependent growth in RASopathy. In conjunction with previously published data showing that RSK3 is important for pathological remodeling of the heart, these data suggest that targeting of this downstream MAP-kinase pathway effector should be considered in the treatment of RASopathy-associated hypertrophic cardiomyopathy. Copyright © 2016 Elsevier Ltd. All rights reserved.

Effects of phytoestrogens on protein turnover in rainbow trout primary myocytes

USDA-ARS?s Scientific Manuscript database

Soybean-derived ingredients used in aquaculture feeds may contain phytoestrogens, but it is unknown if these compounds can mimic the catabolic effects of estradiol in fish muscle. Six day-old rainbow trout primary myocytes were exposed to increasing concentrations (10 nM – 100 µM) of either geniste...

Myotoxic effects of clenbuterol in the rat heart and soleus muscle.

PubMed

Burniston, Jatin G; Ng, Yeelan; Clark, William A; Colyer, John; Tan, Lip-Bun; Goldspink, David F

2002-11-01

Myocyte-specific necrosis in the heart and soleus muscle of adult male Wistar rats was investigated in response to a single subcutaneous injection of the anabolic beta(2)-adrenergic receptor agonist clenbuterol. Necrosis was immunohistochemically detected by administration of a myosin antibody 1 h before the clenbuterol challenge and quantified by using image analysis. Clenbuterol-induced myocyte necrosis occurred against a background of zero damage in control muscles. In the heart, the clenbuterol-induced necrosis was not uniform, being more abundant in the left subendocardium and peaking 2.4 mm from the apex. After position (2.4 mm from the apex), dose (5 mg clenbuterol/kg), and sampling time (12 h) were optimized, maximum cardiomyocyte necrosis was found to be 1.0 +/- 0.2%. In response to the same parameters (i.e., 5 mg of clenbuterol and sampled at 12 h), skeletal myocyte necrosis was 4.4 +/- 0.8% in the soleus. These data show significant myocyte-specific necrosis in the heart and skeletal muscle of the rat. Such irreversible damage in the heart suggests that clenbuterol may be damaging to long-term health.

An abnormal Ca2+ response in mutant sarcomere protein–mediated familial hypertrophic cardiomyopathy

PubMed Central

Fatkin, Diane; McConnell, Bradley K.; Mudd, James O.; Semsarian, Christopher; Moskowitz, Ivan G.P.; Schoen, Frederick J.; Giewat, Michael; Seidman, Christine E.; Seidman, J.G.

2000-01-01

Dominant-negative sarcomere protein gene mutations cause familial hypertrophic cardiomyopathy (FHC), a disease characterized by left-ventricular hypertrophy, angina, and dyspnea that can result in sudden death. We report here that a murine model of FHC bearing a cardiac myosin heavy-chain gene missense mutation (αMHC403/+), when treated with calcineurin inhibitors or a K+-channel agonist, developed accentuated hypertrophy, worsened histopathology, and was at risk for early death. Despite distinct pharmacologic targets, each agent augmented diastolic Ca2+ concentrations in wild-type cardiac myocytes; αMHC403/+ myocytes failed to respond. Pretreatment with a Ca2+-channel antagonist abrogated diastolic Ca2+ changes in wild-type myocytes and prevented the exaggerated hypertrophic response of treated αMHC403/+ mice. We conclude that FHC-causing sarcomere protein gene mutations cause abnormal Ca2+ responses that initiate a hypertrophic response. These data define an important Ca2+-dependent step in the pathway by which mutant sarcomere proteins trigger myocyte growth and remodel the heart, provide definitive evidence that environment influences progression of FHC, and suggest a rational therapeutic approach to this prevalent human disease. PMID:11104788

Infrasound exposure induces apoptosis of rat cardiac myocytes by regulating the expression of apoptosis-related proteins.

PubMed

Pei, Zhao-Hui; Chen, Bao-Ying; Tie, Ru; Zhang, Hai-Feng; Zhao, Ge; Qu, Ping; Zhu, Xiao-Xing; Zhu, Miao-Zhang; Yu, Jun

2011-12-01

It has been reported that exposure to infrasound causes cardiac dysfunction. Allowing for the key role of apoptosis in the pathogenesis of cardiovascular diseases, the objective of this study was to investigate the apoptotic effects of infrasound. Cardiac myocytes cultured from neonatal rats were exposed to infrasound of 5 Hz at 130 dB. The apoptosis was determined by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling. Also, the expression levels of a series of apoptosis-related proteins were detected. As a result, infrasound induced apoptosis of cultured rat cardiac myocytes in a time-dependant manner. The expression of proapoptotic proteins such as Bax, caspase-3, caspase-8, caspase-9, and FAS was significantly up-regulated, with concomitant down-regulated expression of antiapoptotic proteins such as Bcl-x, and the inhibitory apoptosis proteins family proteins including XIAP, cIAP-1, and cIAP-2. The expression of poly (ADP-ribose) polymerase and β-catenin, which are the substrate proteins of caspase-3, was significantly decreased. In conclusion, infrasound is an apoptotic inducer of cardiac myocytes.

[Effect of imidapril on the effective refractory period and sodium current of ventricular noninfarction zone in healed myocardial infarction].

PubMed

Li, Yang; Niu, Hui-Yan; Liu, Nian; Zhang, Cun-Tai; Lu, Zai-Ying; Wang, Shi-Wen

2005-07-01

To investigate the effects of imidapril (IMI) on effective refractory period (ERP) and sodium current (I(Na)) of myocytes in ventricular noninfarction zone of healed myocardial infarction (HMI) in rabbit models. Rabbits with left coronary artery ligation were prepared and IMI (0.625 mg x kg(-1) x d(-1), 8 weeks) was orally administered. The ERP and sodium current were recorded. The ERP in HMI heart was prolonged. The ERP in IMI group was lower significantly than that of HMI group. The I(Na) density of myocyte in HMI ventricle decreased obviously. V 1/2 of steady state inactivation of I(Na) shifted to hyperpolarization, and time constant (tau) of recovery from inactivation in HMI ventricular myocyte was longer than that of sham ventricular myocyte. I(Na) density in IMI group increased markedly as compared with that of HMI group. IMI was shown to reverse the abnormal prolongation of ERP in rabbit heart with the HMI and increase I(Na) density. It may be the mechanism of IMI preventing against antiarrhythmia in healed myocardical infarction.

Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP)-mediated Calcium Signaling and Arrhythmias in the Heart Evoked by β-Adrenergic Stimulation*♦

PubMed Central

Nebel, Merle; Schwoerer, Alexander P.; Warszta, Dominik; Siebrands, Cornelia C.; Limbrock, Ann-Christin; Swarbrick, Joanna M.; Fliegert, Ralf; Weber, Karin; Bruhn, Sören; Hohenegger, Martin; Geisler, Anne; Herich, Lena; Schlegel, Susan; Carrier, Lucie; Eschenhagen, Thomas; Potter, Barry V. L.; Ehmke, Heimo; Guse, Andreas H.

2013-01-01

Nicotinic acid adenine dinucleotide phosphate (NAADP) is the most potent Ca2+-releasing second messenger known to date. Here, we report a new role for NAADP in arrhythmogenic Ca2+ release in cardiac myocytes evoked by β-adrenergic stimulation. Infusion of NAADP into intact cardiac myocytes induced global Ca2+ signals sensitive to inhibitors of both acidic Ca2+ stores and ryanodine receptors and to NAADP antagonist BZ194. Furthermore, in electrically paced cardiac myocytes BZ194 blocked spontaneous diastolic Ca2+ transients caused by high concentrations of the β-adrenergic agonist isoproterenol. Ca2+ transients were recorded both as increases of the free cytosolic Ca2+ concentration and as decreases of the sarcoplasmic luminal Ca2+ concentration. Importantly, NAADP antagonist BZ194 largely ameliorated isoproterenol-induced arrhythmias in awake mice. We provide strong evidence that NAADP-mediated modulation of couplon activity plays a role for triggering spontaneous diastolic Ca2+ transients in isolated cardiac myocytes and arrhythmias in the intact animal. Thus, NAADP signaling appears an attractive novel target for antiarrhythmic therapy. PMID:23564460

Gli3 Regulation of Myogenesis Is Necessary for Ischemia-Induced Angiogenesis

PubMed Central

Renault, Marie-Ange; Vandierdonck, Soizic; Chapouly, Candice; Yu, Yang; Qin, Gangjian; Metras, Alexandre; Couffinhal, Thierry; Losordo, Douglas W.; Yao, Qinyu; Reynaud, Annabel; Jaspard-Vinassa, Béatrice; Belloc, Isabelle; Desgranges, Claude; Gadeau, Alain-Pierre

2015-01-01

Rationale A better understanding of the mechanism underlying skeletal muscle repair is required to develop therapies that promote tissue regeneration in adults. Hedgehog signaling has been shown previously to be involved in myogenesis and angiogenesis: 2 crucial processes for muscle development and regeneration. Objective The objective of this study was to identify the role of the hedgehog transcription factor Gli3 in the crosstalk between angiogenesis and myogenesis in adults. Methods and Results Using conditional knockout mice, we found that Gli3 deficiency in endothelial cells did not affect ischemic muscle repair, whereas in myocytes, Gli3 deficiency resulted in severely delayed ischemia-induced myogenesis. Moreover, angiogenesis was also significantly impaired in HSA-CreERT2; Gli3Flox/Flox mice, demonstrating that impaired myogenesis indirectly affects ischemia-induced angiogenesis. The role of Gli3 in myocytes was then further investigated. We found that Gli3 promotes myoblast differentiation through myogenic factor 5 regulation. In addition, we found that Gli3 regulates several proangiogenic factors, including thymidine phosphorylase and angiopoietin-1 both in vitro and in vivo, which indirectly promote endothelial cell proliferation and arteriole formation. In addition, we found that Gli3 is upregulated in proliferating myoblasts by the cell cycle–associated transcription factor E2F1. Conclusions This study shows for the first time that Gli3-regulated postnatal myogenesis is necessary for muscle repair–associated angiogenesis. Most importantly, it implies that myogenesis drives angiogenesis in the setting of skeletal muscle repair and identifies Gli3 as a potential target for regenerative medicine. PMID:24044950

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

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.

Identification of a New Modulator of the Intercalated Disc in a Zebrafish Model of Arrhythmogenic Cardiomyopathy

PubMed Central

Asimaki, Angeliki; Kapoor, Sudhir; Plovie, Eva; Arndt, Anne Karin; Adams, Edward; Liu, ZhenZhen; James, Cynthia A.; Judge, Daniel P.; Calkins, Hugh; Churko, Jared; Wu, Joseph C.; MacRae, Calum A.; Kléber, André G.; Saffitz, Jeffrey E.

2015-01-01

Arrhythmogenic cardiomyopathy (ACM) is characterized by frequent cardiac arrhythmias. To elucidate the underlying mechanisms and discover potential chemical modifiers, we created a zebrafish model of ACM with cardiac myocyte–specific expression of the human 2057del2 mutation in the gene encoding plakoglobin. A high-throughput screen identified SB216763 as a suppressor of the disease phenotype. Early SB216763 therapy prevented heart failure and reduced mortality in the fish model. Zebrafish ventricular myocytes that expressed 2057del2 plakoglobin exhibited 70 to 80% reductions in INa and IK1 current densities, which were normalized by SB216763. Neonatal rat ventricular myocytes that expressed 2057del2 plakoglobin recapitulated pathobiological features seen in patients with ACM, all of which were reversed or prevented by SB216763. The reverse remodeling observed with SB216763 involved marked subcellular redistribution of plakoglobin, connexin 43, and Nav1.5, but without changes in their total cellular content, implicating a defect in protein trafficking to intercalated discs. In further support of this mechanism, we observed SB216763-reversible, abnormal subcellular distribution of SAP97 (a protein known to mediate forward trafficking of Nav1.5 and Kir2.1) in rat cardiac myocytes expressing 2057del2 plakoglobin and in cardiac myocytes derived from induced pluripotent stem cells from two ACM probands with plakophilin-2 mutations. These observations pinpoint aberrant trafficking of intercalated disc proteins as a central mechanism in ACM myocyte injury and electrical abnormalities. PMID:24920660

Prevention of ventricular arrhythmia and calcium dysregulation in a catecholaminergic polymorphic ventricular tachycardia mouse model carrying calsequestrin-2 mutation.

PubMed

Alcalai, Ronny; Wakimoto, Hiroko; Arad, Michael; Planer, David; Konno, Tetsuo; Wang, Libin; Seidman, Jon G; Seidman, Christine E; Berul, Charles I

2011-03-01

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a familial arrhythmic syndrome caused by mutations in genes encoding the calcium-regulation proteins cardiac ryanodine receptor (RyR2) or calsequestrin-2 (CASQ2). Mechanistic studies indicate that CPVT is mediated by diastolic Ca(2+) overload and increased Ca(2+) leak through the RyR2 channel, implying that treatment targeting these defects might be efficacious in CPVT. CPVT mouse models that lack CASQ2 were treated with Ca(2+) -channel inhibitors, β-adrenergic inhibitors, or Mg(2+) . Treatment effects on ventricular arrhythmia, sarcoplasmic reticulum (SR) protein expression and Ca(2+) transients of isolated myocytes were assessed. Each study agent reduced the frequency of stress-induced ventricular arrhythmia in mutant mice. The Ca(2+) channel blocker verapamil was most efficacious and completely prevented arrhythmia in 85% of mice. Verapamil significantly increased the SR Ca(2+) content in mutant myocytes, diminished diastolic Ca(2+) overload, increased systolic Ca(2+) amplitude, and prevented Ca(2+) oscillations in stressed mutant myocytes. Ca(2+) channel inhibition by verapamil rectified abnormal calcium handling in CPVT myocytes and prevented ventricular arrhythmias. Verapamil-induced partial normalization of SR Ca(2+) content in mutant myocytes implicates CASQ2 as modulator of RyR2 activity, rather than or in addition to, Ca(2+) buffer protein. Agents such as verapamil that attenuate cardiomyocyte calcium overload are appropriate for assessing clinical efficacy in human CPVT. © 2010 Wiley Periodicals, Inc.

Prevention of Ventricular Arrhythmia and Calcium Dysregulation in a Catecholaminergic Polymorphic Ventricular Tachycardia Mouse Model Carrying Calsequestrin-2 Mutation

PubMed Central

Alcalai, Ronny; Wakimoto, Hiroko; Arad, Michael; Planer, David; Konno, Tetsuo; Wang, Libin; Seidman, Jon G.; Seidman, Christine E.; Berul, Charles I

2010-01-01

Background Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a familial arrhythmic syndrome caused by mutations in genes encoding the calcium-regulation proteins cardiac ryanodine receptor (RyR2) or calsequestrin-2 (CASQ2). Mechanistic studies indicate that CPVT is mediated by diastolic Ca2+ overload and increased Ca2+ leak through the RyR2 channel, implying that treatment targeting these defects might be efficacious in CPVT. Method and results CPVT mouse models that lack CASQ2 were treated with Ca2+-channel inhibitors, β-adrenergic inhibitors, or Mg2+. Treatment effects on ventricular arrhythmia, sarcoplasmic reticulum (SR) protein expression and Ca2+ transients of isolated myocytes were assessed. Each study agent reduced the frequency of stress-induced ventricular arrhythmia in mutant mice. The Ca2+ channel blocker verapamil was most efficacious and completely prevented arrhythmia in 85% of mice. Verapamil significantly increased the SR Ca2+ content in mutant myocytes, diminished diastolic Ca2+ overload, increased systolic Ca2+ amplitude, and prevented Ca2+ oscillations in stressed mutant myocytes. Conclusions Ca2+ channel inhibition by verapamil rectified abnormal calcium handling in CPVT myocytes and prevented ventricular arrhythmias. Verapamil-induced partial normalization of SR Ca2+ content in mutant myocytes implicates CASQ2 as modulator of RyR2 activity, rather than or in addition to, Ca2+ buffer protein. Agents such as verapamil that attenuate cardiomyocyte calcium overload are appropriate for assessing clinical efficacy in human CPVT. PMID:20807279

Increased Cardiac Myocyte Progenitors in Failing Human Hearts

PubMed Central

Kubo, Hajime; Jaleel, Naser; Kumarapeli, Asangi; Berretta, Remus M.; Bratinov, George; Shan, Xiaoyin; Wang, Hongmei; Houser, Steven R.; Margulies, Kenneth B.

2009-01-01

Background Increasing evidence, derived mainly from animal models, supports the existence of endogenous cardiac renewal and repair mechanisms in adult mammalian hearts that could contribute to normal homeostasis and the responses to pathological insults. Methods and Results Translating these results, we isolated small c-kit+ cells from 36 of 37 human hearts using primary cell isolation techniques and magnetic cell sorting techniques. The abundance of these cardiac progenitor cells was increased nearly 4-fold in patients with heart failure requiring transplantation compared with nonfailing controls. Polychromatic flow cytometry of primary cell isolates (<30 μm) without antecedent c-kit enrichment confirmed the increased abundance of c-kit+ cells in failing hearts and demonstrated frequent coexpression of CD45 in these cells. Immunocytochemical characterization of freshly isolated, c-kit–enriched human cardiac progenitor cells confirmed frequent coexpression of c-kit and CD45. Primary cardiac progenitor cells formed new human cardiac myocytes at a relatively high frequency after coculture with neonatal rat ventricular myocytes. These contracting new cardiac myocytes exhibited an immature phenotype and frequent electric coupling with the rat myocytes that induced their myogenic differentiation. Conclusions Despite the increased abundance and cardiac myogenic capacity of cardiac progenitor cells in failing human hearts, the need to replace these organs via transplantation implies that adverse features of the local myocardial environment overwhelm endogenous cardiac repair capacity. Developing strategies to improve the success of endogenous cardiac regenerative processes may permit therapeutic myocardial repair without cell delivery per se. PMID:18645055

Hypoxia activates muscle-restricted coiled-coil protein (MURC) expression via transforming growth factor-β in cardiac myocytes.

PubMed

Shyu, Kou-Gi; Cheng, Wen-Pin; Wang, Bao-Wei; Chang, Hang

2014-03-01

The expression of MURC (muscle-restricted coiled-coil protein), a hypertrophy-regulated gene, increases during pressure overload. Hypoxia can cause myocardial hypertrophy; however, how hypoxia affects the regulation of MURC in cardiomyocytes undergoing hypertrophy is still unknown. The aim of the present study was to test the hypothesis that hypoxia induces MURC expression in cardiomyocytes during hypertrophy. The expression of MURC was evaluated in cultured rat neonatal cardiomyocytes subjected to hypoxia and in an in vivo model of AMI (acute myocardial infarction) to induce myocardial hypoxia in adult rats. MURC protein and mRNA expression were significantly enhanced by hypoxia. MURC proteins induced by hypoxia were significantly blocked after the addition of PD98059 or ERK (extracellular-signal-regulated kinase) siRNA 30 min before hypoxia. Gel-shift assay showed increased DNA-binding activity of SRF (serum response factor) after hypoxia. PD98059, ERK siRNA and an anti-TGF-β (transforming growth factor-β) antibody abolished the SRF-binding activity enhanced by hypoxia or exogenous administration of TGF-β. A luciferase promoter assay demonstrated increased transcriptional activity of SRF in cardiomyocytes by hypoxia. Increased βMHC (β-myosin heavy chain) and BNP (B-type natriuretic peptide) protein expression and increased protein synthesis was identified after hypoxia with the presence of MURC in hypertrophic cardiomyocytes. MURC siRNA inhibited the hypertrophic marker protein expression and protein synthesis induced by hypoxia. AMI in adult rats also demonstrated increased MURC protein expression in the left ventricular myocardium. In conclusion, hypoxia in cultured rat neonatal cardiomyocytes increased MURC expression via the induction of TGF-β, SRF and the ERK pathway. These findings suggest that MURC plays a role in hypoxia-induced hypertrophy in cardiomyocytes.

Biosensors to measure inositol 1,4,5-trisphosphate concentration in living cells with spatiotemporal resolution.

PubMed

Remus, Timothy P; Zima, Aleksey V; Bossuyt, Julie; Bare, Dan J; Martin, Jody L; Blatter, Lothar A; Bers, Donald M; Mignery, Gregory A

2006-01-06

Phosphoinositides participate in many signaling cascades via phospholipase C stimulation, which hydrolyzes phosphatidylinositol 4,5-bisphosphate, producing second messengers diacylglycerol and inositol 1,4,5-trisphosphate (InsP3). Destructive chemical approaches required to measure [InsP3] limit spatiotemporal understanding of subcellular InsP3 signaling. We constructed novel fluorescence resonance energy transfer-based InsP3 biosensors called FIRE (fluorescent InsP3-responsive element) by fusing plasmids encoding the InsP3-binding domain of InsP3 receptors (types 1-3) between cyan fluorescent protein and yellow fluorescent protein sequences. FIRE was expressed and characterized in COS-1 cells, cultured neonatal cardiac myocytes, and incorporated into an adenoviral vector for expression in adult cardiac ventricular myocytes. FIRE-1 exhibits an approximately 11% increase in the fluorescence ratio (F530/F480) at saturating [InsP3] (apparent K(d) = 31.3 +/- 6.7 nm InsP3). In COS-1 cells, neonatal rat cardiac myocytes and adult cat ventricular myocytes FIRE-1 exhibited comparable dynamic range and a 10% increase in donor (cyan fluorescent protein) fluorescence upon bleach of yellow fluorescent protein, indicative of fluorescence resonance energy transfer. In FIRE-1 expressing ventricular myocytes endothelin-1, phenylephrine, and angiotensin II all produced rapid and spatially resolved increases in [InsP3] using confocal microscopy (with free [InsP3] rising to approximately 30 nm). Local entry of intracellular InsP3 via membrane rupture by a patch pipette (containing InsP3)in myocytes expressing FIRE-1 allowed detailed spatiotemporal monitoring of intracellular InsP3 diffusion. Both endothelin-1-induced and direct InsP3 application (via pipette rupture) revealed that InsP3 diffusion into the nucleus occurs with a delay and blunted rise of [InsP3] versus cytosolic [InsP3]. These new biosensors allow studying InsP3 dynamics at high temporal and spatial resolution that will be powerful in under-standing InsP3 signaling in intact cells.

A key role for peroxynitrite-mediated inhibition of cardiac ERG (Kv11.1) K+ channels in carbon monoxide–induced proarrhythmic early afterdepolarizations

PubMed Central

Al-Owais, Moza M.; Hettiarachchi, Nishani T.; Kirton, Hannah M.; Hardy, Matthew E.; Boyle, John P.; Scragg, Jason L.; Steele, Derek S.; Peers, Chris

2017-01-01

Exposure to CO causes early afterdepolarization arrhythmias. Previous studies in rats have indicated that arrhythmias arose as a result of augmentation of the late Na+ current. The purpose of the present study was to examine the basis for CO-induced arrhythmias in guinea pig myocytes in which action potentials (APs) more closely resemble those of human myocytes. Whole-cell current- and voltage-clamp recordings were made from isolated guinea pig myocytes as well as from human embryonic kidney 293 (HEK293) cells that express wild-type or a C723S mutant form of ether-a-go-go–related gene (ERG; Kv11.1). We also monitored the formation of peroxynitrite (ONOO−) in HEK293 cells fluorimetrically. CO—applied as the CO-releasing molecule, CORM-2—prolonged the APs and induced early afterdepolarizations in guinea pig myocytes. In HEK293 cells, CO inhibited wild-type, but not C723S mutant, Kv11.1 K+ currents. Inhibition was prevented by an antioxidant, mitochondrial inhibitors, or inhibition of NO formation. CO also raised ONOO− levels, an effect that was reversed by the ONOO− scavenger, FeTPPS [5,10,15,20-tetrakis-(4-sulfonatophenyl)-porphyrinato-iron(III)], which also prevented the CO inhibition of Kv11.1 currents and abolished the effects of CO on Kv11.1 tail currents and APs in guinea pig myocytes. Our data suggest that CO induces arrhythmias in guinea pig cardiac myocytes via the ONOO−-mediated inhibition of Kv11.1 K+ channels.—Al-Owais, M. M., Hettiarachchi, N. T., Kirton, H. M., Hardy, M. E., Boyle, J. P., Scragg, J. L., Steele, D. S., Peers, C. A key role for peroxynitrite-mediated inhibition of cardiac ERG (Kv11.1) K+ channels in carbon monoxide–induced proarrhythmic early afterdepolarizations. PMID:28743763

Validation of an in vitro contractility assay using canine ventricular myocytes

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

Harmer, A.R., E-mail: alex.harmer@astrazeneca.com; Abi-Gerges, N.; Morton, M.J.

Measurement of cardiac contractility is a logical part of pre-clinical safety assessment in a drug discovery project, particularly if a risk has been identified or is suspected based on the primary- or non-target pharmacology. However, there are limited validated assays available that can be used to screen several compounds in order to identify and eliminate inotropic liability from a chemical series. We have therefore sought to develop an in vitro model with sufficient throughput for this purpose. Dog ventricular myocytes were isolated using a collagenase perfusion technique and placed in a perfused recording chamber on the stage of a microscopemore » at ∼ 36 °C. Myocytes were stimulated to contract at a pacing frequency of 1 Hz and a digital, cell geometry measurement system (IonOptix™) was used to measure sarcomere shortening in single myocytes. After perfusion with vehicle (0.1% DMSO), concentration–effect curves were constructed for each compound in 4–30 myocytes taken from 1 or 2 dog hearts. The validation test-set was 22 negative and 8 positive inotropes, and 21 inactive compounds, as defined by their effect in dog, cynolomolgous monkey or humans. By comparing the outcome of the assay to the known in vivo contractility effects, the assay sensitivity was 81%, specificity was 75%, and accuracy was 78%. With a throughput of 6–8 compounds/week from 1 cell isolation, this assay may be of value to drug discovery projects to screen for direct contractility effects and, if a hazard is identified, help identify inactive compounds. -- Highlights: ► Cardiac contractility is an important physiological function of the heart. ► Assessment of contractility is a logical part of pre-clinical drug safety testing. ► There are limited validated assays that predict effects of compounds on contractility. ► Using dog myocytes, we have developed an in vitro cardiac contractility assay. ► The assay predicted the in vivo contractility with a good level of accuracy.« less

Stress-dependent dilated cardiomyopathy in mice with cardiomyocyte-restricted inactivation of cyclic GMP-dependent protein kinase I

PubMed Central

Frantz, Stefan; Klaiber, Michael; Baba, Hideo A.; Oberwinkler, Heike; Völker, Katharina; Gaβner, Birgit; Bayer, Barbara; Abeβer, Marco; Schuh, Kai; Feil, Robert; Hofmann, Franz; Kuhn, Michaela

2013-01-01

Aims Cardiac hypertrophy is a common and often lethal complication of arterial hypertension. Elevation of myocyte cyclic GMP levels by local actions of endogenous atrial natriuretic peptide (ANP) and C-type natriuretic peptide (CNP) or by pharmacological inhibition of phosphodiesterase-5 was shown to counter-regulate pathological hypertrophy. It was suggested that cGMP-dependent protein kinase I (cGKI) mediates this protective effect, although the role in vivo is under debate. Here, we investigated whether cGKI modulates myocyte growth and/or function in the intact organism. Methods and results To circumvent the systemic phenotype associated with germline ablation of cGKI, we inactivated the murine cGKI gene selectively in cardiomyocytes by Cre/loxP-mediated recombination. Mice with cardiomyocyte-restricted cGKI deletion exhibited unaltered cardiac morphology and function under resting conditions. Also, cardiac hypertrophic and contractile responses to β-adrenoreceptor stimulation by isoprenaline (at 40 mg/kg/day during 1 week) were unaltered. However, angiotensin II (Ang II, at 1000 ng/kg/min for 2 weeks) or transverse aortic constriction (for 3 weeks) provoked dilated cardiomyopathy with marked deterioration of cardiac function. This was accompanied by diminished expression of the [Ca2+]i-regulating proteins SERCA2a and phospholamban (PLB) and a reduction in PLB phosphorylation at Ser16, the specific target site for cGKI, resulting in altered myocyte Ca2+i homeostasis. In isolated adult myocytes, CNP, but not ANP, stimulated PLB phosphorylation, Ca2+i-handling, and contractility via cGKI. Conclusion These results indicate that the loss of cGKI in cardiac myocytes compromises the hypertrophic program to pathological stimulation, rendering the heart more susceptible to dysfunction. In particular, cGKI mediates stimulatory effects of CNP on myocyte Ca2+i handling and contractility. PMID:22199120

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

High-fat diet-induced juvenile obesity leads to cardiomyocyte dysfunction and upregulation of Foxo3a transcription factor independent of lipotoxicity and apoptosis.

PubMed

Relling, David P; Esberg, Lucy B; Fang, Cindy X; Johnson, W Thomas; Murphy, Eric J; Carlson, Edward C; Saari, Jack T; Ren, Jun

2006-03-01

Obesity is associated with dyslipidemia, which leads to elevated triglyceride and ceramide levels, apoptosis and compromised cardiac function. To determine the role of high-fat diet-induced obesity on cardiomyocyte function, weanling male Sprague-Dawley rats were fed diets incorporating 10% of kcal or 45% of kcal from fat. Mechanical function of ventricular myocytes was evaluated including peak shortening (PS), time-to-PS (TPS), time-to-90% relengthening (TR90) and maximal velocity of shortening and relengthening (+/- dl/dt). Intracellular Ca properties were assessed using fluorescent microscopy. High-fat diet induced hyperinsulinemic insulin-resistant obesity with depressed PS, +/- dl/dt, prolonged TPS/TR90 reduced intracellular Ca release and Ca clearing rate in the absence of hypertension, diabetes, lipotoxicity and apoptosis. Myocyte responsiveness to increased stimulus frequency and extracellular Ca was compromised. SERCA2a and phospholamban levels were increased, whereas phosphorylated phospholamban and potassium channel (Kv1,2) were reduced in high-fat diet group. High-fat diet upregulated the forkhead transcription factor Foxo3a, and suppressed mitochondrial aconitase activity without affecting expression of the caloric sensitive gene silent information regulator 2 (Sir2), protein nitrotyrosine formation, lipid peroxidation and apoptosis. Levels of endothelial nitric oxide synthase (NOS), inducible NOS, triglycerides and ceramide were similar between the two groups. Collectively, our data show that high-fat diet-induced obesity resulted in impaired cardiomyocyte function, upregulated Foxo3a transcription factor and mitochondrial damage without overt lipotoxicity or apoptosis.

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

Requirement of MEF2A, C, and D for skeletal muscle regeneration

PubMed Central

Liu, Ning; Nelson, Benjamin R.; Bezprozvannaya, Svetlana; Shelton, John M.; Richardson, James A.; Bassel-Duby, Rhonda; Olson, Eric N.

2014-01-01

Regeneration of adult skeletal muscle following injury occurs through the activation of satellite cells, an injury-sensitive muscle stem cell population that proliferates, differentiates, and fuses with injured myofibers. Members of the myocyte enhancer factor 2 (MEF2) family of transcription factors play essential roles in muscle differentiation during embryogenesis, but their potential contributions to adult muscle regeneration have not been systematically explored. To investigate the potential involvement of MEF2 factors in muscle regeneration, we conditionally deleted the Mef2a, c, and d genes, singly and in combination, within satellite cells in mice, using tamoxifen-inducible Cre recombinase under control of the satellite cell-specific Pax7 promoter. We show that deletion of individual Mef2 genes has no effect on muscle regeneration in response to cardiotoxin injury. However, combined deletion of the Mef2a, c, and d genes results in a blockade to regeneration. Satellite cell-derived myoblasts lacking MEF2A, C, and D proliferate normally in culture, but cannot differentiate. The absence of MEF2A, C, and D in satellite cells is associated with aberrant expression of a broad collection of known and unique protein-coding and long noncoding RNA genes. These findings reveal essential and redundant roles of MEF2A, C, and D in satellite cell differentiation and identify a MEF2-dependent transcriptome associated with skeletal muscle regeneration. PMID:24591619

Lamina-associated polypeptide 2alpha loss impairs heart function and stress response in mice.

PubMed

Gotic, Ivana; Leschnik, Michael; Kolm, Ursula; Markovic, Mato; Haubner, Bernhard J; Biadasiewicz, Katarzyna; Metzler, Bernhard; Stewart, Colin L; Foisner, Roland

2010-02-05

Lamina-associated polypeptide (LAP)2alpha is a mammalian chromatin-binding protein that interacts with a fraction of A-type lamins in the nuclear interior. Because mutations in lamins and LAP2alpha lead to cardiac disorders in humans, we hypothesized that these factors may play important roles in heart development and adult tissue homeostasis. We asked whether the presence of LAP2alpha was required for normal cardiac function. To study the molecular mechanisms of the disease, we analyzed heart structure and function in complete and conditional Lap2alpha(-/-) mice as well as Lap2alpha(-/-)/Mdx mutants. Unlike conditional deletion of LAP2alpha in late embryonic striated muscle, its complete knockout caused systolic dysfunction in young mice, accompanied by sporadic fibrosis in old animals, as well as deregulation of major cardiac transcription factors GATA4 and myocyte enhancer factor 2c. Activation of compensatory pathways, including downregulation of beta-adrenergic receptor signaling, resulted in reduced responsiveness of the myocardium to chronic beta-adrenergic stimulation and stalled the progression of LAP2alpha-deficient hearts from hypertrophy toward cardiac failure. Dystrophin deficiency in an Mdx background resulted in a transient rescue of the Lap2alpha(-/-) phenotype. Our data suggest a novel role of LAP2alpha in the maintenance of cardiac function under normal and stress conditions.

The role of luminal Ca2+ in the generation of Ca2+ waves in rat ventricular myocytes

PubMed Central

Lukyanenko, Valeriy; Subramanian, Saisunder; Györke, Inna; Wiesner, Theodore F; Györke, Sandor

1999-01-01

We used confocal Ca2+ imaging and fluo-3 to investigate the transition of localized Ca2+ releases induced by focal caffeine stimulation into propagating Ca2+ waves in isolated rat ventricular myocytes. Self-sustaining Ca2+ waves could be initiated when the cellular Ca2+ load was increased by elevating the extracellular [Ca2+] ([Ca2+]o) and they could also be initiated at normal Ca2+ loads when the sensitivity of the release sites to cytosolic Ca2+ was enhanced by low doses of caffeine. When we prevented the accumulation of extra Ca2+ in the luminal compartment of the sarcoplasmic reticulum (SR) with thapsigargin, focal caffeine pulses failed to trigger self-sustaining Ca2+ waves on elevation of [Ca2+]o. Inhibition of SR Ca2+ uptake by thapsigargin in cells already preloaded with Ca2+ above normal levels did not prevent local Ca2+ elevations from triggering propagating waves. Moreover, wave velocity increased by 20 %. Tetracaine (0·75 mM) caused transient complete inhibition of both local and propagating Ca2+ signals, followed by full recovery of the responses due to increased SR Ca2+ accumulation. Computer simulations using a numerical model with spatially distinct Ca2+ release sites suggested that increased amounts of releasable Ca2+ might not be sufficient to generate self-sustaining Ca2+ waves under conditions of Ca2+ overload unless the threshold of release site Ca2+ activation was set at relatively low levels (< 1·5 μM). We conclude that the potentiation of SR Ca2+ release channels by luminal Ca2+ is an important factor in Ca2+ wave generation. Wave propagation does not require the translocation of Ca2+ from the spreading wave front into the SR. Instead, it relies on luminal Ca2+ sensitizing Ca2+ release channels to cytosolic Ca2+. PMID:10373699

Transgenic over-expression of YY1 induces pathologic cardiac hypertrophy in a sex-specific manner

PubMed Central

Stauffer, Brian L.; Dockstader, Karen; Russell, Gloria; Hijmans, Jamie; Walker, Lisa; Cecil, Mackenzie; Demos-Davies, Kimberly; Medway, Allen; McKinsey, Timothy A.; Sucharov, Carmen C.

2015-01-01

YY1 can activate or repress transcription of various genes. In cardiac myocytes in culture YY1 has been shown to regulate expression of several genes involved in myocyte pathology. YY1 can also acutely protect the heart against detrimental changes in gene expression. In this study we show that cardiac over-expression of YY1 induces pathologic cardiac hypertrophy in male mice, measured by changes in gene expression and lower ejection fraction/fractional shortening. In contrast, female animals are protected against pathologic gene expression changes and cardiac dysfunction. Furthermore, we show that YY1 regulates, in a sex-specific manner, the expression of mammalian enable (Mena), a factor that regulates cytoskeletal actin dynamics and whose expression is increased in several models of cardiac pathology, and that Mena expression in humans with heart failure is sex-dependent. Finally, we show that sex differences in YY1 expression are also observed in human heart failure. In summary, this is the first work to show that YY1 has a sex-specific effect in the regulation of cardiac pathology. PMID:25935483

Selective inhibition of miR-92 in hippocampal neurons alters contextual fear memory.

PubMed

Vetere, Gisella; Barbato, Christian; Pezzola, Silvia; Frisone, Paola; Aceti, Massimiliano; Ciotti, MariaTeresa; Cogoni, Carlo; Ammassari-Teule, Martine; Ruberti, Francesca

2014-12-01

Post-transcriptional gene regulation mediated by microRNAs (miRNAs) is implicated in memory formation; however, the function of miR-92 in this regulation is uncharacterized. The present study shows that training mice in contextual fear conditioning produces a transient increase in miR-92 levels in the hippocampus and decreases several miR-92 gene targets, including: (i) the neuronal Cl(-) extruding K(+) Cl(-) co-transporter 2 (KCC2) protein; (ii) the cytoplasmic polyadenylation protein (CPEB3), an RNA-binding protein regulator of protein synthesis in neurons; and (iii) the transcription factor myocyte enhancer factor 2D (MEF2D), one of the MEF2 genes which negatively regulates memory-induced structural plasticity. Selective inhibition of endogenous miR-92 in CA1 hippocampal neurons, by a sponge lentiviral vector expressing multiple sequences imperfectly complementary to mature miR-92 under the control of the neuronal specific synapsin promoter, leads to up-regulation of KCC2, CPEB3 and MEF2D, impairs contextual fear conditioning, and prevents a memory-induced increase in the spine density. Taken together, the results indicate that neuronal-expressed miR-92 is an endogenous fine regulator of contextual fear memory in mice. © 2014 Wiley Periodicals, Inc.

Cardiac arrhythmia mechanisms in rats with heart failure induced by pulmonary hypertension

PubMed Central

Benoist, David; Stones, Rachel; Drinkhill, Mark J.; Benson, Alan P.; Yang, Zhaokang; Cassan, Cecile; Gilbert, Stephen H.; Saint, David A.; Cazorla, Olivier; Steele, Derek S.; Bernus, Olivier

2012-01-01

Pulmonary hypertension provokes right heart failure and arrhythmias. Better understanding of the mechanisms underlying these arrhythmias is needed to facilitate new therapeutic approaches for the hypertensive, failing right ventricle (RV). The aim of our study was to identify the mechanisms generating arrhythmias in a model of RV failure induced by pulmonary hypertension. Rats were injected with monocrotaline to induce either RV hypertrophy or failure or with saline (control). ECGs were measured in conscious, unrestrained animals by telemetry. In isolated hearts, electrical activity was measured by optical mapping and myofiber orientation by diffusion tensor-MRI. Sarcoplasmic reticular Ca2+ handling was studied in single myocytes. Compared with control animals, the T-wave of the ECG was prolonged and in three of seven heart failure animals, prominent T-wave alternans occurred. Discordant action potential (AP) alternans occurred in isolated failing hearts and Ca2+ transient alternans in failing myocytes. In failing hearts, AP duration and dispersion were increased; conduction velocity and AP restitution were steeper. The latter was intrinsic to failing single myocytes. Failing hearts had greater fiber angle disarray; this correlated with AP duration. Failing myocytes had reduced sarco(endo)plasmic reticular Ca2+-ATPase activity, increased sarcoplasmic reticular Ca2+-release fraction, and increased Ca2+ spark leak. In hypertrophied hearts and myocytes, dysfunctional adaptation had begun, but alternans did not develop. We conclude that increased electrical and structural heterogeneity and dysfunctional sarcoplasmic reticular Ca2+ handling increased the probability of alternans, a proarrhythmic predictor of sudden cardiac death. These mechanisms are potential therapeutic targets for the correction of arrhythmias in hypertensive, failing RVs. PMID:22427523

Direct, Differential Effects of Tamoxifen, 4-Hydroxytamoxifen, and Raloxifene on Cardiac Myocyte Contractility and Calcium Handling

PubMed Central

Asp, Michelle L.; Martindale, Joshua J.; Metzger, Joseph M.

2013-01-01

Tamoxifen (Tam), a selective estrogen receptor modulator, is in wide clinical use for the treatment and prevention of breast cancer. High Tam doses have been used for treatment of gliomas and cancers with multiple drug resistance, but long QT Syndrome is a side effect. Tam is also used experimentally in mice for inducible gene knockout in numerous tissues, including heart; however, the potential direct effects of Tam on cardiac myocyte mechanical function are not known. The goal of this study was to determine the direct, acute effects of Tam, its active metabolite 4-hydroxytamoxifen (4OHT), and related drug raloxifene (Ral) on isolated rat cardiac myocyte mechanical function and calcium handling. Tam decreased contraction amplitude, slowed relaxation, and decreased Ca2+ transient amplitude. Effects were primarily observed at 5 and 10 μM Tam, which is relevant for high dose Tam treatment in cancer patients as well as Tam-mediated gene excision in mice. Myocytes treated with 4OHT responded similarly to Tam-treated cells with regard to both contractility and calcium handling, suggesting an estrogen-receptor independent mechanism is responsible for the effects. In contrast, Ral increased contraction and Ca2+ transient amplitudes. At 10 μM, all drugs had a time-dependent effect to abolish cellular contraction. In conclusion, Tam, 4OHT, and Ral adversely and differentially alter cardiac myocyte contractility and Ca2+ handling. These findings have important implications for understanding the Tam-induced cardiomyopathy in gene excision studies and may be important for understanding effects on cardiac performance in patients undergoing high-dose Tam therapy. PMID:24205315

Minocycline suppresses oxidative stress and attenuates fetal cardiac myocyte apoptosis triggered by in utero cocaine exposure

PubMed Central

Sinha-Hikim, Indrani; Shen, Ruoqing; Nzenwa, Ify; Gelfand, Robert; Mahata, Sushil K.

2015-01-01

This study investigates the molecular mechanisms by which minocycline, a second generation tetracycline, prevents cardiac myocyte death induced by in utero cocaine exposure. Timed mated pregnant Sprague-Dawley (SD) rats received one of the following treatments twice daily from embryonic (E) day 15–21 (E15–E21): (i) intraperitoneal (IP) injections of saline (control); (ii) IP injections of cocaine (15 mg/kg BW); and (iii) IP injections of cocaine + oral administration of 25 mg/kg BW of minocycline. Pups were killed on postnatal day 15 (P15). Additional pregnant dams received twice daily IP injections of cocaine (from E15–E21) + oral administration of a relatively higher (37.5 mg/kg BW) dose of minocycline. Minocycline treatment continued from E15 until the pups were sacrificed on P15. In utero cocaine exposure resulted in an increase in oxidative stress and fetal cardiac myocyte apoptosis through activation of c-Jun-NH2-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK)-mediated mitochondria-dependent apoptotic pathway. Continued minocycline treatment from E15 through P15 significantly prevented oxidative stress, kinase activation, perturbation of BAX/BCL-2 ratio, cytochrome c release, caspase activation, and attenuated fetal cardiac myocyte apoptosis after prenatal cocaine exposure. These results demonstrate in vivo cardioprotective effects of minocycline in preventing fetal cardiac myocyte death after prenatal cocaine exposure. Given its proven clinical safety and ability to cross the placental barrier and enter into the fetal circulation, minocycline may be an effective therapy for preventing cardiac consequences of in utero cocaine exposure. PMID:21424555

Heuristic problems in defining the three-dimensional arrangement of the ventricular myocytes.

PubMed

Anderson, Robert H; Ho, Siew Yen; Sanchez-Quintana, Damian; Redmann, Klaus; Lunkenheimer, Paul P

2006-06-01

There is lack of consensus concerning the three-dimensional arrangement of the myocytes within the ventricular muscle masses. Bioengineers are seeking to model the structure of the heart. Although the success of such models depends on the accuracy of the anatomic evidence, most of them have been based on concepts that are far from anatomical reality, which ignore many significant previous accounts of anatomy presented over the past 400 years. During the 19th century, Pettigrew emphasized that the heart was built on the basis of a modified blood vessel rather than in the form of skeletal muscles. This fact was reemphasized by Lev and Simkins as well as Grant in the 20th century, but the caveats listed by these authors have been ignored by proponents of two current concepts, which state either that the myocardium is arranged in the form of a "unique myocardial band," or that the walls of the ventricles are sequestrated in uniform fashion by laminar sheets of fibrous tissue extending from epicardium to endocardium. These two concepts are themselves incompatible and are further at variance with the majority of anatomic studies, which have emphasized the regional heterogeneity to be found in the three-dimensional packing of the myocytes within a supporting matrix of fibrous tissue. We reemphasize the significance of this three-dimensional muscular mesh, showing how the presence of intruding aggregates of myocytes extending in oblique transmural fashion also contends against the notion that all myocytes are orientated with their long axes parallel to the epicardial and enodcardial surfaces.

Cardioprotection by controlling hyperamylinemia in a "humanized" diabetic rat model.

PubMed

Despa, Sanda; Sharma, Savita; Harris, Todd R; Dong, Hua; Li, Ning; Chiamvimonvat, Nipavan; Taegtmeyer, Heinrich; Margulies, Kenneth B; Hammock, Bruce D; Despa, Florin

2014-08-21

Chronic hypersecretion of the pancreatic hormone amylin is common in humans with obesity or prediabetic insulin resistance and induces amylin aggregation and proteotoxicity in the pancreas. We recently showed that hyperamylinemia also affects the cardiovascular system. Here, we investigated whether amylin aggregates interact directly with cardiac myocytes and whether controlling hyperamylinemia protects the heart. By Western blot, we found abundant amylin aggregates in lysates of cardiac myocytes from obese patients, but not in controls. Aggregated amylin was elevated in failing hearts, suggesting a role in myocyte injury. Using rats overexpressing human amylin in the pancreas (HIP rats) and control myocytes incubated with human amylin, we show that amylin aggregation at the sarcolemma induces oxidative stress and Ca(2+) dysregulation. In time, HIP rats developed cardiac hypertrophy and left-ventricular dilation. We then tested whether metabolites with antiaggregation properties, such as eicosanoid acids, limit myocardial amylin deposition. Rats were treated with an inhibitor of soluble epoxide hydrolase, the enzyme that degrades endogenous eicosanoids. Treatment doubled the blood concentration of eicosanoids, which drastically reduced incorporation of aggregated amylin in cardiac myocytes and blood cells, without affecting pancreatic amylin secretion. Animals in the treated group showed reduced cardiac hypertrophy and left-ventricular dilation. The cardioprotective mechanisms included the mitigation of amylin-induced cardiac oxidative stress and Ca(2+) dysregulation. The results suggest blood amylin as a novel therapeutic target in diabetic heart disease and elevating blood levels of antiaggregation metabolites as a pharmacological strategy to reduce amylin aggregation and amylin-mediated cardiotoxicity. © 2014 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley Blackwell.

Long-term hypoxia increases calcium affinity of BK channels in ovine fetal and adult cerebral artery smooth muscle

PubMed Central

Tao, Xiaoxiao; Lin, Mike T.; Thorington, Glyne U.; Wilson, Sean M.; Longo, Lawrence D.

2015-01-01

Acclimatization to high-altitude, long-term hypoxia (LTH) reportedly alters cerebral artery contraction-relaxation responses associated with changes in K+ channel activity. We hypothesized that to maintain oxygenation during LTH, basilar arteries (BA) in the ovine adult and near-term fetus would show increased large-conductance Ca2+ activated potassium (BK) channel activity. We measured BK channel activity, expression, and cell surface distribution by use of patch-clamp electrophysiology, flow cytometry, and confocal microscopy, respectively, in myocytes from normoxic control and LTH adult and near-term fetus BA. Electrophysiological data showed that BK channels in LTH myocytes exhibited 1) lowered Ca2+ set points, 2) left-shifted activation voltages, and 3) longer dwell times. BK channels in LTH myocytes also appeared to be more dephosphorylated. These differences collectively make LTH BK channels more sensitive to activation. Studies using flow cytometry showed that the LTH fetus exhibited increased BK β1 subunit surface expression. In addition, in both fetal groups confocal microscopy revealed increased BK channel clustering and colocalization to myocyte lipid rafts. We conclude that increased BK channel activity in LTH BA occurred in association with increased channel affinity for Ca2+ and left-shifted voltage activation. Increased cerebrovascular BK channel activity may be a mechanism by which LTH adult and near-term fetal sheep can acclimatize to long-term high altitude hypoxia. Our findings suggest that increasing BK channel activity in cerebral myocytes may be a therapeutic target to ameliorate the adverse effects of high altitude in adults or of intrauterine hypoxia in the fetus. PMID:25599571

Microscopic and ultrastructural changes of Müller's muscle in patients with simple congenital ptosis.

PubMed

Alshehri, Mohammed D; Al-Fakey, Yasser H; Alkhalidi, Hisham M; Mubark, Mohamed A; Alsuhaibani, Adel H

2014-01-01

To study microscopic and ultrastructural changes of Müller's muscle in patients with isolated congenital ptosis. In this prospective, observational case-control study, Müller's muscle specimens were collected during ptosis surgical correction for 18 consecutive patients. Each specimen was divided into 2 parts. One part was embedded in formalin for light microscopy, and the other one was fixed in 3% glutaraldehyde for electron microscopy. A neuropathologist, serving as a masked evaluator, blindly reviewed all the different features for every case and counted the number of myocytes showing distinct myofilaments in the whole grid for every case. Statistical analysis using compare means and correlation tests was conducted to investigate potential associations and/or differences within and across groups. Twelve Müller's muscle specimens from patients with simple congenital ptosis of various severities and 6 specimens from patients with aponeurotic ptosis (controls) were collected and studied. Under light microscopy, congenital ptosis slides showed a small number of dispersed myocytes in a fibrotic background, whereas acquired ptosis slides showed a greater number of well-defined myocytes. Under electron microscopy, all congenital ptosis specimens had only a very small number of myocytes with clear, distinct myofilaments. Most myocytes in the aponeurotic ptosis group showed clear, distinct myofilaments, indicating a well-preserved muscle. No relationship existed between the number of clear, distinct myofilaments observed in the congenital ptosis group by transmission electron microscopy and patient age or ptosis severity. Substantial Müller's muscle atrophy was observed in patients with different severities of isolated congenital ptosis.

Gene transfer, expression, and sarcomeric incorporation of a headless myosin molecule in cardiac myocytes: evidence for a reserve in myofilament motor function

PubMed Central

Vandenboom, Rene; Herron, Todd; Favre, Elizabeth; Albayya, Faris P.

2011-01-01

The purpose of this study was to implement a living myocyte in vitro model system to test whether a motor domain-deleted headless myosin construct could be incorporated into the sarcomere and affect contractility. To this end we used gene transfer to express a “headless” myosin heavy chain (headless-MHC) in complement with the native full-length myosin motors in the cardiac sarcomere. An NH2-terminal Flag epitope was used for unique detection of the motor domain-deleted headless-MHC. Total MHC content (i.e., headless-MHC + endogenous MHC) remained constant, while expression of the headless-MHC in transduced myocytes increased from 24 to 72 h after gene transfer until values leveled off at 96 h after gene transfer, at which time the headless-MHC comprised ∼20% of total MHC. Moreover, immunofluorescence labeling and confocal imaging confirmed expression and demonstrated incorporation of the headless-MHC in the A band of the cardiac sarcomere. Functional measurements in intact myocytes showed that headless-MHC modestly reduced amplitude of dynamic twitch contractions compared with controls (P < 0.05). In chemically permeabilized myocytes, maximum steady-state isometric force and the tension-pCa relationship were unaltered by the headless-MHC. These data suggest that headless-MHC can express to 20% of total myosin and incorporate into the sarcomere yet have modest to no effects on dynamic and steady-state contractile function. This would indicate a degree of functional tolerance in the sarcomere for nonfunctional myosin molecules. PMID:21112946

Cardioprotective activity of urocortin by preventing caspase-independent, non-apoptotic death in cultured neonatal rat cardiomyocytes exposed to ischemia

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

Takatani-Nakase, Tomoka, E-mail: nakase@mukogawa-u.ac.jp; Takahashi, Koichi, E-mail: koichi@mukogawa-u.ac.jp

Research highlights: {yields} Ischemia induces high level of iPLA{sub 2} resulting in caspase-independent myocyte death. {yields} Urocortin causes iPLA{sub 2} down-regulation leading to avoidance of non-apoptotic death. {yields} The survival-promoting effect of urocortin is abrogated by CRH receptor antagonist. -- Abstract: Caspase-independent, non-apoptotic cell death in ischemic heart disease is considered to be one of the important therapeutic targets, however, the detailed mechanisms of this cell death process are not clear. In this study, we investigated the mechanisms of non-apoptotic cell death in cultured neonatal rat cardiomyocytes during ischemia, and the cardioprotection by preventing the mechanisms. We found that ischemiamore » caused elevation of the phospholipase A{sub 2} (iPLA{sub 2}) expression in the myocytes, leading to distinctive non-apoptotic nuclear shrinkage, and cell death. Moreover, we investigated whether the potent cardioprotective corticotropin-releasing hormone (CRH), urocortin, which had been less focused on non-apoptotic cell death, inhibits the ischemic myocyte death. Ischemia-augmented nuclear shrinkage of the myocytes was suppressed by the pretreatment of {approx}10 nM urocortin before the cells were exposed to ischemia. Urocortin could significantly suppress the expression and activity of iPLA{sub 2}, resulting in preventing the ischemia-induced cell death. The survival-promoting effect of urocortin was abrogated by the CRH receptor antagonist astressin. These findings provide the first evidence linking the targets of the urocortin-mediated cardioprotection to the suppression of the caspase-independent, non-apoptotic death in cardiac myocytes exposed to ischemia.« less

The Interplay of Rogue and Clustered Ryanodine Receptors Regulates Ca2+ Waves in Cardiac Myocytes.

PubMed

Chen, Xudong; Feng, Yundi; Huo, Yunlong; Tan, Wenchang

2018-01-01

Ca 2+ waves in cardiac myocytes can lead to arrhythmias owing to delayed after-depolarisations. Based on Ca 2+ regulation from the junctional sarcoplasmic reticulum (JSR), a mathematical model was developed to investigate the interplay of clustered and rogue RyRs on Ca 2+ waves. The model successfully reproduces Ca 2+ waves in cardiac myocytes, which are in agreement with experimental results. A new wave propagation mode of "spark-diffusion-quark-spark" is put forward. It is found that rogue RyRs greatly increase the initiation of Ca 2+ sparks, further contribute to the formation and propagation of Ca 2+ waves when the free Ca 2+ concentration in JSR lumen ([Ca 2+ ] lumen ) is higher than a threshold value of 0.7 mM. Computational results show an exponential increase in the velocity of Ca 2+ waves with [Ca 2+ ] lumen . In addition, more CRUs of rogue RyRs and Ca 2+ release from rogue RyRs result in higher velocity and amplitude of Ca 2+ waves. Distance between CRUs significantly affects the velocity of Ca 2+ waves, but not the amplitude. This work could improve understanding the mechanism of Ca 2+ waves in cardiac myocytes.

The Interplay of Rogue and Clustered Ryanodine Receptors Regulates Ca2+ Waves in Cardiac Myocytes

PubMed Central

Chen, Xudong; Feng, Yundi; Huo, Yunlong; Tan, Wenchang

2018-01-01

Ca2+ waves in cardiac myocytes can lead to arrhythmias owing to delayed after-depolarisations. Based on Ca2+ regulation from the junctional sarcoplasmic reticulum (JSR), a mathematical model was developed to investigate the interplay of clustered and rogue RyRs on Ca2+ waves. The model successfully reproduces Ca2+ waves in cardiac myocytes, which are in agreement with experimental results. A new wave propagation mode of “spark-diffusion-quark-spark” is put forward. It is found that rogue RyRs greatly increase the initiation of Ca2+ sparks, further contribute to the formation and propagation of Ca2+ waves when the free Ca2+ concentration in JSR lumen ([Ca2+]lumen) is higher than a threshold value of 0.7 mM. Computational results show an exponential increase in the velocity of Ca2+ waves with [Ca2+]lumen. In addition, more CRUs of rogue RyRs and Ca2+ release from rogue RyRs result in higher velocity and amplitude of Ca2+ waves. Distance between CRUs significantly affects the velocity of Ca2+ waves, but not the amplitude. This work could improve understanding the mechanism of Ca2+ waves in cardiac myocytes. PMID:29755362

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

Cyclin D2 induces proliferation of cardiac myocytes and represses hypertrophy

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

Busk, Peter K.; Hinrichsen, Rebecca; Bartkova, Jirina

2005-03-10

The myocytes of the adult mammalian heart are considered unable to divide. Instead, mitogens induce cardiomyocyte hypertrophy. We have investigated the effect of adenoviral overexpression of cyclin D2 on myocyte proliferation and morphology. Cardiomyocytes in culture were identified by established markers. Cyclin D2 induced DNA synthesis and proliferation of cardiomyocytes and impaired hypertrophy induced by angiotensin II and serum. At the molecular level, cyclin D2 activated CDK4/6 and lead to pRB phosphorylation and downregulation of the cell cycle inhibitors p21{sup Waf1/Cip1} and p27{sup Kip1}. Expression of the CDK4/6 inhibitor p16 inhibited proliferation and cyclin D2 overexpressing myocytes became hypertrophic undermore » such conditions. Inhibition of hypertrophy by cyclin D2 correlated with downregulation of p27{sup Kip1}. These data show that hypertrophy and proliferation are highly related processes and suggest that cardiomyocyte hypertrophy is due to low amounts of cell cycle activators unable to overcome the block imposed by cell cycle inhibitors. Cell cycle entry upon hypertrophy may be converted to cell division by increased expression of activators such as cyclin D2.« less

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.

Long-term ingestion of Hibiscus sabdariffa calyx extract enhances myocardial capillarization in the spontaneously hypertensive rat.

PubMed

Inuwa, Ibrahim; Ali, Badreldin H; Al-Lawati, Intisar; Beegam, Sumaya; Ziada, Amal; Blunden, Gerald

2012-05-01

The effects of Hibiscus sabdariffa (HS) in lowering blood pressure in human and animal hypertension have been documented. This study investigated the effect of the water extract of the dried calyx of HS and Hibiscus anthocyanins (HAs) on left ventricular myocardial capillary length and surface area in spontaneously hypertensive rats (SHRs). Twelve-week-old male SHRs were divided into eight groups (six rats in each group). Three groups were given three doses; 10%, 15% and 20% of the water extract of HS in lieu of drinking water for 10 consecutive weeks (HS10, HS15 and HS20) with one group kept as control (C). Another three groups were given three doses of the HAs orally at doses of 50, 100 and 200 mg/kg for five consecutive days with one group kept as a control (C). Systolic (SBP) and diastolic (DBP) blood pressures, as well as heart rate (HR), were measured weekly. After the experimental protocols, the left ventricles (LV) of all rats were obtained. Capillary surface area density and length density were determined by unbiased sterological methods on 3 μm LV tissue samples from perfusion-fixed hearts. HS ingestion significantly reduced SBP, DBP and LV mass in a dose-dependent fashion but did not affect the HR. HS significantly increased surface area and length density of myocardial capillaries by 59%, 65% and 86%, and length density by 57%, 77% and 57%, respectively. Myocyte nuclear volume was significantly decreased in HS-treated rats. There was a decrease (although insignificant) in SBP and DBP with HA ingestion compared with controls. These changes suggest that the observed beneficial effect of HS on high BP in SHRs could be mediated through a reduction in the diffusion distance between capillaries and myocytes, as well as new vessel formation. It is proposed that these effects might be beneficial in restoring myocyte normal nutritional status compromised by the hypertrophic state of hypertension.

Total internal reflectance fluorescence imaging of genetically engineered ryanodine receptor-targeted Ca2+ probes in rat ventricular myocytes.

PubMed

Pahlavan, Sara; Morad, Marin

2017-09-01

The details of cardiac Ca 2+ signaling within the dyadic junction remain unclear because of limitations in rapid spatial imaging techniques, and availability of Ca 2+ probes localized to dyadic junctions. To critically monitor ryanodine receptors' (RyR2) Ca 2+ nano-domains, we combined the use of genetically engineered RyR2-targeted pericam probes, (FKBP-YCaMP, K d =150nM, or FKBP-GCaMP6, K d =240nM) with rapid total internal reflectance fluorescence (TIRF) microscopy (resolution, ∼80nm). The punctate z-line patterns of FKBP, 2 -targeted probes overlapped those of RyR2 antibodies and sharply contrasted to the images of probes targeted to sarcoplasmic reticulum (SERCA2a/PLB), or cytosolic Fluo-4 images. FKBP-YCaMP signals were too small (∼20%) and too slow (2-3s) to detect Ca 2+ sparks, but the probe was effective in marking where Fluo-4 Ca 2+ sparks developed. FKBP-GCaMP6, on the other hand, produced rapidly decaying Ca 2+ signals that: a) had faster kinetics and activated synchronous with I Ca 3 but were of variable size at different z-lines and b) were accompanied by spatially confined spontaneous Ca 2+ sparks, originating from a subset of eager sites. The frequency of spontaneously occurring sparks was lower in FKBP-GCaMP6 infected myocytes as compared to Fluo-4 dialyzed myocytes, but isoproterenol enhanced their frequency more effectively than in Fluo-4 dialyzed cells. Nevertheless, isoproterenol failed to dissociate FKBP-GCaMP6 from the z-lines. The data suggests that FKBP-GCaMP6 binds predominantly to junctional RyR2s and has sufficient on-rate efficiency as to monitor the released Ca 2+ in individual dyadic clefts, and supports the idea that β-adrenergic agonists may modulate the stabilizing effects of native FKBP on RyR2. Copyright © 2017 Elsevier Ltd. All rights reserved.

Atrial SERCA2a Overexpression Has No Affect on Cardiac Alternans but Promotes Arrhythmogenic SR Ca2+ Triggers

PubMed Central

Nassal, Michelle M. J.; Wan, Xiaoping; Laurita, Kenneth R.; Cutler, Michael J.

2015-01-01

Background Atrial fibrillation (AF) is the most common arrhythmia in humans, yet; treatment has remained sub-optimal due to poor understanding of the underlying mechanisms. Cardiac alternans precede AF episodes, suggesting an important arrhythmia substrate. Recently, we demonstrated ventricular SERCA2a overexpression suppresses cardiac alternans and arrhythmias. Therefore, we hypothesized that atrial SERCA2a overexpression will decrease cardiac alternans and arrhythmias. Methods Adult rat isolated atrial myocytes where divided into three treatment groups 1) Control, 2) SERCA2a overexpression (Ad.SERCA2a) and 3) SERCA2a inhibition (Thapsigargin, 1μm). Intracellular Ca2+ was measured using Indo-1AM and Ca2+ alternans (Ca-ALT) was induced with a standard ramp pacing protocol. Results As predicted, SR Ca2+ reuptake was enhanced with SERCA2a overexpression (p< 0.05) and reduced with SERCA2a inhibition (p<0.05). Surprisingly, there was no difference in susceptibility to Ca-ALT with either SERCA2a overexpression or inhibition when compared to controls (p = 0.73). In contrast, SERCA2a overexpression resulted in increased premature SR Ca2+ (SCR) release compared to control myocytes (28% and 0%, p < 0.05) and concomitant increase in SR Ca2+ load (p<0.05). Based on these observations we tested in-vivo atrial arrhythmia inducibility in control and Ad.SERCA2a animals using an esophageal atrial burst pacing protocol. There were no inducible atrial arrhythmias in Ad.GFP (n = 4) animals though 20% of Ad.SERCA2a (n = 5) animals had inducible atrial arrhythmias (p = 0.20). Conclusions Our findings suggest that unlike the ventricle, SERCA2a is not a key regulator of cardiac alternans in the atrium. Importantly, SERCA2a overexpression in atrial myocytes can increase SCR, which may be arrhythmogenic. PMID:26352986

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

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.

Fine regulation of RhoA and Rock is required for skeletal muscle differentiation.

PubMed

Castellani, Loriana; Salvati, Erica; Alemà, Stefano; Falcone, Germana

2006-06-02

The RhoA GTPase controls a variety of cell functions such as cell motility, cell growth, and gene expression. Previous studies suggested that RhoA mediates signaling inputs that promote skeletal myogenic differentiation. We show here that levels and activity of RhoA protein are down-regulated in both primary avian myoblasts and mouse satellite cells undergoing differentiation, suggesting that a fine regulation of this GTPase is required. In addition, ectopic expression of activated RhoA in primary quail myocytes, but not in mouse myocytes, inhibits accumulation of muscle-specific proteins and cell fusion. By disrupting RhoA signaling with specific inhibitors, we have shown that this GTPase, although required for cell identity in proliferating myoblasts, is not essential for commitment to terminal differentiation and muscle gene expression. Ectopic expression of an activated form of its downstream effector, Rock, impairs differentiation of both avian and mouse myoblasts. Conversely, Rock inhibition with specific inhibitors and small interfering RNA-mediated gene silencing leads to accelerated progression in the lineage and enhanced cell fusion, underscoring a negative regulatory function of Rock in myogenesis. Finally, we have reported that Rock acts independently from RhoA in preventing myoblast exit from the cell cycle and commitment to differentiation and may receive signaling inputs from Raf-1 kinase.

Woodpecker drumming behavior is linked to the elevated expression of genes that encode calcium handling proteins in the neck musculature.

PubMed

Schuppe, Eric R; Petersen, John O; Fuxjager, Matthew J

2018-05-31

Many animals perform elaborate physical displays for social communication. Identifying molecular mechanisms that co-evolve with these complex behavioral signals can therefore help reveal how forces of selection shape animal design. To study this issue, we examine gene expression profiles in select skeletal muscles that actuate woodpecker drum displays. This remarkable whole-body signal is produced when individuals rapidly hammer their bill against trees. We find that, compared to muscles that play no part in producing this behavior, the main muscle used to drum abundantly expresses two genes that encode proteins that support myocytic calcium (Ca 2+ ) handling dynamics-namely parvalbumin (PV) and sarcoplasmic reticulum Ca 2+ ATPase (SERCA1). Meanwhile, we find no such difference in the expression of another gene similarly vital to Ca 2+ handling, the ryanodine receptor (RYR1). These differences are not present in a non-woodpecker species, which readily produce much slower drum-like movements for foraging (but not social signaling). Our data therefore point to an association between the fast drum displays of woodpeckers and muscle-specific expression of genes whose protein products enhance select aspects of myocytic Ca 2+ handling. © 2018. Published by The Company of Biologists Ltd.

Skeletal muscle inflammation and insulin resistance in obesity.

PubMed

Wu, Huaizhu; Ballantyne, Christie M

2017-01-03

Obesity is associated with chronic inflammation, which contributes to insulin resistance and type 2 diabetes mellitus. Under normal conditions, skeletal muscle is responsible for the majority of insulin-stimulated whole-body glucose disposal; thus, dysregulation of skeletal muscle metabolism can strongly influence whole-body glucose homeostasis and insulin sensitivity. Increasing evidence suggests that inflammation occurs in skeletal muscle in obesity and is mainly manifested by increased immune cell infiltration and proinflammatory activation in intermyocellular and perimuscular adipose tissue. By secreting proinflammatory molecules, immune cells may induce myocyte inflammation, adversely regulate myocyte metabolism, and contribute to insulin resistance via paracrine effects. Increased influx of fatty acids and inflammatory molecules from other tissues, particularly visceral adipose tissue, can also induce muscle inflammation and negatively regulate myocyte metabolism, leading to insulin resistance.

Skeletal muscle inflammation and insulin resistance in obesity

PubMed Central

Wu, Huaizhu; Ballantyne, Christie M.

2017-01-01

Obesity is associated with chronic inflammation, which contributes to insulin resistance and type 2 diabetes mellitus. Under normal conditions, skeletal muscle is responsible for the majority of insulin-stimulated whole-body glucose disposal; thus, dysregulation of skeletal muscle metabolism can strongly influence whole-body glucose homeostasis and insulin sensitivity. Increasing evidence suggests that inflammation occurs in skeletal muscle in obesity and is mainly manifested by increased immune cell infiltration and proinflammatory activation in intermyocellular and perimuscular adipose tissue. By secreting proinflammatory molecules, immune cells may induce myocyte inflammation, adversely regulate myocyte metabolism, and contribute to insulin resistance via paracrine effects. Increased influx of fatty acids and inflammatory molecules from other tissues, particularly visceral adipose tissue, can also induce muscle inflammation and negatively regulate myocyte metabolism, leading to insulin resistance. PMID:28045398

IGF-I binding and receptor signal transduction in primary cell culture of muscle cells of gilthead sea bream: changes throughout in vitro development.

PubMed

Montserrat, N; Sánchez-Gurmaches, J; García de la Serrana, D; Navarro, M I; Gutiérrez, J

2007-12-01

We examined the possibility of culturing muscle cells of gilthead sea bream in vitro and assessed variations in insulin-like growth factor-I (IGF-I) binding during myocyte development. The viability of the cell culture was determined by fluorescence-activated cell-sorting analysis, which showed that the percentage of dead cells decreased with cell differentiation. The intracellular reduction of MTT into formazan pigment was preferentially carried out as cells differentiated (from day 4) indicating an increase in metabolic activity. IGF-I-binding assays demonstrated that the number of receptors increased from 190 +/- 0.09 fmol/mg protein in myocytes at day 5 to 360 +/- 0.09 fmol/mg protein in myotubes at day 12. The affinity of IGF-I receptors did not change significantly during cell development (from 0.89 +/- 0.09 to 0.98 +/- 0.09 nM). The activation of various kinase (ERK 1/2 MAPK and Akt/PKB) proteins by IGFs and insulin was studied by means of Western blot analysis. Levels of MAPK-P increased after IGF and insulin treatment during the first stages of cell culture, with a low response being observed at day 15, whereas IGFs displayed a stimulatory effect on Akt-P throughout the cell culture period, even on day 15. This study thus shows that (1) gilthead sea bream myocytes can be cultured, (2) they express functional IGF-I receptors that increase in number as they differentiate in vitro; (3) IGF signalling transduction through IGF-I receptors stimulates the MAPK and Akt pathways, depending on the development stage of the muscle cell culture.

Role of STAT3 in Angiotensin II-Induced Hypertension and Cardiac Remodeling Revealed by Mice Lacking STAT3 Serine 727 Phosphorylation

PubMed Central

Zouein, Fouad A.; Zgheib, Carlos; Hamza, Shereen; Fuseler, John W.; Hall, John E.; Soljancic, Andrea; Lopez-Ruiz, Arnaldo; Kurdi, Mazen; Booz, George W.

2013-01-01

STAT3 is involved in protection of the heart provided by ischemic preconditioning. However, the role of this transcription factor in the heart in chronic stresses such as hypertension has not been defined. We assessed whether STAT3 is important in hypertension-induced cardiac remodeling using mice with reduced STAT3 activity due to a S727A mutation (SA/SA). Wild type (WT) and SA/SA mice received angiotensin (ANG) II or saline for 17 days. ANG II increased mean arterial and systolic pressure in SA/SA and WT mice, but cardiac levels of cytokines associated with heart failure were increased less in SA/SA mice. Unlike WT mice, hearts of SA/SA mice showed signs of developing systolic dysfunction as evidenced by reduction in ejection fraction and fractional shortening. In the left ventricle of both WT and SA/SA mice, ANG II induced fibrosis. However, fibrosis in SA/SA mice appeared more extensive and was associated with loss of myocytes. Cardiac hypertrophy as indexed by heart to body weight ratio and left ventricular anterior wall dimension during diastole was greater in WT mice. In WT+ANG II mice there was an increase in the mass of individual myofibrils. In contrast, cardiac myocytes of SA/SA+ANG II mice showed a loss in myofibrils and myofibrillar mass density was decreased during ANG II infusion. Our findings reveal that STAT3 transcriptional activity is important for normal cardiac myocyte myofibril morphology. Loss of STAT3 may impair cardiac function in the hypertensive heart due to defective myofibrillar structure and remodeling that may lead to heart failure. PMID:23364341

Plants and their bioactive compounds with the potential to enhance mechanisms of inherited cardiac regeneration.

PubMed

Zhou, Zhen; Li, Dianbin; Zhou, Hua; Lin, Xiaoli; Li, Censing; Tang, Mingfeng; Feng, Zhou; Li, Ming

2015-06-01

This article reviews the current progress and research indications in the application of natural plant compounds with the potential for the treatment of cardiovascular diseases. Our understanding of how to apply natural plant compounds to enhance mechanisms of inherited cardiac regeneration, which is physiologically pertinent to myocyte turnover or minor cardiac repair, for substantial cardiac regeneration to repair pathological heart injuries is discussed. Although significant progress has been made in the application of natural plant compounds for therapy of heart diseases, the understanding or the application of these compounds specifically for enhancing mechanisms of inherited cardiac regeneration for the treatment of cardiovascular diseases is little. Recent recognition of some natural plant compounds that can repair damaged myocardial tissues through enhancing mechanisms of inherited cardiac regeneration has offered an alternative for clinical translation. Application of natural plant compounds, which show the activity of manipulating gene expressions in such a way to enhance mechanisms of inherited cardiac regeneration for cardiac repair, may provide a promising strategy for the reconstruction of damaged cardiac tissues due to cardiovascular diseases. Georg Thieme Verlag KG Stuttgart · New York.

Caffeic acid methyl and ethyl esters exert potential antidiabetic effects on glucose and lipid metabolism in cultured murine insulin-sensitive cells through mechanisms implicating activation of AMPK.

PubMed

Eid, Hoda M; Thong, Farah; Nachar, Abir; Haddad, Pierre S

2017-12-01

Caffeic acid methyl (CAME) and ethyl (CAEE) esters stimulate glucose uptake and AMP-activated protein kinase (AMPK) in C2C12 myocytes (ATCC ® CRL-1772 TM ). Effects of CAME and CAEE were now assessed on myocyte glucose transporter GLUT4 activity and expression, on hepatic gluconeogenesis and on adipogenesis as well as major underlying signaling pathways. GLUT4 protein translocation was studied in L6 GLUT4myc cells, glucose-6-phospatase (G6Pase) in H4IIE hepatocytes and adipogenesis in 3T3-L1 adipocytes. Key modulators were measured using western immunoblot. Cells were treated for 18 h with either CAME or CAEE at various concentrations (12.5-100 μM). Myocyte glucose uptake rose from 10.1 ± 0.5 to 18.7 ± 0.8 and 21.9 ± 1.0 pmol/min/mg protein in DMSO-, CAME- and CAEE-stimulated cells, respectively, similar to insulin (17.7 ± 1.2 pmol/min/mg protein), while GLUT4myc translocation increased significantly by 1.70 ± 0.18, by 1.73 ± 0.18- and by 1.95 ± 0.30-fold (relative to DMSO), following insulin, CAME and CAEE stimulation, respectively. CAME and CAEE suppressed hepatocyte G6Pase by 62.0 ± 6.9% and 62.7 ± 6.0% with IC 50 of 45.93 and 22.64 μM, respectively, comparable to insulin (70.7 ± 2.3% inhibition). Finally, CAME and CAEE almost abrogated adipogenesis (83.3 ± 7.2% and 97.3 ± 3.0% at 100 μM; IC 50 of 13.8 and 12.9 μM, respectively). The compounds inhibited adipogenic factors C/EBP-β and PPAR-γ and stimulated AMPK activity in the three cell-lines. CAME and CAEE exerted antidiabetic activities in insulin-responsive cells through insulin-independent mechanisms involving AMPK and adipogenic factors.

Genetically engineered cardiac pacemaker: Stem cells transfected with HCN2 gene and myocytes—A model

NASA Astrophysics Data System (ADS)

Kanani, S.; Pumir, A.; Krinsky, V.

2008-01-01

One of the successfully tested methods to design genetically engineered cardiac pacemaker cells consists in transfecting a human mesenchymal stem cell (hMSC) with a HCN2 gene and connecting it to a myocyte. We develop and study a mathematical model, describing a myocyte connected to a hMSC transfected with a HCN2 gene. The cardiac action potential is described both with the simple Beeler Reuter model, as well as with the elaborate dynamic Luo Rudy model. The HCN2 channel is described by fitting electrophysiological records, in the spirit of Hodgkin Huxley. The model shows that oscillations can occur in a pair myocyte-stem cell, that was not observed in the experiments yet. The model predicted that: (1) HCN pacemaker channels can induce oscillations only if the number of expressed I channels is low enough. At too high an expression level of I channels, oscillations cannot be induced, no matter how many pacemaker channels are expressed. (2) At low expression levels of I channels, a large domain of values in the parameter space (n, N) exists, where oscillations should be observed. We denote N the number of expressed pacemaker channels in the stem cell, and n the number of gap junction channels coupling the stem cell and the myocyte. (3) The expression levels of I channels observed in ventricular myocytes, both in the Beeler Reuter and in the dynamic Luo Rudy models are too high to allow to observe oscillations. With expression levels below ˜1/4 of the original value, oscillations can be observed. The main consequence of this work is that in order to obtain oscillations in an experiment with a myocyte-stem cell pair, increasing the values of n, N is unlikely to be helpful, unless the expression level of I has been reduced enough. The model also allows us to explore levels of gene expression not yet achieved in experiments, and could be useful to plan new experiments, aimed at improving the robustness of the oscillations.

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

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

A slow-releasing form of prostacyclin agonist (ONO1301SR) enhances endogenous secretion of multiple cardiotherapeutic cytokines and improves cardiac function in a rapid-pacing-induced model of canine heart failure.

PubMed

Shirasaka, Tomonori; Miyagawa, Shigeru; Fukushima, Satsuki; Saito, Atsuhiro; Shiozaki, Motoko; Kawaguchi, Naomasa; Matsuura, Nariaki; Nakatani, Satoshi; Sakai, Yoshiki; Daimon, Takashi; Okita, Yutaka; Sawa, Yoshiki

2013-08-01

Cardiac functional deterioration in dilated cardiomyopathy (DCM) is known to be reversed by intramyocardial up-regulation of multiple cardioprotective factors, whereas a prostacyclin analog, ONO1301, has been shown to paracrinally activate interstitial cells to release a variety of protective factors. We here hypothesized that intramyocardial delivery of a slow-releasing form of ONO1301 (ONO1301SR) might activate regional myocardium to up-regulate cardiotherapeutic factors, leading to regional and global functional recovery in DCM. ONO1301 elevated messenger RNA and protein level of hepatocyte growth factor, vascular endothelial growth factor, and stromal-derived factor-1 of normal human dermal fibroblasts in a dose-dependent manner in vitro. Intramyocardial delivery of ONO1301SR, which is ONO1301 mixed with polylactic and glycolic acid polymer (PLGA), but not that of PLGA only, yielded significant global functional recovery in a canine rapid pacing-induced DCM model, assessed by echocardiography and cardiac catheterization (n = 5 each). Importantly, speckle-tracking echocardiography unveiled significant regional functional recovery in the ONO1301-delivered territory, consistent to significantly increased vascular density, reduced interstitial collagen accumulation, attenuated myocyte hypertrophy, and reversed mitochondrial structure in the corresponding area. Intramyocardial delivery of ONO1301SR, which is a PLGA-coated slow-releasing form of ONO1301, up-regulated multiple cardiotherapeutic factors in the injected territory, leading to region-specific reverse left ventricular remodeling and consequently a global functional recovery in a rapid-pacing-induced canine DCM model, warranting a further preclinical study to optimize this novel drug-delivery system to treat DCM. Copyright © 2013 The American Association for Thoracic Surgery. Published by Mosby, Inc. All rights reserved.

Multimodality of Ca2+ signaling in rat atrial myocytes.

PubMed

Morad, Martin; Javaheri, Ashkan; Risius, Tim; Belmonte, Steve

2005-06-01

It has been suggested that the multiplicity of Ca(2+) signaling pathways in atrial myocytes may contribute to the variability of its function. This article reports on a novel Ca(2+) signaling cascade initiated by mechanical forces induced by "puffing" of solution onto the myocytes. Ca(i) transients were measured in fura-2 acetoxymethyl (AM) loaded cells using alternating 340- and 410-nm excitation waves at 1.2 kHz. Pressurized puffs of bathing solutions, applied by an electronically controlled micro-barrel system, activated slowly (approximately 300 ms) developing Ca(i) transients that lasted 1,693 +/- 68 ms at room temperature. Subsequent second and third puffs, applied at approximately 20 s intervals activated significantly smaller or no Ca(i) transients. Puff-triggered Ca(i) transients could be reactivated once again following caffeine (10 mM)-induced release of Ca(2+) from sarcoplasmic reticulum (SR). Puff-triggered Ca(i) transients were independent of [Ca(2+)](o), and activation of voltage-gated Ca(2+) or cationic stretch channels or influx of Ca(2+) on Na(+)/Ca(2+)exchanger, because puffing solution containing no Ca(2+), 10 microM diltiazem, 1 mM Cd(2+), 5 mM Ni(2+), or 100 microM Gd(3+) failed to suppress them. Puff-triggered Ca(i) transients were enhanced in paced compared to quiescent myocytes. Electrically activated Ca(i) transients triggered during the time course of puff-induced transients were unaltered, suggesting functionally separate Ca(2+) pools. Contribution of inositol 1,4,5-triphosphate (IP(3))-gated or mitochondrial Ca(2+) pools or modulation of SR stores by nitric oxide/nitric oxide synthase (NO/NOS) signaling were evaluated using 0.5 to 500 microM 2-aminoethoxydiphenyl borate (2-APB) and 0.1 to 1 microM carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP), and 1 mM Nomega-Nitro-L-arginine methyl ester (L-NAME) and 7-nitroindizole, respectively. Only FCCP appeared to significantly suppress the puff-triggered Ca(i) transients. It was concluded that neither Ca(2+) influx nor depolarization was required for activation of this signaling pathway. These studies suggest that pressurized puffs of solutions activate a mechanically sensitive receptor, which signals in turn the release of Ca(2+) from a limited Ca(2+) store of mitochondria. How mechanical forces are sensed and transmitted to mitochondria to induce Ca(2+) release and what role such a Ca(2+) signaling pathway plays in the physiology or pathophysiology of the heart remain to be worked out.

Rate-dependent Ca2+ signalling underlying the force-frequency response in rat ventricular myocytes: a coupled electromechanical modeling study

PubMed Central

2013-01-01

Background Rate-dependent effects on the Ca2+ sub-system in a rat ventricular myocyte are investigated. Here, we employ a deterministic mathematical model describing various Ca2+ signalling pathways under voltage clamp (VC) conditions, to better understand the important role of calmodulin (CaM) in modulating the key control variables Ca2+/calmodulin-dependent protein kinase-II (CaMKII), calcineurin (CaN), and cyclic adenosine monophosphate (cAMP) as they affect various intracellular targets. In particular, we study the frequency dependence of the peak force generated by the myofilaments, the force-frequency response (FFR). Methods Our cell model incorporates frequency-dependent CaM-mediated spatially heterogenous interaction of CaMKII and CaN with their principal targets (dihydropyridine (DHPR) and ryanodine (RyR) receptors and the SERCA pump). It also accounts for the rate-dependent effects of phospholamban (PLB) on the SERCA pump; the rate-dependent role of cAMP in up-regulation of the L-type Ca2+ channel (ICa,L); and the enhancement in SERCA pump activity via phosphorylation of PLB. Results Our model reproduces positive peak FFR observed in rat ventricular myocytes during voltage-clamp studies both in the presence/absence of cAMP mediated β-adrenergic stimulation. This study provides quantitative insight into the rate-dependence of Ca2+-induced Ca2+-release (CICR) by investigating the frequency-dependence of the trigger current (ICa,L) and RyR-release. It also highlights the relative role of the sodium-calcium exchanger (NCX) and the SERCA pump at higher frequencies, as well as the rate-dependence of sarcoplasmic reticulum (SR) Ca2+ content. A rigorous Ca2+ balance imposed on our investigation of these Ca2+ signalling pathways clarifies their individual roles. Here, we present a coupled electromechanical study emphasizing the rate-dependence of isometric force developed and also investigate the temperature-dependence of FFR. Conclusions Our model provides mechanistic biophysically based explanations for the rate-dependence of CICR, generating useful and testable hypotheses. Although rat ventricular myocytes exhibit a positive peak FFR in the presence/absence of beta-adrenergic stimulation, they show a characteristic increase in the positive slope in FFR due to the presence of Norepinephrine or Isoproterenol. Our study identifies cAMP-mediated stimulation, and rate-dependent CaMKII-mediated up-regulation of ICa,L as the key mechanisms underlying the aforementioned positive FFR. PMID:24020888

Metoprolol Inhibits Cardiac Apoptosis and Fibrosis in a Canine Model of Chronic Obstructive Sleep Apnea.

PubMed

Li, Wenpeng; Yan, Sen; Zhao, Jing; Ding, Xue; Zhang, Song; Wang, Dingyu; Liu, Lei; Peng, Wenpeng; Li, Hui; Wang, Dongyang; Liu, Zhaorui; Li, Yue

2015-01-01

Emerging evidence suggested that obstructive sleep apnea (OSA) was independently associated with the development of heart failure. In this study, we explored the influence of chronic OSA on left ventricular structural remodeling in canines, and the potential therapeutical role of metoprolol. Chronic OSA model was established by stopping the ventilator and closing the airway for 4 h/day apnea-ventilation cycles every other day for 12 weeks while metoprolol (5 mg· kg(-1)· day(-1)) were administered continuously. Norepinephrine concentration was measured by Enzyme Linked Immunosorbent Assay. Transmission electron microscopy, Hematoxylin and eosin, TUNEL and Masson trichrome staining were employed to detect the morphology, apoptosis and fibrosis of cardiomyocytes. Protein expression of apoptosis and fibrosis-related factors including apoptosis-inducing factor (AIF), caspase 3, Bcl-2, Bax, α-smooth muscle actin (α-SMA), transforming growth factor-β1 (TGF-β1), and p38 mitogen-activated protein kinase (MAPK) were examined by Western blotting. Norepinephrine concentration was markedly increased in chronic OSA dogs and reduced by metoprolol. Both the apoptotic ratio and collagen volume fraction were significantly increased in left ventricular myocytes of chronic OSA dogs, and was reversed by metoprolol. Moreover, chronic OSA-induced upregulation of AIF, cleaved caspase 3, Bax, α-SMA, and TGF-β1 as well as downregulation of Bcl-2 was markedly recovered by metoprolol, which was mediated by p38 MAPK. Metoprolol protects against chronic OSA-induced cardiac apoptosis and fibrosis in left ventricular myocytes of canines, which may provide new potential strategy for drug therapy of OSA. © 2015 S. Karger AG, Basel.

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

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

Formation of functional gap junctions in amniotic fluid-derived stem cells induced by transmembrane co-culture with neonatal rat cardiomyocytes

PubMed Central

Connell, Jennifer Petsche; Augustini, Emily; Moise, Kenneth J; Johnson, Anthony; Jacot, Jeffrey G

2013-01-01

Amniotic fluid-derived stem cells (AFSC) have been reported to differentiate into cardiomyocyte-like cells and form gap junctions when directly mixed and cultured with neonatal rat ventricular myocytes (NRVM). This study investigated whether or not culture of AFSC on the opposite side of a Transwell membrane from NRVM, allowing for contact and communication without confounding factors such as cell fusion, could direct cardiac differentiation and enhance gap junction formation. Results were compared to shared media (Transwell), conditioned media and monoculture media controls. After a 2-week culture period, AFSC did not express cardiac myosin heavy chain or troponin T in any co-culture group. Protein expression of cardiac calsequestrin 2 was up-regulated in direct transmembrane co-cultures and media control cultures compared to the other experimental groups, but all groups were up-regulated compared with undifferentiated AFSC cultures. Gap junction communication, assessed with a scrape-loading dye transfer assay, was significantly increased in direct transmembrane co-cultures compared to all other conditions. Gap junction communication corresponded with increased connexin 43 gene expression and decreased phosphorylation of connexin 43. Our results suggest that direct transmembrane co-culture does not induce cardiomyocyte differentiation of AFSC, though calsequestrin expression is increased. However, direct transmembrane co-culture does enhance connexin-43-mediated gap junction communication between AFSC. PMID:23634988

Predicting changes in cardiac myocyte contractility during early drug discovery with in vitro assays

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

Morton, M.J., E-mail: michael.morton@astrazeneca.com; Armstrong, D.; Abi Gerges, N.

2014-09-01

Cardiovascular-related adverse drug effects are a major concern for the pharmaceutical industry. Activity of an investigational drug at the L-type calcium channel could manifest in a number of ways, including changes in cardiac contractility. The aim of this study was to define which of the two assay technologies – radioligand-binding or automated electrophysiology – was most predictive of contractility effects in an in vitro myocyte contractility assay. The activity of reference and proprietary compounds at the L-type calcium channel was measured by radioligand-binding assays, conventional patch-clamp, automated electrophysiology, and by measurement of contractility in canine isolated cardiac myocytes. Activity inmore » the radioligand-binding assay at the L-type Ca channel phenylalkylamine binding site was most predictive of an inotropic effect in the canine cardiac myocyte assay. The sensitivity was 73%, specificity 83% and predictivity 78%. The radioligand-binding assay may be run at a single test concentration and potency estimated. The least predictive assay was automated electrophysiology which showed a significant bias when compared with other assay formats. Given the importance of the L-type calcium channel, not just in cardiac function, but also in other organ systems, a screening strategy emerges whereby single concentration ligand-binding can be performed early in the discovery process with sufficient predictivity, throughput and turnaround time to influence chemical design and address a significant safety-related liability, at relatively low cost. - Highlights: • The L-type calcium channel is a significant safety liability during drug discovery. • Radioligand-binding to the L-type calcium channel can be measured in vitro. • The assay can be run at a single test concentration as part of a screening cascade. • This measurement is highly predictive of changes in cardiac myocyte contractility.« less

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.

TRPM2 Channels Protect against Cardiac Ischemia-Reperfusion Injury

PubMed Central

Miller, Barbara A.; Hoffman, Nicholas E.; Merali, Salim; Zhang, Xue-Qian; Wang, JuFang; Rajan, Sudarsan; Shanmughapriya, Santhanam; Gao, Erhe; Barrero, Carlos A.; Mallilankaraman, Karthik; Song, Jianliang; Gu, Tongda; Hirschler-Laszkiewicz, Iwona; Koch, Walter J.; Feldman, Arthur M.; Madesh, Muniswamy; Cheung, Joseph Y.

2014-01-01

Cardiac TRPM2 channels were activated by intracellular adenosine diphosphate-ribose and blocked by flufenamic acid. In adult cardiac myocytes the ratio of GCa to GNa of TRPM2 channels was 0.56 ± 0.02. To explore the cellular mechanisms by which TRPM2 channels protect against cardiac ischemia/reperfusion (I/R) injury, we analyzed proteomes from WT and TRPM2 KO hearts subjected to I/R. The canonical pathways that exhibited the largest difference between WT-I/R and KO-I/R hearts were mitochondrial dysfunction and the tricarboxylic acid cycle. Complexes I, III, and IV were down-regulated, whereas complexes II and V were up-regulated in KO-I/R compared with WT-I/R hearts. Western blots confirmed reduced expression of the Complex I subunit and other mitochondria-associated proteins in KO-I/R hearts. Bioenergetic analyses revealed that KO myocytes had a lower mitochondrial membrane potential, mitochondrial Ca2+ uptake, ATP levels, and O2 consumption but higher mitochondrial superoxide levels. Additionally, mitochondrial Ca2+ uniporter (MCU) currents were lower in KO myocytes, indicating reduced mitochondrial Ca2+ uptake was likely due to both lower ψm and MCU activity. Similar to isolated myocytes, O2 consumption and ATP levels were also reduced in KO hearts. Under a simulated I/R model, aberrant mitochondrial bioenergetics was exacerbated in KO myocytes. Reactive oxygen species levels were also significantly higher in KO-I/R compared with WT-I/R heart slices, consistent with mitochondrial dysfunction in KO-I/R hearts. We conclude that TRPM2 channels protect the heart from I/R injury by ameliorating mitochondrial dysfunction and reducing reactive oxygen species levels. PMID:24492610

[Changes in polarization of myometrial cells plasma and internal mitochondrial membranes under calixarenes action as inhibitors of plasma membrane Na+, K+-ATPase].

PubMed

Danylovych, H V; Danylovych, Iu V; Kolomiiets', O V; Kosterin, S O; Rodik, R V; Cherenok, S O; Kal'chenko, V I; Chunikhin, O Iu; Horchev, V F; Karakhim, S O

2012-01-01

The influence of supramolecular macrocyclic compounds--calix[4]arenes C-97, C-99, C-107, which are ouabainomymetic high affinity inhibitors of Na+, K(+)-ATPase, on the polarization level of plasmic and mitochondrial membranes of rat uterine smooth muscle cells was investigated. The influence of these compounds on the myocytes characteristic size was studied. By using a confocal microscopy and specific for mitochondrial MitoTracker Orange CM-H2TMRos dye it was proved that the potential-sensitive fluorescent probe DiOC6(3) interacts with mitochondria. Artificial potential collapse of plasmic membrane in this case was modeled by myocytes preincubation with ouabain (1 mM). Further experiments performed using the method of flow cytometry with DiOC6(3) have shown that the compounds C-97, C-99 and C-107 at concentration 50-100 nM caused depolarization of the plasma membrane (at the level of 30% relative to control values) in conditions of artificial collapse of mitochondrial potential by myocytes preincubation in the presence of 5 mM of sodium azide. Under artificial sarcolemma depolarization by ouabain, calixarenes C-97, C-99 and C-107 at 100 nM concentrations caused a transient increase of mitochondrial membrane potential, that is 40% of the control level and lasted about 5 minutes. Calixarenes C-99 and C-107 caused a significant increase in fluorescence of myocytes in these conditions, which was confirmed by confocal microscopy too. It was proved by photon correlation spectroscopy method that the C-99 and C-107 caused an increase of characteristic size of myocytes.

Minocycline suppresses oxidative stress and attenuates fetal cardiac myocyte apoptosis triggered by in utero cocaine exposure.

PubMed

Sinha-Hikim, Indrani; Shen, Ruoqing; Nzenwa, Ify; Gelfand, Robert; Mahata, Sushil K; Sinha-Hikim, Amiya P

2011-06-01

This study investigates the molecular mechanisms by which minocycline, a second generation tetracycline, prevents cardiac myocyte death induced by in utero cocaine exposure. Timed mated pregnant Sprague-Dawley (SD) rats received one of the following treatments twice daily from embryonic (E) day 15-21 (E15-E21): (i) intraperitoneal (IP) injections of saline (control); (ii) IP injections of cocaine (15 mg/kg BW); and (iii) IP injections of cocaine + oral administration of 25 mg/kg BW of minocycline. Pups were killed on postnatal day 15 (P15). Additional pregnant dams received twice daily IP injections of cocaine (from E15-E21) + oral administration of a relatively higher (37.5 mg/kg BW) dose of minocycline. Minocycline treatment continued from E15 until the pups were sacrificed on P15. In utero cocaine exposure resulted in an increase in oxidative stress and fetal cardiac myocyte apoptosis through activation of c-Jun-NH(2)-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK)-mediated mitochondria-dependent apoptotic pathway. Continued minocycline treatment from E15 through P15 significantly prevented oxidative stress, kinase activation, perturbation of BAX/BCL-2 ratio, cytochrome c release, caspase activation, and attenuated fetal cardiac myocyte apoptosis after prenatal cocaine exposure. These results demonstrate in vivo cardioprotective effects of minocycline in preventing fetal cardiac myocyte death after prenatal cocaine exposure. Given its proven clinical safety and ability to cross the placental barrier and enter into the fetal circulation, minocycline may be an effective therapy for preventing cardiac consequences of in utero cocaine exposure.

L-type Ca(2+) currents overlapping threshold Na(+) currents: could they be responsible for the "slip-mode" phenomenon in cardiac myocytes?

PubMed

Piacentino, Valentino; Gaughan, John P; Houser, Steven R

2002-03-08

Phosphorylation of Na channels has been suggested to increase their Ca permeability. Termed "slip-mode conductance" (SMC), this hypothesis predicts that Ca influx via protein kinase A (PKA)-modified Na channels can induce sarcoplasmic reticulum (SR) Ca release. We tested this hypothesis by determining if SR Ca release is graded with I(Na) in the presence of activated PKA (with Isoproterenol, ISO). V(m), I(m), and [Ca](i) were measured in feline (n=26) and failing human (n=19) ventricular myocytes. Voltage steps from -70 through -40 mV were used to grade I(Na). Na channel antagonists (tetrodotoxin), L-type Ca channel (I(Ca,L)) antagonists (nifedipine, cadmium, verapamil), and agonists (Bay K 8644, FPL 64176) were used to separate SMC from I(Ca,L). In the absence of ISO, I(Na) was associated with SR Ca release in human but not feline myocytes. After ISO, graded I(Na) was associated with small amounts of SR Ca release in feline myocytes and the magnitude of release increased in human myocytes. I(Na)-related SR Ca release was insensitive to tetrodotoxin (n=10) but was blocked by nifedipine (n=10) and cadmium (n=3). SR Ca release was induced over the same voltage range in the absence of ISO with Bay K 8644 and FPL 64176 (n=9). Positive voltage steps (to 0 mV) to fully activate Na channels (SMC) in the presence of ISO and Verapamil only caused SR Ca release when block of I(Ca,L) was incomplete. We conclude that PKA-mediated increases in I(Ca,L) and SR Ca loading can reproduce many of the experimental features of SMC.

Poloxamer 188 protects against ischemia-reperfusion injury in a murine hind-limb model.

PubMed

Murphy, Adrian D; McCormack, Michael C; Bichara, David A; Nguyen, John T; Randolph, Mark A; Watkins, Michael T; Lee, Raphael C; Austen, William G

2010-06-01

Ischemia-reperfusion injury can activate pathways generating reactive oxygen species, which can injure cells by creating holes in the cell membranes. Copolymer surfactants such as poloxamer 188 are capable of sealing defects in cell membranes. The authors postulated that a single-dose administration of poloxamer 188 would decrease skeletal myocyte injury and mortality following ischemia-reperfusion injury. Mice underwent normothermic hind-limb ischemia for 2 hours. Animals were treated with 150 microl of poloxamer 188 or dextran at three time points: (1) 10 minutes before ischemia; (2) 10 minutes before reperfusion; and (3) 2 or 4 hours after reperfusion. After 24 hours of reperfusion, tissues were analyzed for myocyte injury (histology) and metabolic dysfunction (muscle adenosine 5'-triphosphate). Additional groups of mice were followed for 7 days to assess mortality. When poloxamer 188 treatment was administered 10 minutes before ischemia, injury was reduced by 84 percent, from 50 percent injury in the dextran group to 8 percent injury in the poloxamer 188 group (p < 0.001). When administered 10 minutes before reperfusion, poloxamer 188 animals demonstrated a 60 percent reduction in injury compared with dextran controls (12 percent versus 29 percent). Treatment at 2 hours, but not at 4 hours, postinjury prevented substantial myocyte injury. Preservation of muscle adenosine 5'-triphosphate paralleled the decrease in myocyte injury in poloxamer 188-treated animals. Poloxamer 188 treatment significantly reduced mortality following injury (10 minutes before, 75 percent versus 25 percent survival, p = 0.0077; 2 hours after, 50 percent versus 8 percent survival, p = 0.032). Poloxamer 188 administered to animals decreased myocyte injury, preserved tissue adenosine 5'-triphosphate levels, and improved survival following hind-limb ischemia-reperfusion injury.

Long-Term Fish Oil Supplementation Induces Cardiac Electrical Remodeling by Changing Channel Protein Expression in the Rabbit Model

PubMed Central

Xu, Xulin; Jiang, Min; Wang, Yuhong; Smith, Timothy; Baumgarten, Clive M.; Wood, Mark A.; Tseng, Gea-Ny

2010-01-01

Clinical trials and epidemiological studies have suggested that dietary fish oil (FO) supplementation can provide an anti-arrhythmic benefit in some patient populations. The underlying mechanisms are not entirely clear. We wanted to understand how FO supplementation (for 4 weeks) affected the action potential configuration/duration of ventricular myocytes, and the ionic mechanism(s)/molecular basis for these effects. The experiments were conducted on adult rabbits, a widely used animal model for cardiac electrophysiology and pathophysiology. We used gas chromatography - mass spectroscopy to confirm that FO feeding produced a marked increase in the content of n-3 polyunsaturated fatty acids in the phospholipids of rabbit hearts. Left ventricular myocytes were used in current and voltage clamp experiments to monitor action potentials and ionic currents, respectively. Action potentials of myocytes from FO-fed rabbits exhibited much more positive plateau voltages and prolonged durations. These changes could be explained by an increase in the L-type Ca current (ICaL) and a decrease in the transient outward current (Ito) in these myocytes. FO feeding did not change the delayed rectifier or inward rectifier current. Immunoblot experiments showed that the FO-feeding induced changes in ICaL and Ito were associated with corresponding changes in the protein levels of major pore-forming subunits of these channels: increase in Cav1.2 and decrease in Kv4.2 and Kv1.4. There was no change in other channel subunits (Cav1.1, Kv4.3, KChIP2, and ERG1). We conclude that long-term fish oil supplementation can impact on cardiac electrical activity at least partially by changing channel subunit expression in cardiac myocytes. PMID:20405051

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

Comparison of sarcolemmal calcium channel current in rabbit and rat ventricular myocytes.

PubMed Central

Yuan, W; Ginsburg, K S; Bers, D M

1996-01-01

1. Fundamental properties of Ca2+ channel currents in rat and rabbit ventricular myocytes were measured using whole cell voltage clamp. 2. In rat, as compared with rabbit myocytes, Ca2+ channel current (ICa) was half-activated at about 10 mV more negative potential, decayed slower, was half-inactivated (in steady state) at about 5 mV more positive potential, and recovered faster from inactivation. 3. These features result in a larger steady-state window current in rat, and also suggest that under comparable voltage clamp conditions, including action potential (AP) clamp, more Ca2+ influx would be expected in rat myocytes. 4. Ca2+ channel current carried by Na+ and Cs+ in the absence of divalent ions (Ins) also activated at more negative potential and decayed more slowly in rat. 5. The reversal potential for Ins was 6 mV more positive in rabbit, consistent with a larger permeability ratio (PNa/PCs) in rabbit than in rat. ICa also reversed at slightly more positive potentials in rabbit (such that PCa/PCs might also be higher). 6. Ca2+ influx was calculated by integration of ICa evoked by voltage clamp pulses (either square pulses or pulses based on recorded rabbit or rat APs). For a given clamp waveform, the Ca2+ influx was up to 25% greater in rat, as predicted from the fundamental properties of ICa and Ins. 7. However, the longer duration of the AP in rabbit myocytes compensated for the difference in influx, such that the integrated Ca2+ influx via ICa in response to the species-appropriate waveform was about twice as large as that seen in rat. PMID:8799895

Mitochondrial matrix metalloproteinase activation decreases myocyte contractility in hyperhomocysteinemia.

PubMed

Moshal, Karni S; Tipparaju, Srinivas M; Vacek, Thomas P; Kumar, Munish; Singh, Mahavir; Frank, Iluiana E; Patibandla, Phani K; Tyagi, Neetu; Rai, Jayesh; Metreveli, Naira; Rodriguez, Walter E; Tseng, Michael T; Tyagi, Suresh C

2008-08-01

Cardiomyocyte N-methyl-d-aspartate receptor-1 (NMDA-R1) activation induces mitochondrial dysfunction. Matrix metalloproteinase protease (MMP) induction is a negative regulator of mitochondrial function. Elevated levels of homocysteine [hyperhomocysteinemia (HHCY)] activate latent MMPs and causes myocardial contractile abnormalities. HHCY is associated with mitochondrial dysfunction. We tested the hypothesis that HHCY activates myocyte mitochondrial MMP (mtMMP), induces mitochondrial permeability transition (MPT), and causes contractile dysfunction by agonizing NMDA-R1. The C57BL/6J mice were administered homocystinemia (1.8 g/l) in drinking water to induce HHCY. NMDA-R1 expression was detected by Western blot and confocal microscopy. Localization of MMP-9 in the mitochondria was determined using confocal microscopy. Ultrastructural analysis of the isolated myocyte was determined by electron microscopy. Mitochondrial permeability was measured by a decrease in light absorbance at 540 nm using the spectrophotometer. The effect of MK-801 (NMDA-R1 inhibitor), GM-6001 (MMP inhibitor), and cyclosporine A (MPT inhibitor) on myocyte contractility and calcium transients was evaluated using the IonOptix video edge track detection system and fura 2-AM. Our results demonstrate that HHCY activated the mtMMP-9 and caused MPT by agonizing NMDA-R1. A significant decrease in percent cell shortening, maximal rate of contraction (-dL/dt), and maximal rate of relaxation (+dL/dt) was observed in HHCY. The decay of calcium transient amplitude was faster in the wild type compared with HHCY. Furthermore, the HHCY-induced decrease in percent cell shortening, -dL/dt, and +dL/dt was attenuated in the mice treated with MK-801, GM-6001, and cyclosporin A. We conclude that HHCY activates mtMMP-9 and induces MPT, leading to myocyte mechanical dysfunction by agonizing NMDA-R1.

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.

Optimisation of a Generic Ionic Model of Cardiac Myocyte Electrical Activity

PubMed Central

Guo, Tianruo; Al Abed, Amr; Lovell, Nigel H.; Dokos, Socrates

2013-01-01

A generic cardiomyocyte ionic model, whose complexity lies between a simple phenomenological formulation and a biophysically detailed ionic membrane current description, is presented. The model provides a user-defined number of ionic currents, employing two-gate Hodgkin-Huxley type kinetics. Its generic nature allows accurate reconstruction of action potential waveforms recorded experimentally from a range of cardiac myocytes. Using a multiobjective optimisation approach, the generic ionic model was optimised to accurately reproduce multiple action potential waveforms recorded from central and peripheral sinoatrial nodes and right atrial and left atrial myocytes from rabbit cardiac tissue preparations, under different electrical stimulus protocols and pharmacological conditions. When fitted simultaneously to multiple datasets, the time course of several physiologically realistic ionic currents could be reconstructed. Model behaviours tend to be well identified when extra experimental information is incorporated into the optimisation. PMID:23710254

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.

The Kv7 Channel and Cardiovascular Risk Factors.

PubMed

Fosmo, Andreas L; Skraastad, Øyvind B

2017-01-01

Potassium channels play a pivotal role in the regulation of excitability in cells such as neurons, cardiac myocytes, and vascular smooth muscle cells. The KCNQ (Kv7) family of voltage-activated K + channels hyperpolarizes the cell and stabilizes the membrane potential. Here, we outline how Kv7 channel activity may contribute to the development of the cardiovascular risk factors such as hypertension, diabetes, and obesity. Questions and hypotheses regarding previous and future research have been raised. Alterations in the Kv7 channel may contribute to the development of cardiovascular disease (CVD). Pharmacological modification of Kv7 channels may represent a possible treatment for CVD in the future.

The Kv7 Channel and Cardiovascular Risk Factors

PubMed Central

Fosmo, Andreas L.; Skraastad, Øyvind B.

2017-01-01

Potassium channels play a pivotal role in the regulation of excitability in cells such as neurons, cardiac myocytes, and vascular smooth muscle cells. The KCNQ (Kv7) family of voltage-activated K+ channels hyperpolarizes the cell and stabilizes the membrane potential. Here, we outline how Kv7 channel activity may contribute to the development of the cardiovascular risk factors such as hypertension, diabetes, and obesity. Questions and hypotheses regarding previous and future research have been raised. Alterations in the Kv7 channel may contribute to the development of cardiovascular disease (CVD). Pharmacological modification of Kv7 channels may represent a possible treatment for CVD in the future. PMID:29259974

IGF-1 deficiency causes atrophic changes associated with upregulation of VGluT1 and downregulation of MEF2 transcription factors in the mouse cochlear nuclei.

PubMed

Fuentes-Santamaría, V; Alvarado, J C; Rodríguez-de la Rosa, L; Murillo-Cuesta, S; Contreras, J; Juiz, J M; Varela-Nieto, I

2016-03-01

Insulin-like growth factor 1 (IGF-1) is a neurotrophic protein that plays a crucial role in modulating neuronal function and synaptic plasticity in the adult brain. Mice lacking the Igf1 gene exhibit profound deafness and multiple anomalies in the inner ear and spiral ganglion. An issue that remains unknown is whether, in addition to these peripheral abnormalities, IGF-1 deficiency also results in structural changes along the central auditory pathway that may contribute to an imbalance between excitation and inhibition, which might be reflected in abnormal auditory brainstem responses (ABR). To assess such a possibility, we evaluated the morphological and physiological alterations in the cochlear nucleus complex of the adult mouse. The expression and distribution of the vesicular glutamate transporter 1 (VGluT1) and the vesicular inhibitory transporter (VGAT), which were used as specific markers for labeling excitatory and inhibitory terminals, and the involvement of the activity-dependent myocyte enhancer factor 2 (MEF2) transcription factors in regulating excitatory synapses were assessed in a 4-month-old mouse model of IGF-1 deficiency and neurosensorial deafness (Igf1 (-/-) homozygous null mice). The results demonstrate decreases in the cochlear nucleus area and cell size along with cell loss in the cochlear nuclei of the deficient mouse. Additionally, our results demonstrate that there is upregulation of VGluT1, but not VGAT, immunostaining and downregulation of MEF2 transcription factors together with increased wave II amplitude in the ABR recording. Our observations provide evidence of an abnormal neuronal cytoarchitecture in the cochlear nuclei of Igf1 (-/-) null mice and suggest that the increased efficacy of glutamatergic synapses might be mediated by MEF2 transcription factors.

Mitochondrial complex II is a source of the reserve respiratory capacity that is regulated by metabolic sensors and promotes cell survival.

PubMed

Pfleger, J; He, M; Abdellatif, M

2015-07-30

The survival of a cell depends on its ability to meet its energy requirements. We hypothesized that the mitochondrial reserve respiratory capacity (RRC) of a cell is a critical component of its bioenergetics that can be utilized during an increase in energy demand, thereby, enhancing viability. Our goal was to identify the elements that regulate and contribute to the development of RRC and its involvement in cell survival. The results show that activation of metabolic sensors, including pyruvate dehydrogenase and AMP-dependent kinase, increases cardiac myocyte RRC via a Sirt3-dependent mechanism. Notably, we identified mitochondrial complex II (cII) as a target of these metabolic sensors and the main source of RRC. Moreover, we show that RRC, via cII, correlates with enhanced cell survival after hypoxia. Thus, for the first time, we show that metabolic sensors via Sirt3 maximize the cellular RRC through activating cII, which enhances cell survival after hypoxia.

The absence of 2,3-diphosphoglycerate from myocytes, hepatocytes and adipocytes.

PubMed

Reddy, W J; Burns, A H

1976-04-23

Myocytes, hepatocytes and adipocytes were prepared from heart, liver and epididymal fat pad of the rat. No detectable level of 2,3-diphosphoglycerate was found. Evidence is also presented which indicates the absence from these cells of 2,3-diphosphoglycerate mutase and 2,3-diphosphoglycerate phosphatase. Previous findings by others of the presence of 2,3-diphosphoglycerate and 2,3-diphosphoglycerate mutase probably resulted from erythrocytes sequestered in the tissue.

Crebanine inhibits voltage-dependent Na+ current in guinea-pig ventricular myocytes.

PubMed

Xiao-Shan, He; Qing, Lin; Yun-Shu, Ma; Ze-Pu, Yu

2014-01-01

To study the effects of crebanine on voltage-gated Na(+) channels in cardiac tissues. Single ventricular myocytes were enzymatically dissociated from adult guinea-pig heart. Voltage-dependent Na(+) current was recorded using the whole cell voltage-clamp technique. Crebanine reversibly inhibited Na(+) current with an IC50 value of 0.283 mmol·L(-1) (95% confidence range: 0.248-0.318 mmol·L(-1)). Crebanine at 0.262 mmol·L(-1) caused a negative shift (about 12 mV) in the voltage-dependence of steady-state inactivation of Na(+) current, and retarded its recovery from inactivation, but did not affect its activation curve. In addition to blocking other voltage-gated ion channels, crebanine blocked Na(+) channels in guinea-pig ventricular myocytes. Crebanine acted as an inactivation stabilizer of Na(+) channels in cardiac tissues. Copyright © 2014 China Pharmaceutical University. Published by Elsevier B.V. All rights reserved.

Slow [Na+]i dynamics impacts arrhythmogenesis and spiral wave reentry in cardiac myocyte ionic model.

PubMed

Krogh-Madsen, Trine; Christini, David J

2017-09-01

Accumulation of intracellular Na + is gaining recognition as an important regulator of cardiac myocyte electrophysiology. The intracellular Na + concentration can be an important determinant of the cardiac action potential duration, can modulate the tissue-level conduction of excitation waves, and can alter vulnerability to arrhythmias. Mathematical models of cardiac electrophysiology often incorporate a dynamic intracellular Na + concentration, which changes much more slowly than the remaining variables. We investigated the dependence of several arrhythmogenesis-related factors on [Na + ] i in a mathematical model of the human atrial action potential. In cell simulations, we found that [Na + ] i accumulation stabilizes the action potential duration to variations in several conductances and that the slow dynamics of [Na + ] i impacts bifurcations to pro-arrhythmic afterdepolarizations, causing intermittency between different rhythms. In long-lasting tissue simulations of spiral wave reentry, [Na + ] i becomes spatially heterogeneous with a decreased area around the spiral wave rotation center. This heterogeneous region forms a functional anchor, resulting in diminished meandering of the spiral wave. Our findings suggest that slow, physiological, rate-dependent variations in [Na + ] i may play complex roles in cellular and tissue-level cardiac dynamics.

Slow [Na+]i dynamics impacts arrhythmogenesis and spiral wave reentry in cardiac myocyte ionic model

NASA Astrophysics Data System (ADS)

Krogh-Madsen, Trine; Christini, David J.

2017-09-01

Accumulation of intracellular Na+ is gaining recognition as an important regulator of cardiac myocyte electrophysiology. The intracellular Na+ concentration can be an important determinant of the cardiac action potential duration, can modulate the tissue-level conduction of excitation waves, and can alter vulnerability to arrhythmias. Mathematical models of cardiac electrophysiology often incorporate a dynamic intracellular Na+ concentration, which changes much more slowly than the remaining variables. We investigated the dependence of several arrhythmogenesis-related factors on [Na+]i in a mathematical model of the human atrial action potential. In cell simulations, we found that [Na+]i accumulation stabilizes the action potential duration to variations in several conductances and that the slow dynamics of [Na+]i impacts bifurcations to pro-arrhythmic afterdepolarizations, causing intermittency between different rhythms. In long-lasting tissue simulations of spiral wave reentry, [Na+]i becomes spatially heterogeneous with a decreased area around the spiral wave rotation center. This heterogeneous region forms a functional anchor, resulting in diminished meandering of the spiral wave. Our findings suggest that slow, physiological, rate-dependent variations in [Na+]i may play complex roles in cellular and tissue-level cardiac dynamics.

Curvature effects on activation speed and repolarization in an ionic model of cardiac myocytes

NASA Astrophysics Data System (ADS)

Comtois, P.; Vinet, A.

1999-10-01

Reentry is a major mechanism underlying the initiation and perpetuation of many cardiac arrhythmias 12345. Stimulated ventricular myocytes give action potential characterized by a fast upstroke, a long-lasting plateau, and a late repolarization phase. The plateau phase determines the action potential duration (APD) during which the system remains refractory, a property essential to the synchronization of the heart cycle. The APD varies much with prematurity and this change has been shown to be the main determinant of the dynamics in models of paced cells and cable, and during reentry in the one-dimensional loop. Curvature has also been shown to be an important factor for propagation in experimental and theoretical cardiac extended tissue. The objective of this paper is to combine both curvature and prematurity effects in a kinematical model of propagation in cardiac tissue. First, an approximation of the ionic model is used to obtain the effects of curvature and prematurity on the speed of propagation, the APD, and the absolute refractory period. Two versions of the ionic model are studied that differ in their rate of excitability recovery. The functions are used in a kinematical model describing the propagation of period-1 solutions around an annulus.

AFos Dissociates Cardiac Myocyte Hypertrophy and Expression of the Pathological Gene Program

PubMed Central

Jeong, Mark Y.; Kinugawa, Koichiro; Vinson, Charles; Long, Carlin S.

2005-01-01

Background Although induction of activator protein-1 (AP-1) transcription factor activity has been observed in cardiac hypertrophy, a direct role for AP-1 in myocardial growth and gene expression remains obscure. Methods and Results Hypertrophy was induced in cultured neonatal rat cardiomyocytes with phenylephrine or overexpression of a constitutively active MAP3K, MKK6. In both treatment groups, induction of the pathological gene profile was observed, ie, expression of β-myosin heavy chain (βMHC), atrial/brain natriuretic peptides (ANP/BNP), and skeletal α-actin (sACT) was increased, whereas expression for α-myosin heavy chain (αMHC) and the sarcoplasmic reticulum Ca2+-ATPase (SERCA) genes was repressed. The role of AP-1 in the hypertrophic phenotype was evaluated with the use of an adenoviral construct expressing a dominant negative mutant of the c-Fos proto-oncogene (AdAFos). Although AFos did not change the myocyte growth response, it abrogated the gene profile to both agonists, including the upregulation of both αMHC and SERCA expression. Conclusions Although c-Fos/AP-1 is necessary for induction of the pathological/fetal gene program, it does not appear to be critical for cardiomyocyte hypertrophy. PMID:15795322

Adaptor proteins NUMB and NUMBL promote cell cycle withdrawal by targeting ERBB2 for degradation

PubMed Central

Hirai, Maretoshi; Arita, Yoh; McGlade, C. Jane; Lee, Kuo-Fen; Chen, Ju; Evans, Sylvia M.

2017-01-01

Failure of trabecular myocytes to undergo appropriate cell cycle withdrawal leads to ventricular noncompaction and heart failure. Signaling of growth factor receptor ERBB2 is critical for myocyte proliferation and trabeculation. However, the mechanisms underlying appropriate downregulation of trabecular ERBB2 signaling are little understood. Here, we have found that the endocytic adaptor proteins NUMB and NUMBL were required for downregulation of ERBB2 signaling in maturing trabeculae. Loss of NUMB and NUMBL resulted in a partial block of late endosome formation, resulting in sustained ERBB2 signaling and STAT5 activation. Unexpectedly, activated STAT5 overrode Hippo-mediated inhibition and drove YAP1 to the nucleus. Consequent aberrant cardiomyocyte proliferation resulted in ventricular noncompaction that was markedly rescued by heterozygous loss of function of either ERBB2 or YAP1. Further investigations revealed that NUMB and NUMBL interacted with small GTPase Rab7 to transition ERBB2 from early to late endosome for degradation. Our studies provide insight into mechanisms by which NUMB and NUMBL promote cardiomyocyte cell cycle withdrawal and highlight previously unsuspected connections between pathways that are important for cardiomyocyte cell cycle reentry, with relevance to ventricular noncompaction cardiomyopathy and regenerative medicine. PMID:28067668

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.

Disruption of chaperone-mediated autophagy-dependent degradation of MEF2A by oxidative stress-induced lysosome destabilization

PubMed Central

Zhang, Li; Sun, Yang; Fei, Mingjian; Tan, Cheng; Wu, Jing; Zheng, Jie; Tang, Jiqing; Sun, Wei; Lv, Zhaoliang; Bao, Jiandong; Xu, Qiang; Yu, Huixin

2014-01-01

Oxidative stress has been implicated in both normal aging and various neurodegenerative disorders and it may be a major cause of neuronal death. Chaperone-mediated autophagy (CMA) targets selective cytoplasmic proteins for degradation by lysosomes and protects neurons against various extracellular stimuli including oxidative stress. MEF2A (myocyte enhancer factor 2A), a key transcription factor, protects primary neurons from oxidative stress-induced cell damage. However, the precise mechanisms of how the protein stability and the transcriptional activity of MEF2A are regulated under oxidative stress remain unknown. In this study, we report that MEF2A is physiologically degraded through the CMA pathway. In pathological conditions, mild oxidative stress (200 μM H2O2) enhances the degradation of MEF2A as well as its activity, whereas excessive oxidative stress (> 400 μM H2O2) disrupts its degradation process and leads to the accumulation of nonfunctional MEF2A. Under excessive oxidative stress, an N-terminal HDAC4 (histone deacetylase 4) cleavage product (HDAC4-NT), is significantly induced by lysosomal serine proteases released from ruptured lysosomes in a PRKACA (protein kinase, cAMP-dependent, catalytic, α)-independent manner. The production of HDAC4-NT, as a MEF2 repressor, may account for the reduced DNA-binding and transcriptional activity of MEF2A. Our work provides reliable evidence for the first time that MEF2A is targeted to lysosomes for CMA degradation; oxidative stress-induced lysosome destabilization leads to the disruption of MEF2A degradation as well as the dysregulation of its function. These findings may shed light on the underlying mechanisms of pathogenic processes of neuronal damage in various neurodegenerative-related diseases. PMID:24879151

Impaired Intracellular Ca2+ Dynamics in Live Cardiomyocytes Revealed by Rapid Line Scan Confocal Microscopy

NASA Astrophysics Data System (ADS)

Plank, David M.; Sussman, Mark A.

2005-06-01

Altered intracellular Ca2+ dynamics are characteristically observed in cardiomyocytes from failing hearts. Studies of Ca2+ handling in myocytes predominantly use Fluo-3 AM, a visible light excitable Ca2+ chelating fluorescent dye in conjunction with rapid line-scanning confocal microscopy. However, Fluo-3 AM does not allow for traditional ratiometric determination of intracellular Ca2+ concentration and has required the use of mathematic correction factors with values obtained from separate procedures to convert Fluo-3 AM fluorescence to appropriate Ca2+ concentrations. This study describes methodology to directly measure intracellular Ca2+ levels using inactivated, Fluo-3-AM-loaded cardiomyocytes equilibrated with Ca2+ concentration standards. Titration of Ca2+ concentration exhibits a linear relationship to increasing Fluo-3 AM fluorescence intensity. Images obtained from individual myocyte confocal scans were recorded, average pixel intensity values were calculated, and a plot is generated relating the average pixel intensity to known Ca2+ concentrations. These standard plots can be used to convert transient Ca2+ fluorescence obtained with experimental cells to Ca2+ concentrations by linear regression analysis. Standards are determined on the same microscope used for acquisition of unknown Ca2+ concentrations, simplifying data interpretation and assuring accuracy of conversion values. This procedure eliminates additional equipment, ratiometric imaging, and mathematic correction factors and should be useful to investigators requiring a straightforward method for measuring Ca2+ concentrations in live cells using Ca2+-chelating dyes exhibiting variable fluorescence intensity.

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

Nonmyocyte ERK1/2 signaling contributes to load-induced cardiomyopathy in Marfan mice

PubMed Central

MacFarlane, Elena Gallo; Takimoto, Eiki; Chaudhary, Rahul; Nagpal, Varun; Rainer, Peter P.; Bindman, Julia G.; Gerber, Elizabeth E.; Bedja, Djahida; Schiefer, Christopher; Miller, Karen L.; Zhu, Guangshuo; Myers, Loretha; Amat-Alarcon, Nuria; Lee, Dong I.; Koitabashi, Norimichi; Judge, Daniel P.; Dietz, Harry C.

2017-01-01

Among children with the most severe presentation of Marfan syndrome (MFS), an inherited disorder of connective tissue caused by a deficiency of extracellular fibrillin-1, heart failure is the leading cause of death. Here, we show that, while MFS mice (Fbn1C1039G/+ mice) typically have normal cardiac function, pressure overload (PO) induces an acute and severe dilated cardiomyopathy in association with fibrosis and myocyte enlargement. Failing MFS hearts show high expression of TGF-β ligands, with increased TGF-β signaling in both nonmyocytes and myocytes; pathologic ERK activation is restricted to the nonmyocyte compartment. Informatively, TGF-β, angiotensin II type 1 receptor (AT1R), or ERK antagonism (with neutralizing antibody, losartan, or MEK inhibitor, respectively) prevents load-induced cardiac decompensation in MFS mice, despite persistent PO. In situ analyses revealed an unanticipated axis of activation in nonmyocytes, with AT1R-dependent ERK activation driving TGF-β ligand expression that culminates in both autocrine and paracrine overdrive of TGF-β signaling. The full compensation seen in wild-type mice exposed to mild PO correlates with enhanced deposition of extracellular fibrillin-1. Taken together, these data suggest that fibrillin-1 contributes to cardiac reserve in the face of hemodynamic stress, critically implicate nonmyocytes in disease pathogenesis, and validate ERK as a therapeutic target in MFS-related cardiac decompensation. PMID:28768908

Indirect three-dimensional printing: A method for fabricating polyurethane-urea based cardiac scaffolds.

PubMed

Hernández-Córdova, R; Mathew, D A; Balint, R; Carrillo-Escalante, H J; Cervantes-Uc, J M; Hidalgo-Bastida, L A; Hernández-Sánchez, F

2016-08-01

Biomaterial scaffolds are a key part of cardiac tissue engineering therapies. The group has recently synthesized a novel polycaprolactone based polyurethane-urea copolymer that showed improved mechanical properties compared with its previously published counterparts. The aim of this study was to explore whether indirect three-dimensional (3D) printing could provide a means to fabricate this novel, biodegradable polymer into a scaffold suitable for cardiac tissue engineering. Indirect 3D printing was carried out through printing water dissolvable poly(vinyl alcohol) porogens in three different sizes based on a wood-stack model, into which a polyurethane-urea solution was pressure injected. The porogens were removed, leading to soft polyurethane-urea scaffolds with regular tubular pores. The scaffolds were characterized for their compressive and tensile mechanical behavior; and their degradation was monitored for 12 months under simulated physiological conditions. Their compatibility with cardiac myocytes and performance in novel cardiac engineering-related techniques, such as aggregate seeding and bi-directional perfusion, was also assessed. The scaffolds were found to have mechanical properties similar to cardiac tissue, and good biocompatibility with cardiac myocytes. Furthermore, the incorporated cells preserved their phenotype with no signs of de-differentiation. The constructs worked well in perfusion experiments, showing enhanced seeding efficiency. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 1912-1921, 2016. © 2016 Wiley Periodicals, Inc.

Nonmyocyte ERK1/2 signaling contributes to load-induced cardiomyopathy in Marfan mice.

PubMed

Rouf, Rosanne; MacFarlane, Elena Gallo; Takimoto, Eiki; Chaudhary, Rahul; Nagpal, Varun; Rainer, Peter P; Bindman, Julia G; Gerber, Elizabeth E; Bedja, Djahida; Schiefer, Christopher; Miller, Karen L; Zhu, Guangshuo; Myers, Loretha; Amat-Alarcon, Nuria; Lee, Dong I; Koitabashi, Norimichi; Judge, Daniel P; Kass, David A; Dietz, Harry C

2017-08-03

Among children with the most severe presentation of Marfan syndrome (MFS), an inherited disorder of connective tissue caused by a deficiency of extracellular fibrillin-1, heart failure is the leading cause of death. Here, we show that, while MFS mice (Fbn1C1039G/+ mice) typically have normal cardiac function, pressure overload (PO) induces an acute and severe dilated cardiomyopathy in association with fibrosis and myocyte enlargement. Failing MFS hearts show high expression of TGF-β ligands, with increased TGF-β signaling in both nonmyocytes and myocytes; pathologic ERK activation is restricted to the nonmyocyte compartment. Informatively, TGF-β, angiotensin II type 1 receptor (AT1R), or ERK antagonism (with neutralizing antibody, losartan, or MEK inhibitor, respectively) prevents load-induced cardiac decompensation in MFS mice, despite persistent PO. In situ analyses revealed an unanticipated axis of activation in nonmyocytes, with AT1R-dependent ERK activation driving TGF-β ligand expression that culminates in both autocrine and paracrine overdrive of TGF-β signaling. The full compensation seen in wild-type mice exposed to mild PO correlates with enhanced deposition of extracellular fibrillin-1. Taken together, these data suggest that fibrillin-1 contributes to cardiac reserve in the face of hemodynamic stress, critically implicate nonmyocytes in disease pathogenesis, and validate ERK as a therapeutic target in MFS-related cardiac decompensation.

Low-molecular-weight fucoidan regulates myogenic differentiation through the mitogen-activated protein kinase pathway in C2C12 cells.

PubMed

Kim, Kui-Jin; Lee, Ok-Hwan; Lee, Boo-Yong

2011-12-01

Low-molecular-weight fucoidan (LMWF) has been broadly studied in recent years due to its numerous biological properties. Nevertheless, there have been no reports about the effects of LMWF on myogenic differentiation (MyoD). The objective of the present study was to demonstrate the impact of LMWF on myogenesis in C2C12 cells. The ultimate aim was to establish whether LMWF regulates myogenesis similar to heparin as a partial regulator of myogenesis. LMWF was prepared at a minimal size by ultra-filtration compared with crude fucoidan. We treated C2C12 cells with LMWF and/or heparin during MyoD. The data from the present study are the first to suggest that LMWF suppresses the expression of the myogenic regulatory factors and the myocyte enhancer factors as well as the morphological changes that occur during differentiation. Additionally, the expression of the mitogen-activated protein kinase (MAPK) family was significantly inhibited by LMWF when compared with controls. The LMWF-treated group showed significantly decreased expression of reactive oxygen species (ROS) enzymes compared with control cells. Heparin was used as a positive control because it has been reported to activate MyoD. Taken together, these results suggest that LMWF might regulate MyoD through the MAPK pathway and by regulating ROS activity in C2C12 cells.

CIP, a cardiac Isl1-interacting protein, represses cardiomyocyte hypertrophy.

PubMed

Huang, Zhan-Peng; Young Seok, Hee; Zhou, Bin; Chen, Jinghai; Chen, Jian-Fu; Tao, Yazhong; Pu, William T; Wang, Da-Zhi

2012-03-16

Mammalian heart has minimal regenerative capacity. In response to mechanical or pathological stress, the heart undergoes cardiac remodeling. Pressure and volume overload in the heart cause increased size (hypertrophic growth) of cardiomyocytes. Whereas the regulatory pathways that activate cardiac hypertrophy have been well-established, the molecular events that inhibit or repress cardiac hypertrophy are less known. To identify and investigate novel regulators that modulate cardiac hypertrophy. Here, we report the identification, characterization, and functional examination of a novel cardiac Isl1-interacting protein (CIP). CIP was identified from a bioinformatic search for novel cardiac-expressed genes in mouse embryonic hearts. CIP encodes a nuclear protein without recognizable motifs. Northern blotting, in situ hybridization, and reporter gene tracing demonstrated that CIP is highly expressed in cardiomyocytes of developing and adult hearts. Yeast two-hybrid screening identified Isl1, a LIM/homeodomain transcription factor essential for the specification of cardiac progenitor cells in the second heart field, as a cofactor of CIP. CIP directly interacted with Isl1, and we mapped the domains of these two proteins, which mediate their interaction. We show that CIP represses the transcriptional activity of Isl1 in the activation of the myocyte enhancer factor 2C. The expression of CIP was dramatically reduced in hypertrophic cardiomyocytes. Most importantly, overexpression of CIP repressed agonist-induced cardiomyocyte hypertrophy. Our studies therefore identify CIP as a novel regulator of cardiac hypertrophy.

Unlocking Barriers to DNA Vaccine Immunogenicity: A Cross-Species Analysis of Cytosolic DNA Sensing in Skeletal Muscle Myocytes

DTIC Science & Technology

2017-10-01

CRISPR Subtask 1A: i) design and produce mammalian expression plasmids encoding the Cas9 protein and specially...duration in SOW: 2017 Q4 – 2018 Q1 Subtask 2A: i) produce mouse myocyte cell lines that have undergone gene disruption via a technique named CRISPR ii...named CRISPR ii) confirm gene disruption and GFP expression iii) select multiple individual clones characterized with quantitative gene

Silencing GIRK4 expression in human atrial myocytes by adenovirus-delivered small hairpin RNA.

PubMed

Liu, Xiongtao; Yang, Jian; Shang, Fujun; Hong, Changming; Guo, Wangang; Wang, Bing; Zheng, Qiangsun

2009-07-01

GIRK4 has been shown to be a subunit of I(KACh), and the use of GIRK4 in human atrial myocytes to treat arrhythmia remains an important research pursuit. Adenovirus-delivered small hairpin RNA (shRNA) has been used to mediate gene knockdown in mouse cardiocytes, yet there is no information on the successful application of this technique in human cardiocytes. In the current study, we used a siRNA validation system to select the most efficient sequence for silencing GIRK4. To this end, adenovirus-delivered shRNA, which expresses this sequence, was used to silence GIRK4 expression in human atrial myocytes. Finally, the feasibility, challenges, and results of silencing GIRK4 expression were evaluated by RT-PCR, western blotting, and the voltage-clamp technique. The levels of mRNA and protein were depressed significantly in cells infected by adenovirus-delivered shRNA against GIRK4, approximately 86.3% and 51.1% lower than those cells infected by adenovirus-delivered nonsense shRNA, respectively. At the same time, I(KACh) densities were decreased 53% by adenovirus-delivered shRNA against GIRK4. In summary, adenovirus-delivered shRNA against GIRK4 mediated efficient GIRK4 knockdown in human atrial myocytes and decreased I(KACh) densities. As such, these data indicated that adenovirus-delivered shRNA against GIRK4 is a potential tool for treating arrhythmia.

The role of ZFP580, a novel zinc finger protein, in TGF-mediated cytoprotection against chemical hypoxia-induced apoptosis in H9c2 cardiac myocytes

PubMed Central

Mao, Shi-Yun; Meng, Xiang-Yan; Xu, Zhong-Wei; Zhang, Wen-Cheng; Jin, Xiao-Han; Chen, Xi; Zhou, Xin; Li, Yu-Ming; Xu, Rui-Cheng

2017-01-01

Zing finger protein 580 (ZFP580) is a novel Cys2-His2 zinc-finger transcription factor that has an anti-apoptotic role in myocardial cells. It is involved in the endothelial transforming growth factor-β1 (TGF-β1) signal transduction pathway as a mothers against decapentaplegic homolog (Smad)2 binding partner. The aim of the present study was to determine the involvement of ZFP580 in TGF-β1-mediated cytoprotection against chemical hypoxia-induced apoptosis, using H9c2 cardiac myocytes. Hypoxia was chemically induced in H9c2 myocardial cells by exposure to cobalt chloride (CoCl2). In response to hypoxia, cell viability was decreased, whereas the expression levels of hypoxia inducible factor-1α and ZFP580 were increased. Pretreatment with TGF-β1 attenuated CoCl2-induced cell apoptosis and upregulated ZFP580 protein expression; however, these effects could be suppressed by SB431542, an inhibitor of TGF-β type I receptor and Smad2/3 phosphorylation. Furthermore, suppression of ZFP580 expression by RNA interference reduced the anti-apoptotic effects of TGF-β1 and thus increased CoCl2-induced apoptosis. B-cell lymphoma (Bcl)-2-associated X protein/Bcl-2 ratio, reactive oxygen species generation and caspase-3 activation were also increased following ZFP580 inactivation. In conclusion, these results indicate that ZFP580 is a component of the TGF-β1/Smad signaling pathway, and is involved in the protective effects of TGF-β1 against chemical hypoxia-induced cell apoptosis, through inhibition of the mitochondrial apoptotic pathway. PMID:28259939

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.

GDF11 Does not Rescue Aging-Related Pathological Hypertrophy

PubMed Central

Smith, Shavonn C.; Zhang, Xiaoxiao; Zhang, Xiaoying; Gross, Polina; Starosta, Timothy; Mohsin, Sadia; Franti, Michael; Gupta, Priyanka; Hayes, David; Myzithras, Maria; Kahn, Julius; Tanner, James; Weldon, Steven M.; Khalil, Ashraf; Guo, Xinji; Sabri, Abdelkarim; Chen, Xiongwen; MacDonnell, Scott; Houser, Steven R.

2015-01-01

Rationale GDF11 (Growth Differentiation Factor 11) is a member of the transforming growth factor β (TGFβ) super family of secreted factors. A recent study showed that reduced GDF11 blood levels with aging was associated with pathological cardiac hypertrophy (PCH), and restoring GDF11 to normal levels in old mice rescued PCH. Objective To determine if and by what mechanism GDF11 rescues aging dependent PCH. Methods and Results 24-month-old C57BL/6 mice were given a daily injection of either recombinant (r) GDF11 at 0.1mg/kg or vehicle for 28 days. rGDF11 bioactivity was confirmed in-vitro. After treatment, rGDF11 levels were significantly increased but there was no significant effect on either heart weight (HW) or body weight (BW). HW/BW ratios of old mice were not different from 8 or 12 week-old animals, and the PCH marker ANP was not different in young versus old mice. Ejection fraction, internal ventricular dimension, and septal wall thickness were not significantly different between rGDF11 and vehicle treated animals at baseline and remained unchanged at 1, 2 and 4 weeks of treatment. There was no difference in myocyte cross-sectional area rGDF11 versus vehicle-treated old animals. In vitro studies using phenylephrine-treated neonatal rat ventricular myocytes (NRVM), to explore the putative anti-hypertrophic effects of GDF11, showed that GDF11 did not reduce NRVM hypertrophy, but instead induced hypertrophy. Conclusions Our studies show that there is no age-related PCH in disease free 24-month-old C57BL/6 mice and that restoring GDF11 in old mice has no effect on cardiac structure or function. PMID:26383970

Sex Differences in Muscle Wasting.

PubMed

Anderson, Lindsey J; Liu, Haiming; Garcia, Jose M

2017-01-01

With aging and other muscle wasting diseases, men and women undergo similar pathological changes in skeletal muscle: increased inflammation, enhanced oxidative stress, mitochondrial dysfunction, satellite cell senescence, elevated apoptosis and proteasome activity, and suppressed protein synthesis and myocyte regeneration. Decreased food intake and physical activity also indirectly contribute to muscle wasting. Sex hormones also play important roles in maintaining skeletal muscle homeostasis. Testosterone is a potent anabolic factor promoting muscle protein synthesis and muscular regeneration. Estrogens have a protective effect on skeletal muscle by attenuating inflammation; however, the mechanisms of estrogen action in skeletal muscle are less well characterized than those of testosterone. Age- and/or disease-induced alterations in sex hormones are major contributors to muscle wasting. Hence, men and women may respond differently to catabolic conditions because of their hormonal profiles. Here we review the similarities and differences between men and women with common wasting conditions including sarcopenia and cachexia due to cancer, end-stage renal disease/chronic kidney disease, liver disease, chronic heart failure, and chronic obstructive pulmonary disease based on the literature in clinical studies. In addition, the responses in men and women to the commonly used therapeutic agents and their efficacy to improve muscle mass and function are also reviewed.

Reprogramming of mouse fibroblasts into cardiomyocyte-like cells in vitro.

PubMed

Qian, Li; Berry, Emily C; Fu, Ji-dong; Ieda, Masaki; Srivastava, Deepak

2013-06-01

Cardiac fibroblasts can be reprogrammed to cardiomyocyte-like cells by the introduction of three transcription factors: Gata4, Mef2c and Tbx5 (collectively referred to here as GMT). Resident cardiac fibroblasts can be converted in vivo into induced cardiomyocyte-like cells (iCMs) that closely resemble endogenous cardiomyocytes and electrically integrate with the host myocardium. In contrast, in vitro reprogramming yields many partially reprogrammed iCMs, with a few that reprogram fully into contracting myocytes (~3 out of 10,000 GMT-transduced cells). iCMs can be observed as early as 3 d after viral infection, and they continue to mature over 2 months before beating is observed. Despite the success of multiple groups, the inefficiency of in vitro reprogramming has made it challenging for others. However, given the advantages of in vitro iCMs for performing mechanistic studies and, if refined, for testing drugs or small molecules for personalized medicine and modeling cardiac disease in a dish, it is important to standardize the protocol to improve reproducibility and enhance the technology further. Here we describe a detailed step-by-step protocol for in vitro cardiac reprogramming using retroviruses encoding GMT.

Protein kinase D1 phosphorylates HDAC7 and induces its nuclear export after T-cell receptor activation.

PubMed

Parra, Maribel; Kasler, Herbert; McKinsey, Timothy A; Olson, Eric N; Verdin, Eric

2005-04-08

HDAC7, a class II histone deacetylase that is highly expressed in thymocytes, inhibits both transcription of the orphan steroid nuclear receptor Nur77 and induction of apoptosis in response to activation of the T-cell receptor (TCR). Here, we report that HDAC7 is exported to the cytoplasm by a calcium-independent signaling pathway after TCR activation. Protein kinase D1 (PKD1) was activated after TCR engagement, interacted with HDAC7, and phosphorylated three serines (Ser155, Ser318, and Ser448) at its N terminus, leading to its export from the nucleus. Mutation of Ser155, Ser318, and Ser448 blocked the nucleocytoplasmic shuttling of HDAC7 in response to TCR activation, as did overexpression of a kinase-inactive form of PKD1. Consistent with the regulatory role of HDAC7 in Nur77 expression, PKD1 activation led to the transcriptional activation of Nur77 via myocyte enhancer factor 2-binding sites in its promoter. In a mouse model of negative selection, PKD1 was activated during thymocyte activation. These observations indicate that PKD1 regulates the expression of Nur77 during thymocyte activation at least in part by phosphorylating HDAC7.

Embryonic Stem Cell-Based Cardiopatches Improve Cardiac Function in Infarcted Rats

PubMed Central

Vallée, Jean-Paul; Hauwel, Mathieu; Lepetit-Coiffé, Matthieu; Bei, Wang; Montet-Abou, Karin; Meda, Paolo; Gardier, Stephany; Zammaretti, Prisca; Kraehenbuehl, Thomas P.; Herrmann, Francois; Hubbell, Jeffrey A.

2012-01-01

Pluripotent stem cell-seeded cardiopatches hold promise for in situ regeneration of infarcted hearts. Here, we describe a novel cardiopatch based on bone morphogenetic protein 2-primed cardiac-committed mouse embryonic stem cells, embedded into biodegradable fibrin matrices and engrafted onto infarcted rat hearts. For in vivo tracking of the engrafted cardiac-committed cells, superparamagnetic iron oxide nanoparticles were magnetofected into the cells, thus enabling detection and functional evaluation by high-resolution magnetic resonance imaging. Six weeks after transplantation into infarcted rat hearts, both local (p < .04) and global (p < .015) heart function, as well as the left ventricular dilation (p < .0011), were significantly improved (p < .001) as compared with hearts receiving cardiopatches loaded with iron nanoparticles alone. Histological analysis revealed that the fibrin scaffolds had degraded over time and clusters of myocyte enhancer factor 2-positive cardiac-committed cells had colonized most of the infarcted myocardium, including the fibrotic area. De novo CD31-positive blood vessels were formed in the vicinity of the transplanted cardiopatch. Altogether, our data provide evidence that stem cell-based cardiopatches represent a promising therapeutic strategy to achieve efficient cell implantation and improved global and regional cardiac function after myocardial infarction. PMID:23197784

Stimulation of the cardiac myocyte Na+-K+ pump due to reversal of its constitutive oxidative inhibition

PubMed Central

Chia, Karin K. M.; Liu, Chia-Chi; Hamilton, Elisha J.; Garcia, Alvaro; Fry, Natasha A.; Hannam, William; Figtree, Gemma A.

2015-01-01

Protein kinase C can activate NADPH oxidase and induce glutathionylation of the β1-Na+-K+ pump subunit, inhibiting activity of the catalytic α-subunit. To examine if signaling of nitric oxide-induced soluble guanylyl cyclase (sGC)/cGMP/protein kinase G can cause Na+-K+ pump stimulation by counteracting PKC/NADPH oxidase-dependent inhibition, cardiac myocytes were exposed to ANG II to activate NADPH oxidase and inhibit Na+-K+ pump current (Ip). Coexposure to 3-(5′-hydroxymethyl-2′-furyl)-1-benzylindazole (YC-1) to stimulate sGC prevented the decrease of Ip. Prevention of the decrease was abolished by inhibition of protein phosphatases (PP) 2A but not by inhibition of PP1, and it was reproduced by an activator of PP2A. Consistent with a reciprocal relationship between β1-Na+-K+ pump subunit glutathionylation and pump activity, YC-1 decreased ANG II-induced β1-subunit glutathionylation. The decrease induced by YC-1 was abolished by a PP2A inhibitor. YC-1 decreased phosphorylation of the cytosolic p47phox NADPH oxidase subunit and its coimmunoprecipitation with the membranous p22phox subunit, and it decreased O2·−-sensitive dihydroethidium fluorescence of myocytes. Addition of recombinant PP2A to myocyte lysate decreased phosphorylation of p47phox indicating the subunit could be a substrate for PP2A. The effects of YC-1 to decrease coimmunoprecipitation of p22phox and p47phox NADPH oxidase subunits and decrease β1-Na+-K+ pump subunit glutathionylation were reproduced by activation of nitric oxide-dependent receptor signaling. We conclude that sGC activation in cardiac myocytes causes a PP2A-dependent decrease in NADPH oxidase activity and a decrease in β1 pump subunit glutathionylation. This could account for pump stimulation with neurohormonal oxidative stress expected in vivo. PMID:26084308

The AKAP Cypher/Zasp contributes to β-adrenergic/PKA stimulation of cardiac CaV1.2 calcium channels.

PubMed

Yu, Haijie; Yuan, Can; Westenbroek, Ruth E; Catterall, William A

2018-06-04

Stimulation of the L-type Ca 2+ current conducted by Ca V 1.2 channels in cardiac myocytes by the β-adrenergic/protein kinase A (PKA) signaling pathway requires anchoring of PKA to the Ca V 1.2 channel by an A-kinase anchoring protein (AKAP). However, the AKAP(s) responsible for regulation in vivo remain unknown. Here, we test the role of the AKAP Cypher/Zasp in β-adrenergic regulation of Ca V 1.2 channels using physiological studies of cardiac ventricular myocytes from young-adult mice lacking the long form of Cypher/Zasp (LCyphKO mice). These myocytes have increased protein levels of Ca V 1.2, PKA, and calcineurin. In contrast, the cell surface density of Ca V 1.2 channels and the basal Ca 2+ current conducted by Ca V 1.2 channels are significantly reduced without substantial changes to kinetics or voltage dependence. β-adrenergic regulation of these L-type Ca 2+ currents is also significantly reduced in myocytes from LCyphKO mice, whether calculated as a stimulation ratio or as net-stimulated Ca 2+ current. At 100 nM isoproterenol, the net β-adrenergic-Ca 2+ current conducted by Ca V 1.2 channels was reduced to 39 ± 12% of wild type. However, concentration-response curves for β-adrenergic stimulation of myocytes from LCyphKO mice have concentrations that give a half-maximal response similar to those for wild-type mice. These results identify Cypher/Zasp as an important AKAP for β-adrenergic regulation of cardiac Ca V 1.2 channels. Other AKAPs may work cooperatively with Cypher/Zasp to give the full magnitude of β-adrenergic regulation of Ca V 1.2 channels observed in vivo. This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply.

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

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.

Prevention of adenosine A2A receptor activation diminishes beat-to-beat alternation in human atrial myocytes.

PubMed

Molina, Cristina E; Llach, Anna; Herraiz-Martínez, Adela; Tarifa, Carmen; Barriga, Montserrat; Wiegerinck, Rob F; Fernandes, Jacqueline; Cabello, Nuria; Vallmitjana, Alex; Benitéz, Raúl; Montiel, José; Cinca, Juan; Hove-Madsen, Leif

2016-01-01

Atrial fibrillation (AF) has been associated with increased spontaneous calcium release from the sarcoplasmic reticulum and linked to increased adenosine A2A receptor (A2AR) expression and activation. Here we tested whether this may favor atrial arrhythmogenesis by promoting beat-to-beat alternation and irregularity. Patch-clamp and confocal calcium imaging was used to measure the beat-to-beat response of the calcium current and transient in human atrial myocytes. Responses were classified as uniform, alternating or irregular and stimulation of Gs-protein coupled receptors decreased the frequency where a uniform response could be maintained from 1.0 ± 0.1 to 0.6 ± 0.1 Hz; p < 0.01 for beta-adrenergic receptors and from 1.4 ± 0.1 to 0.5 ± 0.1 Hz; p < 0.05 for A2ARs. The latter was linked to increased spontaneous calcium release and after-depolarizations. Moreover, A2AR activation increased the fraction of non-uniformly responding cells in HL-1 myocyte cultures from 19 ± 3 to 51 ± 9 %; p < 0.02, and electrical mapping in perfused porcine atria revealed that adenosine induced electrical alternans at longer cycle lengths, doubled the fraction of electrodes showing alternation, and increased the amplitude of alternations. Importantly, protein kinase A inhibition increased the highest frequency where uniform responses could be maintained from 0.84 ± 0.12 to 1.86 ± 0.11 Hz; p < 0.001 and prevention of A2AR-activation with exogenous adenosine deaminase selectively increased the threshold from 0.8 ± 0.1 to 1.2 ± 0.1 Hz; p = 0.001 in myocytes from patients with AF. In conclusion, A2AR-activation promotes beat-to-beat irregularities in the calcium transient in human atrial myocytes, and prevention of A2AR activation may be a novel means to maintain uniform beat-to-beat responses at higher beating frequencies in patients with atrial fibrillation.

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.

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

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

Nitric oxide-dependent activation of CaMKII increases diastolic sarcoplasmic reticulum calcium release in cardiac myocytes in response to adrenergic stimulation.

PubMed

Curran, Jerry; Tang, Lifei; Roof, Steve R; Velmurugan, Sathya; Millard, Ashley; Shonts, Stephen; Wang, Honglan; Santiago, Demetrio; Ahmad, Usama; Perryman, Matthew; Bers, Donald M; Mohler, Peter J; Ziolo, Mark T; Shannon, Thomas R

2014-01-01

Spontaneous calcium waves in cardiac myocytes are caused by diastolic sarcoplasmic reticulum release (SR Ca(2+) leak) through ryanodine receptors. Beta-adrenergic (β-AR) tone is known to increase this leak through the activation of Ca-calmodulin-dependent protein kinase (CaMKII) and the subsequent phosphorylation of the ryanodine receptor. When β-AR drive is chronic, as observed in heart failure, this CaMKII-dependent effect is exaggerated and becomes potentially arrhythmogenic. Recent evidence has indicated that CaMKII activation can be regulated by cellular oxidizing agents, such as reactive oxygen species. Here, we investigate how the cellular second messenger, nitric oxide, mediates CaMKII activity downstream of the adrenergic signaling cascade and promotes the generation of arrhythmogenic spontaneous Ca(2+) waves in intact cardiomyocytes. Both SCaWs and SR Ca(2+) leak were measured in intact rabbit and mouse ventricular myocytes loaded with the Ca-dependent fluorescent dye, fluo-4. CaMKII activity in vitro and immunoblotting for phosphorylated residues on CaMKII, nitric oxide synthase, and Akt were measured to confirm activity of these enzymes as part of the adrenergic cascade. We demonstrate that stimulation of the β-AR pathway by isoproterenol increased the CaMKII-dependent SR Ca(2+) leak. This increased leak was prevented by inhibition of nitric oxide synthase 1 but not nitric oxide synthase 3. In ventricular myocytes isolated from wild-type mice, isoproterenol stimulation also increased the CaMKII-dependent leak. Critically, in myocytes isolated from nitric oxide synthase 1 knock-out mice this effect is ablated. We show that isoproterenol stimulation leads to an increase in nitric oxide production, and nitric oxide alone is sufficient to activate CaMKII and increase SR Ca(2+) leak. Mechanistically, our data links Akt to nitric oxide synthase 1 activation downstream of β-AR stimulation. Collectively, this evidence supports the hypothesis that CaMKII is regulated by nitric oxide as part of the adrenergic cascade leading to arrhythmogenesis.

Immunogold labeling study of the distribution of GLUT-1 and GLUT-4 in cardiac tissue following stimulation by insulin or ischemia.

PubMed

Davey, Katherine A B; Garlick, Pamela B; Warley, Alice; Southworth, Richard

2007-04-01

Whereas glucose transporter 1 (GLUT-1) is thought to be responsible for basal glucose uptake in cardiac myocytes, little is known about its relative distribution between the different plasma membranes and cell types in the heart. GLUT-4 translocates to the myocyte surface to increase glucose uptake in response to a number of stimuli. The mechanisms underlying ischemia- and insulin-mediated GLUT-4 translocation are known to be different, raising the possibility that the intracellular destinations of GLUT-4 following these stimuli also differ. Using immunogold labeling, we describe the cellular localization of these two transporters and investigate whether insulin and ischemia induce differential translocation of GLUT-4 to different cardiac membranes. Immunogold labeling of GLUT-1 and GLUT-4 was performed on left ventricular sections from isolated hearts following 30 min of either insulin, ischemia, or control perfusion. In control tissue, GLUT-1 was predominantly (76%) localized in the capillary endothelial cells, with only 24% of total cardiac GLUT-1 present in myocytes. GLUT-4 was found predominantly in myocytes, distributed between sarcolemmal and T tubule membranes (1.84 +/- 0.49 and 1.54 +/- 0.33 golds/microm, respectively) and intracellular vesicles (127 +/- 18 golds/microm(2)). Insulin increased T tubule membrane GLUT-4 content (2.8 +/- 0.4 golds/microm, P < 0.05) but had less effect on sarcolemmal GLUT-4 (1.72 +/- 0.53 golds/microm). Ischemia induced greater GLUT-4 translocation to both membrane types (4.25 +/- 0.84 and 4.01 +/- 0.27 golds/microm, respectively P < 0.05). The localization of GLUT-1 suggests a significant role in transporting glucose across the capillary wall before myocyte uptake via GLUT-1 and GLUT-4. We demonstrate independent spatial translocation of GLUT-4 under insulin or ischemic stimulation and propose independent roles for T-tubular and sarcolemmal GLUT-4.

Histone methylations in heart development, congenital and adult heart diseases.

PubMed

Zhang, Qing-Jun; Liu, Zhi-Ping

2015-01-01

Heart development comprises myocyte specification, differentiation and cardiac morphogenesis. These processes are regulated by a group of core cardiac transcription factors in a coordinated temporal and spatial manner. Histone methylation is an emerging epigenetic mechanism for regulating gene transcription. Interplay among cardiac transcription factors and histone lysine modifiers plays important role in heart development. Aberrant expression and mutation of the histone lysine modifiers during development and in adult life can cause either embryonic lethality or congenital heart diseases, and influences the response of adult hearts to pathological stresses. In this review, we describe current body of literature on the role of several common histone methylations and their modifying enzymes in heart development, congenital and adult heart diseases.

Eliminating spiral waves pinned to an anatomical obstacle in cardiac myocytes by high-frequency stimuli.

PubMed

Isomura, Akihiro; Hörning, Marcel; Agladze, Konstantin; Yoshikawa, Kenichi

2008-12-01

The unpinning of spiral waves by the application of high-frequency wave trains was studied in cultured cardiac myocytes. Successful unpinning was observed when the frequency of the paced waves exceeded a critical level. The unpinning process was analyzed by a numerical simulation with a model of cardiac tissue. The mechanism of unpinning by high-frequency stimuli is discussed in terms of local entrainment failure, through a reduction of the two-dimensional spatial characteristics into one dimension.

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.

Quantitative analysis of the Ca2+ -dependent regulation of delayed rectifier K+ current IKs in rabbit ventricular myocytes.

PubMed

Bartos, Daniel C; Morotti, Stefano; Ginsburg, Kenneth S; Grandi, Eleonora; Bers, Donald M

2017-04-01

[Ca 2+ ] i enhanced rabbit ventricular slowly activating delayed rectifier K + current (I Ks ) by negatively shifting the voltage dependence of activation and slowing deactivation, similar to perfusion of isoproterenol. Rabbit ventricular rapidly activating delayed rectifier K + current (I Kr ) amplitude and voltage dependence were unaffected by high [Ca 2+ ] i . When measuring or simulating I Ks during an action potential, I Ks was not different during a physiological Ca 2+ transient or when [Ca 2+ ] i was buffered to 500 nm. The slowly activating delayed rectifier K + current (I Ks ) contributes to repolarization of the cardiac action potential (AP). Intracellular Ca 2+ ([Ca 2+ ] i ) and β-adrenergic receptor (β-AR) stimulation modulate I Ks amplitude and kinetics, but details of these important I Ks regulators and their interaction are limited. We assessed the [Ca 2+ ] i dependence of I Ks in steady-state conditions and with dynamically changing membrane potential and [Ca 2+ ] i during an AP. I Ks was recorded from freshly isolated rabbit ventricular myocytes using whole-cell patch clamp. With intracellular pipette solutions that controlled free [Ca 2+ ] i , we found that raising [Ca 2+ ] i from 100 to 600 nm produced similar increases in I Ks as did β-AR activation, and the effects appeared additive. Both β-AR activation and high [Ca 2+ ] i increased maximally activated tail I Ks , negatively shifted the voltage dependence of activation, and slowed deactivation kinetics. These data informed changes in our well-established mathematical model of the rabbit myocyte. In both AP-clamp experiments and simulations, I Ks recorded during a normal physiological Ca 2+ transient was similar to I Ks measured with [Ca 2+ ] i clamped at 500-600 nm. Thus, our study provides novel quantitative data as to how physiological [Ca 2+ ] i regulates I Ks amplitude and kinetics during the normal rabbit AP. Our results suggest that micromolar [Ca 2+ ] i , in the submembrane or junctional cleft space, is not required to maximize [Ca 2+ ] i -dependent I Ks activation during normal Ca 2+ transients. © 2016 The Authors. The Journal of Physiology © 2016 The Physiological Society.

Quantitative analysis of the Ca2+‐dependent regulation of delayed rectifier K+ current I Ks in rabbit ventricular myocytes

PubMed Central

Bartos, Daniel C.; Morotti, Stefano; Ginsburg, Kenneth S.; Grandi, Eleonora

2017-01-01

Key points [Ca2+]i enhanced rabbit ventricular slowly activating delayed rectifier K+ current (I Ks) by negatively shifting the voltage dependence of activation and slowing deactivation, similar to perfusion of isoproterenol.Rabbit ventricular rapidly activating delayed rectifier K+ current (I Kr) amplitude and voltage dependence were unaffected by high [Ca2+]i.When measuring or simulating I Ks during an action potential, I Ks was not different during a physiological Ca2+ transient or when [Ca2+]i was buffered to 500 nm. Abstract The slowly activating delayed rectifier K+ current (I Ks) contributes to repolarization of the cardiac action potential (AP). Intracellular Ca2+ ([Ca2+]i) and β‐adrenergic receptor (β‐AR) stimulation modulate I Ks amplitude and kinetics, but details of these important I Ks regulators and their interaction are limited. We assessed the [Ca2+]i dependence of I Ks in steady‐state conditions and with dynamically changing membrane potential and [Ca2+]i during an AP. I Ks was recorded from freshly isolated rabbit ventricular myocytes using whole‐cell patch clamp. With intracellular pipette solutions that controlled free [Ca2+]i, we found that raising [Ca2+]i from 100 to 600 nm produced similar increases in I Ks as did β‐AR activation, and the effects appeared additive. Both β‐AR activation and high [Ca2+]i increased maximally activated tail I Ks, negatively shifted the voltage dependence of activation, and slowed deactivation kinetics. These data informed changes in our well‐established mathematical model of the rabbit myocyte. In both AP‐clamp experiments and simulations, I Ks recorded during a normal physiological Ca2+ transient was similar to I Ks measured with [Ca2+]i clamped at 500–600 nm. Thus, our study provides novel quantitative data as to how physiological [Ca2+]i regulates I Ks amplitude and kinetics during the normal rabbit AP. Our results suggest that micromolar [Ca2+]i, in the submembrane or junctional cleft space, is not required to maximize [Ca2+]i‐dependent I Ks activation during normal Ca2+ transients. PMID:28008618

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.

Lopinavir Impairs Protein Synthesis and Induces eEF2 Phosphorylation via the Activation of AMP-Activated Protein Kinase

PubMed Central

Hong-Brown, Ly Q.; Brown, C. Randell; Huber, Danuta S.; Lang, Charles H.

2008-01-01

HIV anti-retroviral drugs decrease protein synthesis, although the underlying regulatory mechanisms of this process are not fully established. Therefore, we investigated the effects of the HIV protease inhibitor lopinavir (LPV) on protein metabolism. We also characterized the mechanisms that mediate the effects of this drug on elongation factor-2 (eEF2), a key component of the translational machinery. Treatment of C2C12 myocytes with LPV produced a dose-dependent inhibitory effect on protein synthesis. This effect was observed at 15 min and was maintained for at least 4 h. Mechanistically, LPV increased the phosphorylation of eEF2 and thereby decreased the activity of this protein. Increased phosphorylation of eEF2 was associated with increased activity of its upstream regulators AMP-activated protein kinase (AMPK) and eEF2 kinase (eEF2K). Both AMPK and eEF2K directly phosphorylated eEF2 in an in vitro kinase assay suggesting two distinct paths lead to eEF2 phosphorylation. To verify this connection, myocytes were treated with the AMPK inhibitor compound C. Compound C blocked eEF2K and eEF2 phosphorylation, demonstrating that LPV affects eEF2 activity via an AMPK-eEF2K dependent pathway. In contrast, incubation of myocytes with rottlerin suppressed eEF2K, but not eEF2 phosphorylation, suggesting that eEF2 can be regulated independent of eEF2K. Finally, LPV did not affect PP2A activity when either eEF2 or peptide was used as the substrate. Collectively, these results indicate that LPV decreases protein synthesis, at least in part, via inhibition of eEF2. This appears regulated by AMPK which can act directly on eEF2 or indirectly via the action of eEF2K. PMID:18712774

Cartilage intermediate layer protein-1 alleviates pressure overload-induced cardiac fibrosis via interfering TGF-β1 signaling.

PubMed

Zhang, Cheng-Lin; Zhao, Qian; Liang, Hui; Qiao, Xue; Wang, Jin-Yu; Wu, Dan; Wu, Li-Ling; Li, Li

2018-03-01

Cardiac fibrosis is characterized by excessive deposition of extracellular matrix (ECM) proteins in the myocardium and results in decreased ventricular compliance and diastolic dysfunction. Cartilage intermediate layer protein-1 (CILP-1), a novel identified cardiac matricellular protein, is upregulated in most conditions associated with cardiac remodeling, however, whether CILP-1 is involved in pressure overload-induced fibrotic response is unknown. Here, we investigated whether CILP-1 was critically involved in the fibrotic remodeling induced by pressure overload. Western blot analysis and immunofluorescence staining showed that CILP-1 was predominantly detected in cardiac myocytes and to a less extent in the interstitium. In isolated adult mouse ventricular myocytes and nonmyocytes, CILP-1 was found to be mainly synthesized by myocytes. CILP-1 expression in left ventricles was upregulated in C57BL/6 mice undergoing transverse aortic constriction (TAC). Myocardial CILP-1 knockdown aggravated whereas CILP-1 overexpression attenuated TAC-induced ventricular remodeling and dysfunction, as measured by echocardiography test, morphological examination, and gene expressions of fibrotic molecules. Incubation of cardiac fibroblasts with the conditioned medium containing full-length, N-terminal, or C-terminal CILP-1 inhibited transforming growth factor (TGF)-β1-induced Smad3 phosphorylation and the subsequent profibrotic events. We first demonstrated that C-terminal CILP-1 increased Akt phosphorylation, promoted the interaction between Akt and Smad3, and suppressed Smad3 phosphorylation. Blockade of PI3K-Akt pathway attenuated the inhibitory effect of C-CILP-1 on TGF-β1-induced Smad3 activation. We conclude that CILP-1 is a novel ECM protein possessing anti-fibrotic ability in pressure overload-induced fibrotic remodeling. This anti-fibrotic effect of CILP-1 attributes to interfering TGF-β1 signaling through its N- and C- terminal fragments. Copyright © 2018 Elsevier Ltd. All rights reserved.

Temporal responses to intrinsically coupled calcium and zinc dyshomeostasis in cardiac myocytes and mitochondria during aldosteronism

PubMed Central

Kamalov, German; Ahokas, Robert A.; Zhao, Wenyuan; Shahbaz, Atta U.; Bhattacharya, Syamal K.; Sun, Yao; Gerling, Ivan C.

2010-01-01

Intracellular Ca2+ overloading, coupled to induction of oxidative stress, is present at 4-wk aldosterone/salt treatment (ALDOST). This prooxidant reaction in cardiac myocytes and mitochondria accounts for necrotic cell death and subsequent myocardial scarring. It is intrinsically linked to increased intracellular zinc concentration ([Zn2+]i) serving as an antioxidant. Herein, we addressed the temporal responses in coupled Ca2+ and Zn2+ dyshomeostasis, reflecting the prooxidant-antioxidant equilibrium, by examining preclinical (week 1) and pathological (week 4) stages of ALDOST to determine whether endogenous antioxidant defenses would be ultimately overwhelmed to account for this delay in cardiac remodeling. We compared responses in cardiomyocyte free [Ca2+]i and [Zn2+]i and mitochondrial total [Ca2+]m and [Zn2+]m, together with biomarkers of oxidative stress and antioxidant defenses, during 1- and 4-wk ALDOST. At week 1 and compared with controls, we found: 1) elevations in [Ca2+]i and [Ca2+]m were coupled with [Zn2+]i and [Zn2+]m; 2) increased mitochondrial H2O2 production, cardiomyocyte xanthine oxidase activity, and cardiac and mitochondrial 8-isoprostane levels, counterbalanced by increased activity of antioxidant proteins, enzymes, and the nonenzymatic antioxidants that can be considered as cumulative antioxidant capacity; some of these enzymes and proteins (e.g., metallothionein-1, Cu/Zn-superoxide, glutathione synthase) are regulated by metal-responsive transcription factor-1; and 3) although these augmented antioxidant defenses were sustained at week 4, they fell short in combating the persistent intracellular Ca2+ overloading and marked rise in cardiac tissue 8-isoprostane and mitochondrial transition pore opening. Thus a coupled Ca2+ and Zn2+ dyshomeostasis occurs early during ALDOST in cardiac myocytes and mitochondria that regulate redox equilibrium until week 4 when ongoing intracellular Ca2+ overloading and prooxidants overwhelm antioxidant defenses. PMID:19915175

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

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.

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

Morphometry of right ventricular hypertrophy induced by myocardial infarction in the rat.

PubMed Central

Anversa, P.; Beghi, C.; McDonald, S. L.; Levicky, V.; Kikkawa, Y.; Olivetti, G.

1984-01-01

The growth response of the right ventricle was studied in rats following ligation of the left coronary artery, which produced infarcts comprising approximately 40% of the left ventricle. A month after surgery the weight of the right ventricle was increased 30%, and this hypertrophic change was characterized by a 17% wall thickening, consistent with the 13% greater diameter of myocytes. Myocardial hypertrophy was accompanied by an inadequate growth of the microvasculature that supports tissue oxygenation. This was seen by relative decreases in capillary luminal volume density (-27%) and capillary luminal surface density (-21%) and by an increase in the average maximum distance from the capillary wall to the mitochondria of myocytes (19%). In contrast, measurements of the mean myocyte volume per nucleus showed a proportional enlargement of these cells (32%), from 16,300 cu mu in control animals to 21,500 cu mu in experimental rats. Quantitative analysis of the right coronary artery revealed a 33% increase in its luminal area, commensurate with the magnitude of ventricular hypertrophy. PMID:6236695

Stimulation of the p38 Mitogen-activated Protein Kinase Pathway in Neonatal Rat Ventricular Myocytes by the G Protein–coupled Receptor Agonists, Endothelin-1 and Phenylephrine: A Role in Cardiac Myocyte Hypertrophy?

PubMed Central

Clerk, Angela; Michael, Ashour; Sugden, Peter H.

1998-01-01

We examined the activation of the p38 mitogen-activated protein kinase (p38-MAPK) pathway by the G protein–coupled receptor agonists, endothelin-1 and phenylephrine in primary cultures of cardiac myocytes from neonatal rat hearts. Both agonists increased the phosphorylation (activation) of p38-MAPK by ∼12-fold. A p38-MAPK substrate, MAPK-activated protein kinase 2 (MAPKAPK2), was activated approximately fourfold and 10 μM SB203580, a p38-MAPK inhibitor, abolished this activation. Phosphorylation of the MAPKAPK2 substrate, heat shock protein 25/27, was also increased. Using selective inhibitors, activation of the p38-MAPK pathway by endothelin-1 was shown to involve protein kinase C but not Gi/Go nor the extracellularly responsive kinase (ERK) pathway. SB203580 failed to inhibit the morphological changes associated with cardiac myocyte hypertrophy induced by endothelin-1 or phenylephrine between 4 and 24 h. However, it decreased the myofibrillar organization and cell profile at 48 h. In contrast, inhibition of the ERK cascade with PD98059 prevented the increase in myofibrillar organization but not cell profile. These data are not consistent with a role for the p38-MAPK pathway in the immediate induction of the morphological changes of hypertrophy but suggest that it may be necessary over a longer period to maintain the response. PMID:9679149

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

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

Calreticulin reveals a critical Ca2+ checkpoint in cardiac myofibrillogenesis

PubMed Central

Li, Jian; Pucéat, Michel; Perez-Terzic, Carmen; Mery, Annabelle; Nakamura, Kimitoshi; Michalak, Marek; Krause, Karl-Heinz; Jaconi, Marisa E.

2002-01-01

Calreticulin (crt) is an ubiquitously expressed and multifunctional Ca2+-binding protein that regulates diverse vital cell functions, including Ca2+ storage in the ER and protein folding. Calreticulin deficiency in mice is lethal in utero due to defects in heart development and function. Herein, we used crt − / − embryonic stem (ES) cells differentiated in vitro into cardiac cells to investigate the molecular mechanisms underlying heart failure of knockout embryos. After 8 d of differentiation, beating areas were prominent in ES-derived wild-type (wt) embryoid bodies (EBs), but not in ES-derived crt − / − EBs, despite normal expression levels of cardiac transcription factors. Crt − / − EBs exhibited a severe decrease in expression and a lack of phosphorylation of ventricular myosin light chain 2 (MLC2v), resulting in an impaired organization of myofibrils. Crt − / − phenotype could be recreated in wt cells by chelating extracellular or cytoplasmic Ca2+ with EGTA or BAPTA, or by inhibiting Ca2+/calmodulin-dependent kinases (CaMKs). An imposed ionomycin-triggered cystolic-free Ca2+ concentration ([Ca2+]c) elevation restored the expression, phosphorylation, and insertion of MLC2v into sarcomeric structures and in turn the myofibrillogenesis. The transcription factor myocyte enhancer factor C2 failed to accumulate into nuclei of crt − / − cardiac cells in the absence of ionomycin-triggered [Ca2+]c increase. We conclude that the absence of calreticulin interferes with myofibril formation. Most importantly, calreticulin deficiency revealed the importance of a Ca2+-dependent checkpoint critical for early events during cardiac myofibrillogenesis. PMID:12105184

Apoglobin Stability Is the Major Factor Governing both Cell-free and in Vivo Expression of Holomyoglobin*♦

PubMed Central

Samuel, Premila P.; Smith, Lucian P.; Phillips, George N.; Olson, John S.

2015-01-01

Expression levels in animal muscle tissues and in Escherichia coli vary widely for naturally occurring mammalian myoglobins (Mb). To explore this variation, we developed an in vitro transcription and wheat germ extract-based translation assay to examine quantitatively the factors that govern expression of holoMb. We constructed a library of naturally occurring Mbs from two terrestrial and four deep-diving aquatic mammals and three distal histidine mutants designed to enhance apoglobin stability but decrease hemin affinity. A strong linear correlation is observed between cell-free expression levels of holo-metMb variants and their corresponding apoglobin stabilities, which were measured independently by guanidine HCl-induced unfolding titrations using purified proteins. In contrast, there is little dependence of expression on hemin affinity. Our results confirm quantitatively that deep diving mammals have highly stable Mbs that express to higher levels in animal myocytes, E. coli, and the wheat germ cell-free system than Mbs from terrestrial mammals. Our theoretical analyses show that the rate of aggregation of unfolded apoMb is very large, and as a result, the key factor for high level expression of holoMb, and presumably other heme proteins, is an ultra high fraction of folded, native apoglobin that is capable of rapidly binding hemin. This fraction is determined by the overall equilibrium folding constant and not hemin affinity. These results also demonstrate that the cell-free transcription/translation system can be used as a high throughput platform to screen for apoglobin stability without the need to generate large amounts of protein for in vitro unfolding measurements. PMID:26205820

Inhibition of potassium currents is involved in antiarrhythmic effect of moderate ethanol on atrial fibrillation.

PubMed

Yang, Baode; Li, Chenxing; Sun, Junyi; Wang, Xinghui; Liu, Xinling; Yang, Chun; Chen, Lina; Zhou, Jun; Hu, Hao

2017-05-01

Excessive consumption of alcohol is a well-established risk factor of atrial fibrillation (AF). However, the effects of moderate alcohol drinking remain to be elucidated. This study was designed to determine the effects of moderate ethanol ingestion on atrial fibrillation and the electrophysiological mechanisms. In acetylcholine-induced canine and mouse AF models, the moderate ethanol prevented the generation and persistence of AF through prolonging the latent period of AF and shortening the duration of AF. The action potential duration (APD) was remarkably prolonged under the concentration range of 12.5-50.0mM ethanol in guinea pig atrial myocytes. Ultra-rapid delayed rectified potassium currents (I Kv1.5 ) were markedly inhibited by 12.5-50.0mM ethanol in a concentration-dependent manner. Ethanol with 50.0mM could inhibit rapid delayed rectifier potassium currents (I hERG ). Ethanol under 6.25-50.0mM did not affect on inward rectifier potassium currents (I Kir2.1 ). Collectively, the present study provided an evidence that moderate ethanol intake can prolong the APD of atrial myocytes by inhibition of I Kv1.5 and I hERG , which contributed to preventing the development and duration of AF. Copyright © 2017 Elsevier Inc. All rights reserved.

The interdependence of Ca2+ activation, sarcomere length, and power output in the heart.

PubMed

McDonald, Kerry S

2011-07-01

Myocardium generates power to perform external work on the circulation; yet, many questions regarding intermolecular mechanisms regulating power output remain unresolved. Power output equals force × shortening velocity, and some interesting new observations regarding control of these two factors have arisen. While it is well established that sarcomere length tightly controls myocyte force, sarcomere length-tension relationships also appear to be markedly modulated by PKA-mediated phosphorylation of myofibrillar proteins. Concerning loaded shortening, historical models predict independent cross-bridge mechanics; however, it seems that the mechanical state of one population of cross-bridges affects the activity of other cross-bridges by, for example, recruitment of cross-bridges from the non-cycling pool to the cycling force-generating pool during submaximal Ca(2+) activation. This is supported by the findings that Ca(2+) activation levels, myofilament phosphorylation, and sarcomere length are all modulators of loaded shortening and power output independent of their effects on force. This fine tuning of power output probably helps optimize myocardial energetics and to match ventricular supply with peripheral demand; yet, the discernment of the chemo-mechanical signals that modulate loaded shortening needs further clarification since power output may be a key convergent point and feedback regulator of cytoskeleton and cellular signals that control myocyte growth and survival.

Genetic determinants of cardiometabolic risk: a proposed model for phenotype association and interaction.

PubMed

Blackett, Piers R; Sanghera, Dharambir K

2013-01-01

This review provides a translational and unifying summary of metabolic syndrome genetics and highlights evidence that genetic studies are starting to unravel and untangle origins of the complex and challenging cluster of disease phenotypes. The associated genes effectively express in the brain, liver, kidney, arterial endothelium, adipocytes, myocytes, and β cells. Progression of syndrome traits has been associated with ectopic lipid accumulation in the arterial wall, visceral adipocytes, myocytes, and liver. Thus, it follows that the genetics of dyslipidemia, obesity, and nonalcoholic fatty liver disease are central in triggering progression of the syndrome to overt expression of disease traits and have become a key focus of interest for early detection and for designing prevention and treatments. To support the "birds' eye view" approach, we provide a road-map depicting commonality and interrelationships between the traits and their genetic and environmental determinants based on known risk factors, metabolic pathways, pharmacologic targets, treatment responses, gene networks, pleiotropy, and association with circadian rhythm. Although only a small portion of the known heritability is accounted for and there is insufficient support for clinical application of gene-based prediction models, there is direction and encouraging progress in a rapidly moving field that is beginning to show clinical relevance. Copyright © 2013 National Lipid Association. Published by Elsevier Inc. All rights reserved.

Genetic Determinants of Cardio-Metabolic Risk: A Proposed Model for Phenotype Association and Interaction

PubMed Central

Blackett, Piers R; Sanghera, Dharambir K

2012-01-01

This review provides a translational and unifying summary of metabolic syndrome genetics and highlights evidence that genetic studies are starting to unravel and untangle origins of the complex and challenging cluster of disease phenotypes. The associated genes effectively express in the brain, liver, kidney, arterial endothelium, adipocytes, myocytes and β cells. Progression of syndrome traits has been associated with ectopic lipid accumulation in the arterial wall, visceral adipocytes, myocytes, and liver. Thus it follows that the genetics of dyslipidemia, obesity, and non-alcoholic fatty liver (NAFLD) disease are central in triggering progression of the syndrome to overt expression of disease traits, and have become a key focus of interest for early detection and for designing prevention and treatments. To support the “birds’ eye view” approach we provide a road-map depicting commonality and interrelationships between the traits and their genetic and environmental determinants based on known risk factors, metabolic pathways, pharmacological targets, treatment responses, gene networks, pleiotropy, and association with circadian rhythm. Although only a small portion of the known heritability is accounted for and there is insufficient support for clinical application of gene-based prediction models, there is direction and encouraging progress in a rapidly moving field that is beginning to show clinical relevance. PMID:23351585

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

Effects of a traditional Chinese medicine formula and its extraction on muscle fiber characteristics in finishing pigs, porcine cell proliferation and isoforms of myosin heavy chain gene expression in myocytes

PubMed Central

Yu, Qin Ping; Feng, Ding Yuan; He, Xiao Jun; Wu, Fan; Xia, Min Hao; Dong, Tao; Liu, Yi Hua; Tan, Hui Ze; Zou, Shi Geng; Zheng, Tao; Ou, Xian Hua; Zuo, Jian Jun

2017-01-01

Objective This study evaluated the effects of a traditional Chinese medicine formula (TCMF) on muscle fiber characteristics in finishing pigs and the effects of the formula’s extract (distilled water, ethyl acetate and petroleum ether extraction) on porcine cell proliferation and isoforms of myosin heavy chain (MyHC) gene expression in myocytes. Methods In a completely randomized design, ninety pigs were assigned to three diets with five replications per treatment and six pigs per pen. The diets included the basal diet (control group), TCMF1 (basal diet+2.5 g/kg TCMF) and TCMF2 (basal diet+5 g/kg TCMF). The psoas major muscle was obtained from pigs at the end of the experiment. Muscle fiber characteristics in the psoas major muscle were analyzed using myosin ATPase staining. Cell proliferation was measured using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) dye and cytometry. Isoforms of MyHC gene expression were detected by real-time quantitative polymerase chain reaction. Results The final body weight and carcass weight of finishing pigs were increased by TCMF1 (p<0.05), while the psoas major muscle cross-sectional area was increased by TCMF (p<0.05). The cross-sectional area and diameter of psoas major muscle fiber I, IIA, and IIB were increased by TCMF2 (p<0.05). The cross-sectional area and fiber diameter of psoas major muscle fiber IIA and IIB were increased by diet supplementation with TCMF1 (p<0.05). Psoas major muscle fiber IIA and IIB fiber density from the pigs fed the TCMF1 diet and the type IIB fiber density from the pigs fed the TCMF2 diet were lower compared to pigs fed the control diet (p<0.05). Pigs fed TCMF2 had a higher composition of type I fiber and a lower percentage of type IIB fiber in the psoas major muscle (p<0.05). The expression levels of MyHC I, MyHC IIa, and MyHC IIx mRNA increased and the amount of MyHC IIb mRNA decreased in the psoas major muscle from TCMF2, whereas MyHC I and MyHC IIx mRNA increased in the psoas major muscle from TCMF1 (p<0.05). Peroxisome proliferator-activated receptor γ coactivator-1α and CaN mRNA expression in the psoas major muscle were up-regulated by TCMF (p<0.05). Porcine skeletal muscle satellite cell proliferation was promoted by 4 μg/mL and 20 μg/mL TCMF water extraction (p<0.05). Both 1 μg/mL and 5 μg/mL of TCMF water extraction increased MyHC IIa, MyHC IIb, and MyHC IIx mRNA expression in porcine myocytes (p<0.05), while MyHC I mRNA expression in porcine myocytes was decreased by 5 μg/mL TCMF water extraction (p<0.05). Porcine myocyte MyHC I and MyHC IIx mRNA expression were increased, and MyHC IIa and MyHC IIb mRNA expression were down-regulated by 5 μg/mL TCMF ethyl acetate extraction (p<0.05). MyHC I and MyHC IIa mRNA expression in porcine myocytes were increased, and the MyHC IIb mRNA expression was decreased by 1 μg/mL TCMF ethyl acetate extraction (p<0.05). Four isoforms of MyHC mRNA expression in porcine myocytes were reduced by 5 μg/mL TCMF petroleum ether extraction (p<0.05). MyHC IIa mRNA expression in porcine myocytes increased and MyHC IIb mRNA expression decreased by 1 μg/mL in a TCMF petroleum ether extraction (p<0.05). Conclusion These results indicated that TCMF amplified the psoas major muscle cross-sectional area through changing muscle fiber characteristics in finishing pigs. This effect was confirmed as TCMF extraction promoted porcine cell proliferation and affected isoforms of MyHC gene expression in myocytes. PMID:28728382

A novel mechanism of tandem activation of ryanodine receptors by cytosolic and SR luminal Ca2+ during excitation-contraction coupling in atrial myocytes.

PubMed

Maxwell, Joshua T; Blatter, Lothar A

2017-06-15

In atrial myocytes excitation-contraction coupling is strikingly different from ventricle because atrial myocytes lack a transverse tubule membrane system: Ca 2+ release starts in the cell periphery and propagates towards the cell centre by Ca 2+ -induced Ca 2+ release from the sarcoplasmic reticulum (SR) Ca 2+ store. The cytosolic Ca 2+ sensitivity of the ryanodine receptor (RyRs) Ca 2+ release channel is low and it is unclear how Ca 2+ release can be activated in the interior of atrial cells. Simultaneous confocal imaging of cytosolic and intra-SR calcium revealed a transient elevation of store Ca 2+ that we termed 'Ca 2+ sensitization signal'. We propose a novel paradigm of atrial ECC that is based on tandem activation of the RyRs by cytosolic and luminal Ca 2+ through a 'fire-diffuse-uptake-fire' (or FDUF) mechanism: Ca 2+ uptake by SR Ca 2+ pumps at the propagation front elevates Ca 2+ inside the SR locally, leading to luminal RyR sensitization and lowering of the cytosolic Ca 2+ activation threshold. In atrial myocytes Ca 2+ release during excitation-contraction coupling (ECC) is strikingly different from ventricular myocytes. In many species atrial myocytes lack a transverse tubule system, dividing the sarcoplasmic reticulum (SR) Ca 2+ store into the peripheral subsarcolemmnal junctional (j-SR) and the much more abundant central non-junctional (nj-SR) SR. Action potential (AP)-induced Ca 2+ entry activates Ca 2+ -induced Ca 2+ release (CICR) from j-SR ryanodine receptor (RyR) Ca 2+ release channels. Peripheral elevation of [Ca 2+ ] i initiates CICR from nj-SR and sustains propagation of CICR to the cell centre. Simultaneous confocal measurements of cytosolic ([Ca 2+ ] i ; with the fluorescent Ca 2+ indicator rhod-2) and intra-SR ([Ca 2+ ] SR ; fluo-5N) Ca 2+ in rabbit atrial myocytes revealed that Ca 2+ release from j-SR resulted in a cytosolic Ca 2+ transient of higher amplitude compared to release from nj-SR; however, the degree of depletion of j-SR [Ca 2+ ] SR was smaller than nj-SR [Ca 2+ ] SR . Similarly, Ca 2+ signals from individual release sites of the j-SR showed a larger cytosolic amplitude (Ca 2+ sparks) but smaller depletion (Ca 2+ blinks) than release from nj-SR. During AP-induced Ca 2+ release the rise of [Ca 2+ ] i detected at individual release sites of the nj-SR preceded the depletion of [Ca 2+ ] SR , and during this latency period a transient elevation of [Ca 2+ ] SR occurred. We propose that Ca 2+ release from nj-SR is activated by cytosolic and luminal Ca 2+ (tandem RyR activation) via a novel 'fire-diffuse-uptake-fire' (FDUF) mechanism. This novel paradigm of atrial ECC predicts that Ca 2+ uptake by sarco-endoplasmic reticulum Ca 2+ -ATPase (SERCA) at the propagation front elevates local [Ca 2+ ] SR , leading to luminal RyR sensitization and lowering of the activation threshold for cytosolic CICR. © 2016 The Authors. The Journal of Physiology © 2016 The Physiological Society.

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

Allogenic Myocytes and Mesenchymal Stem Cells Partially Improve Fatty Rotator Cuff Degeneration in a Rat Model.

PubMed

Güleçyüz, Mehmet F; Macha, Konstanze; Pietschmann, Matthias F; Ficklscherer, Andreas; Sievers, Birte; Roßbach, Björn P; Jansson, Volkmar; Müller, Peter E

2018-05-31

Rotator cuff (RC) tears result not only in functional impairment but also in RC muscle atrophy, muscle fattening and eventually to muscle fibrosis. We hypothesized that allogenic bone marrow derived mesenchymal stem cells (MSC) and myocytes can be utilized to improve the rotator cuff muscle fattening and increase the atrophied muscle mass in a rat model. The right supraspinatus (SSP) tendons of 105 inbred rats were detached and muscle fattening was provoked over 4 weeks; the left side remained untouched (control group). The animals (n = 25) of the output group were euthanized after 4 weeks for reference purposes. The SSP-tendon of one group (n = 16) was left unoperated to heal spontaneously. The SSP-tendons of the remaining 64 rats (4 groups with n = 16) were repaired with transosseous sutures. One group received a saline solution injection in the SSP muscle belly, two other groups received 5 × 10 6 allogenic myocytes and 5 × 10 6 allogenic MSC injections from donor rats, respectively, and one group received no additional treatment. After 4 weeks of healing, the supraspinatus muscle mass was compared quantitatively and histologically to all the treated groups and to the untreated contralateral side. In the end of the experiments at week 8, the myocyte and MCS treated groups showed a significantly higher muscle mass with 0.2322 g and 0.2257 g, respectively, in comparison to the output group (0.1911 g) at week 4 with p < 0.05. There was no statistical difference between the repaired, treated, or spontaneous healing groups at week 8. Supraspinatus muscle mass of all experimental groups of the right side was significantly lower compared to the untreated contralateral muscle mass. This defect model shows that the injection of allogenic mycocytes and MSC in fatty infiltrated SSP muscles is better than no treatment and can partially improve the SSP muscle belly fattening. Nevertheless, a full restoration of the degenerated and fattened rotator cuff muscle to its original condition is not possible using myocytes and MSC in this model.

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.

Triggered intracellular calcium waves in dog and human left atrial myocytes from normal and failing hearts.

PubMed

Aistrup, Gary L; Arora, Rishi; Grubb, Søren; Yoo, Shin; Toren, Benjamin; Kumar, Manvinder; Kunamalla, Aaron; Marszalec, William; Motiwala, Tej; Tai, Shannon; Yamakawa, Sean; Yerrabolu, Satya; Alvarado, Francisco J; Valdivia, Hector H; Cordeiro, Jonathan M; Shiferaw, Yohannes; Wasserstrom, John Andrew

2017-11-01

Abnormal intracellular Ca2+ cycling contributes to triggered activity and arrhythmias in the heart. We investigated the properties and underlying mechanisms for systolic triggered Ca2+ waves in left atria from normal and failing dog hearts. Intracellular Ca2+ cycling was studied using confocal microscopy during rapid pacing of atrial myocytes (36 °C) isolated from normal and failing canine hearts (ventricular tachypacing model). In normal atrial myocytes (NAMs), Ca2+ waves developed during rapid pacing at rates ≥ 3.3 Hz and immediately disappeared upon cessation of pacing despite high sarcoplasmic reticulum (SR) load. In heart failure atrial myocytes (HFAMs), triggered Ca2+ waves (TCWs) developed at a higher incidence at slower rates. Because of their timing, TCW development relies upon action potential (AP)-evoked Ca2+ entry. The distribution of Ca2+ wave latencies indicated two populations of waves, with early events representing TCWs and late events representing conventional spontaneous Ca2+ waves. Latency analysis also demonstrated that TCWs arise after junctional Ca2+ release has occurred and spread to non-junctional (cell core) SR. TCWs also occurred in intact dog atrium and in myocytes from humans and pigs. β-adrenergic stimulation increased Ca2+ release and abolished TCWs in NAMs but was ineffective in HFAMs making this a potentially effective adaptive mechanism in normals but potentially arrhythmogenic in HF. Block of Ca-calmodulin kinase II also abolished TCWs, suggesting a role in TCW formation. Pharmacological manoeuvres that increased Ca2+ release suppressed TCWs as did interventions that decreased Ca2+ release but these also severely reduced excitation-contraction coupling. TCWs develop during the atrial AP and thus could affect AP duration, producing repolarization gradients and creating a substrate for reentry, particularly in HF where they develop at slower rates and a higher incidence. TCWs may represent a mechanism for the initiation of atrial fibrillation particularly in HF. Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2017. For permissions please email: journals.permissions@oup.com.

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.

Extraction of the 3D local orientation of myocytes in human cardiac tissue using X-ray phase-contrast micro-tomography and multi-scale analysis.

PubMed

Varray, François; Mirea, Iulia; Langer, Max; Peyrin, Françoise; Fanton, Laurent; Magnin, Isabelle E

2017-05-01

This paper presents a methodology to access the 3D local myocyte arrangements in fresh human post-mortem heart samples. We investigated the cardiac micro-structure at a high and isotropic resolution of 3.5 µm in three dimensions using X-ray phase micro-tomography at the European Synchrotron Radiation Facility. We then processed the reconstructed volumes to extract the 3D local orientation of the myocytes using a multi-scale approach with no segmentation. We created a simplified 3D model of tissue sample made of simulated myocytes with known size and orientations, to evaluate our orientation extraction method. Afterwards, we applied it to 2D histological cuts and to eight 3D left ventricular (LV) cardiac tissue samples. Then, the variation of the helix angles, from the endocardium to the epicardium, was computed at several spatial resolutions ranging from 3.6 3  mm 3 to 112 3  µm 3 . We measure an increased range of 20° to 30° from the coarsest resolution level to the finest level in the experimental samples. This result is in line with the higher values measured from histology. The displayed tractography demonstrates a rather smooth evolution of the transmural helix angle in six LV samples and a sudden discontinuity of the helix angle in two septum samples. These measurements bring a new vision of the human heart architecture from macro- to micro-scale. Copyright © 2017 Elsevier B.V. All rights reserved.

Glutamine reduces myocardial cell apoptosis in a rat model of sepsis by promoting expression of heat shock protein 90.

PubMed

Li, Wanxia; Tao, Shaoyu; Wu, Qinghua; Wu, Tao; Tao, Ran; Fan, Jun

2017-12-01

Myocardial cell injury and cardiac myocyte apoptosis are associated with sepsis. Glutamine (Gln) has been reported to repair myocardial cell injury. The aim of this study was to explore the role of Gln on cardiac myocytes in a cecal ligation and puncture (CLP) model of sepsis in Wistar rats. Following induction of sepsis in a CLP rat model, viral encoding heat shock protein 90 (Hsp90) gene and Hsp90dsDNA were designed to express and knockdown Hsp90, respectively. Rat cardiac tissues were examined histologically, and apoptosis was detected by terminal deoxynucleotidyl transferase dUTP nick end labeling staining. The expression of B-cell lymphoma-2 (Bcl-2), Bcl-2-associated X protein, Hsp90, p53 upregulated modulator of apoptosis, and p53 was measured by western blotting and real-time polymerase chain reaction. Caspase-3, caspase-8, and caspase-9 were detected by enzyme-linked immunosorbent assay. Rat cardiac myocyte damage induced by CLP was reduced by Gln treatment and Hsp90 overexpression, and these changes were reversed by Hsp90 knockdown. Bcl-2 expression, Bcl-2-associated X protein, p53, p53 upregulated modulator of apoptosis, caspase-8, caspase-9, and caspase-3 activities were significantly upregulated in the CLP model, which were reduced by Gln treatment and Hsp90 overexpression. Gln reduced apoptosis of cardiac myocytes in a rat model of sepsis, by promoting Hsp90 expression. Further studies are needed to determine the possible therapeutic action of Gln in sepsis in human tissue. Copyright © 2017 Elsevier Inc. All rights reserved.

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.

Abolition of reperfusion-induced arrhythmias in hearts from thiamine-deficient rats.

PubMed

Oliveira, Fernando A; Guatimosim, Silvia; Castro, Carlos H; Galan, Diogo T; Lauton-Santos, Sandra; Ribeiro, Angela M; Almeida, Alvair P; Cruz, Jader S

2007-07-01

Extensive work has been done regarding the impact of thiamine deprivation on the nervous system. In cardiac tissue, chronic thiamine deficiency is described to cause changes in the myocardium that can be associated with arrhythmias. However, compared with the brain, very little is known about the effects of thiamine deficiency on the heart. Thus this study was undertaken to explore whether thiamine deprivation has a role in cardiac arrhythmogenesis. We examined hearts isolated from thiamine-deprived and control rats. We measured heart rate, diastolic and systolic tension, and contraction and relaxation rates. Whole cell voltage clamp was performed in rat isolated cardiac myocytes to measure L-type Ca(2+) current. In addition, we investigated the global intracellular calcium transients by using confocal microscopy in the line-scan mode. The hearts from thiamine-deficient rats did not degenerate into ventricular fibrillation during 30 min of reperfusion after 15 min of coronary occlusion. The antiarrhythmogenic effects were characterized by the arrhythmia severity index. Our results suggest that hearts from thiamine-deficient rats did not experience irreversible arrhythmias. There was no change in L-type Ca(2+) current density. Inactivation kinetics of this current in Ca(2+)-buffered cells was retarded in thiamine-deficient cardiac myocytes. The global Ca(2+) release was significantly reduced in thiamine-deficient cardiac myocytes. The amplitude of caffeine-releasable Ca(2+) was lower in thiamine-deficient myocytes. In summary, we have found that thiamine deprivation attenuates the incidence and severity of postischemic arrhythmias, possibly through a mechanism involving a decrease in global Ca(2+) release.

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

Quantitative determination of adenovirus-mediated gene delivery to rat cardiac myocytes in vitro and in vivo.

PubMed Central

Kass-Eisler, A; Falck-Pedersen, E; Alvira, M; Rivera, J; Buttrick, P M; Wittenberg, B A; Cipriani, L; Leinwand, L A

1993-01-01

To optimize the use of modified adenoviruses as vectors for gene delivery to the myocardium, we have characterized infection of cultured fetal and adult rat cardiac myocytes in vitro and of adult cardiac myocytes in vivo by using a replication-defective adenovirus carrying the chloramphenicol acetyltransferase (CAT) reporter gene driven by the cytomegalovirus promoter (AdCMVCATgD). In vitro, virtually all fetal or adult cardiocytes express the CAT gene when infected with 1 plaque-forming unit of virus per cell. CAT enzymatic activity can be detected in these cells as early as 4 hr after infection, reaching near-maximal levels at 48 hr. In fetal cells, CAT expression was maintained without a loss in activity for at least 1 week. Using in vitro studies as a guide, we introduced the AdCMVCATgD virus directly into adult rat myocardium and compared the expression results obtained from virus injection with those obtained by direct injection of pAdCMVCATgD plasmid DNA. The amount of CAT activity resulting from adenovirus infection of the myocardium was orders of magnitude higher than that seen from DNA injection and was proportional to the amount of input virus. Immunostaining for CAT protein in cardiac tissue sections following adenovirus injection demonstrated large numbers of positive cells, reaching nearly 100% of the myocytes in many regions of the heart. Expression of genes introduced by adenovirus peaked at 5 days but was still detectable 55 days following infection. Adenoviruses are therefore a very useful tool for high-efficiency gene transfer into the cardiovascular system. Images Fig. 1 Fig. 5 PMID:8265580

The changes of potassium currents in rabbit ventricle with healed myocardial infarction.

PubMed

Liu, Nian; Niu, Huiyan; Li, Yang; Zhang, Cuntai; Zhou, Qiang; Ruan, Yanfei; Pu, Jun; Lu, Zaiying

2004-01-01

To elucidate the mechanism of arrhythmia in healed myocardial infarction (HMI), the changes of action potential duration (APD), transient outward potassium current (Ito), delayed rectifier potassium current (IK) and inward rectifier potassium current (IK1) of left ventricular myocytes in non-infarcted zone of HMI were investigated. Rabbits were randomly assigned into two groups: HMI group, in which animals were subjected to thoracotomy and ligation of the circumflex coronary and sham-operated group, in which rabbits underwent thoracotomy but no conorary ligation. 3 months after the operation, the whole myocyte patch clamp technique was used to record APD, Ito, IK, and IK1 of ventricular myocytes in non-infarcted zone. Our results showed that the membrane capacitance was larger in HMI group than in sham-operated group. Action potential duration was significantly lengthened in HMI group and early afterdepolarization (EAD) appeared in HMI group. The densities of Ito, I(K, tail), and IK1 were reduced significantly in HMI group, from 6.72 +/- 0.42 pA/pF, 1.54 +/- 0.13 pA/pF and 25.6 +/- 2.6 pA/pF in sham-operated group to 4.03 +/- 0.33 pA/pF, 1.14 +/- 0.11 pA/pF and 17.6 +/- 2.3 pA/pF, respectively. It is concluded that the reduced densities of Ito, I(K, tail) and IK1 in ventricular myocytes of non-infarcted zone in HMI were responsible for the prolongation of APD and the presentation of EAD which played important roles in the development of malignant arrhythmia in HMI.

Alternative Polyadenylation in Glioblastoma Multiforme and Changes in Predicted RNA Binding Protein Profiles

PubMed Central

Shao, Jiaofang; Zhang, Jing; Zhang, Zengming; Jiang, Huawei; Lou, Xiaoyan; Foltz, Gregory; Lan, Qing; Huang, Qiang

2013-01-01

Abstract Alternative polyadenylation (APA) is widely present in the human genome and plays a key role in carcinogenesis. We conducted a comprehensive analysis of the APA products in glioblastoma multiforme (GBM, one of the most lethal brain tumors) and normal brain tissues and further developed a computational pipeline, RNAelements (http://sysbio.zju.edu.cn/RNAelements/), using covariance model from known RNA binding protein (RBP) targets acquired by RNA Immunoprecipitation (RIP) analysis. We identified 4530 APA isoforms for 2733 genes in GBM, and found that 182 APA isoforms from 148 genes showed significant differential expression between normal and GBM brain tissues. We then focused on three genes with long and short APA isoforms that show inconsistent expression changes between normal and GBM brain tissues. These were myocyte enhancer factor 2D, heat shock factor binding protein 1, and polyhomeotic homolog 1 (Drosophila). Using the RNAelements program, we found that RBP binding sites were enriched in the alternative regions between the first and the last polyadenylation sites, which would result in the short APA forms escaping regulation from those RNA binding proteins. To the best of our knowledge, this report is the first comprehensive APA isoform dataset for GBM and normal brain tissues. Additionally, we demonstrated a putative novel APA-mediated mechanism for controlling RNA stability and translation for APA isoforms. These observations collectively lay a foundation for novel diagnostics and molecular mechanisms that can inform future therapeutic interventions for GBM. PMID:23421905

Association of class II histone deacetylases with heterochromatin protein 1: potential role for histone methylation in control of muscle differentiation.

PubMed

Zhang, Chun Li; McKinsey, Timothy A; Olson, Eric N

2002-10-01

Class II histone deacetylases (HDACs) 4, 5, 7, and 9 repress muscle differentiation through associations with the myocyte enhancer factor 2 (MEF2) transcription factor. MEF2-interacting transcription repressor (MITR) is an amino-terminal splice variant of HDAC9 that also potently inhibits MEF2 transcriptional activity despite lacking a catalytic domain. Here we report that MITR, HDAC4, and HDAC5 associate with heterochromatin protein 1 (HP1), an adaptor protein that recognizes methylated lysines within histone tails and mediates transcriptional repression by recruiting histone methyltransferase. Promyogenic signals provided by calcium/calmodulin-dependent kinase (CaMK) disrupt the interaction of MITR and HDACs with HP1. Since the histone methyl-lysine residues recognized by HP1 also serve as substrates for deacetylation by HDACs, the interaction of MITR and HDACs with HP1 provides an efficient mechanism for silencing MEF2 target genes by coupling histone deacetylation and methylation. Indeed, nucleosomal histones surrounding a MEF2-binding site in the myogenin gene promoter are highly methylated in undifferentiated myoblasts, when the gene is silent, and become acetylated during muscle differentiation, when the myogenin gene is expressed at high levels. The ability of MEF2 to recruit a histone methyltransferase to target gene promoters via HP1-MITR and HP1-HDAC interactions and of CaMK signaling to disrupt these interactions provides an efficient mechanism for signal-dependent regulation of the epigenetic events controlling muscle differentiation.

Association of Class II Histone Deacetylases with Heterochromatin Protein 1: Potential Role for Histone Methylation in Control of Muscle Differentiation

PubMed Central

Zhang, Chun Li; McKinsey, Timothy A.; Olson, Eric N.

2002-01-01

Class II histone deacetylases (HDACs) 4, 5, 7, and 9 repress muscle differentiation through associations with the myocyte enhancer factor 2 (MEF2) transcription factor. MEF2-interacting transcription repressor (MITR) is an amino-terminal splice variant of HDAC9 that also potently inhibits MEF2 transcriptional activity despite lacking a catalytic domain. Here we report that MITR, HDAC4, and HDAC5 associate with heterochromatin protein 1 (HP1), an adaptor protein that recognizes methylated lysines within histone tails and mediates transcriptional repression by recruiting histone methyltransferase. Promyogenic signals provided by calcium/calmodulin-dependent kinase (CaMK) disrupt the interaction of MITR and HDACs with HP1. Since the histone methyl-lysine residues recognized by HP1 also serve as substrates for deacetylation by HDACs, the interaction of MITR and HDACs with HP1 provides an efficient mechanism for silencing MEF2 target genes by coupling histone deacetylation and methylation. Indeed, nucleosomal histones surrounding a MEF2-binding site in the myogenin gene promoter are highly methylated in undifferentiated myoblasts, when the gene is silent, and become acetylated during muscle differentiation, when the myogenin gene is expressed at high levels. The ability of MEF2 to recruit a histone methyltransferase to target gene promoters via HP1-MITR and HP1-HDAC interactions and of CaMK signaling to disrupt these interactions provides an efficient mechanism for signal-dependent regulation of the epigenetic events controlling muscle differentiation. PMID:12242305

A Postpartum Model in Rat: Behavioral and Gene Expression Changes Induced by Ovarian Steroid Deprivation

PubMed Central

Suda, Shiro; Segi-Nishida, Eri; Newton, Samuel S.; Duman, Ronald S.

2013-01-01

Background Postpartum depression (PPD) affects approximately 10% to 20% of women during the first 4 weeks of the postpartum period and is characterized by labile mood with prominent anxiety and irritability, insomnia,and depressive mood. During the postpartum period, elevated ovarian hormones abruptly decrease to the early follicular phase levels that are postulated to play a major role in triggering PPD. However, the underlying neurobiological mechanisms that contribute to PPD have not been determined. Methods In the present study, we examined the effect of ovarian steroids, administered at levels that occur during human pregnancy followed by rapid withdrawal to simulate postpartum conditions, on behavior and gene expression in the rat. Results The results of behavioral testing reveal that the hormone-simulated postpartum treatment results in the development of a phenotype relevant to PPD, including vulnerability for helplessness, increased anxiety, and aggression. Real-time quantitative polymerase chain reaction (PCR) demonstrated transient regulation of several genes, including Ca2+/calmodulin-dependent protein kinase II (CAMKII), serotonin transporter (SERT), myocyte enhancer factor 2A (MEF2A), brain-derived neurotrophic factor (BDNF), gamma-aminobutyric acid type A receptor α4 (GABAARA4), mothers against decapentaplegic homolog 4 (SMAD4), and aquaporin 4 (AQP4) that could underlie these behavioral effects. Conclusions These studies provide an improved understanding of the effects of withdrawal from high doses of ovarian hormones on behavior and gene expression changes in the brain that could contribute to the pathophysiology of PPD. PMID:18471802

Cell Electrical Impedance as a Novel Approach for Studies on Senescence Not Based on Biomarkers

PubMed Central

Cha, Jung-Joon; Park, Yangkyu; Yun, Joho; Kim, Hyeon Woo; Park, Chang-Ju; Kang, Giseok; Jung, Minhyun; Pak, Boryeong; Jin, Suk-Won

2016-01-01

Senescence of cardiac myocytes is frequently associated with heart diseases. To analyze senescence in cardiac myocytes, a number of biomarkers have been isolated. However, due to the complex nature of senescence, multiple markers are required for a single assay to accurately depict complex physiological changes associated with senescence. In single cells, changes in both cytoplasm and cell membrane during senescence can affect the changes in electrical impedance. Based on this phenomenon, we developed MEDoS, a novel microelectrochemical impedance spectroscopy for diagnosis of senescence, which allows us to precisely measure quantitative changes in electrical properties of aging cells. Using cardiac myocytes isolated from 3-, 6-, and 18-month-old isogenic zebrafish, we examined the efficacy of MEDoS and showed that MEDoS can identify discernible changes in electrical impedance. Taken together, our data demonstrated that electrical impedance in cells at different ages is distinct with quantitative values; these results were comparable with previously reported ones. Therefore, we propose that MEDoS be used as a new biomarker-independent methodology to obtain quantitative data on the biological senescence status of individual cells. PMID:27812531

Expression of microRNA-122 contributes to apoptosis in H9C2 myocytes

PubMed Central

Huang, Xiaoyan; Huang, Fang; Yang, Deye; Dong, Fengquan; Shi, Xiangxiang; Wang, Hongyu; Zhou, Xi; Wang, Suyun; Dai, Shengchuan

2012-01-01

The microRNAs (miRNAs) can post-transcriptionally regulate gene expression and heart development. The Pax-8 gene knockout mice have apparent heart abnormalities. This study investigated the role of miRNAs in regulation of cardiac apoptosis and development in the knockout mice. MicroRNA microarrays demonstrated differential expression of microRNAs between Pax-8−/− and Pax-8+/− mice, confirmed by real-time PCR. The miR-122 was up-regulated by 1.92 folds in Pax-8−/− mice. There were ventricular septum defects in Pax-8−/− mice, and increased numbers of apoptotic cells in the left ventricular wall and interventricular septum in Pax-8−/− mice. In H9C2 myocytes, treatment with miR-122 mimics or miR-122 inhibitor affects the expression of CCK-8 and activity of Caspase-3. The miR-122 is up-regulated in the myocytes of Pax-8−/− mice and may participate in the apoptotic gene expression and pathogenesis of heart development defect. PMID:22453009

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.

Expression of androgen-binding protein (ABP) in human cardiac myocytes.

PubMed

Schock, H W; Herbert, Z; Sigusch, H; Figulla, H R; Jirikowski, G F; Lotze, U

2006-04-01

Cardiomyocytes are known to be androgen targets. Changing systemic steroid levels are thought to be linked to various cardiac ailments, including dilated cardiomyopathy (DCM). The mode of action of gonadal steroid hormones on the human heart is unknown to date. In the present study, we used high-resolution immunocytochemistry on semithin sections (1 microm thick), IN SITU hybridization, and mass spectrometry to investigate the expression of androgen-binding protein (ABP) in human myocardial biopsies taken from male patients with DCM. We observed distinct cytoplasmic ABP immunoreactivity in a fraction of the myocytes. IN SITU hybridization with synthetic oligonucleotide probes revealed specific hybridization signals in these cells. A portion of the ABP-positive cells contained immunostaining for androgen receptor. With SELDI TOF mass spectrometry of affinity purified tissue extracts of human myocardium, we confirmed the presence of a 50 kDa protein similar to ABP. Our observations provide evidence of an intrinsic expression of ABP in human heart. ABP may be secreted from myocytes in a paracrine manner perhaps to influence the bioavailabity of gonadal steroids in myocardium.

Vascular Function, Insulin Action and Exercise: An Intricate Interplay

PubMed Central

Zheng, Chao; Liu, Zhenqi

2015-01-01

Insulin enhances the compliance of conduit arteries, relaxes resistance arterioles to increase tissue blood flow and dilates precapillary arterioles to expand muscle microvascular blood volume. These actions are impaired in the insulin resistant states. Exercise ameliorates endothelial dysfunction and improves insulin responses in insulin resistant patients, but the precise underlying mechanisms remain unclear. The microvasculature critically regulates insulin action in muscle by modulating insulin delivery to the capillaries nurturing the myocytes and trans-endothelial insulin transport. Recent data suggest that exercise may exert its insulin-sensitizing effect via recruiting muscle microvasculature to increase insulin delivery to and action in muscle. The current review focuses on how the interplay among exercise, insulin action and the vasculature contributes to exercise-mediated insulin sensitization in muscle. PMID:25735473

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

The single nucleotide polymorphism Gly482Ser in the PGC-1α gene impairs exercise-induced slow-twitch muscle fibre transformation in humans.

PubMed

Steinbacher, Peter; Feichtinger, René G; Kedenko, Lyudmyla; Kedenko, Igor; Reinhardt, Sandra; Schönauer, Anna-Lena; Leitner, Isabella; Sänger, Alexandra M; Stoiber, Walter; Kofler, Barbara; Förster, Holger; Paulweber, Bernhard; Ring-Dimitriou, Susanne

2015-01-01

PGC-1α (peroxisome proliferator-activated receptor γ co-activator 1α) is an important regulator of mitochondrial biogenesis and a master regulator of enzymes involved in oxidative phosphorylation. Recent evidence demonstrated that the Gly482Ser single nucleotide polymorphism (SNP) in the PGC-1α gene affects insulin sensitivity, blood lipid metabolism and binding to myocyte enhancer factor 2 (MEF2). Individuals carrying this SNP were shown to have a reduced cardiorespiratory fitness and a higher risk to develop type 2 diabetes. Here, we investigated the responses of untrained men with the Gly482Ser SNP to a 10 week programme of endurance training (cycling, 3 x 60 min/week, heart rate at 70-90% VO2peak). Quantitative data from analysis of biopsies from vastus lateralis muscle revealed that the SNP group, in contrast to the control group, lacked a training-induced increase in content of slow contracting oxidative fibres. Capillary supply, mitochondrial density, mitochondrial enzyme activities and intramyocellular lipid content increased similarly in both groups. These results indicate that the impaired binding of MEF2 to PGC-1α in humans with this SNP impedes exercise-induced fast-to-slow muscle fibre transformation.

The Single Nucleotide Polymorphism Gly482Ser in the PGC-1α Gene Impairs Exercise-Induced Slow-Twitch Muscle Fibre Transformation in Humans

PubMed Central

Steinbacher, Peter; Feichtinger, René G.; Kedenko, Lyudmyla; Kedenko, Igor; Reinhardt, Sandra; Schönauer, Anna-Lena; Leitner, Isabella; Sänger, Alexandra M.; Stoiber, Walter; Kofler, Barbara; Förster, Holger; Paulweber, Bernhard; Ring-Dimitriou, Susanne

2015-01-01

PGC-1α (peroxisome proliferator-activated receptor γ co-activator 1α) is an important regulator of mitochondrial biogenesis and a master regulator of enzymes involved in oxidative phosphorylation. Recent evidence demonstrated that the Gly482Ser single nucleotide polymorphism (SNP) in the PGC-1α gene affects insulin sensitivity, blood lipid metabolism and binding to myocyte enhancer factor 2 (MEF2). Individuals carrying this SNP were shown to have a reduced cardiorespiratory fitness and a higher risk to develop type 2 diabetes. Here, we investigated the responses of untrained men with the Gly482Ser SNP to a 10 week programme of endurance training (cycling, 3 x 60 min/week, heart rate at 70-90% VO2peak). Quantitative data from analysis of biopsies from vastus lateralis muscle revealed that the SNP group, in contrast to the control group, lacked a training-induced increase in content of slow contracting oxidative fibres. Capillary supply, mitochondrial density, mitochondrial enzyme activities and intramyocellular lipid content increased similarly in both groups. These results indicate that the impaired binding of MEF2 to PGC-1α in humans with this SNP impedes exercise-induced fast-to-slow muscle fibre transformation. PMID:25886402

[Metabolic effects of static physical load under the conditions of pharmakologicheskoĭ mobilization of physical endurance].

PubMed

Nagornev, S N; Kalinkin, S V; Bobrovnitskiĭ, I P; Sytnik, S I; Petrova, T V; Orlova, T A

2000-01-01

The model of static physical loading (SPL) was used to study the biochemical effects of graded static tension and potentiality for pharmacological mobilization of physical endurance with participation of male volunteers. A close pathogenetic linkage between the established metabolic effects of the model and their adaptive adequacy to the stressing factor show that there is every reason to arrange the observed shifts in a SPL syndrome. The SPL syndrome is primarily manifested by exaggerated tone of the adrenoactive structures, inhibition of insulin production by the pancreas, activation of the neuropeptide anti-stress mechanisms, predominant utilization of the lipid substrate in energy production, intensification of protein catabolism, and increase in myocyte membrane permeability due to energy deficit. The investigation demonstrated that improvement of static physical endurance can be attained with a mobilizing stimulator (sidnocarb) and a combination of sidnocarb with a nonmediatory preparation (bemytil). This pharmacological combination levels side-effects of exorbitant activation of the adrenal system. On the contrary, a metabolic vitamin-microelements complex ("cocktail C") perceivably enhances SPL endurance (sidnocarb dose was lowered in three times), possesses the stress-protective effect, the ability to moderate the intensity of free (uninvolved in phosphorylation) oxidation and to optimize energy-plastic processes with predominant utilization of the lipid substrate.

Vitamin D inhibits growth of human airway smooth muscle cells through growth factor-induced phosphorylation of retinoblastoma protein and checkpoint kinase 1

PubMed Central

Damera, G; Fogle, HW; Lim, P; Goncharova, EA; Zhao, H; Banerjee, A; Tliba, O; Krymskaya, VP; Panettieri, RA

2009-01-01

Background and purpose: Airway remodelling in asthma is manifested, in part, as increased airway smooth muscle (ASM) mass, reflecting myocyte proliferation. We hypothesized that calcitriol, a secosteroidal vitamin D receptor (VDR) modulator, would inhibit growth factor-induced myocyte proliferation. Experimental approach: Human ASM cell cultures were derived from bronchial samples taken during surgery. ASM cells were treated with platelet-derived growth factor (PDGF) (10 ng·mL−1) for 24 h in the presence of calcitriol, dexamethasone or a checkpoint kinase 1 (Chk1) inhibitor (SB218078). The effects of calcitriol on PDGF-mediated cell proliferation were assessed by thymidine incorporation assay, propidium iodide-based cell cycle analysis, caspase-3 assay and immunoblotting for specific cell cycle modulators. Key results: Calcitriol, but not dexamethasone, inhibited PDGF-induced ASM DNA synthesis concentration dependently (IC50= 520 ± 52 nM). These effects were associated with VDR-mediated expression of cytochrome CYP24A1 with no effects on ASM apoptosis. Calcitriol substantially inhibited (P < 0.01) PDGF-stimulated cell growth in ASM derived from both normal (59 ± 8%) and asthmatic subjects (57 ± 9%). Calcitriol inhibited PDGF-induced phosphorylation of retinoblastoma protein (Rb) and Chk1, with no effects on PDGF-mediated activation of extracellular signal-regulated kinases 1/2, PI3-kinase and S6 kinase, or expression of p21Waf/Cip-1, p27Kip1, cyclin D and E2F-1. Consistent with these observations, SB218078 also inhibited (IC50= 450 ± 100 pM) PDGF-induced cell cycle progression. Conclusions and implications: Calcitriol decreased PDGF-induced ASM cell growth by inhibiting Rb and Chk1 phosphorylation. This Research Paper is the subject of a Commentary in this issue by Clifford and Knox (pp. 1426–1428). To view this article visit http://www3.interscience.wiley.com/journal/121548564/issueyear?year=2009 PMID:19814732

Increased Susceptibility to Atrial Fibrillation Secondary to Atrial Fibrosis in Transgenic Goats Expressing Transforming Growth Factor-β1

PubMed Central

Davies, Christopher J.; Regouski, Misha; Hall, Justin; Olsen, Aaron L.; Meng, Qinggang; Rutigliano, Heloisa M.; Dosdall, Derek J.; Angel, Nathan A.; Sachse, Frank B.; Seidel, Thomas; Thomas, Aaron J.; Stott, Rusty; Panter, Kip E.; Lee, Pamela M.; Van Wettere, Arnaud J.; Stevens, John R.; Wang, Zhongde; MacLeod, Rob S.; Marrouche, Nassir F.; White, Kenneth L.

2016-01-01

Introduction Large animal models of progressive atrial fibrosis would provide an attractive platform to study relationship between structural and electrical remodeling in atrial fibrillation (AF). Here we established a new transgenic goat model of AF with cardiac specific overexpression of TGF-β1 and investigated the changes in the cardiac structure and function leading to AF. Methods and Results Transgenic goats with cardiac specific overexpression of constitutively active TGF-β1 were generated by somatic cell nuclear transfer. We examined myocardial tissue, ECGs, echocardiographic data, and AF susceptibility in transgenic and wild-type control goats. Transgenic goats exhibited significant increase in fibrosis and myocyte diameters in the atria compared to controls, but not in the ventricles. P-wave duration was significantly greater in transgenic animals starting at 12-month of age, but no significant chamber enlargement was detected, suggesting conduction slowing in the atria. Furthermore, this transgenic goat model exhibited a significant increase in AF vulnerability. Six of 8 transgenic goats (75%) were susceptible to AF induction and exhibited sustained AF (>2 minutes), whereas, none of 6 controls displayed sustained AF (P<0.01). Length of induced AF episodes was also significantly greater in the transgenic group compared to controls (687±212.02 vs. 2.50±0.88 seconds, P<0.0001), but no persistent or permanent AF was observed. Conclusion A novel transgenic goat model with a substrate for AF was generated. In this model, cardiac overexpression of TGF-β1 led to an increase in fibrosis and myocyte size in the atria, and to progressive P-wave prolongation. We suggest that these factors underlie increased AF susceptibility. PMID:27447370

Impact of Modifiable Risk Factors on B-type Natriuretic Peptide and Cardiac Troponin T Concentrations

PubMed Central

Srivastava, Pratyaksh K.; Pradhan, Aruna D.; Cook, Nancy R.; Ridker, Paul M.; Everett, Brendan M.

2015-01-01

Alcohol use, physical activity, diet, and cigarette smoking are modifiable cardiovascular risk factors that have a substantial impact on the risk of myocardial infarction, stroke, and cardiovascular death. We hypothesized that these behaviors may alter concentrations of cardiac troponin, a marker of myocyte injury, and B-type natriuretic peptide, a marker of myocyte stress. Both markers have shown strong association with adverse cardiovascular outcomes. In 519 women with no evidence of cardiovascular disease, we measured circulating concentrations of cardiac troponin T, using a high-sensitivity assay (hsTnT), and the amino-terminal fragment of B-type natriuretic peptide (NT-proBNP). We used logistic regression to determine if these behaviors were associated with detectable hsTnT (>3 ng/L) or with NT-proBNP in the highest quartile (≥127.3 ng/L). The median (Q1-Q3) NT-proBNP of the cohort was 68.8 (40.3–127.3), and 30.8% (160/519) of the cohort had detectable circulating hsTnT. In adjusted models, women who drank 1–6 drinks per week had lower odds of having a detectable hsTnT (0.58, 95% CI: 0.34–0.96) and lower odds of having an elevated NT-proBNP (OR 0.55, 95% CI 0.32–0.96). We were subsequently able to validate the results for B-type natriuretic peptide in a large independent cohort. In conclusion, our results suggest that regular alcohol consumption is associated with lower concentrations of hsTnT and NT-proBNP, two cardiovascular biomarkers associated with cardiovascular risk, and raise the hypothesis that the beneficial effects of alcohol consumption may be mediated by direct effects on the myocardium. PMID:26739393

Fibroblast growth factor-21 is positively associated with atrial fibrosis in atrial fibrillation patients with rheumatic heart disease.

PubMed

Wang, Rui; Yi, Xin; Li, Xiaoyan; Jiang, Xuejun

2015-01-01

Fibroblast growth factor-21 (FGF-21) has been discovered as a strong hormone, plays an important role in lipid metabolism, glucose metabolism, associated with several diseases such as obesity, metabolic syndrome, diabetes mellitus, and cardiovascular events; however, no evidence is available concerning the relationship of FGF-21 and atrial fibrosis in patients with atrial fibrillation (AF) and rheumatic heart disease (RHD). Twenty-four rheumatic heart disease patients were divided into two groups, 12 cases with AF and 12 cases with sinus rhythm (SR). Clinical characteristics and blood samples were collected before surgery; right atrial appendage samples were taken in the surgery of valve replacement. HE staining was performed to determine cross-sectional area of atrial myocytes; Masson stained sections and mRNA levels of cardiac fibrosis biomarkers were used to evaluate the degree of cardiac fibrosis; the level of FGF-21 was evaluated via enzyme-linked immunosorbent assay (ELISA), immunohistochemistry, and real-time polymerase chain reaction (PCR). Compared with SR group, cross-sectional area of atrial myocytes and collagen volume fraction were significantly increased in the atrial tissue of AF group. The distribution of FGF-21 in the AF group was remarkably higher than SR group. In addition, plasma and mRNA levels of FGF-21 in atrial tissue of AF showed the same trend as the result of immunohistochemistry. Using linear correlation analysis, the expression level of FGF-21 was found to be positively related to the degree of atrial fibrosis. FGF-21 might involve in the development and maintenance of atrial fibrosis in atrial fibrillation with rheumatic heart disease, and FGF-21 could be used as a novel biomarker to evaluate myocardial fibrosis in the future.

Increased Susceptibility to Atrial Fibrillation Secondary to Atrial Fibrosis in Transgenic Goats Expressing Transforming Growth Factor-β1.

PubMed

Polejaeva, Irina A; Ranjan, Ravi; Davies, Christopher J; Regouski, Misha; Hall, Justin; Olsen, Aaron L; Meng, Qinggang; Rutigliano, Heloisa M; Dosdall, Derek J; Angel, Nathan A; Sachse, Frank B; Seidel, Thomas; Thomas, Aaron J; Stott, Rusty; Panter, Kip E; Lee, Pamela M; Van Wettere, Arnaud J; Stevens, John R; Wang, Zhongde; MacLeod, Rob S; Marrouche, Nassir F; White, Kenneth L

2016-10-01

Large animal models of progressive atrial fibrosis would provide an attractive platform to study relationship between structural and electrical remodeling in atrial fibrillation (AF). Here we established a new transgenic goat model of AF with cardiac specific overexpression of TGF-β1 and investigated the changes in the cardiac structure and function leading to AF. Transgenic goats with cardiac specific overexpression of constitutively active TGF-β1 were generated by somatic cell nuclear transfer. We examined myocardial tissue, ECGs, echocardiographic data, and AF susceptibility in transgenic and wild-type control goats. Transgenic goats exhibited significant increase in fibrosis and myocyte diameters in the atria compared to controls, but not in the ventricles. P-wave duration was significantly greater in transgenic animals starting at 12 months of age, but no significant chamber enlargement was detected, suggesting conduction slowing in the atria. Furthermore, this transgenic goat model exhibited a significant increase in AF vulnerability. Six of 8 transgenic goats (75%) were susceptible to AF induction and exhibited sustained AF (>2 minutes), whereas none of 6 controls displayed sustained AF (P < 0.01). Length of induced AF episodes was also significantly greater in the transgenic group compared to controls (687 ± 212.02 seconds vs. 2.50 ± 0.88 seconds, P < 0.0001), but no persistent or permanent AF was observed. A novel transgenic goat model with a substrate for AF was generated. In this model, cardiac overexpression of TGF-β1 led to an increase in fibrosis and myocyte size in the atria, and to progressive P-wave prolongation. We suggest that these factors underlie increased AF susceptibility. © 2016 Wiley Periodicals, Inc.

Valsartan Attenuates KIR2.1 by Downregulating the Th1 Immune Response in Rats Following Myocardial Infarction.

PubMed

Li, Xinran; Hu, Hesheng; Wang, Ye; Xue, Mei; Li, Xiaolu; Cheng, Wenjuan; Xuan, Yongli; Yin, Jie; Yang, Na; Yan, Suhua

2016-03-01

Myocardial infarction (MI) results in decreased inward-rectifier K⁺ current (IK1), which is mediated primarily by the Kir2.1 protein and is accompanied by upregulated T cells. Interferon γ (IFN-γ), secreted predominantly by Th1 cells, causes a decrease in IK1 in microglia. Whether Th1 cells can induce IK1/Kir2.1 remodeling following MI and whether valsartan can ameliorate this phenomenon remain unclear. Rats experiencing MI received either valsartan or saline for 7 days. Th1-enriched lymphocytes and myocytes were cocultured with or without valsartan treatment. Th1 cells were monitored by flow cytometry. The protein levels of Kir2.1 were detected by Western blot analyses. IK1 was recorded through whole-cell patch clamping. The plasma levels of IFN-γ, interleukin 2, and tumor necrosis factor α were detected by enzyme-linked immunosorbent assay. Th1 cell number and cytokine expression levels were higher following MI, and the Kir2.1 protein level was decreased. In MI rats, valsartan reduced Th1 cell number and cytokine expression levels and increased the Kir2.1 expression and the IK1 current compared with the rats that received saline treatment; these results are consistent with the effect of valsartan in cocultured lymphocytes and myocytes. In vitro, IFN-γ overexpression suppressed the IK1 current, whereas interleukin 2 and tumor necrosis factor α had no significant effect on the current, establishing that Th1 cell regulation of IK1/Kir2.1 expression is mainly dependent on IFN-γ. Valsartan ameliorates IK1/Kir2.1 remodeling by downregulating the Th1 immune response following MI.

Egr-1 mediated cardiac miR-99 family expression diverges physiological hypertrophy from pathological hypertrophy.

PubMed

Ramasamy, Subbiah; Velmurugan, Ganesan; Rekha, Balakrishnan; Anusha, Sivakumar; Shanmugha Rajan, K; Shanmugarajan, Suresh; Ramprasath, Tharmarajan; Gopal, Pandi; Tomar, Dhanendra; Karthik, Karuppusamy V; Verma, Suresh Kumar; Garikipati, Venkata Naga Srikanth; Sudarsan, Rajan

2018-04-01

The physiological cardiac hypertrophy is an adaptive condition without myocyte cell death, while pathological hypertrophy is a maladaptive condition associated with myocyte cell death. This study explores the miRNome of α-2M-induced physiologically hypertrophied cardiomyocytes and the role of miRNA-99 family during cardiac hypertrophy. Physiological and pathological cardiac hypertrophy was induced in H9c2 cardiomyoblast cell lines using α-2M and isoproterenol respectively. Total RNA isolation and small RNA sequencing were executed for physiological hypertrophy model. The differentially expressed miRNAs and its target mRNAs were validated in animal models. Transcription factor binding sites were predicted in the promoter of specific miRNAs and validated by ChIP-PCR. Subsequently, the selected miRNA was functionally characterized by overexpression and silencing. The effects of silencing of upstream regulator and downstream target gene were studied. Analysis of small RNA reads revealed the differential expression of a large set of miRNAs during hypertrophy, of which miR-99 family was highly downregulated upon α-2M treatment. However, this miR-99 family expression was upregulated during pathological hypertrophy and confirmed in animal models. ChIP-PCR confirms the binding of Egr-1 transcription factor to the miR-99 promoter. Further, silencing of Egr-1 decreased the expression of miR-99. The overexpression or silencing of miR-99 diverges the physiological hypertrophy to pathological hypertrophy and vice versa by regulating Akt-1 pathway. Silencing of Akt-1 replicates the effect of overexpression of miR-99. The results proved Egr-1 mediated regulation of miR-99 family that plays a key role in determining the fate of cardiac hypertrophy by regulating Akt-1 signaling. Copyright © 2018 Elsevier Inc. All rights reserved.

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

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

Regional distribution of T-tubule density in left and right atria in dogs.

PubMed

Arora, Rishi; Aistrup, Gary L; Supple, Stephen; Frank, Caleb; Singh, Jasleen; Tai, Shannon; Zhao, Anne; Chicos, Laura; Marszalec, William; Guo, Ang; Song, Long-Sheng; Wasserstrom, J Andrew

2017-02-01

The peculiarities of transverse tubule (T-tubule) morphology and distribution in the atrium-and how they contribute to excitation-contraction coupling-are just beginning to be understood. The objectives of this study were to determine T-tubule density in the intact, live right and left atria in a large animal and to determine intraregional differences in T-tubule organization within each atrium. Using confocal microscopy, T-tubules were imaged in both atria in intact, Langendorf-perfused normal dog hearts loaded with di-4-ANEPPS. T-tubules were imaged in large populations of myocytes from the endocardial surface of each atrium. Computerized data analysis was performed using a new MatLab (Mathworks, Natick, MA) routine, AutoTT. There was a large percentage of myocytes that had no T-tubules in both atria with a higher percentage in the right atrium (25.1%) than in the left atrium (12.5%) (P < .02). The density of transverse and longitudinal T-tubule elements was low in cells that did contain T-tubules, but there were no significant differences in density between the left atrial appendage, the pulmonary vein-posterior left atrium, the right atrial appendage, and the right atrial free wall. In contrast, there were significant differences in sarcomere spacing and cell width between different regions of the atria. There is a sparse T-tubule network in atrial myocytes throughout both dog atria, with significant numbers of myocytes in both atria-the right atrium more so than the left atrium-having no T-tubules at all. These regional differences in T-tubule distribution, along with differences in cell width and sarcomere spacing, may have implications for the emergence of substrate for atrial fibrillation. Copyright © 2016 Heart Rhythm Society. Published by Elsevier Inc. All rights reserved.

Effect of trimetazidine treatment on the transient outward potassium current of the left ventricular myocytes of rats with streptozotocin-induced type 1 diabetes mellitus.

PubMed

Xiang, Yu-luan; He, Li; Xiao, Jun; Xia, Shuang; Deng, Song-bai; Xiu, Yun; She, Qiang

2012-03-01

Cardiovascular complications are a leading cause of mortality in patients with diabetes mellitus (DM). The present study was designed to investigate the effects of trimetazidine (TMZ), an anti-angina drug, on transient outward potassium current (Ito) remodeling in ventricular myocytes and the plasma contents of free fatty acid (FFA) and glucose in DM. Sprague-Dawley rats, 8 weeks old and weighing 200-250 g, were randomly divided into three groups of 20 animals each. The control group was injected with vehicle (1 mM citrate buffer), the DM group was injected with 65 mg/kg streptozotocin (STZ) for induction of type 1 DM, and the DM + TMZ group was injected with the same dose of STZ followed by a 4-week treatment with TMZ (60 mg·kg-1·day-1). All animals were then euthanized and their hearts excised and subjected to electrophysiological measurements or gene expression analyses. TMZ exposure significantly reversed the increased plasma FFA level in diabetic rats, but failed to change the plasma glucose level. The amplitude of Ito was significantly decreased in left ventricular myocytes from diabetic rats relative to control animals (6.25 ± 1.45 vs 20.72 ± 2.93 pA/pF at +40 mV). The DM-associated Ito reduction was attenuated by TMZ. Moreover, TMZ treatment reversed the increased expression of the channel-forming alpha subunit Kv1.4 and the decreased expression of Kv4.2 and Kv4.3 in diabetic rat hearts. These data demonstrate that TMZ can normalize, or partially normalize, the increased plasma FFA content, the reduced Ito of ventricular myocytes, and the altered expression Kv1.4, Kv4.2, and Kv4.3 in type 1 DM.

Activation of NADPH oxidase mediates increased endoplasmic reticulum stress and left ventricular remodeling after myocardial infarction in rabbits.

PubMed

Li, Bao; Tian, Jing; Sun, Yi; Xu, Tao-Rui; Chi, Rui-Fang; Zhang, Xiao-Li; Hu, Xin-Ling; Zhang, Yue-An; Qin, Fu-Zhong; Zhang, Wei-Fang

2015-05-01

Nicotinamide adenine dinucleotide 3-phosphate (NADPH) oxidase activity and endoplasmic reticulum (ER) stress are increased after myocardial infarction (MI). In this study, we proposed to test whether activation of the NADPH oxidase in the remote non-infarcted myocardium mediates ER stress and left ventricular (LV) remodeling after MI. Rabbits with MI or sham operation were randomly assigned to orally receive an NADPH oxidase inhibitor apocynin or placebo for 30 days. The agents were administered beginning at 1 week after surgery. MI rabbits exhibited decreases in LV fractional shortening, LV ejection fraction and the first derivative of the LV pressure rise, which were abolished by apocynin treatment. NADPH oxidase Nox2 protein and mRNA expressions were increased in the remote non-infarcted myocardium after MI. Immunolabeling further revealed that Nox2 was increased in cardiac myocytes in the remote myocardium. The apocynin treatment prevented increases in the Nox2 expression, NADPH oxidase activity, oxidative stress, myocyte apoptosis and GRP78, CHOP and cleaved caspase 12 protein expression in the remote myocardium. The apocynin treatment also attenuated increases in myocyte diameter and cardiac fibrosis. In cultured H9C2 cardiomyocytes exposed to angiotensin II, an important stimulus for post-MI remodeling, Nox2 knockdown with siRNA significantly inhibited angiotensin II-induced NADPH oxidase activation, reactive oxygen species and GRP78 and CHOP protein expression. We conclude that NADPH oxidase inhibition attenuates increased ER stress in the remote non-infarcted myocardium and LV remodeling late after MI in rabbits. These findings suggest that the activation of NADPH oxidase in the remote non-infarcted myocardium mediates increased ER stress, contributing to myocyte apoptosis and LV remodeling after MI. Copyright © 2015 Elsevier B.V. All rights reserved.

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

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

Role of Ca2+/calmodulin-stimulated cyclic nucleotide phosphodiesterase 1 in mediating cardiomyocyte hypertrophy

PubMed Central

Miller, Clint L.; Oikawa, Masayoshi; Cai, Yujun; Wojtovich, Andrew P.; Nagel, David J.; Xu, Xiangbin; Xu, Haodong; Florio, Vince; Rybalkin, Sergei D.; Beavo, Joseph A.; Chen, Yiu-Fai; Li, Jian-Dong; Blaxall, Burns C.; Abe, Jun-ichi; Yan, Chen

2009-01-01

Rationale Cyclic nucleotide phosphodiesterases (PDE) through the degradation of second messenger cyclic guanosine monophosphate (cGMP) play critical roles in maintaining cardiomyocyte homeostasis. Ca2+/CaM-activated cGMP-hydrolyzing PDE1 family may play a pivotal role in balancing intracellular Ca2+/CaM and cGMP signaling, however its function in cardiomyocytes is unknown. Objective Herein we investigate the role of Ca2+/CaM-stimulated PDE1 in regulating pathological cardiomyocyte hypertrophy in neonatal and adult rat ventricular myocytes (NRVM and ARVM) and in the heart in vivo. Methods and Results Inhibition of PDE1 activity using a PDE1 selective inhibitor IC86340 or downregulation of PDE1A using siRNA prevented phenylephrine (PE) induced pathological myocyte hypertrophy and hypertrophic marker expression in neonatal (NRVM) and adult (ARVM) rat ventricular myocytes. Importantly, administration of the PDE1 inhibitor IC86340 attenuated cardiac hypertrophy induced by chronic ISO infusion in vivo. Both PDE1A and PDE1C mRNA and protein were detected in human hearts, however PDE1A expression was conserved in rodent hearts. Moreover, PDE1A expression was significantly upregulated in vivo in the heart and myocytes from various pathological hypertrophy animal models and in vitro in isolated NRVM and ARVM treated with neurohumoral stimuli such as angiotensin II (Ang II) and ISO. Further, PDE1A plays a critical role in PE-induced reduction of intracellular cGMP and PKG activity, and thereby cardiomyocyte hypertrophy in vitro. Conclusions These results elucidate a novel role for Ca2+/CaM-stimulated PDE1, particularly PDE1A, in regulating pathological cardiomyocyte hypertrophy via a cGMP/PKG-dependent mechanism, thereby demonstrating Ca2+ and cGMP signaling cross-talk during cardiac hypertrophy. PMID:19797176

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.