Enhanced basal late sodium current appears to underlie the age-related prolongation of action potential duration in guinea pig ventricular myocytes.

PubMed

Song, Yejia; Belardinelli, Luiz

2017-12-14

Aging hearts have prolonged QT interval and are vulnerable to oxidative stress. Because the QT interval indirectly reflects the action potential duration (APD), we examined the hypotheses that 1) the APD of ventricular myocytes increases with age; 2) the age-related prolongation of APD is due to an enhancement of basal late Na + current (I NaL ); 3) inhibition of I NaL may protect aging hearts from arrhythmogenic effects of hydrogen peroxide (H 2 O 2 ). Experiments were performed on ventricular myocytes isolated from one-month (young) and one-year (old) guinea pigs (GPs). The APD of myocytes from old GPs was significantly longer than that from young GPs and was shortened by the I NaL inhibitors GS967 and tetrodotoxin. The magnitude of I NaL was significantly larger in myocytes from old than from young GPs. The CaMKII inhibitors KN-93 and AIP and the Na V 1.5-channel blocker MTSEA blocked the I NaL . There were no significant differences between myocytes from young and old GPs in L-type Ca 2+ current and the rapidly- and slowly-activating delayed rectifier K + currents, although the inward rectifier K + current was slightly decreased in myocytes from old GPs. H 2 O 2 induced more early afterdepolarizations in myocytes from old than from young GPs. The effect of H 2 O 2 was attenuated by GS967. The results suggest that 1) the APD of myocytes from old GPs is prolonged, 2) a CaMKII-mediated increase in Na V 1.5-channel I NaL is responsible for the prolongation of APD, and 3) Inhibition of I NaL may be beneficial for maintaining electrical stability under oxidative stress in myocytes of old GPs.

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

PubMed

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

2003-11-28

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

Modeling beta-adrenergic control of cardiac myocyte contractility in silico

NASA Technical Reports Server (NTRS)

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

2003-01-01

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

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

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

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

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

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

PubMed

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

2001-05-01

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

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

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

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.

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.

Transcriptional deregulation of oncogenic myocyte enhancer factor 2C in T-cell acute lymphoblastic leukemia.

PubMed

Nagel, Stefan; Venturini, Letizia; Meyer, Corinna; Kaufmann, Maren; Scherr, Michaela; Drexler, Hans G; Macleod, Roderick A F

2011-02-01

Myocyte enhancer factor 2C (MEF2C) encodes a transcription factor which is ectopically expressed in T-cell acute lymphoblastic leukemia (T-ALL) cell lines, deregulated directly by ectopically expressed homeodomain protein NKX2-5 or by loss of promoter regions via del(5)(q14). Here, we analyzed the MEF2C 5'-region, thus identifying potential regulatory binding sites for GFI1B, basic helix-loop-helix proteins, STAT5, and HOXA9/HOXA10. Chromatin immunoprecipitation and overexpression analyses demonstrated direct activation by GFI1B and LYL1 and inhibition by STAT5. HOXA9/HOXA10 activated expression of NMYC which in turn mediated MEF2C repression, indicating an indirect mode of regulation via NMYC interactor (NMI) and STAT5. Lacking comma: Chromosomal deletion of the STAT5 binding site in LOUCY cells reduced protein levels of STAT5 in some MEF2C-positve T-ALL cell lines, and the presence of inhibitory IL7-JAK-STAT5 signaling highlighted the repressive impact of this factor in MEF2C regulation. Taken together, our results indicate that the expression of MEF2C in T-ALL cells is principally deregulated via activating leukemic transcription factors GFI1B or NKX2-5 and by escaping inhibitory developmental STAT5 signaling.

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

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.

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.

Syzygium aromaticum L. (Clove) extract regulates energy metabolism in myocytes.

PubMed

Tu, Zheng; Moss-Pierce, Tijuana; Ford, Paul; Jiang, T Alan

2014-09-01

The prevalence of metabolic syndrome and type 2 diabetes is increasing worldwide. Herbs and spices have been used for the treatment of diabetes for centuries in folk medicine. Syzygium aromaticum L. (Clove) extracts (SE) have been shown to perform comparably to insulin by significantly reducing blood glucose levels in animal models; however, the mechanisms are not well understood. We investigated the effects of clove on metabolism in C2C12 myocytes and demonstrated that SE significantly increases glucose consumption. The phosphorylation of AMP-activated protein kinase (AMPK), as well as its substrate, acetyl-CoA carboxylase (ACC) was increased by SE treatment. SE also transcriptionally regulates genes involved in metabolism, including sirtuin 1 (SIRT1) and PPARγ coactivator 1α (PGC1α). Nicotinamide, an SIRT1 inhibitor, diminished SE's effects on glucose consumption. Furthermore, treatment with SE dose-dependently increases muscle glycolysis and mitochondrial spare respiratory capacity. Overall, our study suggests that SE has the potential to increase muscle glycolysis and mitochondria function by activating both AMPK and SIRT1 pathways.

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.

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.

Nitrate-containing beetroot enhances myocyte metabolism and mitochondrial content

PubMed Central

Vaughan, Roger A.; Gannon, Nicholas P.; Carriker, Colin R.

2015-01-01

Beetroot (甜菜 tián cài) juice consumption is of current interest for improving aerobic performance by acting as a vasodilator and possibly through alterations in skeletal muscle metabolism and physiology. This work explored the effects of a commercially available beetroot supplement on metabolism, gene expression, and mitochondrial content in cultured myocytes. C2C12 myocytes were treated with various concentrations of the beetroot supplement for various durations. Glycolytic metabolism and oxidative metabolism were quantified via measurement of extracellular acidification and oxygen consumption, respectively. Metabolic gene expression was measured using quantitative reverse transcription–polymerase chain reaction, and mitochondrial content was assessed with flow cytometry and confocal microscopy. Cells treated with beetroot exhibited significantly increased oxidative metabolism, concurrently with elevated metabolic gene expression including peroxisome proliferator-activated receptor gamma coactivator-1 alpha, nuclear respiratory factor 1, mitochondrial transcription factor A, and glucose transporter 4, leading to increased mitochondrial biogenesis. Our data show that treatment with a beetroot supplement increases basal oxidative metabolism. Our observations are also among the first to demonstrate that beetroot extract is an inducer of metabolic gene expression and mitochondrial biogenesis. These observations support the need for further investigation into the therapeutic and pharmacological effects of nitrate-containing supplements for health and athletic benefits. PMID:26870674

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

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

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.

Neutrophil adherence to isolated adult canine myocytes. Evidence for a CD18-dependent mechanism.

PubMed

Entman, M L; Youker, K; Shappell, S B; Siegel, C; Rothlein, R; Dreyer, W J; Schmalstieg, F C; Smith, C W

1990-05-01

Cardiac myocytes were isolated from adult dogs and incubated with isolated canine neutrophils (PMN). Intercellular adhesion was low and unchanged by stimulation of the PMN with zymosan activated serum or platelet activating factor (PAF) at concentrations that significantly enhance PMN adhesion to protein-coated glass and canine endothelial cell monolayers. Intercellular adhesion was significantly increased only when both myocytes and PMN were stimulated (e.g., myocytes incubated with IL-1, tumor necrosis factor, or phorbol myristate acetate, and PMN were chemotactically stimulated). Inhibitors of protein synthesis diminished the IL-1 beta-induced effect by greater than 80%. The IL-1 beta, PAF-stimulated PMN-myocyte adhesion was associated with substantial H2O2 production. Under conditions with low PMN-myocyte adhesion (i.e., IL-1 beta alone, PAF alone, or no stimulus) H2O2 production was generally less than 5% of that occurring with high adhesion. An anti-CD18 monoclonal antibody (R15.7) inhibited stimulated PMN-myocyte adhesion by greater than 95% and reduced H2O2 production by greater than 90%. Control isotype-matched, binding, and nonbinding antibodies were without effect on adherence or H2O2 production. The results indicate that cytokine stimulation of adult myocytes induces expression of a ligand involved in CD18-dependent adherence of canine neutrophils.

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

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...

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.

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

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.

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

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-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

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

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

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.

Cell contact as an independent factor modulating cardiac myocyte hypertrophy and survival in long-term primary culture

NASA Technical Reports Server (NTRS)

Clark, W. A.; Decker, M. L.; Behnke-Barclay, M.; Janes, D. M.; Decker, R. S.

1998-01-01

Cardiac myocytes maintained in cell culture develop hypertrophy both in response to mechanical loading as well as to receptor-mediated signaling mechanisms. However, it has been shown that the hypertrophic response to these stimuli may be modulated through effects of intercellular contact achieved by maintaining cells at different plating densities. In this study, we show that the myocyte plating density affects not only the hypertrophic response and features of the differentiated phenotype of isolated adult myocytes, but also plays a significant role influencing myocyte survival in vitro. The native rod-shaped phenotype of freshly isolated adult myocytes persists in an environment which minimizes myocyte attachment and spreading on the substratum. However, these conditions are not optimal for long-term maintenance of cultured adult cardiac myocytes. Conditions which promote myocyte attachment and spreading on the substratum, on the other hand, also promote the re-establishment of new intercellular contacts between myocytes. These contacts appear to play a significant role in the development of spontaneous activity, which enhances the redevelopment of highly differentiated contractile, junctional, and sarcoplasmic reticulum structures in the cultured adult cardiomyocyte. Although it has previously been shown that adult cardiac myocytes are typically quiescent in culture, the addition of beta-adrenergic agonists stimulates beating and myocyte hypertrophy, and thereby serves to increase the level of intercellular contact as well. However, in densely-plated cultures with intrinsically high levels of intercellular contact, spontaneous contractile activity develops without the addition of beta-adrenergic agonists. In this study, we compare the function, morphology, and natural history of adult feline cardiomyocytes which have been maintained in cultures with different levels of intercellular contact, with and without the addition of beta-adrenergic agonists

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.

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.

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.

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

PubMed

Fearon, Ian M

2006-03-01

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

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.

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

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.

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.

The AMPK-related kinase SNARK regulates muscle mass and myocyte survival

PubMed Central

Lessard, Sarah J.; Rivas, Donato A.; So, Kawai; Koh, Ho-Jin; Queiroz, André Lima; Hirshman, Michael F.; Fielding, Roger A.; Goodyear, Laurie J.

2015-01-01

The maintenance of skeletal muscle mass is critical for sustaining health; however, the mechanisms responsible for muscle loss with aging and chronic diseases, such as diabetes and obesity, are poorly understood. We found that expression of a member of the AMPK-related kinase family, the SNF1-AMPK-related kinase (SNARK, also known as NUAK2), increased with muscle cell differentiation. SNARK expression increased in skeletal muscles from young mice exposed to metabolic stress and in muscles from healthy older human subjects. The regulation of SNARK expression in muscle with differentiation and physiological stress suggests that SNARK may function in the maintenance of muscle mass. Consistent with this hypothesis, decreased endogenous SNARK expression (using siRNA) in cultured muscle cells resulted in increased apoptosis and decreased cell survival under conditions of metabolic stress. Likewise, muscle-specific transgenic animals expressing a SNARK dominant-negative inactive mutant (SDN) had increased myonuclear apoptosis and activation of apoptotic mediators in muscle. Moreover, animals expressing SDN had severe, age-accelerated muscle atrophy and increased adiposity, consistent with sarcopenic obesity. Reduced SNARK activity, in vivo and in vitro, caused downregulation of the Rho kinase signaling pathway, a key mediator of cell survival. These findings reveal a critical role for SNARK in myocyte survival and the maintenance of muscle mass with age. PMID:26690705

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.

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

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

Systolic [Ca2+]i regulates diastolic levels in rat ventricular myocytes

PubMed Central

Sankaranarayanan, Rajiv; Kistamás, Kornél; Greensmith, David J.; Venetucci, Luigi A.

2017-01-01

Key points For the heart to function as a pump, intracellular calcium concentration ([Ca2+]i) must increase during systole to activate contraction and then fall, during diastole, to allow the myofilaments to relax and the heart to refill with blood.The present study investigates the control of diastolic [Ca2+]i in rat ventricular myocytes.We show that diastolic [Ca2+]i is increased by manoeuvres that decrease sarcoplasmic reticulum function. This is accompanied by a decrease of systolic [Ca2+]i such that the time‐averaged [Ca2+]i remains constant.We report that diastolic [Ca2+]i is controlled by the balance between Ca2+ entry and Ca2+ efflux during systole.The results of the present study identify a novel mechanism by which changes of the amplitude of the systolic Ca transient control diastolic [Ca2+]i. Abstract The intracellular Ca concentration ([Ca2+]i) must be sufficently low in diastole so that the ventricle is relaxed and can refill with blood. Interference with this will impair relaxation. The factors responsible for regulation of diastolic [Ca2+]i, in particular the relative roles of the sarcoplasmic reticulum (SR) and surface membrane, are unclear. We investigated the effects on diastolic [Ca2+]i that result from the changes of Ca cycling known to occur in heart failure. Experiments were performed using Fluo‐3 in voltage clamped rat ventricular myocytes. Increasing stimulation frequency increased diastolic [Ca2+]i. This increase of [Ca2+]i was larger when SR function was impaired either by making the ryanodine receptor leaky (with caffeine or ryanodine) or by decreasing sarco/endoplasmic reticulum Ca‐ATPase activity with thapsigargin. The increase of diastolic [Ca2+]i produced by interfering with the SR was accompanied by a decrease of the amplitude of the systolic Ca transient, such that there was no change of time‐averaged [Ca2+]i. Time‐averaged [Ca2+]i was increased by β‐adrenergic stimulation with isoprenaline and increased in a saturating

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

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

Invited review: Regulation of skeletal muscle GLUT-4 expression by exercise.

PubMed

Dohm, G Lynis

2002-08-01

The amount of GLUT-4 protein is a primary factor in determining the maximal rate of glucose transport into skeletal muscle. Therefore, it is important that we understand how exercise regulates GLUT-4 expression so that therapeutic strategies can be designed to increase muscle glucose disposal as a treatment for diabetes. Muscle contraction increases the rates of GLUT-4 transcription and translation. Transcriptional control likely requires at least two DNA binding proteins, myocyte enhancer factor-2 and GLUT-4 enhancer factor, which bind to the promoter. Increased GLUT-4 expression may be mediated by the enzyme AMP-activated kinase, which is activated during exercise and has been demonstrated to increase GLUT-4 transcription. Further research needs to be done to investigate whether AMP-activated kinase activates myocyte enhancer factor-2 and GLUT-4 enhancer factor to increase transcription of the GLUT-4 gene.

Repressive histone methylation regulates cardiac myocyte cell cycle exit.

PubMed

El-Nachef, Danny; Oyama, Kyohei; Wu, Yun-Yu; Freeman, Miles; Zhang, Yiqiang; Robb MacLellan, W

2018-05-22

Mammalian cardiac myocytes (CMs) stop proliferating soon after birth and subsequent heart growth comes from hypertrophy, limiting the adult heart's regenerative potential after injury. The molecular events that mediate CM cell cycle exit are poorly understood. To determine the epigenetic mechanisms limiting CM cycling in adult CMs (ACMs) and whether trimethylation of lysine 9 of histone H3 (H3K9me3), a histone modification associated with repressed chromatin, is required for the silencing of cell cycle genes, we developed a transgenic mouse model where H3K9me3 is specifically removed in CMs by overexpression of histone demethylase, KDM4D. Although H3K9me3 is found across the genome, its loss in CMs preferentially disrupts cell cycle gene silencing. KDM4D binds directly to cell cycle genes and reduces H3K9me3 levels at these promotors. Loss of H3K9me3 preferentially leads to increased cell cycle gene expression resulting in enhanced CM cycling. Heart mass was increased in KDM4D overexpressing mice by postnatal day 14 (P14) and continued to increase until 9-weeks of age. ACM number, but not size, was significantly increased in KDM4D expressing hearts, suggesting CM hyperplasia accounts for the increased heart mass. Inducing KDM4D after normal development specifically in ACMs resulted in increased cell cycle gene expression and cycling. We demonstrated that H3K9me3 is required for CM cell cycle exit and terminal differentiation in ACMs. Depletion of H3K9me3 in adult hearts prevents and reverses permanent cell cycle exit and allows hyperplastic growth in adult hearts in vivo. Copyright © 2017. Published by Elsevier Ltd.

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

Is depressed myocyte contractility centrally involved in heart failure?

PubMed

Houser, Steven R; Margulies, Kenneth B

2003-03-07

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

Enhanced effect of VEGF165 on L-type calcium currents in guinea-pig cardiac ventricular myocytes.

PubMed

Xing, Wenlu; Gao, Chuanyu; Qi, Datun; Zhang, You; Hao, Peiyuan; Dai, Guoyou; Yan, Ganxin

2017-01-01

The mechanisms of vascular endothelial growth factor 165 (VEGF165) on electrical properties of cardiomyocytes have not been fully elucidated. The aim of this study is to test the hypothesis that VEGF165, an angiogenesis-initiating factor, affects L-type calcium currents (I Ca,L ) and cell membrane potential in cardiac myocytes by acting on VEGF type-2 receptors (VEGFR2). I Ca,L and action potentials (AP) were recorded by the whole-cell patch clamp method in isolated guinea-pig ventricular myocytes treated with different concentrations of VEGF165 proteins. Using a VEGFR2 inhibitor, we also tested the receptor of VEGF165 in cardiomyocytes. We found that VEGF165 increased I Ca,L in a concentration-dependent manner. SU5416, a VEGFR2 inhibitor, almost completely eliminated VEGF165-induced I Ca,L increase. VEGF165 had no significant influence on action potential 90 (APD90) and other properties of AP. We conclude that in guinea-pig ventricular myocytes, I Ca,L can be increased by VEGF165 in a concentration-dependent manner through binding to VEGFR2 without causing any significant alteration to action potential duration. Results of this study may further expound the safety of VEGF165 when used in the intervention of heart diseases. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

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.

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.

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

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

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

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

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

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

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

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.

[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.

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

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.

Redox signaling in cardiac myocytes

PubMed Central

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

2011-01-01

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

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

Cloning and characterization of the mouse alpha1C/A-adrenergic receptor gene and analysis of an alpha1C promoter in cardiac myocytes: role of an MCAT element that binds transcriptional enhancer factor-1 (TEF-1).

PubMed

O'Connell, T D; Rokosh, D G; Simpson, P C

2001-05-01

alpha1-Adrenergic receptor (AR) subtypes in the heart are expressed by myocytes but not by fibroblasts, a feature that distinguishes alpha1-ARs from beta-ARs. Here we studied myocyte-specific expression of alpha1-ARs, focusing on the subtype alpha1C (also called alpha1A), a subtype implicated in cardiac hypertrophic signaling in rat models. We first cloned the mouse alpha1C-AR gene, which consisted of two exons with an 18 kb intron, similar to the alpha1B-AR gene. The receptor coding sequence was >90% homologous to that of rat and human. alpha1C-AR transcription in mouse heart was initiated from a single Inr consensus sequence at -588 from the ATG; this and a putative polyadenylation sequence 8.5 kb 3' could account for the predominant 11 kb alpha1C mRNA in mouse heart. A 5'-nontranscribed fragment of 4.4 kb was active as a promoter in cardiac myocytes but not in fibroblasts. Promoter activity in myocytes required a single muscle CAT (MCAT) element, and this MCAT bound in vitro to recombinant and endogenous transcriptional enhancer factor-1. Thus, alpha1C-AR transcription in cardiac myocytes shares MCAT dependence with other cardiac-specific genes, including the alpha- and beta-myosin heavy chains, skeletal alpha-actin, and brain natriuretic peptide. However, the mouse alpha1C gene was not transcribed in the neonatal heart and was not activated by alpha1-AR and other hypertrophic agonists in rat myocytes, and thus differed from other MCAT-dependent genes and the rat alpha1C gene.

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.

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

Interactions between inflammatory signals and the progesterone receptor in regulating gene expression in pregnant human uterine myocytes

PubMed Central

Lee, Yun; Sooranna, Suren R; Terzidou, Vasso; Christian, Mark; Brosens, Jan; Huhtinen, Kaisa; Poutanen, Matti; Barton, Geraint; Johnson, Mark R; Bennett, Phillip R

2012-01-01

The absence of a fall in circulating progesterone levels has led to the concept that human labour is associated with ‘functional progesterone withdrawal’ caused through changes in the expression or function of progesterone receptor (PR). At the time of labour, the human uterus is heavily infiltrated with inflammatory cells, which release cytokines to create a ‘myometrial inflammation’ via NF-κB activation. The negative interaction between NF-κB and PR, may represent a mechanism to account for ‘functional progesterone withdrawal’ at term. Conversely, PR may act to inhibit NF-κB function and so play a role in inhibition of myometrial inflammation during pregnancy. To model this inter-relationship, we have used small interfering (si) RNA-mediated knock-down of PR in human pregnant myocytes and whole genome microarray analysis to identify genes regulated through PR. We then activated myometrial inflammation using IL-1β stimulation to determine the role of PR in myometrial inflammation regulation. Through PR-knock-down, we found that PR regulates gene networks involved in myometrial quiescence and extracellular matrix integrity. Activation of myometrial inflammation was found to antagonize PR-induced gene expression, of genes normally upregulated via PR. We found that PR does not play a role in repression of pro-inflammatory gene networks induced by IL-1β and that only MMP10 was significantly regulated in opposite directions by IL-1β and PR. We conclude that progesterone acting through PR does not generally inhibit myometrial inflammation. Activation of myometrial inflammation does cause ‘functional progesterone withdrawal’ but only in the context of genes normally upregulated via PR. PMID:22435466

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

PubMed

Clegg, C H; Hauschka, S D

1987-08-01

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

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

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.

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

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.

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

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

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

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.

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.

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.

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

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

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

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

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

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

Effects of rogue ryanodine receptors on Ca2+ sparks in cardiac myocytes

PubMed Central

Chen, Xudong; Feng, Yundi; Tan, Wenchang

2018-01-01

Ca2+ sparks and Ca2+ quarks, arising from clustered and rogue ryanodine receptors (RyRs), are significant Ca2+ release events from the junctional sarcoplasmic reticulum (JSR). Based on the anomalous subdiffusion of Ca2+ in the cytoplasm, a mathematical model was developed to investigate the effects of rogue RyRs on Ca2+ sparks in cardiac myocytes. Ca2+ quarks and sparks from the stochastic opening of rogue and clustered RyRs are numerically reproduced and agree with experimental measurements. It is found that the stochastic opening Ca2+ release units (CRUs) of clustered RyRs are regulated by free Ca2+ concentration in the JSR lumen (i.e. [Ca2+]lumen). The frequency of spontaneous Ca2+ sparks is remarkably increased by the rogue RyRs opening at high [Ca2+]lumen, but not at low [Ca2+]lumen. Hence, the opening of rogue RyRs contributes to the formation of Ca2+ sparks at high [Ca2+]lumen. The interplay of Ca2+ sparks and Ca2+ quarks has been discussed in detail. This work is of significance to provide insight into understanding Ca2+ release mechanisms in cardiac myocytes. PMID:29515864

Effects of rogue ryanodine receptors on Ca2+ sparks in cardiac myocytes.

PubMed

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

2018-02-01

Ca 2+ sparks and Ca 2+ quarks, arising from clustered and rogue ryanodine receptors (RyRs), are significant Ca 2+ release events from the junctional sarcoplasmic reticulum (JSR). Based on the anomalous subdiffusion of Ca 2+ in the cytoplasm, a mathematical model was developed to investigate the effects of rogue RyRs on Ca 2+ sparks in cardiac myocytes. Ca 2+ quarks and sparks from the stochastic opening of rogue and clustered RyRs are numerically reproduced and agree with experimental measurements. It is found that the stochastic opening Ca 2+ release units (CRUs) of clustered RyRs are regulated by free Ca 2+ concentration in the JSR lumen (i.e. [Ca 2+ ] lumen ). The frequency of spontaneous Ca 2+ sparks is remarkably increased by the rogue RyRs opening at high [Ca 2+ ] lumen , but not at low [Ca 2+ ] lumen . Hence, the opening of rogue RyRs contributes to the formation of Ca 2+ sparks at high [Ca 2+ ] lumen . The interplay of Ca 2+ sparks and Ca 2+ quarks has been discussed in detail. This work is of significance to provide insight into understanding Ca 2+ release mechanisms in cardiac myocytes.

Computational Modeling and Numerical Methods for Spatiotemporal Calcium Cycling in Ventricular Myocytes

PubMed Central

Nivala, Michael; de Lange, Enno; Rovetti, Robert; Qu, Zhilin

2012-01-01

Intracellular calcium (Ca) cycling dynamics in cardiac myocytes is regulated by a complex network of spatially distributed organelles, such as sarcoplasmic reticulum (SR), mitochondria, and myofibrils. In this study, we present a mathematical model of intracellular Ca cycling and numerical and computational methods for computer simulations. The model consists of a coupled Ca release unit (CRU) network, which includes a SR domain and a myoplasm domain. Each CRU contains 10 L-type Ca channels and 100 ryanodine receptor channels, with individual channels simulated stochastically using a variant of Gillespie’s method, modified here to handle time-dependent transition rates. Both the SR domain and the myoplasm domain in each CRU are modeled by 5 × 5 × 5 voxels to maintain proper Ca diffusion. Advanced numerical algorithms implemented on graphical processing units were used for fast computational simulations. For a myocyte containing 100 × 20 × 10 CRUs, a 1-s heart time simulation takes about 10 min of machine time on a single NVIDIA Tesla C2050. Examples of simulated Ca cycling dynamics, such as Ca sparks, Ca waves, and Ca alternans, are shown. PMID:22586402

Cellular Trafficking of Phospholamban and Formation of Functional Sarcoplasmic Reticulum During Myocyte DIfferentiation

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

Stenoien, David L.; Knyushko, Tatyana V.; Londono, Monica P.

2007-06-01

The sarco/endoplasmic reticulum Ca-ATPase (SERCA) family members are transmembrane proteins that play an essential role in regulating intracellular calcium levels. Phospholamban (PLB), a 52 amino acid phosphoprotein, regulates SERCA activity in adult heart and skeletal muscle. Using the C2C12 myocyte cell line, we find endogenous PLB constitutively expressed in both myoblasts and myotubes, whereas SERCA expression coincides with activation of the differentiation program. PLB has a punctuate distribution in myoblasts changing to a reticular distribution in myotubes where it colocalizes with SERCAs. To examine the distribution and dynamics of PLB and SERCA, we expressed fluorescent fusion proteins (GFP, CFP, andmore » YFP) of PLB and SERCA in myoblasts. Coexpressed PLB and SERCA localize to distinct cellular compartments in myoblasts but begin to colocalize as cells differentiate. Fluorescence Recovery After Photobleaching (FRAP) studies show different recovery patterns for each protein in myoblasts confirming their localization to distinct compartments. To extend these studies, we created stable cell lines expressing O6-alkylguanine-DNA alkyltransferase (AGT) fusions with PLB or SERCA to track their localization as myocytes differentiate. These experiments demonstrate that PLB localizes to punctate vesicles in myoblasts and adopts a reticular distribution that coincides with SERCA distribution after differentiation. Colocalization experiments indicate that a subset of PLB in myoblasts colocalizes with endosomes, Golgi, and the plasma membrane however PLB also localizes to other, as yet unidentified vesicles. Our results indicate that differentiation plays a critical role in regulating PLB distribution to ensure its colocalization within the same cellular compartment as SERCA in differentiated cells. The presence and altered distribution of PLB in undifferentiated myoblasts raises the possibility that this protein has additional functions distinct from SERCA

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

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.

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

NASA Technical Reports Server (NTRS)

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

1995-01-01

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

Modeling CICR in rat ventricular myocytes: voltage clamp studies

PubMed Central

2010-01-01

Background The past thirty-five years have seen an intense search for the molecular mechanisms underlying calcium-induced calcium-release (CICR) in cardiac myocytes, with voltage clamp (VC) studies being the leading tool employed. Several VC protocols including lowering of extracellular calcium to affect Ca2+ loading of the sarcoplasmic reticulum (SR), and administration of blockers caffeine and thapsigargin have been utilized to probe the phenomena surrounding SR Ca2+ release. Here, we develop a deterministic mathematical model of a rat ventricular myocyte under VC conditions, to better understand mechanisms underlying the response of an isolated cell to calcium perturbation. Motivation for the study was to pinpoint key control variables influencing CICR and examine the role of CICR in the context of a physiological control system regulating cytosolic Ca2+ concentration ([Ca2+]myo). Methods The cell model consists of an electrical-equivalent model for the cell membrane and a fluid-compartment model describing the flux of ionic species between the extracellular and several intracellular compartments (cell cytosol, SR and the dyadic coupling unit (DCU), in which resides the mechanistic basis of CICR). The DCU is described as a controller-actuator mechanism, internally stabilized by negative feedback control of the unit's two diametrically-opposed Ca2+ channels (trigger-channel and release-channel). It releases Ca2+ flux into the cyto-plasm and is in turn enclosed within a negative feedback loop involving the SERCA pump, regulating[Ca2+]myo. Results Our model reproduces measured VC data published by several laboratories, and generates graded Ca2+ release at high Ca2+ gain in a homeostatically-controlled environment where [Ca2+]myo is precisely regulated. We elucidate the importance of the DCU elements in this process, particularly the role of the ryanodine receptor in controlling SR Ca2+ release, its activation by trigger Ca2+, and its refractory characteristics

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

Exploring Regulatory Mechanisms of Atrial Myocyte Hypertrophy of Mitral Regurgitation through Gene Expression Profiling Analysis: Role of NFAT in Cardiac Hypertrophy

PubMed Central

Chang, Tzu-Hao; Chen, Mien-Cheng; Chang, Jen-Ping; Huang, Hsien-Da; Ho, Wan-Chun; Lin, Yu-Sheng; Pan, Kuo-Li; Huang, Yao-Kuang; Liu, Wen-Hao; Wu, Chia-Chen

2016-01-01

Background Left atrial enlargement in mitral regurgitation (MR) predicts a poor prognosis. The regulatory mechanisms of atrial myocyte hypertrophy of MR patients remain unknown. Methods and Results This study comprised 14 patients with MR, 7 patients with aortic valve disease (AVD), and 6 purchased samples from normal subjects (NC). We used microarrays, enrichment analysis and quantitative RT-PCR to study the gene expression profiles in the left atria. Microarray results showed that 112 genes were differentially up-regulated and 132 genes were differentially down-regulated in the left atria between MR patients and NC. Enrichment analysis of differentially expressed genes demonstrated that “NFAT in cardiac hypertrophy” pathway was not only one of the significant associated canonical pathways, but also the only one predicted with a non-zero score of 1.34 (i.e. activated) through Ingenuity Pathway Analysis molecule activity predictor. Ingenuity Pathway Analysis Global Molecular Network analysis exhibited that the highest score network also showed high association with cardiac related pathways and functions. Therefore, 5 NFAT associated genes (PPP3R1, PPP3CB, CAMK1, MEF2C, PLCE1) were studies for validation. The mRNA expressions of PPP3CB and MEF2C were significantly up-regulated, and CAMK1 and PPP3R1 were significantly down-regulated in MR patients compared to NC. Moreover, MR patients had significantly increased mRNA levels of PPP3CB, MEF2C and PLCE1 compared to AVD patients. The atrial myocyte size of MR patients significantly exceeded that of the AVD patients and NC. Conclusions Differentially expressed genes in the “NFAT in cardiac hypertrophy” pathway may play a critical role in the atrial myocyte hypertrophy of MR patients. PMID:27907007

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

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.

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

MiR-130a inhibition protects rat cardiac myocytes from hypoxia-triggered apoptosis by targeting Smad4.

PubMed

Li, Yuanshi; Du, Yingrong; Cao, Junxian; Gao, Qianping; Li, Hongjuan; Chen, Yangjun; Lu, Nihong

2018-02-05

Cardiomyocyte death facilitates the pathological process underlying ischemic heart diseases, such as myocardial infarction. Emerging evidence suggests that microRNAs play a critical role in the pathological process underlying myocardial infarction by regulating cardiomyocyte apoptosis. However, the relevance of miR-130a in regulating cardiomyocyte apoptosis and the mechanism of regulation is still uncertain. This study aimed to explore the regulatory effect of miR-130a on hypoxic cardiomyocyte apoptosis. The expression of miR-130a was measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Cell survival was determined by the MTT assay. The lactate dehydrogenase (LDH) assay was performed to determine the severity of hypoxia-induced cell injury. Apoptosis was assessed via caspase-3 analysis. Protein expression level was determined by Western blotting. The genes targeted by miR-130a were predicted using bioinformatics and were validated via the dual-luciferase reporter assay. We found that miR-130a expression was greatly increased in hypoxic cardiac myocytes, and that the downregulation of miR-130a effectively shielded cardiac myocytes from hypoxia-triggered apoptosis. The results of our bioinformatic analysis predicted the Smad4 gene to be the target of miR-130a. This finding was validated through the Western blot assay, dual-luciferase reporter gene assay, and qRT-PCR. MiR-130a inhibition significantly promoted the activation of Smad4 in hypoxic cardiomyocytes. Interestingly, knockdown of Smad4 markedly reversed the protective effects induced by miR-130a inhibition. Moreover, we found that the inhibition of miR-130a promoted the activation of TGF-β signaling. Blocking Smad4 signaling significantly abrogated the protective effects of miR-130a inhibition. Overall, these findings indicate that inhibition of miR-130a, which targets the Smad4 gene, shields cardiac myocytes from hypoxic apoptosis. This study offers a novel perspective of the

β1-adrenergic regulation of rapid component of delayed rectifier K+ currents in guinea-pig cardiac myocytes.

PubMed

Wang, Sen; Xu, Di; Wu, Ting-Ting; Guo, Yan; Chen, Yan-Hong; Zou, Jian-Gang

2014-05-01

Human ether-à-go-go-related gene (hERG) potassium channels conduct the rapid component of the delayed rectifier potassium current (IKr), which is crucial for repolarization of cardiac action potential. Patients with hERG‑associated long QT syndrome usually develop tachyarrhythmias during physical and/or emotional stress, both known to stimulate adrenergic receptors. The present study aimed to investigate a putative functional link between β1-adrenergic stimulation and IKr in guinea-pig left ventricular myocytes and to analyze how IKr is regulated following activation of the β1-adrenergic signaling pathway. The IKr current was measured using a whole-cell patch-clamp technique. A selective β1-adrenergic receptor agonist, xamoterol, at concentrations of 0.01-100 µM decreased IKr in a concentration-dependent manner. The 10 µM xamoterol-induced inhibition of IKr was attenuated by the protein kinase A (PKA) inhibitor KT5720, the protein kinase C (PKC) inhibitor chelerythrine, and the phospholipase (PLC) inhibitor U73122, indicating involvement of PKA, PKC and PLC in β1-adrenergic inhibition of IKr. The results of the present study indicate an association between IKr and the β1-adrenergic receptor in arrhythmogenesis, involving the activation of PKA, PKC and PLC.

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

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

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

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.

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.

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

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

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.

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.

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.

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

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

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.

Model study of ATP and ADP buffering, transport of Ca(2+) and Mg(2+), and regulation of ion pumps in ventricular myocyte

NASA Technical Reports Server (NTRS)

Michailova, A.; McCulloch, A.

2001-01-01

We extended the model of the ventricular myocyte by Winslow et al. (Circ. Res 1999, 84:571-586) by incorporating equations for Ca(2+) and Mg(2+) buffering and transport by ATP and ADP and equations for MgATP regulation of ion transporters (Na(+)-K(+) pump, sarcolemmal and sarcoplasmic Ca(2+) pumps). The results indicate that, under normal conditions, Ca(2+) binding by low-affinity ATP and diffusion of CaATP may affect the amplitude and time course of intracellular Ca(2+) signals. The model also suggests that a fall in ATP/ADP ratio significantly reduces sarcoplasmic Ca(2+) content, increases diastolic Ca(2+), lowers systolic Ca(2+), increases Ca(2+) influx through L-type channels, and decreases the efficiency of the Na(+)/Ca(2+) exchanger in extruding Ca(2+) during periodic voltage-clamp stimulation. The analysis suggests that the most important reason for these changes during metabolic inhibition is the down-regulation of the sarcoplasmic Ca(2+)-ATPase pump by reduced diastolic MgATP levels. High Ca(2+) concentrations developed near the membrane might have a greater influence on Mg(2+), ATP, and ADP concentrations than that of the lower Ca(2+) concentrations in the bulk myoplasm. The model predictions are in general agreement with experimental observations measured under normal and pathological conditions.

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

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

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

Computational analysis of the regulation of Ca2+ dynamics in rat ventricular myocytes

NASA Astrophysics Data System (ADS)

Bugenhagen, Scott M.; Beard, Daniel A.

2015-10-01

Force-frequency relationships of isolated cardiac myocytes show complex behaviors that are thought to be specific to both the species and the conditions associated with the experimental preparation. Ca2+ signaling plays an important role in shaping the force-frequency relationship, and understanding the properties of the force-frequency relationship in vivo requires an understanding of Ca2+ dynamics under physiologically relevant conditions. Ca2+ signaling is itself a complicated process that is best understood on a quantitative level via biophysically based computational simulation. Although a large number of models are available in the literature, the models are often a conglomeration of components parameterized to data of incompatible species and/or experimental conditions. In addition, few models account for modulation of Ca2+ dynamics via β-adrenergic and calmodulin-dependent protein kinase II (CaMKII) signaling pathways even though they are hypothesized to play an important regulatory role in vivo. Both protein-kinase-A and CaMKII are known to phosphorylate a variety of targets known to be involved in Ca2+ signaling, but the effects of these pathways on the frequency- and inotrope-dependence of Ca2+ dynamics are not currently well understood. In order to better understand Ca2+ dynamics under physiological conditions relevant to rat, a previous computational model is adapted and re-parameterized to a self-consistent dataset obtained under physiological temperature and pacing frequency and updated to include β-adrenergic and CaMKII regulatory pathways. The necessity of specific effector mechanisms of these pathways in capturing inotrope- and frequency-dependence of the data is tested by attempting to fit the data while including and/or excluding those effector components. We find that: (1) β-adrenergic-mediated phosphorylation of the L-type calcium channel (LCC) (and not of phospholamban (PLB)) is sufficient to explain the inotrope-dependence; and (2) that

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

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

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.

TGF-β1, released by myofibroblasts, differentially regulates transcription and function of sodium and potassium channels in adult rat ventricular myocytes.

PubMed

Kaur, Kuljeet; Zarzoso, Manuel; Ponce-Balbuena, Daniela; Guerrero-Serna, Guadalupe; Hou, Luqia; Musa, Hassan; Jalife, José

2013-01-01

Cardiac injury promotes fibroblasts activation and differentiation into myofibroblasts, which are hypersecretory of multiple cytokines. It is unknown whether any of such cytokines are involved in the electrophysiological remodeling of adult cardiomyocytes. We cultured adult cardiomyocytes for 3 days in cardiac fibroblast conditioned medium (FCM) from adult rats. In whole-cell voltage-clamp experiments, FCM-treated myocytes had 41% more peak inward sodium current (I(Na)) density at -40 mV than myocytes in control medium (p<0.01). In contrast, peak transient outward current (I(to)) was decreased by ∼55% at 60 mV (p<0.001). Protein analysis of FCM demonstrated that the concentration of TGF-β1 was >3 fold greater in FCM than control, which suggested that FCM effects could be mediated by TGF-β1. This was confirmed by pre-treatment with TGF-β1 neutralizing antibody, which abolished the FCM-induced changes in both I(Na) and I(to). In current-clamp experiments TGF-β1 (10 ng/ml) prolonged the action potential duration at 30, 50, and 90 repolarization (p<0.05); at 50 ng/ml it gave rise to early afterdepolarizations. In voltage-clamp experiments, TGF-β1 increased I(Na) density in a dose-dependent manner without affecting voltage dependence of activation or inactivation. I(Na) density was -36.25±2.8 pA/pF in control, -59.17±6.2 pA/pF at 0.1 ng/ml (p<0.01), and -58.22±6.6 pA/pF at 1 ng/ml (p<0.01). In sharp contrast, I(to) density decreased from 22.2±1.2 pA/pF to 12.7±0.98 pA/pF (p<0.001) at 10 ng/ml. At 1 ng/ml TGF-β1 significantly increased SCN5A (Na(V)1.5) (+73%; p<0.01), while reducing KCNIP2 (Kchip2; -77%; p<0.01) and KCND2 (K(V)4.2; -50% p<0.05) mRNA levels. Further, the TGF-β1-induced increase in I(Na) was mediated through activation of the PI3K-AKT pathway via phosphorylation of FOXO1 (a negative regulator of SCN5A). TGF-β1 released by myofibroblasts differentially regulates transcription and function of the main cardiac sodium channel and of the channel

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

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.

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.

MicroRNA-133 regulates the expression of GLUT4 by targeting KLF15 and is involved in metabolic control in cardiac myocytes.

PubMed

Horie, Takahiro; Ono, Koh; Nishi, Hitoo; Iwanaga, Yoshitaka; Nagao, Kazuya; Kinoshita, Minako; Kuwabara, Yasuhide; Takanabe, Rieko; Hasegawa, Koji; Kita, Toru; Kimura, Takeshi

2009-11-13

GLUT4 shows decreased levels in failing human adult hearts. We speculated that GLUT4 expression in cardiac muscle may be fine-tuned by microRNAs. Forced expression of miR-133 decreased GLUT4 expression and reduced insulin-mediated glucose uptake in cardiomyocytes. A computational miRNA target prediction algorithm showed that KLF15 is one of the targets of miR-133. It was confirmed that over-expression of miR-133 reduced the protein level of KLF15, which reduced the level of the downstream target GLUT4. Cardiac myocytes infected with lenti-decoy, in which the 3'UTR with tandem sequences complementary to miR-133 was linked to the luciferase reporter gene, had decreased miR-133 levels and increased levels of GLUT4. The expression levels of KLF15 and GLUT4 were decreased at the left ventricular hypertrophy and congestive heart failure stage in a rat model. The present results indicated that miR-133 regulates the expression of GLUT4 by targeting KLF15 and is involved in metabolic control in cardiomyocytes.

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.

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

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

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

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.

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

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.

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

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.

[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

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.

From syncitium to regulated pump: a cardiac muscle cellular update

PubMed Central

2011-01-01

The primary purpose of this article is to present a basic overview of some key teaching concepts that should be considered for inclusion in an six- to eight-lecture introductory block on the regulation of cardiac performance for graduate students. Within the context of cardiac excitation-contraction coupling, this review incorporates information on Ca2+ microdomains and local control theory, with particular emphasis on the role of Ca2+ sparks as a key regulatory component of ventricular myocyte contraction dynamics. Recent information pertaining to local Ca2+ cycling in sinoatrial nodal cells (SANCs) as a mechanism underlying cardiac automaticity is also presented as part of the recently described coupled-clock pacemaker system. The details of this regulation are emerging; however, the notion that the sequestration and release of Ca2+ from internal stores in SANCs (similar to that observed in ventricular myocytes) regulates the rhythmic excitation of the heart (i.e., membrane ion channels) is an important advancement in this area. The regulatory role of cardiac adrenergic receptors on cardiac rate and function is also included, and fundamental concepts related to intracellular signaling are discussed. An important point of emphasis is that whole organ cardiac dynamics can be traced back to cellular events regulating intracellular Ca2+ homeostasis and, as such, provides an important conceptual framework from which students can begin to think about whole organ physiology in health and disease. Greater synchrony of Ca2+-regulatory mechanisms between ventricular and pacemaker cells should enhance student comprehension of complex regulatory phenomenon in cardiac muscle. PMID:21385997

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.

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.

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.

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

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

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.

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.

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

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

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

Spatial enhancer clustering and regulation of enhancer-proximal genes by cohesin

PubMed Central

Ing-Simmons, Elizabeth; Seitan, Vlad C.; Faure, Andre J.; Flicek, Paul; Carroll, Thomas; Dekker, Job; Fisher, Amanda G.; Lenhard, Boris

2015-01-01

In addition to mediating sister chromatid cohesion during the cell cycle, the cohesin complex associates with CTCF and with active gene regulatory elements to form long-range interactions between its binding sites. Genome-wide chromosome conformation capture had shown that cohesin's main role in interphase genome organization is in mediating interactions within architectural chromosome compartments, rather than specifying compartments per se. However, it remains unclear how cohesin-mediated interactions contribute to the regulation of gene expression. We have found that the binding of CTCF and cohesin is highly enriched at enhancers and in particular at enhancer arrays or “super-enhancers” in mouse thymocytes. Using local and global chromosome conformation capture, we demonstrate that enhancer elements associate not just in linear sequence, but also in 3D, and that spatial enhancer clustering is facilitated by cohesin. The conditional deletion of cohesin from noncycling thymocytes preserved enhancer position, H3K27ac, H4K4me1, and enhancer transcription, but weakened interactions between enhancers. Interestingly, ∼50% of deregulated genes reside in the vicinity of enhancer elements, suggesting that cohesin regulates gene expression through spatial clustering of enhancer elements. We propose a model for cohesin-dependent gene regulation in which spatial clustering of enhancer elements acts as a unified mechanism for both enhancer-promoter “connections” and “insulation.” PMID:25677180

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.

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.

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.

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

PubMed Central

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

2008-01-01

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

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

Toward an Integrative Computational Model of the Guinea Pig Cardiac Myocyte

PubMed Central

Gauthier, Laura Doyle; Greenstein, Joseph L.; Winslow, Raimond L.

2012-01-01

The local control theory of excitation-contraction (EC) coupling asserts that regulation of calcium (Ca2+) release occurs at the nanodomain level, where openings of single L-type Ca2+ channels (LCCs) trigger openings of small clusters of ryanodine receptors (RyRs) co-localized within the dyad. A consequence of local control is that the whole-cell Ca2+ transient is a smooth continuous function of influx of Ca2+ through LCCs. While this so-called graded release property has been known for some time, its functional importance to the integrated behavior of the cardiac ventricular myocyte has not been fully appreciated. We previously formulated a biophysically based model, in which LCCs and RyRs interact via a coarse-grained representation of the dyadic space. The model captures key features of local control using a low-dimensional system of ordinary differential equations. Voltage-dependent gain and graded Ca2+ release are emergent properties of this model by virtue of the fact that model formulation is closely based on the sub-cellular basis of local control. In this current work, we have incorporated this graded release model into a prior model of guinea pig ventricular myocyte electrophysiology, metabolism, and isometric force production. The resulting integrative model predicts the experimentally observed causal relationship between action potential (AP) shape and timing of Ca2+ and force transients, a relationship that is not explained by models lacking the graded release property. Model results suggest that even relatively subtle changes in AP morphology that may result, for example, from remodeling of membrane transporter expression in disease or spatial variation in cell properties, may have major impact on the temporal waveform of Ca2+ transients, thus influencing tissue level electromechanical function. PMID:22783206

Toward an integrative computational model of the Guinea pig cardiac myocyte.

PubMed

Gauthier, Laura Doyle; Greenstein, Joseph L; Winslow, Raimond L

2012-01-01

The local control theory of excitation-contraction (EC) coupling asserts that regulation of calcium (Ca(2+)) release occurs at the nanodomain level, where openings of single L-type Ca(2+) channels (LCCs) trigger openings of small clusters of ryanodine receptors (RyRs) co-localized within the dyad. A consequence of local control is that the whole-cell Ca(2+) transient is a smooth continuous function of influx of Ca(2+) through LCCs. While this so-called graded release property has been known for some time, its functional importance to the integrated behavior of the cardiac ventricular myocyte has not been fully appreciated. We previously formulated a biophysically based model, in which LCCs and RyRs interact via a coarse-grained representation of the dyadic space. The model captures key features of local control using a low-dimensional system of ordinary differential equations. Voltage-dependent gain and graded Ca(2+) release are emergent properties of this model by virtue of the fact that model formulation is closely based on the sub-cellular basis of local control. In this current work, we have incorporated this graded release model into a prior model of guinea pig ventricular myocyte electrophysiology, metabolism, and isometric force production. The resulting integrative model predicts the experimentally observed causal relationship between action potential (AP) shape and timing of Ca(2+) and force transients, a relationship that is not explained by models lacking the graded release property. Model results suggest that even relatively subtle changes in AP morphology that may result, for example, from remodeling of membrane transporter expression in disease or spatial variation in cell properties, may have major impact on the temporal waveform of Ca(2+) transients, thus influencing tissue level electromechanical function.

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

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.

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

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.

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

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.

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

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

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

Robust generation and expansion of skeletal muscle progenitors and myocytes from human pluripotent stem cells.

PubMed

Shelton, Michael; Kocharyan, Avetik; Liu, Jun; Skerjanc, Ilona S; Stanford, William L

2016-05-15

Human pluripotent stem cells provide a developmental model to study early embryonic and tissue development, tease apart human disease processes, perform drug screens to identify potential molecular effectors of in situ regeneration, and provide a source for cell and tissue based transplantation. Highly efficient differentiation protocols have been established for many cell types and tissues; however, until very recently robust differentiation into skeletal muscle cells had not been possible unless driven by transgenic expression of master regulators of myogenesis. Nevertheless, several breakthrough protocols have been published in the past two years that efficiently generate cells of the skeletal muscle lineage from pluripotent stem cells. Here, we present an updated version of our recently described 50-day protocol in detail, whereby chemically defined media are used to drive and support muscle lineage development from initial CHIR99021-induced mesoderm through to PAX7-expressing skeletal muscle progenitors and mature skeletal myocytes. Furthermore, we report an optional method to passage and expand differentiating skeletal muscle progenitors approximately 3-fold every 2weeks using Collagenase IV and continued FGF2 supplementation. Both protocols have been optimized using a variety of human pluripotent stem cell lines including patient-derived induced pluripotent stem cells. Taken together, our differentiation and expansion protocols provide sufficient quantities of skeletal muscle progenitors and myocytes that could be used for a variety of studies. Copyright © 2015 The Authors. Published by Elsevier Inc. 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

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.

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

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.

TVP1022 Protects Neonatal Rat Ventricular Myocytes against Doxorubicin-Induced Functional Derangements

PubMed Central

Berdichevski, Alexandra; Meiry, Gideon; Milman, Felix; Reiter, Irena; Sedan, Oshra; Eliyahu, Sivan; Duffy, Heather S.; Youdim, Moussa B.; Binah, Ofer

2010-01-01

Our recent studies demonstrated that propargylamine derivatives such as rasagiline (Azilect, Food and Drug Administration-approved anti-Parkinson drug) and its S-isomer TVP1022 protect cardiac and neuronal cell cultures against apoptotic-inducing stimuli. Studies on structure-activity relationship revealed that their neuroprotective effect is associated with the propargylamine moiety, which protects mitochondrial viability and prevents apoptosis by activating Bcl-2 and protein kinase C-ε and by down-regulating the proapoptotic protein Bax. Based on the established cytoprotective and neuroprotective efficacies of propargylamine derivatives, as well as on our recent study showing that TVP1022 attenuates serum starvation-induced and doxorubicin-induced apoptosis in neonatal rat ventricular myocytes (NRVMs), we tested the hypothesis that TVP1022 will also provide protection against doxorubicin-induced NRVM functional derangements. The present study demonstrates that pretreatment of NRVMs with TVP1022 (1 μM, 24 h) prevented doxorubicin (0.5 μM, 24 h)-induced elevation of diastolic [Ca2+]i, the slowing of [Ca2+]i relaxation kinetics, and the decrease in the rates of myocyte contraction and relaxation. Furthermore, pretreatment with TVP1022 attenuated the doxorubicin-induced reduction in the protein expression of sarco/endoplasmic reticulum calcium (Ca2+) ATPase, Na+/Ca2+ exchanger 1, and total connexin 43. Finally, TVP1022 diminished the inhibitory effect of doxorubicin on gap junctional intercellular coupling (measured by means of Lucifer yellow transfer) and on conduction velocity, the amplitude of the activation phase, and the maximal rate of activation (dv/dtmax) measured by the Micro-Electrode-Array system. In summary, our results indicate that TVP1022 acts as a novel cardioprotective agent against anthracycline cardiotoxicity, and therefore potentially can be coadmhence, the inistered with doxorubicin in the treatment of malignancies in humans. PMID:19915070

Growth hormone-releasing hormone promotes survival of cardiac myocytes in vitro and protects against ischaemia-reperfusion injury in rat heart.

PubMed

Granata, Riccarda; Trovato, Letizia; Gallo, Maria Pia; Destefanis, Silvia; Settanni, Fabio; Scarlatti, Francesca; Brero, Alessia; Ramella, Roberta; Volante, Marco; Isgaard, Jorgen; Levi, Renzo; Papotti, Mauro; Alloatti, Giuseppe; Ghigo, Ezio

2009-07-15

The hypothalamic neuropeptide growth hormone-releasing hormone (GHRH) stimulates GH synthesis and release in the pituitary. GHRH also exerts proliferative effects in extrapituitary cells, whereas GHRH antagonists have been shown to suppress cancer cell proliferation. We investigated GHRH effects on cardiac myocyte cell survival and the underlying signalling mechanisms. Reverse transcriptase-polymerase chain reaction analysis showed GHRH receptor (GHRH-R) mRNA in adult rat ventricular myocytes (ARVMs) and in rat heart H9c2 cells. In ARVMs, GHRH prevented cell death and caspase-3 activation induced by serum starvation and by the beta-adrenergic receptor agonist isoproterenol. The GHRH-R antagonist JV-1-36 abolished GHRH survival action under both experimental conditions. GHRH-induced cardiac cell protection required extracellular signal-regulated kinase (ERK)1/2 and phosphoinositide-3 kinase (PI3K)/Akt activation and adenylyl cyclase/cAMP/protein kinase A signalling. Isoproterenol strongly upregulated the mRNA and protein of the pro-apoptotic inducible cAMP early repressor, whereas GHRH completely blocked this effect. Similar to ARVMs, in H9c2 cardiac cells, GHRH inhibited serum starvation- and isoproterenol-induced cell death and apoptosis through the same signalling pathways. Finally, GHRH improved left ventricular recovery during reperfusion and reduced infarct size in Langendorff-perfused rat hearts, subjected to ischaemia-reperfusion (I/R) injury. These effects involved PI3K/Akt signalling and were inhibited by JV-1-36. Our findings suggest that GHRH promotes cardiac myocyte survival through multiple signalling mechanisms and protects against I/R injury in isolated rat heart, indicating a novel cardioprotective role of this hormone.

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.

Lethal effect of electric fields on isolated ventricular myocytes.

PubMed

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

2008-11-01

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

Regulators of Autophagosome Formation in Drosophila Muscles

PubMed Central

Zirin, Jonathan; Nieuwenhuis, Joppe; Samsonova, Anastasia; Tao, Rong; Perrimon, Norbert

2015-01-01

Given the diversity of autophagy targets and regulation, it is important to characterize autophagy in various cell types and conditions. We used a primary myocyte cell culture system to assay the role of putative autophagy regulators in the specific context of skeletal muscle. By treating the cultures with rapamycin (Rap) and chloroquine (CQ) we induced an autophagic response, fully suppressible by knockdown of core ATG genes. We screened D. melanogaster orthologs of a previously reported mammalian autophagy protein-protein interaction network, identifying several proteins required for autophagosome formation in muscle cells, including orthologs of the Rab regulators RabGap1 and Rab3Gap1. The screen also highlighted the critical roles of the proteasome and glycogen metabolism in regulating autophagy. Specifically, sustained proteasome inhibition inhibited autophagosome formation both in primary culture and larval skeletal muscle, even though autophagy normally acts to suppress ubiquitin aggregate formation in these tissues. In addition, analyses of glycogen metabolic genes in both primary cultured and larval muscles indicated that glycogen storage enhances the autophagic response to starvation, an important insight given the link between glycogen storage disorders, autophagy, and muscle function. PMID:25692684

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

PubMed

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

2012-01-01

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

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.

[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.

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

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.

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

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

PubMed Central

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

2017-01-01

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

Protein tyrosine phosphatase 1B is a mediator of cyclic ADP ribose-induced Ca2+ signaling in ventricular myocytes.

PubMed

Park, Seon-Ah; Hong, Bing-Zhe; Ha, Ki-Chan; Kim, Uh-Hyun; Han, Myung-Kwan; Kwak, Yong-Geun

2017-06-02

Cyclic ADP-ribose (cADPR) releases Ca 2+ from ryanodine receptor (RyR)-sensitive calcium pools in various cell types. In cardiac myocytes, the physiological levels of cADPR transiently increase the amplitude and frequency of Ca 2+ (that is, a rapid increase and decrease of calcium within one second) during the cardiac action potential. In this study, we demonstrated that cADPR levels higher than physiological levels induce a slow and gradual increase in the resting intracellular Ca 2+ ([Ca 2+ ] i ) level over 10 min by inhibiting the sarcoendoplasmic reticulum Ca 2+ ATPase (SERCA). Higher cADPR levels mediate the tyrosine-dephosphorylation of α-actin by protein tyrosine phosphatase 1B (PTP1B) present in the endoplasmic reticulum. The tyrosine dephosphorylation of α-actin dissociates phospholamban, the key regulator of SERCA, from α-actin and results in SERCA inhibition. The disruption of the integrity of α-actin by cytochalasin B and the inhibition of α-actin tyrosine dephosphorylation by a PTP1B inhibitor block cADPR-mediated Ca 2+ increase. Our results suggest that levels of cADPR that are relatively higher than normal physiological levels modify calcium homeostasis through the dephosphorylation of α-actin by PTB1B and the subsequent inhibition of SERCA in cardiac myocytes.

Choosing to regulate: does choice enhance craving regulation?

PubMed Central

Mobasser, Arian; Zeithamova, Dagmar; Pfeifer, Jennifer H

2018-01-01

Abstract Goal-directed behavior and lifelong well-being often depend on the ability to control appetitive motivations, such as cravings. Cognitive reappraisal is an effective way to modulate emotional states, including cravings, but is often studied under explicit instruction to regulate. Despite the strong prediction from Self-Determination Theory that choice should enhance task engagement and regulation success, little is known empirically about whether and how regulation is different when participants choose (vs are told) to exert control. To investigate how choice affects neural activity and regulation success, participants reappraised their responses to images of personally-craved foods while undergoing functional neuroimaging. Participants were either instructed to view or reappraise (‘no-choice’) or chose freely to view or reappraise (‘yes-choice’). Choice increased activity in the frontoparietal control network. We expected this activity would be associated with increased task engagement, resulting in better regulation success. However, contrary to this prediction, choice slightly reduced regulation success. Follow-up multivariate functional neuroimaging analyses indicated that choice likely disrupted allocation of limited cognitive resources during reappraisal. While unexpected, these results highlight the importance of studying upstream processes such as regulation choice, as they may affect the ability to regulate cravings and other emotional states. PMID:29462475

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...

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

Diadenosine tetraphosphate-induced inhibition of ATP-sensitive K+ channels in patches excised from ventricular myocytes.

PubMed Central

Jovanovic, A.; Terzic, A.

1996-01-01

Diadenosine 5',5''-P1,P4-tetraphosphate (Ap4A) has been termed 'alarmone' due to its role in intracellular signaling during metabolic stress. It is not known whether Ap4A could modulate ATP-sensitive K+ (KATP) channels, a family of channels regulated by the metabolic status of a cell. We applied the single-channel patch-clamp technique to measure the effect of Ap4A on KATP channels. When applied to the intracellular side of patches, excised from guinea-pig ventricular myocytes, Ap4A inhibited KATP channel activity, in a reversible and concentration-dependent (half-maximal concentration approximately 17 microM) manner. We conclude that Ap4A, a naturally occurring diadenosine polyphosphate, is actually an inhibitor of the myocardial KATP channel. PMID:8789372

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

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.

Out-of-equilibrium pH transients in the guinea-pig ventricular myocyte

PubMed Central

Leem, Chae-Hun; Vaughan-Jones, Richard D

1998-01-01

Following an intracellular alkali load (imposed by acetate prepulsing in CO2/HCO3− buffer), intracellular pH (pHi) of the guinea-pig ventricular myocyte (recorded from intracellular SNARF fluorescence) recovers to control levels. Recovery has two phases. An initial rapid phase (lasting up to 2 min) is followed by a later slow phase (several minutes). Inhibition of sarcolemmal acid-loading carriers (by removal of extracellular Cl−) inhibits the later, slow phase but the initial rapid recovery phase persists. It also persists in the absence of extracellular Na+ and in the presence of the HCO3− transport inhibitor DIDS (4,4-di-isothiocyanatostilbene-2,2-disulphonic acid). The rapid recovery phase is not evident if the alkali load has been induced by reducing PCO2 (from 10 to 5 %), and it is inhibited in the absence of CO2/HCO3− buffer (i.e. Hepes buffer). It is also slowed by the carbonic anhydrase (CA) inhibitor acetazolamide (ATZ). We conclude that it is caused by buffering of the alkali load through the hydration of intracellular CO2 (CO2-dependent buffering). The time course of rapid recovery is consistent with an intracellular CO2 hydration rate constant (k1) of 0.36 s−1 in the presence of CA activity, and 0.14 s−1 in the absence of CA activity. This latter k1 value matches the literature value for uncatalysed CO2 hydration in free solution. Natural CO2 hydration is accelerated 2.6-fold in the ventricular myocyte by endogenous CA. The rapid recovery phase represents a period when the intracellular CO2/HCO3− buffer is out of equilibrium (OOE). Modelling of the recovery phase using our k1 value, indicates that OOE conditions will normally extend for at least 2 min following a step rise in pHi (at constant PCO2). If CA is inactive, this period can be as long as 5 min. During normal pHi regulation, the recovery rate during these periods cannot be used as a measure of sarcolemmal acid loading since it is a mixture of slow CO2-dependent buffering and

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.

Epigenetic regulation of cardiac fibrosis

PubMed Central

Stratton, Matthew S.; McKinsey, Timothy A.

2016-01-01

Fibrosis is defined as excess deposition of extracellular matrix (ECM), resulting in tissue scarring and organ dysfunction. In the heart, fibrosis may be reparative, replacing areas of myocyte loss with a structural scar following infarction, or reactive, which is triggered in the absence of cell death and involves interstitial ECM deposition in response to long-lasting stress. Interstitial fibrosis can increase the passive stiffness of the myocardium, resulting in impaired relaxation and diastolic dysfunction. Additionally, fibrosis can lead to disruption of electrical conduction in the heart, causing arrhythmias, and can limit myocyte oxygen availability and thus exacerbate myocardial ischemia. Here, we review recent studies that have illustrated key roles for epigenetic events in the control of pro-fibrotic gene expression, and highlight the potential of small molecules that target epigenetic regulators as a means of treating fibrotic cardiac diseases. PMID:26876451

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

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

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

PubMed Central

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

2014-01-01

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

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

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.

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.

Autonomic regulation therapy to enhance myocardial function in heart failure patients: the ANTHEM‐HFpEF study

PubMed Central

DiCarlo, Lorenzo A.; Libbus, Imad; Kumar, H. Uday; Mittal, Sanjay; Premchand, Rajendra K.; Amurthur, Badri; KenKnight, Bruce H.; Ardell, Jeffrey L.

2017-01-01

Abstract Background Approximately half of the patients presenting with new‐onset heart failure (HF) have HF with preserved left ventricular ejection fraction (HFpEF) and HF with mid‐range left ventricular ejection fraction (HFmrEF). These patients have neurohormonal activation like that of HF with reduced ejection fraction; however, beta‐blockers and angiotensin‐converting enzyme inhibitors have not been shown to improve their outcomes, and current treatment for these patients is symptom based and empiric. Sympathoinhibition using parasympathetic stimulation has been shown to improve central and peripheral aspects of the cardiac nervous system, reflex control, induce myocyte cardioprotection, and can lead to regression of left ventricular hypertrophy. Beneficial effects of autonomic regulation therapy (ART) using vagus nerve stimulation (VNS) have also been observed in several animal models of HFpEF, suggesting a potential role for ART in patients with this disease. Methods The Autonomic Neural Regulation Therapy to Enhance Myocardial Function in Patients with Heart Failure and Preserved Ejection Fraction (ANTHEM‐HFpEF) study is designed to evaluate the feasibility, tolerability, and safety of ART using right cervical VNS in patients with chronic, stable HFpEF and HFmrEF. Patients with symptomatic HF and HFpEF or HFmrEF fulfilling the enrolment criteria will receive chronic ART with a subcutaneous VNS system attached to the right cervical vagus nerve. Safety parameters will be continuously monitored, and cardiac function and HF symptoms will be assessed every 3 months during a post‐titration follow‐up period of at least 12 months. Conclusions The ANTHEM‐HFpEF study is likely to provide valuable information intended to expand our understanding of the potential role of ART in patients with chronic symptomatic HFpEF and HFmrEF. PMID:29283224

Reduced density and altered regulation of rat atrial L-type Ca2+ current in heart failure.

PubMed

Bond, Richard C; Bryant, Simon M; Watson, Judy J; Hancox, Jules C; Orchard, Clive H; James, Andrew F

2017-03-01

Constitutive regulation by PKA has recently been shown to contribute to L-type Ca 2+ current ( I CaL ) at the ventricular t-tubule in heart failure. Conversely, reduction in constitutive regulation by PKA has been proposed to underlie the downregulation of atrial I CaL in heart failure. The hypothesis that downregulation of atrial I CaL in heart failure involves reduced channel phosphorylation was examined. Anesthetized adult male Wistar rats underwent surgical coronary artery ligation (CAL, N =10) or equivalent sham-operation (Sham, N =12). Left atrial myocytes were isolated ~18 wk postsurgery and whole cell currents recorded (holding potential=-80 mV). I CaL activated by depolarizing pulses to voltages from -40 to +50 mV were normalized to cell capacitance and current density-voltage relations plotted. CAL cell capacitances were ~1.67-fold greater than Sham ( P ≤ 0.0001). Maximal I CaL conductance ( G max ) was downregulated more than 2-fold in CAL vs. Sham myocytes ( P < 0.0001). Norepinephrine (1 μmol/l) increased G max >50% more effectively in CAL than in Sham so that differences in I CaL density were abolished. Differences between CAL and Sham G max were not abolished by calyculin A (100 nmol/l), suggesting that increased protein dephosphorylation did not account for I CaL downregulation. Treatment with either H-89 (10 μmol/l) or AIP (5 μmol/l) had no effect on basal currents in Sham or CAL myocytes, indicating that, in contrast to ventricular myocytes, neither PKA nor CaMKII regulated basal I CaL Expression of the L-type α 1C -subunit, protein phosphatases 1 and 2A, and inhibitor-1 proteins was unchanged. In conclusion, reduction in PKA-dependent regulation did not contribute to downregulation of atrial I CaL in heart failure. NEW & NOTEWORTHY Whole cell recording of L-type Ca 2+ currents in atrial myocytes from rat hearts subjected to coronary artery ligation compared with those from sham-operated controls reveals marked reduction in current density

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

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

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

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.

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.

[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.

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

Ca2+ paradox injury mediated through TRPC channels in mouse ventricular myocytes

PubMed Central

Kojima, Akiko; Kitagawa, Hirotoshi; Omatsu-Kanbe, Mariko; Matsuura, Hiroshi; Nosaka, Shuichi

2010-01-01

BACKGROUND AND PURPOSE The Ca2+ paradox is an important phenomenon associated with Ca2+ overload-mediated cellular injury in myocardium. The present study was undertaken to elucidate molecular and cellular mechanisms for the development of the Ca2+ paradox. EXPERIMENTAL APPROACH Fluorescence imaging was performed on fluo-3 loaded quiescent mouse ventricular myocytes using confocal laser scanning microscope. KEY RESULTS The Ca2+ paradox was readily evoked by restoration of the extracellular Ca2+ following 10–20 min of nominally Ca2+-free superfusion. The Ca2+ paradox was significantly reduced by blockers of transient receptor potential canonical (TRPC) channels (2-aminoethoxydiphenyl borate, Gd3+, La3+) and anti-TRPC1 antibody. The sarcoplasmic reticulum (SR) Ca2+ content, assessed by caffeine application, gradually declined during Ca2+-free superfusion, which was further accelerated by metabolic inhibition. Block of SR Ca2+ leak by tetracaine prevented Ca2+ paradox. The Na+/Ca2+ exchange (NCX) blocker KB-R7943 significantly inhibited Ca2+ paradox when applied throughout superfusion period, but had little effect when added for a period of 3 min before and during Ca2+ restoration. The SR Ca2+ content was better preserved during Ca2+ depletion by KB-R7943. Immunocytochemistry confirmed the expression of TRPC1, in addition to TRPC3 and TRPC4, in mouse ventricular myocytes. CONCLUSIONS AND IMPLICATIONS These results provide evidence that (i) the Ca2+ paradox is primarily mediated by Ca2+ entry through TRPC (probably TRPC1) channels that are presumably activated by SR Ca2+ depletion; and (ii) reverse mode NCX contributes little to the Ca2+ paradox, whereas inhibition of NCX during Ca2+ depletion improves SR Ca2+ loading, and is associated with reduced incidence of Ca2+ paradox in mouse ventricular myocytes. PMID:20718730

Liganded Peroxisome Proliferator-Activated Receptors (PPARs) Preserve Nuclear Histone Deacetylase 5 Levels in Endothelin-Treated Sprague-Dawley Rat Cardiac Myocytes

PubMed Central

Zhang, Haining; Shao, Zongjun; Alibin, Caroline P.; Acosta, Crystal; Anderson, Hope D.

2014-01-01

Ligand activation of peroxisome proliferator-activated receptors (PPARs) prevents cardiac myocyte hypertrophy, and we previously reported that diacylglycerol kinase zeta (DGKζ) is critically involved. DGKζ is an intracellular lipid kinase that catalyzes phosphorylation of diacylglycerol; by attenuating DAG signaling, DGKζ suppresses protein kinase C (PKC) and G-protein signaling. Here, we investigated how PPAR-DGKζ signaling blocks activation of the hypertrophic gene program. We focused on export of histone deacetylase 5 (HDAC5) from the nucleus, a key event during hypertrophy, since crosstalk occurs between PPARs and other members of the HDAC family. Using cardiac myocytes isolated from Sprague-Dawley rats, we determined that liganded PPARs disrupt endothelin-1 (ET1)-induced nuclear export of HDAC5 in a manner that is dependent on DGKζ. When DGKζ-mediated PKC inhibition was circumvented using a constitutively-active PKCε mutant, PPARs failed to block ET1-induced nuclear retention of HDAC5. Liganded PPARs also prevented (i) activation of protein kinase D (the downstream effector of PKC), (ii) HDAC5 phosphorylation at 14-3-3 protein chaperone binding sites (serines 259 and 498), and (iii) physical interaction between HDAC5 and 14-3-3, all of which are consistent with blockade of nucleo-cytoplasmic shuttling of HDAC5. Finally, the ability of PPARs to prevent neutralization of HDAC5 activity was associated with transcriptional repression of hypertrophic genes. This occurred by first, reduced MEF2 transcriptional activity and second, augmented deacetylation of histone H3 associated with hypertrophic genes expressing brain natriuretic peptide, β-myosin heavy chain, skeletal muscle α-actin, and cardiac muscle α-actin. Our findings identify spatial regulation of HDAC5 as a target for liganded PPARs, and to our knowledge, are the first to describe a mechanistic role for nuclear DGKζ in cardiac myocytes. In conclusion, these results implicate modulation of HDAC5

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

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.

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

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.

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.

Vagus nerve stimulation mitigates intrinsic cardiac neuronal and adverse myocyte remodeling postmyocardial infarction

PubMed Central

Beaumont, Eric; Southerland, Elizabeth M.; Hardwick, Jean C.; Wright, Gary L.; Ryan, Shannon; Li, Ying; KenKnight, Bruce H.; Armour, J. Andrew

2015-01-01

This paper aims to determine whether chronic vagus nerve stimulation (VNS) mitigates myocardial infarction (MI)-induced remodeling of the intrinsic cardiac nervous system (ICNS), along with the cardiac tissue it regulates. Guinea pigs underwent VNS implantation on the right cervical vagus. Two weeks later, MI was produced by ligating the ventral descending coronary artery. VNS stimulation started 7 days post-MI (20 Hz, 0.9 ± 0.2 mA, 14 s on, 48 s off; VNS-MI, n = 7) and was compared with time-matched MI animals with sham VNS (MI n = 7) vs. untreated controls (n = 8). Echocardiograms were performed before and at 90 days post-MI. At termination, IC neuronal intracellular voltage recordings were obtained from whole-mount neuronal plexuses. MI increased left ventricular end systolic volume (LVESV) 30% (P = 0.027) and reduced LV ejection fraction (LVEF) 6.5% (P < 0.001) at 90 days post-MI compared with baseline. In the VNS-MI group, LVESV and LVEF did not differ from baseline. IC neurons showed depolarization of resting membrane potentials and increased input resistance in MI compared with VNS-MI and sham controls (P < 0.05). Neuronal excitability and sensitivity to norepinephrine increased in MI and VNS-MI groups compared with controls (P < 0.05). Synaptic efficacy, as determined by evoked responses to stimulating input axons, was reduced in VNS-MI compared with MI or controls (P < 0.05). VNS induced changes in myocytes, consistent with enhanced glycogenolysis, and blunted the MI-induced increase in the proapoptotic Bcl-2-associated X protein (P < 0.05). VNS mitigates MI-induced remodeling of the ICNS, correspondingly preserving ventricular function via both neural and cardiomyocyte-dependent actions. PMID:26276818

Vagus nerve stimulation mitigates intrinsic cardiac neuronal and adverse myocyte remodeling postmyocardial infarction.

PubMed

Beaumont, Eric; Southerland, Elizabeth M; Hardwick, Jean C; Wright, Gary L; Ryan, Shannon; Li, Ying; KenKnight, Bruce H; Armour, J Andrew; Ardell, Jeffrey L

2015-10-01

This paper aims to determine whether chronic vagus nerve stimulation (VNS) mitigates myocardial infarction (MI)-induced remodeling of the intrinsic cardiac nervous system (ICNS), along with the cardiac tissue it regulates. Guinea pigs underwent VNS implantation on the right cervical vagus. Two weeks later, MI was produced by ligating the ventral descending coronary artery. VNS stimulation started 7 days post-MI (20 Hz, 0.9 ± 0.2 mA, 14 s on, 48 s off; VNS-MI, n = 7) and was compared with time-matched MI animals with sham VNS (MI n = 7) vs. untreated controls (n = 8). Echocardiograms were performed before and at 90 days post-MI. At termination, IC neuronal intracellular voltage recordings were obtained from whole-mount neuronal plexuses. MI increased left ventricular end systolic volume (LVESV) 30% (P = 0.027) and reduced LV ejection fraction (LVEF) 6.5% (P < 0.001) at 90 days post-MI compared with baseline. In the VNS-MI group, LVESV and LVEF did not differ from baseline. IC neurons showed depolarization of resting membrane potentials and increased input resistance in MI compared with VNS-MI and sham controls (P < 0.05). Neuronal excitability and sensitivity to norepinephrine increased in MI and VNS-MI groups compared with controls (P < 0.05). Synaptic efficacy, as determined by evoked responses to stimulating input axons, was reduced in VNS-MI compared with MI or controls (P < 0.05). VNS induced changes in myocytes, consistent with enhanced glycogenolysis, and blunted the MI-induced increase in the proapoptotic Bcl-2-associated X protein (P < 0.05). VNS mitigates MI-induced remodeling of the ICNS, correspondingly preserving ventricular function via both neural and cardiomyocyte-dependent actions. Copyright © 2015 the American Physiological Society.

Model of excitation-contraction coupling of rat neonatal ventricular myocytes.

PubMed

Korhonen, Topi; Hänninen, Sandra L; Tavi, Pasi

2009-02-01

The neonatal rat ventricular myocyte culture is one of the most popular experimental cardiac cell models. To our knowledge, the excitation-contraction coupling (ECC) of these cells, i.e., the process linking the electrical activity to the cytosolic Ca2+ transient and contraction, has not been previously analyzed, nor has it been presented as a complete system in detail. Neonatal cardiomyocytes are in the postnatal developmental stage, and therefore, the features of their ECC differ vastly from those of adult ventricular myocytes. We present the first complete analysis of ECC in these cells by characterizing experimentally the action potential and calcium signaling and developing the first mathematical model of ECC in neonatal cardiomyocytes that we know of. We show that in comparison to adult cardiomyocytes, neonatal cardiomyocytes have long action potentials, heterogeneous cytosolic Ca2+ signals, weaker sarcoplasmic reticulum Ca2+ handling, and stronger sarcolemmal Ca2+ handling, with a significant contribution by the Na+/Ca2+ exchanger. The developed model reproduces faithfully the ECC of rat neonatal cardiomyocytes with a novel description of spatial cytosolic [Ca2+] signals. Simulations also demonstrate how an increase in the cell size (hypertrophy) affects the ECC in neonatal cardiomyocytes. This model of ECC in developing cardiomyocytes provides a platform for developing future models of cardiomyocytes at different developmental stages.

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.

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

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.

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 (...

Identification of the functional domain in the transcription factor RTEF-1 that mediates alpha 1-adrenergic signaling in hypertrophied cardiac myocytes.

PubMed

Ueyama, T; Zhu, C; Valenzuela, Y M; Suzow, J G; Stewart, A F

2000-06-09

Cardiac myocytes respond to alpha(1)-adrenergic receptor stimulation by a progressive hypertrophy accompanied by the activation of many fetal genes, including skeletal muscle alpha-actin. The skeletal muscle alpha-actin gene is activated by signaling through an MCAT element, the binding site of the transcription enhancer factor-1 (TEF-1) family of transcription factors. Previously, we showed that overexpression of the TEF-1-related factor (RTEF-1) increased the alpha(1)-adrenergic response of the skeletal muscle alpha-actin promoter, whereas TEF-1 overexpression did not. Here, we identified the functional domains and specific sequences in RTEF-1 that mediate the alpha(1)-adrenergic response. Chimeric TEF-1 and RTEF-1 expression constructs localized the region responsible for the alpha(1)-adrenergic response to the carboxyl-terminal domain of RTEF-1. Site-directed mutagenesis was used to inactivate eight serine residues of RTEF-1, not present in TEF-1, that are putative targets of alpha(1)-adrenergic-dependent kinases. Mutation of a single serine residue, Ser-322, reduced the alpha(1)-adrenergic activation of RTEF-1 by 70% without affecting protein stability, suggesting that phosphorylation at this serine residue accounts for most of the alpha(1)-adrenergic response. Thus, these results demonstrate that RTEF-1 is a direct target of alpha(1)-adrenergic signaling in hypertrophied cardiac myocytes.

Regional acidosis locally inhibits but remotely stimulates Ca2+ waves in ventricular myocytes

PubMed Central

Ford, Kerrie L.; Moorhouse, Emma L.; Bortolozzi, Mario; Richards, Mark A.; Swietach, Pawel; Vaughan-Jones, Richard D.

2017-01-01

Abstract Aims Spontaneous Ca2+ waves in cardiomyocytes are potentially arrhythmogenic. A powerful controller of Ca2+ waves is the cytoplasmic H+ concentration ([H+]i), which fluctuates spatially and temporally in conditions such as myocardial ischaemia/reperfusion. H+-control of Ca2+ waves is poorly understood. We have therefore investigated how [H+]i co-ordinates their initiation and frequency. Methods and results Spontaneous Ca2+ waves were imaged (fluo-3) in rat isolated ventricular myocytes, subjected to modest Ca2+-overload. Whole-cell intracellular acidosis (induced by acetate-superfusion) stimulated wave frequency. Pharmacologically blocking sarcolemmal Na+/H+ exchange (NHE1) prevented this stimulation, unveiling inhibition by H+. Acidosis also increased Ca2+ wave velocity. Restricting acidosis to one end of a myocyte, using a microfluidic device, inhibited Ca2+ waves in the acidic zone (consistent with ryanodine receptor inhibition), but stimulated wave emergence elsewhere in the cell. This remote stimulation was absent when NHE1 was selectively inhibited in the acidic zone. Remote stimulation depended on a locally evoked, NHE1-driven rise of [Na+]i that spread rapidly downstream. Conclusion Acidosis influences Ca2+ waves via inhibitory Hi+ and stimulatory Nai+ signals (the latter facilitating intracellular Ca2+-loading through modulation of sarcolemmal Na+/Ca2+ exchange activity). During spatial [H+]i-heterogeneity, Hi+-inhibition dominates in acidic regions, while rapid Nai+ diffusion stimulates waves in downstream, non-acidic regions. Local acidosis thus simultaneously inhibits and stimulates arrhythmogenic Ca2+-signalling in the same myocyte. If the principle of remote H+-stimulation of Ca2+ waves also applies in multicellular myocardium, it raises the possibility of electrical disturbances being driven remotely by adjacent ischaemic areas, which are known to be intensely acidic. PMID:28339694

Regional acidosis locally inhibits but remotely stimulates Ca2+ waves in ventricular myocytes.

PubMed

Ford, Kerrie L; Moorhouse, Emma L; Bortolozzi, Mario; Richards, Mark A; Swietach, Pawel; Vaughan-Jones, Richard D

2017-07-01

Spontaneous Ca2+ waves in cardiomyocytes are potentially arrhythmogenic. A powerful controller of Ca2+ waves is the cytoplasmic H+ concentration ([H+]i), which fluctuates spatially and temporally in conditions such as myocardial ischaemia/reperfusion. H+-control of Ca2+ waves is poorly understood. We have therefore investigated how [H+]i co-ordinates their initiation and frequency. Spontaneous Ca2+ waves were imaged (fluo-3) in rat isolated ventricular myocytes, subjected to modest Ca2+-overload. Whole-cell intracellular acidosis (induced by acetate-superfusion) stimulated wave frequency. Pharmacologically blocking sarcolemmal Na+/H+ exchange (NHE1) prevented this stimulation, unveiling inhibition by H+. Acidosis also increased Ca2+ wave velocity. Restricting acidosis to one end of a myocyte, using a microfluidic device, inhibited Ca2+ waves in the acidic zone (consistent with ryanodine receptor inhibition), but stimulated wave emergence elsewhere in the cell. This remote stimulation was absent when NHE1 was selectively inhibited in the acidic zone. Remote stimulation depended on a locally evoked, NHE1-driven rise of [Na+]i that spread rapidly downstream. Acidosis influences Ca2+ waves via inhibitory Hi+ and stimulatory Nai+ signals (the latter facilitating intracellular Ca2+-loading through modulation of sarcolemmal Na+/Ca2+ exchange activity). During spatial [H+]i-heterogeneity, Hi+-inhibition dominates in acidic regions, while rapid Nai+ diffusion stimulates waves in downstream, non-acidic regions. Local acidosis thus simultaneously inhibits and stimulates arrhythmogenic Ca2+-signalling in the same myocyte. If the principle of remote H+-stimulation of Ca2+ waves also applies in multicellular myocardium, it raises the possibility of electrical disturbances being driven remotely by adjacent ischaemic areas, which are known to be intensely acidic. © The Author 2017. Published by Oxford University Press on behalf of the European Society of Cardiology.

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

PubMed

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

2016-07-01

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

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

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

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

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

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.

Substance P modulates localized calcium transients and membrane current responses in murine colonic myocytes

PubMed Central

Bayguinov, Orline; Hagen, Brian; Sanders, Kenton M

2003-01-01

Neurokinins contribute to the neural regulation of gastrointestinal (GI) smooth muscles. We studied responses of murine colonic smooth muscle cells to substance P (SP) and NK1 and NK2 agonists using confocal microscopy and the patch clamp technique. Colonic myocytes generated localized Ca2+ transients that were coupled to spontaneous transient outward currents (STOCs). SP (10−10 M) increased Ca2+ transients and STOCs. Higher concentrations of SP (10−6 M) increased basal Ca2+ and inhibited Ca2+ transients and STOCs. Effects of SP were due to increased Ca2+ entry via L-type Ca2+ channels, and were mediated by protein kinase C (PKC). Nifedipine (10−6 M) and the PKC inhibitor, GF 109203X (10−6 M) reduced L-type Ca2+ current and blocked the effects of SP. SP responses depended upon parallel stimulation of NK1 and NK2 receptors. NK1 agonist ([Sar9,Met(O2)11]-substance P; SSP) and NK2 agonists (neurokinin A (NKA) or GR-64349) did not mimic the effects of SP alone, but NK1 and NK2 agonists were effective when added in combination (10−10–10−6 M). Consistent with this, either an NK1-specific antagonist (GR-82334; 10−7 M) or an NK2-specific antagonist (MEN 10,627; 10−7 M) blocked responses to SP (10−6 M). Ryanodine (10−5 M) blocked the increase in Ca2+ transients and STOCs in response to SP (10−10 M). Our findings show that low concentrations of SP, via PKC-dependent enhancement of L-type Ca2+ current and recruitment of ryanodine receptors, stimulate Ca2+ transients. At higher concentrations of SP (10−6 M), basal Ca2+ increases and spontaneous Ca2+ transients and STOCs are inhibited. PMID:12711623

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.

Genome organization and long-range regulation of gene expression by enhancers

PubMed Central

Smallwood, Andrea; Ren, Bing

2014-01-01

It is now well accepted that cell-type specific gene regulation is under the purview of enhancers. Great strides have been made recently to characterize and identify enhancers both genetically and epigenetically for multiple cell types and species, but efforts have just begun to link enhancers to their target promoters. Mapping these interactions and understanding how the 3D landscape of the genome constrains such interactions is fundamental to our understanding of mammalian gene regulation. Here, we review recent progress in mapping long-range regulatory interactions in mammalian genomes, focusing on transcriptional enhancers and chromatin organization principles. PMID:23465541

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

PubMed

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

2018-06-18

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

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.

Simulation of Atrial Fibrosis Using Coupled Myocyte-Fibroblast Cellular and Human Atrial Models

PubMed Central

Gao, Yuan

2017-01-01

Atrial fibrosis is characterized by expansion of extracellular matrix and increase in the number of fibroblasts which has been associated with the development and maintenance of atrial arrhythmias. However, the mechanisms how the fibrosis contributes to atrial arrhythmia remain incompletely understood. In this study, we used a proposed fibroblast model coupled with the human atrial myocyte to investigate the effects of fibrosis on atrial excitability and repolarization at both cellular and macroscopic levels. The 12-lead electrocardiogram (ECG) was also simulated to explore the index of clinical diagnosis for fibrosis. The simulation results showed that the fibrosis can modify action potential morphology of human atrial myocyte, slow down wave propagation, and have rate adaptation, thus causing the atrial electrical heterogeneity. The fibrosis alone was sufficient to cause arrhythmia, induce reentry wave, and result in low amplitude and wide P waves at normal heart rate and significant prolonged and inverse P waves at high heart rate. All these symptoms aggravated when the level of fibrosis increased. Our simulations demonstrated that fibrosis is the substrate of atrial arrhythmia and thereby may be a potential target in the treatment of atrial arrhythmias. PMID:29441121

Regulation of the instantaneous inward rectifier and the delayed outward rectifier potassium channels by Captopril and Angiotensin II via the Phosphoinositide-3 kinase pathway in volume-overload-induced hypertrophied cardiac myocytes.

PubMed

Alvin, Zikiar V; Laurence, Graham G; Coleman, Bernell R; Zhao, Aiqiu; Hajj-Moussa, Majd; Haddad, Georges E

2011-07-01

Early development of cardiac hypertrophy may be beneficial but sustained hypertrophic activation leads to myocardial dysfunction. Regulation of the repolarizing currents can be modulated by the activation of humoral factors, such as angiotensin II (ANG II) through protein kinases. The aim of this work is to assess the regulation of IK and IK1 by ANG II through the PI3-K pathway in hypertrophied ventricular myocytes. Cardiac eccentric hypertrophy was induced through volume-overload in adult male rats by aorto-caval shunt (3 weeks). After one week half of the rats were given captopril (2 weeks; 0.5 g/l/day) and the other half served as control. The voltage-clamp and western blot techniques were used to measure the delayed outward rectifier potassium current (IK) and the instantaneous inward rectifier potassium current (IK1) and Akt activity, respectively. Hypertrophied cardiomyocytes showed reduction in IK and IK1. Treatment with captopril alleviated this difference seen between sham and shunt cardiomyocytes. Acute administration of ANG II (10-6M) to cardiocytes treated with captopril reduced IK and IK1 in shunts, but not in sham. Captopril treatment reversed ANG II effects on IK and IK1 in a PI3-K-independent manner. However in the absence of angiotensin converting enzyme inhibition, ANG II increased both IK and IK1 in a PI3-K-dependent manner in hypertrophied cardiomyocytes. Thus, captopril treatment reveals a negative effect of ANG II on IK and IK1, which is PI3-K independent, whereas in the absence of angiotensin converting enzyme inhibition IK and IK1 regulation is dependent upon PI3-K.

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

Genome organization and long-range regulation of gene expression by enhancers.

PubMed

Smallwood, Andrea; Ren, Bing

2013-06-01

It is now well accepted that cell-type specific gene regulation is under the purview of enhancers. Great strides have been made recently to characterize and identify enhancers both genetically and epigenetically for multiple cell types and species, but efforts have just begun to link enhancers to their target promoters. Mapping these interactions and understanding how the 3D landscape of the genome constrains such interactions is fundamental to our understanding of mammalian gene regulation. Here, we review recent progress in mapping long-range regulatory interactions in mammalian genomes, focusing on transcriptional enhancers and chromatin organization principles. Copyright © 2013. Published by Elsevier Ltd.

Expression of a mutation causing hypertrophic cardiomyopathy disrupts sarcomere assembly in adult feline cardiac myocytes.

PubMed

Marian, A J; Yu, Q T; Mann, D L; Graham, F L; Roberts, R

1995-07-01

Mutations in the beta-myosin heavy chain (beta MyHC) induce hypertrophic cardiomyopathy (HCM), cardiac hypertrophy, and sarcomere disarray, with the latter being the characteristic hallmark. Thus, we sought to determine whether expression of mutant beta MyHC in adult feline cardiac myocytes, a species known to develop HCM with a phenotype identical to that in humans, induces sarcomere disarray. A full-length beta MyHC cDNA was cloned from a human heart cDNA library, and an HCM-causing mutation (Arg403Gln) was induced in the beta MyHC cDNA by site-directed mutagenesis using polymerase chain reaction (PCR). The normal and mutant beta MyHC cDNAs were cloned into p delta E1spIB shuttle vector, downstream from a cytomegalovirus (CMV) promoter. Replication-deficient recombinant adenoviral constructs (Ad5/CMV/beta MyHC-N and Ad5/CMV/beta MyHC-403) were generated through homologous recombination of p delta E1spIB/CMV/beta MyHC-N or Ad5/CMV/beta MyHC-403 and pBHG10 after cotransfection in 293 host cells. Infection of COS-1 cells with the beta MyHC construct resulted in the expression of a full-length myosin protein. Efficiency of infection of isolated adult cardiac myocytes was > 95%. Expression of the beta MyHC constructs into mRNA at 48 hours after infection of feline cardiac myocytes was confirmed by reverse transcription-PCR. The net total protein and beta-myosin synthesis were determined by using the amount of incorporation of [3H]phenylalanine into total protein and beta-myosin, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

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

Intrinsic H+ ion mobility in the rabbit ventricular myocyte

PubMed Central

Vaughan-Jones, R D; Peercy, B E; Keener, J P; Spitzer, K W

2002-01-01

The intrinsic mobility of intracellular H+ ions was investigated by confocally imaging the longitudinal movement of acid inside rabbit ventricular myocytes loaded with the acetoxymethyl ester (AM) form of carboxy-seminaphthorhodafluor-1 (carboxy-SNARF-1). Acid was diffused into one end of the cell through a patch pipette filled with an isotonic KCl solution of pH 3.0. Intracellular H+ mobility was low, acid taking 20-30 s to move 40 μm down the cell. Inhibiting sarcolemmal Na+-H+ exchange with 1 mm amiloride had no effect on this time delay. Net Hi+ movement was associated with a longitudinal intracellular pH (pHi) gradient of up to 0.4 pH units. Hi+ movement could be modelled using the equations for diffusion, assuming an apparent diffusion coefficient for H+ ions (DappH) of 3.78 × 10−7 cm2 s−1, a value more than 300-fold lower than the H+ diffusion coefficient in a dilute, unbuffered solution. Measurement of the intracellular concentration of SNARF (≈400 μM) and its intracellular diffusion coefficient (0.9 × 10−7 cm2 s−1) indicated that the fluorophore itself exerted an insignificant effect (between 0.6 and 3.3 %) on the longitudinal movement of H+ equivalents inside the cell. The longitudinal movement of intracellular H+ is discussed in terms of a diffusive shuttling of H+ equivalents on high capacity mobile buffers which comprise about half (≈11 mm) of the total intrinsic buffering capacity within the myocyte (the other half being fixed buffer sites on low mobility, intracellular proteins). Intrinsic Hi+ mobility is consistent with an average diffusion coefficient for the intracellular mobile buffers (Dmob) of ≈9 × 10−7 cm2 s−1. PMID:12015426

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

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.

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

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.

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

Early intracellular signaling events induced by in vitro metreleptin administration in cardiac myocytes and uterine smooth muscle cells.

PubMed

Choi, S K; Park, S; Choi, Y; Moon, H-S

2015-08-05

Intracellular signaling pathways regulated by leptin have largely been studied in metabolically important organs such as adipose tissue and peripheral blood mononuclear cells, suggesting that leptin plays a key role in pathophysiology of insulin resistance. However, whether synthetic analog of leptin, metreleptin, has similar effects on cardiac myocytes (CM) and uterine smooth muscle cells (USMC) has not yet been studied. Hence, in order to address these questions, we extended previous observations and investigated in vitro signaling study whether metreleptin may activate key signaling pathways. We observed that metreleptin activates Jak2 and STAT3 signaling pathways in dose- and time-dependent manner in CM and USMC. Also, we found that metreleptin increases ERK1/2, JNK and/or p38 phosphorylation in CM. In vitro metreleptin administration also increased ERK1/2 and/or p38 phosphorylation in USMC. By contrast, JNK was not regulated by in vitro metreleptin administration in USMC. Moreover, metreleptin-activated all signaling pathways were blocked by pre-treatment of PD98095 (ERK inhibitor), SB203580 (p38 inhibitor) and/or SP600125 (JNK inhibitor), respectively. Finally, metreleptin increased cell size (hypertrophy) in both CM and USMC. Our data provide novel insights into the role of Jak2, STAT3, ERK1/2, JNK and/or p38 as probable mediators of the action of leptin in regulating hypertrophy in CM and USMC.

Benchmarking electrophysiological models of human atrial myocytes

PubMed Central

Wilhelms, Mathias; Hettmann, Hanne; Maleckar, Mary M.; Koivumäki, Jussi T.; Dössel, Olaf; Seemann, Gunnar

2013-01-01

Mathematical modeling of cardiac electrophysiology is an insightful method to investigate the underlying mechanisms responsible for arrhythmias such as atrial fibrillation (AF). In past years, five models of human atrial electrophysiology with different formulations of ionic currents, and consequently diverging properties, have been published. The aim of this work is to give an overview of strengths and weaknesses of these models depending on the purpose and the general requirements of simulations. Therefore, these models were systematically benchmarked with respect to general mathematical properties and their ability to reproduce certain electrophysiological phenomena, such as action potential (AP) alternans. To assess the models' ability to replicate modified properties of human myocytes and tissue in cardiac disease, electrical remodeling in chronic atrial fibrillation (cAF) was chosen as test case. The healthy and remodeled model variants were compared with experimental results in single-cell, 1D and 2D tissue simulations to investigate AP and restitution properties, as well as the initiation of reentrant circuits. PMID:23316167

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

UNCOUPLING THE COUPLED CALCIUM AND ZINC DYSHOMEOSTASIS IN CARDIAC MYOCYTES AND MITOCHONDRIA SEEN IN ALDOSTERONISM

PubMed Central

Kamalov, German; Ahokas, Robert A.; Zhao, Wenyuan; Zhao, Tieqiang; Shahbaz, Atta U.; Johnson, Patti L.; Bhattacharya, Syamal K.; Sun, Yao; Gerling, Ivan C.; Weber, Karl T.

2010-01-01

Intracellular [Ca2+]i overloading in cardiomyocytes is a fundamental pathogenic event associated with chronic aldosterone/salt treatment (ALDOST) and accounts for an induction of oxidative stress that leads to necrotic cell death and consequent myocardial scarring. This prooxidant response to Ca2+ overloading in cardiac myocytes and mitochondria is intrinsically coupled to simultaneous increased Zn2+ entry serving as an antioxidant. Herein, we investigated whether Ca2+ and Zn2+ dyshomeostasis and prooxidant:antioxidant dysequilibrium seen at 4 wks, the pathologic stage of ALDOST, could be uncoupled in favor of antioxidants, using cotreatment with a ZnSO4 supplement, pyrrolidine dithiocarbamate (PDTC), a Zn2+ ionophore, or ZnSO4 in combination with amlodipine (Amlod), a Ca2+ channel blocker. We monitored and compared responses in cardiomyocyte free [Ca2+]i and [Zn2+]i together with biomarkers of oxidative stress in cardiac myocytes and mitochondria. At wk 4 ALDOST and compared to controls, we found: i) an elevation in [Ca2+]i coupled with [Zn2+]i; and ii) increased mitochondrial H2O2 production, and increased mitochondrial and cardiac 8-isoprostane levels. Cotreatment with the ZnSO4 supplement alone, PDTC, or ZnSO4+Amlod augmented the rise in cardiomyocyte [Zn2+]i beyond that seen with ALDOST alone, while attenuating the rise in [Ca2+]i which together served to reduce oxidative stress. Thus, a coupled dyshomeostasis of intracellular Ca2+ and Zn2+ was demonstrated in cardiac myocytes and mitochondria during 4 wks ALDOST, where prooxidants overwhelm antioxidant defenses. This intrinsically coupled Ca2+ and Zn2+ dyshomeostasis could be uncoupled in favor of antioxidant defenses by selectively increasing free [Zn2+]i and/or reducing [Ca2+]i using cotreatment with ZnSO4 or PDTC alone or ZnSO4+Amlod in combination. PMID:20051880

Selective activation of heteromeric SK channels contributes to action potential repolarization in mouse atrial myocytes.

PubMed

Hancock, Jane M; Weatherall, Kate L; Choisy, Stéphanie C; James, Andrew F; Hancox, Jules C; Marrion, Neil V

2015-05-01

Activation of small conductance calcium-activated potassium (SK) channels is proposed to contribute to repolarization of the action potential in atrial myocytes. This role is controversial, as these cardiac SK channels appear to exhibit an uncharacteristic pharmacology. The objectives of this study were to resolve whether activation of SK channels contributes to atrial action potential repolarization and to determine the likely subunit composition of the channel. The effect of 2 SK channel inhibitors was assessed on outward current evoked in voltage clamp and on action potential duration in perforated patch and whole-cell current clamp recording from acutely isolated mouse atrial myocytes. The presence of SK channel subunits was assessed using immunocytochemistry. A significant component of outward current was reduced by the SK channel blockers apamin and UCL1684. Block by apamin displayed a sensitivity indicating that this current was carried by homomeric SK2 channels. Action potential duration was significantly prolonged by UCL1684, but not by apamin. This effect was accompanied by an increase in beat-to-beat variability and action potential triangulation. This pharmacology was matched by that of expressed heteromeric SK2-SK3 channels in HEK293 cells. Immunocytochemistry showed that atrial myocytes express both SK2 and SK3 channels with an overlapping expression pattern. Only proposed heteromeric SK2-SK3 channels are physiologically activated to contribute to action potential repolarization, which is indicated by the difference in pharmacology of evoked outward current and prolongation of atrial action potential duration. The effect of blocking this channel on the action potential suggests that SK channel inhibition during cardiac function has the potential to be proarrhythmic. Copyright © 2015 Heart Rhythm Society. Published by Elsevier Inc. All rights reserved.

An integrative model of the cardiac ventricular myocyte incorporating local control of Ca2+ release.

PubMed Central

Greenstein, Joseph L; Winslow, Raimond L

2002-01-01

The local control theory of excitation-contraction (EC) coupling in cardiac muscle asserts that L-type Ca(2+) current tightly controls Ca(2+) release from the sarcoplasmic reticulum (SR) via local interaction of closely apposed L-type Ca(2+) channels (LCCs) and ryanodine receptors (RyRs). These local interactions give rise to smoothly graded Ca(2+)-induced Ca(2+) release (CICR), which exhibits high gain. In this study we present a biophysically detailed model of the normal canine ventricular myocyte that conforms to local control theory. The model formulation incorporates details of microscopic EC coupling properties in the form of Ca(2+) release units (CaRUs) in which individual sarcolemmal LCCs interact in a stochastic manner with nearby RyRs in localized regions where junctional SR membrane and transverse-tubular membrane are in close proximity. The CaRUs are embedded within and interact with the global systems of the myocyte describing ionic and membrane pump/exchanger currents, SR Ca(2+) uptake, and time-varying cytosolic ion concentrations to form a model of the cardiac action potential (AP). The model can reproduce both the detailed properties of EC coupling, such as variable gain and graded SR Ca(2+) release, and whole-cell phenomena, such as modulation of AP duration by SR Ca(2+) release. Simulations indicate that the local control paradigm predicts stable APs when the L-type Ca(2+) current is adjusted in accord with the balance between voltage- and Ca(2+)-dependent inactivation processes as measured experimentally, a scenario where common pool models become unstable. The local control myocyte model provides a means for studying the interrelationship between microscopic and macroscopic behaviors in a manner that would not be possible in experiments. PMID:12496068

An anomalous subdiffusion model for calcium spark in cardiac myocytes

NASA Astrophysics Data System (ADS)

Tan, Wenchang; Fu, Chaoqi; Fu, Ceji; Xie, Wenjun; Cheng, Heping

2007-10-01

The elementary events of excitation-contraction coupling in heart muscle are Ca2+ sparks, which arise from ryanodine receptors in the sarcoplasmic reticulum (SR). Here, an anomalous subdiffusion model is developed to explore Ca2+ spark formation in cardiac myocytes. Numerical simulations reproduce the brightness, the time course, and spatial size of a typical cardiac Ca2+ spark. It is suggested that the diffusion of Ca2+ spark in the cytoplasm may no longer obey Fickian second law, but the anomalous space subdiffusion. The physical reason is perhaps due to the effects of the electric field of the calcium ions and the viscoelasticity of the cytoplasm and its complex structures.

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.

Mapping of Human FOXP2 Enhancers Reveals Complex Regulation.

PubMed

Becker, Martin; Devanna, Paolo; Fisher, Simon E; Vernes, Sonja C

2018-01-01

Mutations of the FOXP2 gene cause a severe speech and language disorder, providing a molecular window into the neurobiology of language. Individuals with FOXP2 mutations have structural and functional alterations affecting brain circuits that overlap with sites of FOXP2 expression, including regions of the cortex, striatum, and cerebellum. FOXP2 displays complex patterns of expression in the brain, as well as in non-neuronal tissues, suggesting that sophisticated regulatory mechanisms control its spatio-temporal expression. However, to date, little is known about the regulation of FOXP2 or the genomic elements that control its expression. Using chromatin conformation capture (3C), we mapped the human FOXP2 locus to identify putative enhancer regions that engage in long-range interactions with the promoter of this gene. We demonstrate the ability of the identified enhancer regions to drive gene expression. We also show regulation of the FOXP2 promoter and enhancer regions by candidate regulators - FOXP family and TBR1 transcription factors. These data point to regulatory elements that may contribute to the temporal- or tissue-specific expression patterns of human FOXP2 . Understanding the upstream regulatory pathways controlling FOXP2 expression will bring new insight into the molecular networks contributing to human language and related disorders.

Mapping of Human FOXP2 Enhancers Reveals Complex Regulation

PubMed Central

Becker, Martin; Devanna, Paolo; Fisher, Simon E.; Vernes, Sonja C.

2018-01-01

Mutations of the FOXP2 gene cause a severe speech and language disorder, providing a molecular window into the neurobiology of language. Individuals with FOXP2 mutations have structural and functional alterations affecting brain circuits that overlap with sites of FOXP2 expression, including regions of the cortex, striatum, and cerebellum. FOXP2 displays complex patterns of expression in the brain, as well as in non-neuronal tissues, suggesting that sophisticated regulatory mechanisms control its spatio-temporal expression. However, to date, little is known about the regulation of FOXP2 or the genomic elements that control its expression. Using chromatin conformation capture (3C), we mapped the human FOXP2 locus to identify putative enhancer regions that engage in long-range interactions with the promoter of this gene. We demonstrate the ability of the identified enhancer regions to drive gene expression. We also show regulation of the FOXP2 promoter and enhancer regions by candidate regulators – FOXP family and TBR1 transcription factors. These data point to regulatory elements that may contribute to the temporal- or tissue-specific expression patterns of human FOXP2. Understanding the upstream regulatory pathways controlling FOXP2 expression will bring new insight into the molecular networks contributing to human language and related disorders. PMID:29515369

Thyroxine-induced cardiac hypertrophy: influence of adrenergic nervous system versus renin-angiotensin system on myocyte remodeling.

PubMed

Hu, L W; Benvenuti, L A; Liberti, E A; Carneiro-Ramos, M S; Barreto-Chaves, M L M

2003-12-01

The present study assessed the possible involvement of the renin-angiotensin system (RAS) and the sympathetic nervous system (SNS) in thyroxine (T4)-induced cardiac hypertrophy. Hemodynamic parameters, heart weight (HW), ratio of HW to body weight (HW/BW), and myocyte width were evaluated in absence of thyroid hormone (hypothyroidism) and after T4 administration. Male Wistar rats were used. Some were subjected to thyroidectomies, whereas hyperthyroidism was induced in others via daily intraperitoneal injection of T4 (25 or 100 microg x 100 g BW(-1) x day(-1)) for 7 days. In some cases, T4 administration was combined with the angiotensin I-converting enzyme inhibitor enalapril (Ena), with the angiotensin type 1 (AT1) receptor blocker losartan (Los) or with the beta-adrenergic blocker propanolol (Prop). Hemodynamics and morphology were then evaluated. Systolic blood pressure (SBP) was not altered by administration of either T4 alone or T4 in combination with the specific inhibitors. However, SBP decreased significantly in hypothyroid rats. An increased heart rate was seen after administration of either T4 alone or T4 in combination with either Los or Ena. Although the higher dose of T4 significantly increased HW, HW/BW increased in both T4-treated groups. Ena and Prop inhibited the increase in HW or HW/BW in hyperthyroid rats. Morphologically, both T4 dose levels significantly increased myocyte width, an occurrence prevented by RAS or SNS blockers. There was a good correlation between changes in HW/BW and myocyte width. These results indicate that T4-induced cardiac hypertrophy is associated with both the SNS and the RAS.

Quantifying the Release of Biomarkers of Myocardial Necrosis from Cardiac Myocytes and Intact Myocardium.

PubMed

Marjot, Jack; Kaier, Thomas E; Martin, Eva D; Reji, Shiney S; Copeland, O'Neal; Iqbal, Mohammed; Goodson, Bob; Hamren, Sarah; Harding, Sian E; Marber, Michael S

2017-05-01

Myocardial infarction is diagnosed when biomarkers of cardiac necrosis exceed the 99th centile, although guidelines advocate even lower concentrations for early rule-out. We examined how many myocytes and how much myocardium these concentrations represent. We also examined if dietary troponin can confound the rule-out algorithm. Individual rat cardiac myocytes, rat myocardium, ovine myocardium, or human myocardium were spiked into 400-μL aliquots of human serum. Blood was drawn from a volunteer after ingestion of ovine myocardium. High-sensitivity assays were used to measure cardiac troponin T (cTnT; Roche, Elecsys), cTnI (Abbott, Architect), and cardiac myosin-binding protein C (cMyC; EMD Millipore, Erenna ® ). The cMyC assay could only detect the human protein. For each rat cardiac myocyte added to 400 μL of human serum, cTnT and cTnI increased by 19.0 ng/L (95% CI, 16.8-21.2) and 18.9 ng/L (95% CI, 14.7-23.1), respectively. Under identical conditions cTnT, cTnI, and cMyC increased by 3.9 ng/L (95% CI, 3.6-4.3), 4.3 ng/L (95% CI, 3.8-4.7), and 41.0 ng/L (95% CI, 38.0-44.0) per μg of human myocardium. There was no detectable change in cTnI or cTnT concentration after ingestion of sufficient ovine myocardium to increase cTnT and cTnI to approximately 1 × 10 8 times their lower limits of quantification. Based on pragmatic assumptions regarding cTn and cMyC release efficiency, circulating species, and volume of distribution, 99th centile concentrations may be exceeded by necrosis of 40 mg of myocardium. This volume is much too small to detect by noninvasive imaging. © 2017 American Association for Clinical Chemistry.

Enhancers and super-enhancers have an equivalent regulatory role in embryonic stem cells through regulation of single or multiple genes

PubMed Central

Moorthy, Sakthi D.; Davidson, Scott; Shchuka, Virlana M.; Singh, Gurdeep; Malek-Gilani, Nakisa; Langroudi, Lida; Martchenko, Alexandre; So, Vincent; Macpherson, Neil N.; Mitchell, Jennifer A.

2017-01-01

Transcriptional enhancers are critical for maintaining cell-type–specific gene expression and driving cell fate changes during development. Highly transcribed genes are often associated with a cluster of individual enhancers such as those found in locus control regions. Recently, these have been termed stretch enhancers or super-enhancers, which have been predicted to regulate critical cell identity genes. We employed a CRISPR/Cas9-mediated deletion approach to study the function of several enhancer clusters (ECs) and isolated enhancers in mouse embryonic stem (ES) cells. Our results reveal that the effect of deleting ECs, also classified as ES cell super-enhancers, is highly variable, resulting in target gene expression reductions ranging from 12% to as much as 92%. Partial deletions of these ECs which removed only one enhancer or a subcluster of enhancers revealed partially redundant control of the regulated gene by multiple enhancers within the larger cluster. Many highly transcribed genes in ES cells are not associated with a super-enhancer; furthermore, super-enhancer predictions ignore 81% of the potentially active regulatory elements predicted by cobinding of five or more pluripotency-associated transcription factors. Deletion of these additional enhancer regions revealed their robust regulatory role in gene transcription. In addition, select super-enhancers and enhancers were identified that regulated clusters of paralogous genes. We conclude that, whereas robust transcriptional output can be achieved by an isolated enhancer, clusters of enhancers acting on a common target gene act in a partially redundant manner to fine tune transcriptional output of their target genes. PMID:27895109

34 CFR 225.3 - What regulations apply to the Credit Enhancement for Charter School Facilities Program?

Code of Federal Regulations, 2013 CFR

2013-07-01

... Charter School Facilities Program? 225.3 Section 225.3 Education Regulations of the Offices of the... ENHANCEMENT FOR CHARTER SCHOOL FACILITIES PROGRAM General § 225.3 What regulations apply to the Credit Enhancement for Charter School Facilities Program? The following regulations apply to the Credit Enhancement...

34 CFR 225.3 - What regulations apply to the Credit Enhancement for Charter School Facilities Program?

Code of Federal Regulations, 2014 CFR

2014-07-01

... Charter School Facilities Program? 225.3 Section 225.3 Education Regulations of the Offices of the... ENHANCEMENT FOR CHARTER SCHOOL FACILITIES PROGRAM General § 225.3 What regulations apply to the Credit Enhancement for Charter School Facilities Program? The following regulations apply to the Credit Enhancement...

34 CFR 225.3 - What regulations apply to the Credit Enhancement for Charter School Facilities Program?

Code of Federal Regulations, 2012 CFR

2012-07-01

... Charter School Facilities Program? 225.3 Section 225.3 Education Regulations of the Offices of the... ENHANCEMENT FOR CHARTER SCHOOL FACILITIES PROGRAM General § 225.3 What regulations apply to the Credit Enhancement for Charter School Facilities Program? The following regulations apply to the Credit Enhancement...

34 CFR 225.3 - What regulations apply to the Credit Enhancement for Charter School Facilities Program?

Code of Federal Regulations, 2011 CFR

2011-07-01

... Charter School Facilities Program? 225.3 Section 225.3 Education Regulations of the Offices of the... ENHANCEMENT FOR CHARTER SCHOOL FACILITIES PROGRAM General § 225.3 What regulations apply to the Credit Enhancement for Charter School Facilities Program? The following regulations apply to the Credit Enhancement...

34 CFR 225.3 - What regulations apply to the Credit Enhancement for Charter School Facilities Program?

Code of Federal Regulations, 2010 CFR

2010-07-01

... Charter School Facilities Program? 225.3 Section 225.3 Education Regulations of the Offices of the... ENHANCEMENT FOR CHARTER SCHOOL FACILITIES PROGRAM General § 225.3 What regulations apply to the Credit Enhancement for Charter School Facilities Program? The following regulations apply to the Credit Enhancement...

Lysine and Leucine Deficiencies Affect Myocytes Development and IGF Signaling in Gilthead Sea Bream (Sparus aurata)

PubMed Central

Azizi, Sheida; Nematollahi, Mohammad Ali; Mojazi Amiri, Bagher; Vélez, Emilio J.; Lutfi, Esmail; Navarro, Isabel; Capilla, Encarnación; Gutiérrez, Joaquim

2016-01-01

Optimizing aquaculture production requires better knowledge of growth regulation and improvement in diet formulation. A great effort has been made to replace fish meal for plant protein sources in aquafeeds, making necessary the supplementation of such diets with crystalline amino acids (AA) to cover the nutritional requirements of each species. Lysine and Leucine are limiting essential AA in fish, and it has been demonstrated that supplementation with them improves growth in different species. However, the specific effects of AA deficiencies in myogenesis are completely unknown and have only been studied at the level of hepatic metabolism. It is well-known that the TOR pathway integrates the nutritional and hormonal signals to regulate protein synthesis and cell proliferation, to finally control muscle growth, a process also coordinated by the expression of myogenic regulatory factors (MRFs). This study aimed to provide new information on the impact of Lysine and Leucine deficiencies in gilthead sea bream cultured myocytes examining their development and the response of insulin-like growth factors (IGFs), MRFs, as well as key molecules involved in muscle growth regulation like TOR. Leucine deficiency did not cause significant differences in most of the molecules analyzed, whereas Lysine deficiency appeared crucial in IGFs regulation, decreasing significantly IGF-I, IGF-II and IGF-IRb mRNA levels. This treatment also down-regulated the gene expression of different MRFs, including Myf5, Myogenin and MyoD2. These changes were also corroborated by a significant decrease in proliferation and differentiation markers in the Lysine-deficient treatment. Moreover, both Lysine and Leucine limitation induced a significant down-regulation in FOXO3 gene expression, which deserves further investigation. We believe that these results will be relevant for the production of a species as appreciated for human consumption as it is gilthead sea bream and demonstrates the importance of

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

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.

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.

Regulation of Cardiac Stress Signaling by Protein Kinase D1

PubMed Central

Harrison, Brooke C.; Kim, Mi-Sung; van Rooij, Eva; Plato, Craig F.; Papst, Philip J.; Vega, Rick B.; McAnally, John A.; Richardson, James A.; Bassel-Duby, Rhonda; Olson, Eric N.; McKinsey, Timothy A.

2006-01-01

In response to pathological stresses such as hypertension or myocardial infarction, the heart undergoes a remodeling process that is associated with myocyte hypertrophy, myocyte death, and fibrosis. Histone deacetylase 5 (HDAC5) is a transcriptional repressor of cardiac remodeling that is subject to phosphorylation-dependent neutralization in response to stress signaling. Recent studies have suggested a role for protein kinase C (PKC) and its downstream effector, protein kinase D1 (PKD1), in the control of HDAC5 phosphorylation. While PKCs are well-documented regulators of cardiac signaling, the function of PKD1 in heart muscle remains unclear. Here, we demonstrate that PKD1 catalytic activity is stimulated in cardiac myocytes by diverse hypertrophic agonists that signal through G protein-coupled receptors (GPCRs) and Rho GTPases. PKD1 activation in cardiomyocytes occurs through PKC-dependent and -independent mechanisms. In vivo, cardiac PKD1 is activated in multiple rodent models of pathological cardiac remodeling. PKD1 activation correlates with phosphorylation-dependent nuclear export of HDAC5, and reduction of endogenous PKD1 expression with small interfering RNA suppresses HDAC5 shuttling and associated cardiomyocyte growth. Conversely, ectopic overexpression of constitutively active PKD1 in mouse heart leads to dilated cardiomyopathy. These findings support a role for PKD1 in the control of pathological remodeling of the heart via its ability to phosphorylate and neutralize HDAC5. PMID:16648482

Structural correlates of the creatine transporter function regulation: the undiscovered country.

PubMed

Santacruz, Lucia; Jacobs, Danny O

2016-08-01

Creatine (Cr) and phosphocreatine constitute an energy shuttle that links ATP production in mitochondria to subcellular locations of ATP consumption. Cells in tissues that are reliant on this energy shuttle, such as myocytes and neurons, appear to have very limited ability to synthesize creatine. Therefore, these cells depend on Cr uptake across the cell membrane by a specialized creatine transporter (CrT solute carrier SLC6A8) in order to maintain intracellular creatine levels. Cr supplementation has been shown to have a beneficial effect in numerous in vitro and in vivo models, particularly in cases of oxidative stress, and is also widely used by athletes as a performance enhancement nutraceutical. Intracellular creatine content is maintained within narrow limits. However, the physiological and cellular mechanisms that mediate Cr transport during health and disease (such as cardiac failure) are not understood. In this narrative mini-review, we summarize the last three decades of research on CrT structure, function and regulation.

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.

Compartmentalized cAMP Signaling Associated With Lipid Raft and Non-raft Membrane Domains in Adult Ventricular Myocytes.

PubMed

Agarwal, Shailesh R; Gratwohl, Jackson; Cozad, Mia; Yang, Pei-Chi; Clancy, Colleen E; Harvey, Robert D

2018-01-01

Aim: Confining cAMP production to discrete subcellular locations makes it possible for this ubiquitous second messenger to elicit unique functional responses. Yet, factors that determine how and where the production of this diffusible signaling molecule occurs are incompletely understood. The fluid mosaic model originally proposed that signal transduction occurs through random interactions between proteins diffusing freely throughout the plasma membrane. However, it is now known that the movement of membrane proteins is restricted, suggesting that the plasma membrane is segregated into distinct microdomains where different signaling proteins can be concentrated. In this study, we examined what role lipid raft and non-raft membrane domains play in compartmentation of cAMP signaling in adult ventricular myocytes. Methods and Results: The freely diffusible fluorescence resonance energy transfer-based biosensor Epac2-camps was used to measure global cytosolic cAMP responses, while versions of the probe targeted to lipid raft (Epac2-MyrPalm) and non-raft (Epac2-CAAX) domains were used to monitor local cAMP production near the plasma membrane. We found that β-adrenergic receptors, which are expressed in lipid raft and non-raft domains, produce cAMP responses near the plasma membrane that are distinctly different from those produced by E-type prostaglandin receptors, which are expressed exclusively in non-raft domains. We also found that there are differences in basal cAMP levels associated with lipid raft and non-raft domains, and that this can be explained by differences in basal adenylyl cyclase activity associated with each of these membrane environments. In addition, we found evidence that phosphodiesterases 2, 3, and 4 work together in regulating cAMP activity associated with both lipid raft and non-raft domains, while phosphodiesterase 3 plays a more prominent role in the bulk cytoplasmic compartment. Conclusion: These results suggest that different membrane

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.

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

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

The regulation of cognitive enhancement devices: extending the medical model

PubMed Central

Maslen, Hannah; Douglas, Thomas; Cohen Kadosh, Roi; Levy, Neil; Savulescu, Julian

2014-01-01

This article presents a model for regulating cognitive enhancement devices (CEDs). Recently, it has become very easy for individuals to purchase devices which directly modulate brain function. For example, transcranial direct current stimulators are increasingly being produced and marketed online as devices for cognitive enhancement. Despite posing risks in a similar way to medical devices, devices that do not make any therapeutic claims do not have to meet anything more than basic product safety standards. We present the case for extending existing medical device legislation to cover CEDs. Medical devices and CEDs operate by the same or similar mechanisms and pose the same or similar risks. This fact coupled with the arbitrariness of the line between treatment and enhancement count in favour of regulating these devices in the same way. In arguing for this regulatory model, the paper highlights potential challenges to its implementation, and suggests solutions. PMID:25243073

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

Differences in transient outward currents of feline endocardial and epicardial myocytes.

PubMed

Furukawa, T; Myerburg, R J; Furukawa, N; Bassett, A L; Kimura, S

1990-11-01

Whole-cell voltage-clamp experiments were performed on enzymatically dissociated single ventricular myocytes harvested from feline endocardial and epicardial surfaces. The studies were designed to test the hypothesis that the differences in the amplitude of transient outward current (Ito) contribute to the difference in action potential configuration between endocardial and epicardial myocytes. In the control state, action potentials recorded from epicardial cells demonstrated a prominent notch between phases 1 and 2, and membrane current recordings displayed a prominent Ito, whereas in endocardial cells the notch in action potentials and Ito were small. External application of 4-aminopyridine (2 mM) reduced the amplitudes of notch and Ito in epicardial cells but not in endocardial cells. After application of 4-aminopyridine (2 mM) and caffeine (5 mM), the notch and Ito were abolished completely in both endocardial and epicardial cells. The first component of Ito (Ito1) was present in all epicardial cells studied (n = 20); it was absent in 12 of the 20 endocardial cells, and a small Ito1 was present in the remaining eight endocardial cells. The mean amplitude of Ito1 was significantly greater in epicardial than in endocardial cells. At a test voltage of +80 mV, the amplitude of Ito1 was 102.0 +/- 47.7 pA/pF in epicardial cells and 3.3 +/- 3.3 pA/pF in endocardial cells (p less than 0.01). The second component of Ito (Ito2) was present in all endocardial (n = 30) and epicardial (n = 30) cells studied. The amplitude of Ito2 was significantly greater in epicardial than in endocardial cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Crataegus extract blocks potassium currents in guinea pig ventricular cardiac myocytes.

PubMed

Müller, A; Linke, W; Klaus, W

1999-05-01

Crataegus extract is used in cardiology for the treatment of mild to moderate heart failure (NYHA II) in Germany. However, little is known about the electrophysiological actions of Crataegus extract in the heart. Recently, it was shown that Crataegus extract prolongs the refractory period in isolated perfused hearts and increases action potential duration in guinea pig papillary muscle. It was the aim of this study to find out the mechanism of the increase in action potential duration caused by Crataegus extract. Using the patch-clamp technique, we measured the effects of Crataegus extract (10 mg/l; flavonoid content: 2.25%, total procyanidin content: 11.3 +/- 0.4%) on the inward rectifier and the delayed rectifier potassium current in isolated guinea pig ventricular myocytes. To get some insight into the mechanism underlying the positive inotropic effect of Crataegus extract, we also looked for effects on the L-type calcium current. Crataegus extract slightly blocked both the delayed and the inward rectifier potassium current. The inhibition amounted to 25% and about 15%, respectively. This amount of inhibition of these repolarising currents is sufficient to explain the prolongation of action potential duration caused by Crataegus extract. To our surprise we could not detect any influence of Crataegus extract on the L-type calcium current. In summary, our results show that Crataegus extract blocks repolarising potassium currents in ventricular myocytes. This effect is similar to the action of class III antiarrhythmic drugs and might be the basis of the antiarrhythmic effects described for Crataegus extract. Our measurements of the L-type calcium current indicate that Crataegus extract's positive inotropic effect is not caused by phosphodiesterase inhibition or a beta-sympathomimetic effect.

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.

Spiral-wave dynamics in a mathematical model of human ventricular tissue with myocytes and fibroblasts.

PubMed

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

2013-01-01

Cardiac fibroblasts, when coupled functionally with myocytes, can modulate the electrophysiological properties of cardiac tissue. We present systematic numerical studies of such modulation of electrophysiological properties in mathematical models for (a) single myocyte-fibroblast (MF) units and (b) two-dimensional (2D) arrays of such units; our models build on earlier ones and allow for zero-, one-, and two-sided MF couplings. Our studies of MF units elucidate the dependence of the action-potential (AP) morphology on parameters such as [Formula: see text], the fibroblast resting-membrane potential, the fibroblast conductance [Formula: see text], and the MF gap-junctional coupling [Formula: see text]. Furthermore, we find that our MF composite can show autorhythmic and oscillatory behaviors in addition to an excitable response. Our 2D studies use (a) both homogeneous and inhomogeneous distributions of fibroblasts, (b) various ranges for parameters such as [Formula: see text], and [Formula: see text], and (c) intercellular couplings that can be zero-sided, one-sided, and two-sided connections of fibroblasts with myocytes. We show, in particular, that the plane-wave conduction velocity [Formula: see text] decreases as a function of [Formula: see text], for zero-sided and one-sided couplings; however, for two-sided coupling, [Formula: see text] decreases initially and then increases as a function of [Formula: see text], and, eventually, we observe that conduction failure occurs for low values of [Formula: see text]. In our homogeneous studies, we find that the rotation speed and stability of a spiral wave can be controlled either by controlling [Formula: see text] or [Formula: see text]. Our studies with fibroblast inhomogeneities show that a spiral wave can get anchored to a local fibroblast inhomogeneity. We also study the efficacy of a low-amplitude control scheme, which has been suggested for the control of spiral-wave turbulence in mathematical models for cardiac

Paracrine Engineering of Human Explant-Derived Cardiac Stem Cells to Over-Express Stromal-Cell Derived Factor 1α Enhances Myocardial Repair.

PubMed

Tilokee, Everad L; Latham, Nicholas; Jackson, Robyn; Mayfield, Audrey E; Ye, Bin; Mount, Seth; Lam, Buu-Khanh; Suuronen, Erik J; Ruel, Marc; Stewart, Duncan J; Davis, Darryl R

2016-07-01

First generation cardiac stem cell products provide indirect cardiac repair but variably produce key cardioprotective cytokines, such as stromal-cell derived factor 1α, which opens the prospect of maximizing up-front paracrine-mediated repair. The mesenchymal subpopulation within explant derived human cardiac stem cells underwent lentiviral mediated gene transfer of stromal-cell derived factor 1α. Unlike previous unsuccessful attempts to increase efficacy by boosting the paracrine signature of cardiac stem cells, cytokine profiling revealed that stromal-cell derived factor 1α over-expression prevented lv-mediated "loss of cytokines" through autocrine stimulation of CXCR4+ cardiac stem cells. Stromal-cell derived factor 1α enhanced angiogenesis and stem cell recruitment while priming cardiac stem cells to readily adopt a cardiac identity. As compared to injection with unmodified cardiac stem cells, transplant of stromal-cell derived factor 1α enhanced cells into immunodeficient mice improved myocardial function and angiogenesis while reducing scarring. Increases in myocardial stromal-cell derived factor 1α content paralleled reductions in myocyte apoptosis but did not influence long-term engraftment or the fate of transplanted cells. Transplantation of stromal-cell derived factor 1α transduced cardiac stem cells increased the generation of new myocytes, recruitment of bone marrow cells, new myocyte/vessel formation and the salvage of reversibly damaged myocardium to enhance cardiac repair after experimental infarction. Stem Cells 2016;34:1826-1835. © 2016 AlphaMed Press.

Should we use Commitment Contracts to Regulate Student use of Cognitive Enhancing Drugs?

PubMed

Danaher, John

2016-10-01

Are universities justified in trying to regulate student use of cognitive enhancing drugs? In this article I argue that they can be, but that the most appropriate kind of regulatory intervention is likely to be voluntary in nature. To be precise, I argue that universities could justifiably adopt a commitment contract system of regulation wherein students are encouraged to voluntarily commit to not using cognitive enhancing drugs (or to using them in a specific way). If they are found to breach that commitment, they should be penalized by, for example, forfeiting a number of marks on their assessments. To defend this model of regulation, I adopt a recently-proposed evaluative framework for determining the appropriateness of enhancement in specific domains of activity, and I focus on particular existing types of cognitive enhancement drugs, not hypothetical or potential forms. In this way, my argument is tailored to the specific features of university education, and common patterns of usage among students. It is not concerned with the general ethical propriety of using cognitive enhancing drugs. © 2016 John Wiley & Sons Ltd.

Does Documenting the Regulation Process on a Blog Enhance Pre-Service Teachers' Self- and Co-Regulation in a Collaborative Project?

ERIC Educational Resources Information Center

Cho, Moon-Heum; Lim, Seongmi; Lee, Kyeonghwa

2017-01-01

Students often complain about unsatisfactory experiences resulting from disproportionate contributions to collaborative projects. To improve the experience, we applied regulation theory to design a process to document regulation on a blog and examined whether such documentation enhanced students' self-regulation and co-regulation skills while…

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

Mechanism of block by tedisamil of transient outward current in human ventricular subepicardial myocytes

PubMed Central

Wettwer, Erich; Himmel, Herbert M; Amos, Gregory J; Li, Qi; Metzger, Franz; Ravens, Ursula

1998-01-01

Tedisamil is a new antiarrhythmic drug with predominant class III action. The aim of the present study was to investigate the blocking pattern of the compound on the transient outward current (Ito) in human subepicardial myocytes isolated from explanted left ventricles. Using the single electrode whole cell voltage clamp technique, Ito was analysed after appropriate voltage inactivation of sodium current and block of calcium current.Tedisamil reduced the amplitude of peak Ito, but did not affect the amplitude of non-inactivating outward current. The drug accelerated the apparent rate of Ito inactivation. The reduction in time constant of Ito inactivation depended on drug concentration, the apparent IC50 value was 4.4 μM.Tedisamil affected Ito amplitude in a use-dependent manner. After 2 min at −80 mV, maximum block of Ito was reached after 4–5 clamp steps either at the frequency of 0.2 or 2 Hz, indicating that the block was not frequency-dependent in an experimentally relevant range. Recovery from block was very slow and proceeded with a time constant of 12.1±1.8 s. Also in the presence of drug, a fraction of channels recovered from inactivation with a similar time constant as in control myocytes (i.e. 81±40 ms and 51±8 ms, respectively, n.s.).From the onset of fractional block of Ito by tedisamil during the initial 60 ms of a clamp step, we calculated k1=9×106 mol−1 s−1 for the association rate constant, and k2=23 s−1 for the dissociation rate constant. The resulting apparent KD was 2.6 μM and is similar to the IC50 value.The effects of tedisamil on Ito could be simulated by assuming a four state channel model where the drug binds to the channel in an open (activated) conformation. It is concluded that in human subepicardial myocytes tedisamil is an open channel blocker of Ito and that this effect probably contributes to the antiarrhythmic potential of this drug. PMID:9831899

LRP8-Reelin-regulated Neuronal (LRN) Enhancer Signature Underlying Learning and Memory Formation

PubMed Central

Telese, Francesca; Ma, Qi; Perez, Patricia Montilla; Notani, Dimple; Oh, Soohwan; Li, Wenbo; Comoletti, Davide; Ohgi, Kenneth A.; Taylor, Havilah; Rosenfeld, Michael G.

2015-01-01

Summary One of the exceptional properties of the brain is its ability to acquire new knowledge through learning and to store that information through memory. The epigenetic mechanisms linking changes in neuronal transcriptional programs to behavioral plasticity remain largely unknown. Here, we identify the epigenetic signature of the neuronal enhancers required for transcriptional regulation of synaptic plasticity genes during memory formation, linking this to Reelin signaling. The binding of Reelin to its receptor, LRP8, triggers activation of this cohort of LRP8-Reelin-regulated-Neuronal (LRN) enhancers that serve as the ultimate convergence point of a novel synapse-to-nucleus pathway. Reelin simultaneously regulates NMDA-receptor transmission, which reciprocally permits the required, γ-secretase-dependent cleavage of LRP8, revealing an unprecedented role for its intracellular domain in the regulation of synaptically generated signals. These results uncover an in vivo enhancer code serving as a critical molecular component of cognition and relevant to psychiatric disorders linked to defects in Reelin signaling. PMID:25892301

S-Nitrosoglutathione Reductase Deficiency Enhances the Proliferative Expansion of Adult Heart Progenitors and Myocytes Post Myocardial Infarction

PubMed Central

Hatzistergos, Konstantinos E; Paulino, Ellena C; Dulce, Raul A; Takeuchi, Lauro M; Bellio, Michael A; Kulandavelu, Shathiyah; Cao, Yenong; Balkan, Wayne; Kanashiro-Takeuchi, Rosemeire M; Hare, Joshua M

2015-01-01

Background Mammalian heart regenerative activity is lost before adulthood but increases after cardiac injury. Cardiac repair mechanisms, which involve both endogenous cardiac stem cells (CSCs) and cardiomyocyte cell-cycle reentry, are inadequate to achieve full recovery after myocardial infarction (MI). Mice deficient in S-nitrosoglutathione reductase (GSNOR−⁄−), an enzyme regulating S-nitrosothiol turnover, have preserved cardiac function after MI. Here, we tested the hypothesis that GSNOR activity modulates cardiac cell proliferation in the post-MI adult heart. Methods and Results GSNOR−⁄− and C57Bl6/J (wild-type [WT]) mice were subjected to sham operation (n=3 GSNOR−⁄−; n=3 WT) or MI (n=41 GSNOR−⁄−; n=65 WT). Compared with WT,GSNOR−⁄− mice exhibited improved survival, cardiac performance, and architecture after MI, as demonstrated by higher ejection fraction (P<0.05), lower endocardial volumes (P<0.001), and diminished scar size (P<0.05). In addition, cardiomyocytes from post-MI GSNOR−⁄− hearts exhibited faster calcium decay and sarcomeric relaxation times (P<0.001). Immunophenotypic analysis illustrated that post-MI GSNOR−⁄− hearts demonstrated enhanced neovascularization (P<0.001), c-kit+ CSC abundance (P=0.013), and a ≈3-fold increase in proliferation of adult cardiomyocytes and c-kit+/CD45− CSCs (P<0.0001 and P=0.023, respectively) as measured by using 5-bromodeoxyuridine. Conclusions Loss of GSNOR confers enhanced post-MI cardiac regenerative activity, characterized by enhanced turnover of cardiomyocytes and CSCs. Endogenous denitrosylases exert an inhibitory effect over cardiac repair mechanisms and therefore represents a potential novel therapeutic target. PMID:26178404

Cognitive emotion regulation enhances aversive prediction error activity while reducing emotional responses.

PubMed

Mulej Bratec, Satja; Xie, Xiyao; Schmid, Gabriele; Doll, Anselm; Schilbach, Leonhard; Zimmer, Claus; Wohlschläger, Afra; Riedl, Valentin; Sorg, Christian

2015-12-01

Cognitive emotion regulation is a powerful way of modulating emotional responses. However, despite the vital role of emotions in learning, it is unknown whether the effect of cognitive emotion regulation also extends to the modulation of learning. Computational models indicate prediction error activity, typically observed in the striatum and ventral tegmental area, as a critical neural mechanism involved in associative learning. We used model-based fMRI during aversive conditioning with and without cognitive emotion regulation to test the hypothesis that emotion regulation would affect prediction error-related neural activity in the striatum and ventral tegmental area, reflecting an emotion regulation-related modulation of learning. Our results show that cognitive emotion regulation reduced emotion-related brain activity, but increased prediction error-related activity in a network involving ventral tegmental area, hippocampus, insula and ventral striatum. While the reduction of response activity was related to behavioral measures of emotion regulation success, the enhancement of prediction error-related neural activity was related to learning performance. Furthermore, functional connectivity between the ventral tegmental area and ventrolateral prefrontal cortex, an area involved in regulation, was specifically increased during emotion regulation and likewise related to learning performance. Our data, therefore, provide first-time evidence that beyond reducing emotional responses, cognitive emotion regulation affects learning by enhancing prediction error-related activity, potentially via tegmental dopaminergic pathways. Copyright © 2015 Elsevier Inc. All rights reserved.

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

Effects of itopride hydrochloride on the delayed rectifier K+ and L-type CA2+ currents in guinea-pig ventricular myocytes.

PubMed

Morisawa, T; Hasegawa, J; Hama, R; Kitano, M; Kishimoto, Y; Kawasaki, H

1999-01-01

The effects of itopride hydrochloride, a new drug used to regulate motility in the gastrointestinal tract, on the delayed rectifier K+ current (I(K)) and the L-type Ca2+ current (I(Ca)) were evaluated in guinea-pig ventricular myocytes at concentrations of 1, 10 and 100 microM to determine whether the drug has a proarrhythmic effect through blockade of I(K). Itopride did not affect I(K) at concentrations of 100 microM or less, and no significant effects of 1, 10 or 100 microM itopride were observed on the inward rectifier K+ current (I(K1)) responsible for the resting potential and final repolarization phase of the action potential. We next investigated the effects of itopride on L-type Ca2+ current (I(Ca)). Significant inhibition of I(Ca) was observed at itopride concentrations greater than 10 microM. These results suggested that itopride hydrochloride has an inhibitory effect on I(Ca) at concentrations much higher than those in clinical use.

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.

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

PubMed Central

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

2013-01-01

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

Collagen and the myocardium: fibrillar structure, biosynthesis and degradation in relation to hypertrophy and its regression.

PubMed

Eghbali, M; Weber, K T

1990-07-17

The extracellular matrix of the myocardium contains an elaborate structural matrix composed mainly of fibrillar types I and III collagen. This matrix is responsible for the support and alignment of myocytes and capillaries. Because of its alignment, location, configuration and tensile strength, relative to cardiac myocytes, the collagen matrix represents a major determinant of myocardial stiffness. Cardiac fibroblasts, not myocytes, contain the mRNA for these fibrillar collagens. In the hypertrophic remodeling of the myocardium that accompanies arterial hypertension, a progressive structural and biochemical remodeling of the matrix follows enhanced collagen gene expression. The resultant significant accumulation of collagen in the interstitium and around intramyocardial coronary arteries, or interstitial and perivascular fibrosis, represents a pathologic remodeling of the myocardium that compromises this normally efficient pump. This report reviews the structural nature, biosynthesis and degradation of collagen in the normal and hypertrophied myocardium. It suggests that interstitial heart disease, or the disproportionate growth of the extracellular matrix relative to myocyte hypertrophy, is an entity that merits greater understanding, particularly the factors regulating types I and III collagen gene expression and their degradation.

Both endothelin-A and endothelin-B receptors are present on adult rat cardiac ventricular myocytes.

PubMed

Allen, Bruce G; Phuong, Luu Lien; Farhat, Hala; Chevalier, Dominique

2003-02-01

Endothelin-A (ET(A)) and endothelin-B (ET(B)) receptors have been demonstrated in intact heart and cardiac membranes. ET(A) receptors have been demonstrated on adult ventricular myocytes. The aim of the present study was to determine the presence of ET(B) and the relative contribution of this receptor subtype to total endothelin-1 (ET-1) binding on adult ventricular myocytes. Saturation binding experiments indicated that ET-1 bound to a single population of receptors (Kd = 0.52 +/- 0.13 nM, n = 4) with an apparent maximum binding (Bmax) of 2.10 +/- 0.25 sites (x 10(5))/cell (n = 4). Competition experiments using 40 pM [125I]ET-1 and nonradioactive ET-1 revealed a Ki of 660 +/- 71 pM (n = 10) and a Hill coefficient (nH) of 0.99 +/- 0.10 (n = 10). A selective ET(A) antagonist, BQ610, displaced 80% of the bound [125I]ET-1. No displacement was observed by concentrations of an ET(B)-selective antagonist, BQ788, up to 1.0 microM. However, in the presence of 1.0 microM BQ610, BQ788 inhibited the remaining [125I]ET-1 binding. Similarly, in the presence of 1.0 microM BQ788, BQ610 inhibited the remaining specific [125I]ET-1 binding. Binding of an ET(B1)-selective agonist, [125I]IRL-1620, confirmed the presence of ET(B). ET(B) bound to ET-1 irreversibly, whereas binding to ET(A) demonstrated both reversible and irreversible components, and BQ610 and BQ788 bound reversibly. Reducing the incubation temperature to 0 degrees C did not alter the irreversible component of ET-1 binding. Hence, both ET(A) and ET(B) receptors are present on intact adult rat ventricular myocytes, and the ratio of ET(A):ET(B) binding sites is 4:1. Both receptor subtypes bind to ET-1 by a two-step association involving the formation of a tight receptor-ligand complex; however, the kinetics of ET-1 binding to ET(A) versus ET(B) differ.

Modulation of cytosolic and intra-sarcoplasmic reticulum calcium waves by calsequestrin in rat cardiac myocytes

PubMed Central

Kubalova, Zuzana; Györke, Inna; Terentyeva, Radmila; Viatchenko-Karpinski, Serge; Terentyev, Dmitry; Williams, Simon C; Györke, Sandor

2004-01-01

Waves of Ca2+-induced Ca2+ release occur in various cell types and are involved in the pathology of certain forms of cardiac arrhythmia. These arrhythmias include catecholaminergic polymorphic ventricular tachycardia (CPVT), certain cases of which are associated with mutations in the cardiac calsequestrin gene (CASQ2). To explore the mechanisms of Ca2+ wave generation and unravel the underlying causes of CPVT, we investigated the effects of adenoviral-mediated changes in CASQ2 protein levels on the properties of cytosolic and sarcoplasmic reticulum (SR) Ca2+ waves in permeabilized rat ventricular myocytes. The free [Ca2+] inside the sarcoplasmic reticulum ([Ca2+]SR) was monitored by fluo-5N entrapped into the SR, and cytosolic Ca2+ was imaged using fluo-3. Overexpression of CASQ2 resulted in significant increases in the amplitude of Ca2+ waves and interwave intervals, whereas reduced CASQ2 levels caused drastic reductions in the amplitude and period of Ca2+ waves. CASQ2 abundance had no impact on resting diastolic [Ca2+]SR or on the amplitude of the [Ca2+]SR depletion signal during the Ca2+ wave. However, the recovery dynamics of [Ca2+]SR following Ca2+ release were dramatically altered as the rate of [Ca2+]SR recovery increased ∼3-fold in CASQ2-overexpressing myocytes and decreased to 30% of control in CASQ2-underexpressing myocytes. There was a direct linear relationship between Ca2+ wave period and the half-time of basal [Ca2+]SR recovery following Ca2+ release. Loading the SR with the low affinity exogenous Ca2+ buffer citrate exerted effects quantitatively similar to those observed on overexpressing CASQ2. We conclude that free intra-SR [Ca2+] is a critical determinant of cardiac Ca2+ wave generation. Our data indicate that reduced intra-SR Ca2+ binding activity promotes the generation of Ca2+ waves by accelerating the dynamics of attaining a threshold free [Ca2+]SR required for Ca2+ wave initiation, potentially accounting for arrythmogenesis in CPVT linked

Substance P and neurokinin A metabolism by cultured human skeletal muscle myocytes and fibroblasts.

PubMed

Russell, J S; Chi, H; Lantry, L E; Stephens, R E; Ward, P E

1996-01-01

A recent study determined that cultured human skeletal muscle adult myoblasts, myotubes, and fibroblasts degraded angiotensins and kinins via neutral endopeptidase-24.11 (NEP-24.11: EC 3.4.24.11) and aminopeptidase N (APN: EC 3.4.11.2). Due to the possible importance of other peptides to skeletal muscle blood flow and function, the present study looked specifically at the metabolism of the neurokinins substance P (SP) and neurokinin A (NKA) by skeletal muscle peptidases. The results show that SP is degraded not only by NEP-24.11, but also sequentially by dipeptidyl(amino)peptidase IV (DAP IV: EC 3.4.14.5)/APN. NKA is unaffected by DAP IV but is metabolized by NEP-24.11 and APN. NEP-24.11 was inhibited by phosphoramidon (IC50 = 80 nM), thiorphan and ZINCOV, DAP IV by diprotin A (IC50 = 8 microM), and APN by amastatin (IC50 = 50 nM) and bestatin (IC50 = 100 microM). Skeletal muscle myocyte and fibroblast metabolism of SP and NKA may regulate local skeletal muscle vascular and extravascular functions including SP- and NKA-mediated nerve-induced vasodilation. Inhibition of both NEP-24.11 and DAP IV/APN may increase skeletal muscle blood flow and decrease peripheral vascular resistance via potentiation of local neurokinin levels.

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.

Use of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes (hiPSC-CMs) to Monitor Compound Effects on Cardiac Myocyte Signaling Pathways.

PubMed

Guo, Liang; Eldridge, Sandy; Furniss, Mike; Mussio, Jodie; Davis, Myrtle

2015-09-01

There is a need to develop mechanism-based assays to better inform risk of cardiotoxicity. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are rapidly gaining acceptance as a biologically relevant in vitro model for use in drug discovery and cardiotoxicity screens. Utilization of hiPSC-CMs for mechanistic investigations would benefit from confirmation of the expression and activity of cellular pathways that are known to regulate cardiac myocyte viability and function. This unit describes an approach to demonstrate the presence and function of signaling pathways in hiPSC-CMs and the effects of treatments on these pathways. We present a workflow that employs protocols to demonstrate protein expression and functional integrity of signaling pathway(s) of interest and to characterize biological consequences of signaling modulation. These protocols utilize a unique combination of structural, functional, and biochemical endpoints to interrogate compound effects on cardiomyocytes. Copyright © 2015 John Wiley & Sons, Inc.

Alpha 1-acid glycoprotein reverses cocaine-induced sodium channel blockade in cardiac myocytes.

PubMed

Ma, Yu-Ling; Peters, Nicholas S; Henry, John A

2006-03-01

Alpha 1-acid glycoprotein (AAG) is an acute phase protein capable of binding basic drugs. This action explains its reversal of sodium channel blockade by drugs such as amitriptyline and quinidine. We report here the reversal of cocaine-induced sodium channel blockade by AAG. The sodium channel blocking property of cocaine is a major mechanism behind cocaine-induced sudden cardiac death, since sodium channels play a key role in the initiation and regulation of the heart beat. Voltage-gated sodium current (I(Na)) was recorded using whole-cell patch-clamp techniques. Guinea-pig cardiac ventricular myocytes were isolated and continuously perfused at room temperature with physiological solutions. At concentrations ranging from 5 to 320 microM cocaine showed a dose-dependent and reversible blockade of I(Na) with an IC50 of 45.9 microM. The addition of equimolar amounts of AAG to cocaine produced almost complete reversal of cocaine's effects, suggesting a single binding site for cocaine on the AAG molecule. With changes of peak I(Na) normalized against control as 1, cocaine at 20 and 40 microM reduced I(Na) to 0.62+/-0.042 (n = 6) and 0.57+/-0.052 (n = 9), respectively, and the addition of an equimolar concentration of AAG reversed I(Na) to 0.86+/-0.022 and 0.91+/-0.060, respectively. AAG reverses cocaine-induced sodium channel blockade in a dose-dependent manner, indicating a therapeutic potential to reverse acute cocaine cardiac toxicity.

Executioner Caspase-3 and 7 Deficiency Reduces Myocyte Number in the Developing Mouse Heart

PubMed Central

Cardona, Maria; López, Juan Antonio; Serafín, Anna; Rongvaux, Anthony; Inserte, Javier; García-Dorado, David; Flavell, Richard; Llovera, Marta; Cañas, Xavier; Vázquez, Jesús; Sanchis, Daniel

2015-01-01

Executioner caspase-3 and -7 are proteases promoting cell death but non-apoptotic roles are being discovered. The heart expresses caspases only during development, suggesting they contribute to the organ maturation process. Therefore, we aimed at identifying novel functions of caspases in heart development. We induced simultaneous deletion of executioner caspase-3 and -7 in the mouse myocardium and studied its effects. Caspase knockout hearts are hypoplastic at birth, reaching normal weight progressively through myocyte hypertrophy. To identify the molecular pathways involved in these effects, we used microarray-based transcriptomics and multiplexed quantitative proteomics to compare wild type and executioner caspase-deficient myocardium at different developmental stages. Transcriptomics showed reduced expression of genes promoting DNA replication and cell cycle progression in the neonatal caspase-deficient heart suggesting reduced myocyte proliferation, and expression of non-cardiac isoforms of structural proteins in the adult null myocardium. Proteomics showed reduced abundance of proteins involved in oxidative phosphorylation accompanied by increased abundance of glycolytic enzymes underscoring retarded metabolic maturation of the caspase-null myocardium. Correlation between mRNA expression and protein abundance of relevant genes was confirmed, but transcriptomics and proteomics indentified complementary molecular pathways influenced by caspases in the developing heart. Forced expression of wild type or proteolytically inactive caspases in cultured cardiomyocytes induced expression of genes promoting cell division. The results reveal that executioner caspases can modulate heart’s cellularity and maturation during development, contributing novel information about caspase biology and heart development. PMID:26121671

Modeling the Effects of β1-Adrenergic Receptor Blockers and Polymorphisms on Cardiac Myocyte Ca2+ Handling

PubMed Central

Amanfu, Robert K.

2014-01-01

β-Adrenergic receptor blockers (β-blockers) are commonly used to treat heart failure, but the biologic mechanisms governing their efficacy are still poorly understood. The complexity of β-adrenergic signaling coupled with the influence of receptor polymorphisms makes it difficult to intuit the effect of β-blockers on cardiac physiology. While some studies indicate that β-blockers are efficacious by inhibiting β-adrenergic signaling, other studies suggest that they work by maintaining β-adrenergic responsiveness. Here, we use a systems pharmacology approach to test the hypothesis that in ventricular myocytes, these two apparently conflicting mechanisms for β-blocker efficacy can occur concurrently. We extended a computational model of the β1-adrenergic pathway and excitation-contraction coupling to include detailed receptor interactions for 19 ligands. Model predictions, validated with Ca2+ and Förster resonance energy transfer imaging of adult rat ventricular myocytes, surprisingly suggest that β-blockers can both inhibit and maintain signaling depending on the magnitude of receptor stimulation. The balance of inhibition and maintenance of β1-adrenergic signaling is predicted to depend on the specific β-blocker (with greater responsiveness for metoprolol than carvedilol) and β1-adrenergic receptor Arg389Gly polymorphisms. PMID:24867460

Regulation of Urea Transporters by Tonicity-responsive Enhancer Binding Protein

PubMed Central

Kwon, H. Moo; Kim, Jim

2007-01-01

Urea accumulation in the renal inner medulla plays a key role in the maintenance of maximal urinary concentrating ability. Urea transport in the kidney is mediated by transporter proteins that include renal urea transporter (UT-A) and erythrocyte urea transporter (UT-B). UT-A1 and UT-A2 are produced from the same gene. There is an active tonicity-responsive enhancer (TonE) in the promoter of UT-A1, and the UT-A1 promoter is stimulated by hypertonicity via tonicity-responsive enhancer binding protein (TonEBP). The downregulation of UT-A2 raises the possibility that TonEBP also regulates its promoter. There is some evidence that TonEBP regulates expression of UT-A in vivo; (1) during the renal development of the urinary concentrating ability, expression of TonEBP precedes that of UT-A1; (2) in transgenic mice expressing a dominant negative form of TonEBP, expression of UT-A1 and UT-A2 is severely impaired; (3) in treatment with cyclosporine A, TonEBP was significantly downregulated after 28 days. This downregulation involves mRNA levels of UT-A2; (4) in hypokalemic animals, downregulation of TonEBP contributed to the down regulation of UT-A in the inner medulla. These data support that TonEBP directly contributes to the urinary concentration and renal urea recycling by the regulation of urea transporters. PMID:24459497

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

Simulation of action potentials from metabolically impaired cardiac myocytes. Role of ATP-sensitive K+ current.

PubMed

Ferrero, J M; Sáiz, J; Ferrero, J M; Thakor, N V

1996-08-01

The role of the ATP-sensitive K+ current (IK-ATP) and its contribution to electrophysiological changes that occur during metabolic impairment in cardiac ventricular myocytes is still being discussed. The aim of this work was to quantitatively study this issue by using computer modeling. A model of IK-ATP is formulated and incorporated into the Luo-Rudy ionic model of the ventricular action potential. Action potentials under different degrees of activation of IK-ATP are simulated. Our results show that in normal ionic concentrations, only approximately 0.6% of the KATP channels, when open, should account for a 50% reduction in action potential duration. However, increased levels of intracellular Mg2+ counteract this shortening. Under conditions of high [K+]0, such as those found in early ischemia, the activation of only approximately 0.4% of the KATP channels could account for a 50% reduction in action potential duration. Thus, our results suggest that opening of IK-ATP channels should play a significant role in action potential shortening during hypoxic/ischemic episodes, with the fraction of open channels involved being very low ( < 1%). However, the results of the model suggest that activation of IK-ATP alone does not quantitatively account for the observed K+ efflux in metabolically impaired cardiac myocytes. Mechanisms other than KATP channel activation should be responsible for a significant part of the K+ efflux measured in hypoxic/ischemic situations.

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

Medical regulation of cognitive enhancement devices: some concerns

PubMed Central

King, Mike; Gavaghan, Colin; McMillan, John

2014-01-01

The authors present a cogent and detailed case for altering the Medical Devices Directive to allow regulation of cognitive enhancement devices (CEDs). Protection against significant risk of harm, especially for the vulnerable, and promotion of benefit through informed use of CEDs are all good features of the proposal. However, the pre-market approval process has limitations, which we explore. We raise the possibility of ‘risk compensation’ in response to the introduction of safety measures, which could alter its effectiveness. The proposal alludes to use of ‘formally trained practitioners,’ which provide a further tier of regulation for CEDs within the proposal. We consider some positive and negative implications of this aspect of the proposal that might warrant further consideration. PMID:27774173

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

Federal regulation of vision enhancement devices for normal and abnormal vision

NASA Astrophysics Data System (ADS)

Drum, Bruce

2006-09-01

The Food and Drug Administration (FDA) evaluates the safety and effectiveness of medical devices and biological products as well as food and drugs. The FDA defines a device as a product that is intended, by physical means, to diagnose, treat, or prevent disease, or to affect the structure or function of the body. All vision enhancement devices fulfill this definition because they are intended to affect a function (vision) of the body. In practice, however, FDA historically has drawn a distinction between devices that are intended to enhance low vision as opposed to normal vision. Most low vision aids are therapeutic devices intended to compensate for visual impairment, and are actively regulated according to their level of risk to the patient. The risk level is usually low (e.g. Class I, exempt from 510(k) submission requirements for magnifiers that do not touch the eye), but can be as high as Class III (requiring a clinical trial and Premarket Approval (PMA) application) for certain implanted and prosthetic devices (e.g. intraocular telescopes and prosthetic retinal implants). In contrast, the FDA usually does not actively enforce its regulations for devices that are intended to enhance normal vision, are low risk, and do not have a medical intended use. However, if an implanted or prosthetic device were developed for enhancing normal vision, the FDA would likely decide to regulate it actively, because its intended use would entail a substantial medical risk to the user. Companies developing such devices should contact the FDA at an early stage to clarify their regulatory status.

Enhancer Sharing Promotes Neighborhoods of Transcriptional Regulation Across Eukaryotes

PubMed Central

Quintero-Cadena, Porfirio; Sternberg, Paul W.

2016-01-01

Enhancers physically interact with transcriptional promoters, looping over distances that can span multiple regulatory elements. Given that enhancer–promoter (EP) interactions generally occur via common protein complexes, it is unclear whether EP pairing is predominantly deterministic or proximity guided. Here, we present cross-organismic evidence suggesting that most EP pairs are compatible, largely determined by physical proximity rather than specific interactions. By reanalyzing transcriptome datasets, we find that the transcription of gene neighbors is correlated over distances that scale with genome size. We experimentally show that nonspecific EP interactions can explain such correlation, and that EP distance acts as a scaling factor for the transcriptional influence of an enhancer. We propose that enhancer sharing is commonplace among eukaryotes, and that EP distance is an important layer of information in gene regulation. PMID:27799341

Comprehensive Analyses of Ventricular Myocyte Models Identify Targets Exhibiting Favorable Rate Dependence

PubMed Central

Bugana, Marco; Severi, Stefano; Sobie, Eric A.

2014-01-01

Reverse rate dependence is a problematic property of antiarrhythmic drugs that prolong the cardiac action potential (AP). The prolongation caused by reverse rate dependent agents is greater at slow heart rates, resulting in both reduced arrhythmia suppression at fast rates and increased arrhythmia risk at slow rates. The opposite property, forward rate dependence, would theoretically overcome these parallel problems, yet forward rate dependent (FRD) antiarrhythmics remain elusive. Moreover, there is evidence that reverse rate dependence is an intrinsic property of perturbations to the AP. We have addressed the possibility of forward rate dependence by performing a comprehensive analysis of 13 ventricular myocyte models. By simulating populations of myocytes with varying properties and analyzing population results statistically, we simultaneously predicted the rate-dependent effects of changes in multiple model parameters. An average of 40 parameters were tested in each model, and effects on AP duration were assessed at slow (0.2 Hz) and fast (2 Hz) rates. The analysis identified a variety of FRD ionic current perturbations and generated specific predictions regarding their mechanisms. For instance, an increase in L-type calcium current is FRD when this is accompanied by indirect, rate-dependent changes in slow delayed rectifier potassium current. A comparison of predictions across models identified inward rectifier potassium current and the sodium-potassium pump as the two targets most likely to produce FRD AP prolongation. Finally, a statistical analysis of results from the 13 models demonstrated that models displaying minimal rate-dependent changes in AP shape have little capacity for FRD perturbations, whereas models with large shape changes have considerable FRD potential. This can explain differences between species and between ventricular cell types. Overall, this study provides new insights, both specific and general, into the determinants of AP duration

Comprehensive analyses of ventricular myocyte models identify targets exhibiting favorable rate dependence.

PubMed

Cummins, Megan A; Dalal, Pavan J; Bugana, Marco; Severi, Stefano; Sobie, Eric A

2014-03-01

Reverse rate dependence is a problematic property of antiarrhythmic drugs that prolong the cardiac action potential (AP). The prolongation caused by reverse rate dependent agents is greater at slow heart rates, resulting in both reduced arrhythmia suppression at fast rates and increased arrhythmia risk at slow rates. The opposite property, forward rate dependence, would theoretically overcome these parallel problems, yet forward rate dependent (FRD) antiarrhythmics remain elusive. Moreover, there is evidence that reverse rate dependence is an intrinsic property of perturbations to the AP. We have addressed the possibility of forward rate dependence by performing a comprehensive analysis of 13 ventricular myocyte models. By simulating populations of myocytes with varying properties and analyzing population results statistically, we simultaneously predicted the rate-dependent effects of changes in multiple model parameters. An average of 40 parameters were tested in each model, and effects on AP duration were assessed at slow (0.2 Hz) and fast (2 Hz) rates. The analysis identified a variety of FRD ionic current perturbations and generated specific predictions regarding their mechanisms. For instance, an increase in L-type calcium current is FRD when this is accompanied by indirect, rate-dependent changes in slow delayed rectifier potassium current. A comparison of predictions across models identified inward rectifier potassium current and the sodium-potassium pump as the two targets most likely to produce FRD AP prolongation. Finally, a statistical analysis of results from the 13 models demonstrated that models displaying minimal rate-dependent changes in AP shape have little capacity for FRD perturbations, whereas models with large shape changes have considerable FRD potential. This can explain differences between species and between ventricular cell types. Overall, this study provides new insights, both specific and general, into the determinants of AP duration

PDE4 and mAKAPβ are nodal organizers of β2-ARs nuclear PKA signaling in cardiac myocytes.

PubMed

Bedioune, Ibrahim; Lefebvre, Florence; Lechêne, Patrick; Varin, Audrey; Domergue, Valérie; Kapiloff, Michael S; Fischmeister, Rodolphe; Vandecasteele, Grégoire

2018-05-03

β1- and β2-adrenergic receptors (β-ARs) produce different acute contractile effects on the heart partly because they impact on different cytosolic pools of cAMP-dependent protein kinase (PKA). They also exert different effects on gene expression but the underlying mechanisms remain unknown. The aim of this study was to understand the mechanisms by which β1- and β2-ARs regulate nuclear PKA activity in cardiomyocytes. We used cytoplasmic and nuclear targeted biosensors to examine cAMP signals and PKA activity in adult rat ventricular myocytes upon selective β1- or β2-ARs stimulation. Both β1- and β2-AR stimulation increased cAMP and activated PKA in the cytoplasm. While the two receptors also increased cAMP in the nucleus, only β1-ARs increased nuclear PKA activity and up-regulated the PKA target gene and pro-apoptotic factor, inducible cAMP element repressor (ICER). Inhibition of PDE4, but not Gi, PDE3, GRK2 nor caveolae disruption disclosed nuclear PKA activation and ICER induction by β2-ARs. Both nuclear and cytoplasmic PKI prevented nuclear PKA activation and ICER induction by β1-ARs, indicating that PKA activation outside the nucleus is required for subsequent nuclear PKA activation and ICER mRNA expression. Cytoplasmic PKI also blocked ICER induction by β2-AR stimulation (with concomitant PDE4 inhibition). However, in this case nuclear PKI decreased ICER up-regulation by only 30%, indicating that other mechanisms are involved. Down-regulation of mAKAPβ partially inhibited nuclear PKA activation upon β1-AR stimulation, and drastically decreased nuclear PKA activation upon β2-AR stimulation in the presence of PDE4 inhibition. β1- and β2-ARs differentially regulate nuclear PKA activity and ICER expression in cardiomyocytes. PDE4 insulates a mAKAPβ-targeted PKA pool at the nuclear envelope that prevents nuclear PKA activation upon β2-AR stimulation.

Glucose oxidation positively regulates glucose uptake and improves cardiac function recovery after myocardial reperfusion.

PubMed

Li, Tingting; Xu, Jie; Qin, Xinghua; Hou, Zuoxu; Guo, Yongzheng; Liu, Zhenhua; Wu, Jianjiang; Zheng, Hong; Zhang, Xing; Gao, Feng

2017-11-01

Myocardial reperfusion decreases glucose oxidation and uncouples glucose oxidation from glycolysis. Therapies that increase glucose oxidation lessen myocardial ischemia-reperfusion (I/R) injury. However, the regulation of glucose uptake during reperfusion remains poorly understood. We found that glucose uptake was remarkably diminished in the myocardium following reperfusion in Sprague-Dawley rats as detected by 18 F-labeled and fluorescent-labeled glucose analogs, even though GLUT1 was upregulated by threefold and GLUT4 translocation remained unchanged compared with those of sham-treated rats. The decreased glucose uptake was accompanied by suppressed glucose oxidation. Interestingly, stimulating glucose oxidation by inhibition of pyruvate dehydrogenase kinase 4 (PDK4), a rate-limiting enzyme for glucose oxidation, increased glucose uptake and alleviated I/R injury. In vitro data in neonatal myocytes showed that PDK4 overexpression decreased glucose uptake, whereas its knockdown increased glucose uptake, suggesting that PDK4 has a role in regulating glucose uptake. Moreover, inhibition of PDK4 increased myocardial glucose uptake with concomitant enhancement of cardiac insulin sensitivity following myocardial I/R. These results showed that the suppressed glucose oxidation mediated by PDK4 contributes to the reduced glucose uptake in the myocardium following reperfusion, and enhancement of glucose uptake exerts cardioprotection. The findings suggest that stimulating glucose oxidation via PDK4 could be an efficient approach to improve recovery from myocardial I/R injury. Copyright © 2017 the American Physiological Society.

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.

A Systematic Survey and Characterization of Enhancers that Regulate Sox3 in Neuro-Sensory Development in Comparison with Sox2 Enhancers.

PubMed

Nishimura, Naoko; Kamimura, Yoshifumi; Ishida, Yoshiko; Takemoto, Tatsuya; Kondoh, Hisato; Uchikawa, Masanori

2012-11-22

Development of neural and sensory primordia at the early stages of embryogenesis depends on the activity of two B1 Sox transcription factors, Sox2 and Sox3. The embryonic expression patterns of the Sox2 and Sox3 genes are similar, yet they show gene-unique features. We screened for enhancers of the 231-kb genomic region encompassing Sox3 of chicken, and identified 13 new enhancers that showed activity in different domains of the neuro-sensory primordia. Combined with the three Sox3-proximal enhancers determined previously, at least 16 enhancers were involved in Sox3 regulation. Starting from the NP1 enhancer, more enhancers with different specificities are activated in sequence, resulting in complex overlapping patterns of enhancer activities. NP1 was activated in the caudal lateral epiblast adjacent to the posterior growing end of neural plate, and by the combined action of Wnt and Fgf signaling, similar to the Sox2 N1 enhancer involved in neural/mesodermal dichotomous cell lineage segregation. The Sox3 D5 enhancer and Sox2 N3 enhancer were also activated similarly in the diencephalon, optic vesicle and lens placode, suggesting analogies in their regulation. In general, however, the specificities of the enhancers were not identical between Sox3 and Sox2, including the cases of the NP1 and D5 enhancers.

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

Statistical Metamodeling and Sequential Design of Computer Experiments to Model Glyco-Altered Gating of Sodium Channels in Cardiac Myocytes.

PubMed

Du, Dongping; Yang, Hui; Ednie, Andrew R; Bennett, Eric S

2016-09-01

Glycan structures account for up to 35% of the mass of cardiac sodium ( Nav ) channels. To question whether and how reduced sialylation affects Nav activity and cardiac electrical signaling, we conducted a series of in vitro experiments on ventricular apex myocytes under two different glycosylation conditions, reduced protein sialylation (ST3Gal4(-/-)) and full glycosylation (control). Although aberrant electrical signaling is observed in reduced sialylation, realizing a better understanding of mechanistic details of pathological variations in INa and AP is difficult without performing in silico studies. However, computer model of Nav channels and cardiac myocytes involves greater levels of complexity, e.g., high-dimensional parameter space, nonlinear and nonconvex equations. Traditional linear and nonlinear optimization methods have encountered many difficulties for model calibration. This paper presents a new statistical metamodeling approach for efficient computer experiments and optimization of Nav models. First, we utilize a fractional factorial design to identify control variables from the large set of model parameters, thereby reducing the dimensionality of parametric space. Further, we develop the Gaussian process model as a surrogate of expensive and time-consuming computer models and then identify the next best design point that yields the maximal probability of improvement. This process iterates until convergence, and the performance is evaluated and validated with real-world experimental data. Experimental results show the proposed algorithm achieves superior performance in modeling the kinetics of Nav channels under a variety of glycosylation conditions. As a result, in silico models provide a better understanding of glyco-altered mechanistic details in state transitions and distributions of Nav channels. Notably, ST3Gal4(-/-) myocytes are shown to have higher probabilities accumulated in intermediate inactivation during the repolarization and yield a

78 FR 60169 - Federal Acquisition Regulation; Pilot Program for Enhancement of Contractor Employee...

Federal Register 2010, 2011, 2012, 2013, 2014

2013-09-30

... 9000-AM56 Federal Acquisition Regulation; Pilot Program for Enhancement of Contractor Employee... program enhancing whistleblower protections for contractor employees. DATES: Effective: September 30, 2013... contractor employees at FAR subpart 3.9. The pilot program is mandated by section 828, entitled ``Pilot...

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

Regulation of Six1 expression by evolutionarily conserved enhancers in tetrapods.

PubMed

Sato, Shigeru; Ikeda, Keiko; Shioi, Go; Nakao, Kazuki; Yajima, Hiroshi; Kawakami, Kiyoshi

2012-08-01

The Six1 homeobox gene plays critical roles in vertebrate organogenesis. Mice deficient for Six1 show severe defects in organs such as skeletal muscle, kidney, thymus, sensory organs and ganglia derived from cranial placodes, and mutations in human SIX1 cause branchio-oto-renal syndrome, an autosomal dominant developmental disorder characterized by hearing loss and branchial defects. The present study was designed to identify enhancers responsible for the dynamic expression pattern of Six1 during mouse embryogenesis. The results showed distinct enhancer activities of seven conserved non-coding sequences (CNSs) retained in tetrapod Six1 loci. The activities were detected in all cranial placodes (excluding the lens placode), dorsal root ganglia, somites, nephrogenic cord, notochord and cranial mesoderm. The major Six1-expression domains during development were covered by the sum of activities of these enhancers, together with the previously identified enhancer for the pre-placodal region and foregut endoderm. Thus, the eight CNSs identified in a series of our study represent major evolutionarily conserved enhancers responsible for the expression of Six1 in tetrapods. The results also confirmed that chick electroporation is a robust means to decipher regulatory information stored in vertebrate genomes. Mutational analysis of the most conserved placode-specific enhancer, Six1-21, indicated that the enhancer integrates a variety of inputs from Sox, Pax, Fox, Six, Wnt/Lef1 and basic helix-loop-helix proteins. Positive autoregulation of Six1 is achieved through the regulation of Six protein-binding sites. The identified Six1 enhancers provide valuable tools to understand the mechanism of Six1 regulation and to manipulate gene expression in the developing embryo, particularly in the sensory organs. Copyright © 2012 Elsevier Inc. All rights reserved.

Memory Enhancement by Targeting Cdk5 Regulation of NR2B

PubMed Central

Plattner, Florian; Hernandéz, Adan; Kistler, Tara M.; Pozo, Karine; Zhong, Ping; Yuen, Eunice Y.; Tan, Chunfeng; Hawasli, Ammar H.; Cooke, Sam F.; Nishi, Akinori; Guo, Ailan; Wiederhold, Thorsten; Yan, Zhen; Bibb, James A.

2014-01-01

SUMMARY Many psychiatric and neurological disorders are characterized by learning and memory deficits, for which cognitive enhancement is considered a valid treatment strategy. The N-methyl-D-aspartate receptor (NMDAR) is a prime target for the development of cognitive enhancers due to its fundamental role in learning and memory. In particular, the NMDAR subunit NR2B improves synaptic plasticity and memory when over-expressed in neurons. However, NR2B regulation is not well understood and no therapies potentiating NMDAR function have been developed. Here, we show that serine 1116 of NR2B is phosphorylated by cyclin-dependent kinase 5 (Cdk5). Cdk5-dependent NR2B phosphorylation is regulated by neuronal activity and controls the receptor’s cell surface expression. Disrupting NR2B-Cdk5 interaction using a small interfering peptide (siP) increases NR2B surface levels, facilitates synaptic transmission, and improves memory formation in vivo. Our results reveal a novel regulatory mechanism critical to NR2B function that can be targeted for the development of cognitive enhancers. PMID:24607229

Regulation of hepatitis B virus ENI enhancer activity by hepatocyte-enriched transcription factor HNF3.

PubMed

Chen, M; Hieng, S; Qian, X; Costa, R; Ou, J H

1994-11-15

Hepatitis B virus (HBV) ENI enhancer can activate the expression of HBV and non-HBV genes in a liver-specific manner. By performing the electrophoretic mobility-shift assays, we demonstrated that the three related, liver-enriched, transcription factors, HNF3 alpha, HNF3 beta, and HNF3 gamma could all bind to the 2c site of HBV ENI enhancer. Mutations introduced in the 2c site to abolish the binding by HNF3 reduced the enhancer activity approximately 15-fold. Moreover, expression of HNF3 antisense sequences to suppress the expression of HNF3 in Huh-7 hepatoma cells led to reduction of the ENI enhancer activity. These results indicate that HNF3 positively regulates the ENI enhancer activity and this regulation is most likely mediated through the 2c site. The requirement of HNF3 for the ENI enhancer activity could explain the liver specificity of this enhancer element.

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

Regulation of ATP sensitive potassium channel of isolated guinea pig ventricular myocytes by sarcolemmal monocarboxylate transport.

PubMed

Coetzee, W A

1992-11-01

The aim was to describe the effects of extracellular application of monocarboxylates (pyruvate, lactate, or acetate) on current through KATP channels (iK,ATP) in isolated guinea pig ventricular myocytes. The iK,ATP was elicited during whole cell voltage clamping by application of metabolic poisons, 2,4-dinitrophenol (150 microM) or glucose free cyanide (1 mM) and could be blocked by glibenclamide (3 microM). Extracellular application of monocarboxylates, pyruvate (0.1-10 mM), L-lactate (0.1-10 mM), and acetate (10 mM) led to a rapid inhibition of iK,ATP--an effect which was fully reversible upon washout. Substances without any effect on iK,ATP were (10 mM each) gluconate, citrate, glutamate, creatine, succinate, and glycine. The mechanism underlying the effects of monocarboxylates on iK,ATP was unlikely to be related to an increased ATP production, since D-lactate (10 mM) essentially had the same effect on iK,ATP as the L-isomer of lactate. Furthermore, with intracellular dialysis of alpha-cyano-4-hydroxycinnamate (0.1-0.5 mM), which inhibits pyruvate uptake into mitochondria, extracellular pyruvate exerted the same inhibitory effect on iK,ATP. High concentrations of extracellular alpha-cyano-4-hydroxycinnamate (4 mM), which blocks the sarcolemmal monocarboxylate carrier, prevented the effects on iK,ATP by pyruvate, L-lactate, D-lactate, and acetate. Furthermore, intracellular dialysis with D-lactate (10 mM) led to a more rapid onset of iK,ATP when activated by ATP free dialysis. Activity of isolated KATP channels, measured in isolated membrane patches in the inside out or outside out configuration, typically had a single channel conductance of around 80 pS and was blocked by glibenclamide (3-9 microM). No significant effect of pyruvate was observed in either patch configuration. In cardiac tissue there may be some modulatory role involving monocarboxylate transport on KATP channel activity, the nature of which is unclear at present but which may involve cytosolic

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.

A Technology-Enhanced Intervention for Self-Regulated Learning in Science

ERIC Educational Resources Information Center

Berglas-Shapiro, Tali; Eylon, Bat-Sheva; Scherz, Zahava

2017-01-01

This article describes the development of a technology-enhanced self-regulated learning (Te- SRL) environment designed to foster students' SRL of complex science topics. The environment consists of three components, one of which is a specially designed computerized system that offers students a choice between different types of scaffolding and…

A class of circadian long non-coding RNAs mark enhancers modulating long-range circadian gene regulation

PubMed Central

Fan, Zenghua; Zhao, Meng; Joshi, Parth D.; Li, Ping; Zhang, Yan; Guo, Weimin; Xu, Yichi; Wang, Haifang; Zhao, Zhihu

2017-01-01

Abstract Circadian rhythm exerts its influence on animal physiology and behavior by regulating gene expression at various levels. Here we systematically explored circadian long non-coding RNAs (lncRNAs) in mouse liver and examined their circadian regulation. We found that a significant proportion of circadian lncRNAs are expressed at enhancer regions, mostly bound by two key circadian transcription factors, BMAL1 and REV-ERBα. These circadian lncRNAs showed similar circadian phases with their nearby genes. The extent of their nuclear localization is higher than protein coding genes but less than enhancer RNAs. The association between enhancer and circadian lncRNAs is also observed in tissues other than liver. Comparative analysis between mouse and rat circadian liver transcriptomes showed that circadian transcription at lncRNA loci tends to be conserved despite of low sequence conservation of lncRNAs. One such circadian lncRNA termed lnc-Crot led us to identify a super-enhancer region interacting with a cluster of genes involved in circadian regulation of metabolism through long-range interactions. Further experiments showed that lnc-Crot locus has enhancer function independent of lnc-Crot's transcription. Our results suggest that the enhancer-associated circadian lncRNAs mark the genomic loci modulating long-range circadian gene regulation and shed new lights on the evolutionary origin of lncRNAs. PMID:28335007

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.

Bacterial RNA induces myocyte cellular dysfunction through the activation of PKR

PubMed Central

Bleiblo, Farag; Michael, Paul; Brabant, Danielle; Ramana, Chilakamarti V.; Tai, TC; Saleh, Mazen; Parrillo, Joseph E.; Kumar, Anand

2012-01-01

Severe sepsis and the ensuing septic shock are serious life threatening conditions. These diseases are triggered by the host's over exuberant systemic response to the infecting pathogen. Several surveillance mechanisms have evolved to discriminate self from foreign RNA and accordingly trigger effective cellular responses to target the pathogenic threats. The RNA-dependent protein kinase (PKR) is a key component of the cytoplasmic RNA sensors involved in the recognition of viral double-stranded RNA (dsRNA). Here, we identify bacterial RNA as a distinct pathogenic pattern recognized by PKR. Our results indicate that natural RNA derived from bacteria directly binds to and activates PKR. We further show that bacterial RNA induces human cardiac myocyte apoptosis and identify the requirement for PKR in mediating this response. In addition to bacterial immunity, the results presented here may also have implications in cardiac pathophysiology. PMID:22833816

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

PubMed Central

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

2014-01-01

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

The effect of PKA-mediated phosphorylation of ryanodine receptor on SR Ca2+ leak in ventricular myocytes.

PubMed

Bovo, Elisa; Huke, Sabine; Blatter, Lothar A; Zima, Aleksey V

2017-03-01

Functional impact of cardiac ryanodine receptor (type 2 RyR or RyR2) phosphorylation by protein kinase A (PKA) remains highly controversial. In this study, we characterized a functional link between PKA-mediated RyR2 phosphorylation level and sarcoplasmic reticulum (SR) Ca 2+ release and leak in permeabilized rabbit ventricular myocytes. Changes in cytosolic [Ca 2+ ] and intra-SR [Ca 2+ ] SR were measured with Fluo-4 and Fluo-5N, respectively. Changes in RyR2 phosphorylation at two PKA sites, serine-2031 and -2809, were measured with phospho-specific antibodies. cAMP (10μM) increased Ca 2+ spark frequency approximately two-fold. This effect was associated with an increase in SR Ca 2+ load from 0.84 to 1.24mM. PKA inhibitory peptide (PKI; 10μM) abolished the cAMP-dependent increase of SR Ca 2+ load and spark frequency. When SERCA was completely blocked by thapsigargin, cAMP did not affect RyR2-mediated Ca 2+ leak. The lack of a cAMP effect on RyR2 function can be explained by almost maximal phosphorylation of RyR2 at serine-2809 after sarcolemma permeabilization. This high RyR2 phosphorylation level is likely the consequence of a balance shift between protein kinase and phosphatase activity after permeabilization. When RyR2 phosphorylation at serine-2809 was reduced to its "basal" level (i.e. RyR2 phosphorylation level in intact myocytes) using kinase inhibitor staurosporine, SR Ca 2+ leak was significantly reduced. Surprisingly, further dephosphorylation of RyR2 with protein phosphatase 1 (PP1) markedly increased SR Ca 2+ leak. At the same time, phosphorylation of RyR2 at serine 2031 did not significantly change under identical experimental conditions. These results suggest that RyR2 phosphorylation by PKA has a complex effect on SR Ca 2+ leak in ventricular myocytes. At an intermediate level of RyR2 phosphorylation SR Ca 2+ leak is minimal. However, complete dephosphorylation and maximal phosphorylation of RyR2 increases SR Ca 2+ leak. Copyright © 2017 Elsevier

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.

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.

A transient-enhanced NMOS low dropout voltage regulator with parallel feedback compensation

NASA Astrophysics Data System (ADS)

Han, Wang; Lin, Tan

2016-02-01

This paper presents a transient-enhanced NMOS low-dropout regulator (LDO) for portable applications with parallel feedback compensation. The parallel feedback structure adds a dynamic zero to get an adequate phase margin with a load current variation from 0 to 1 A. A class-AB error amplifier and a fast charging/discharging unit are adopted to enhance the transient performance. The proposed LDO has been implemented in a 0.35 μm BCD process. From experimental results, the regulator can operate with a minimum dropout voltage of 150 mV at a maximum 1 A load and IQ of 165 μA. Under the full range load current step, the voltage undershoot and overshoot of the proposed LDO are reduced to 38 mV and 27 mV respectively.

Pre-Conditioning with CDP-Choline Attenuates Oxidative Stress-Induced Cardiac Myocyte Death in a Hypoxia/Reperfusion Model

PubMed Central

González-Pacheco, Héctor; Méndez-Domínguez, Aurelio; Hernández, Salomón; López-Marure, Rebeca; Vazquez-Mellado, Maria J.; Aguilar, Cecilia; Rocha-Zavaleta, Leticia

2014-01-01

Background. CDP-choline is a key intermediate in the biosynthesis of phosphatidylcholine, which is an essential component of cellular membranes, and a cell signalling mediator. CDP-choline has been used for the treatment of cerebral ischaemia, showing beneficial effects. However, its potential benefit for the treatment of myocardial ischaemia has not been explored yet. Aim. In the present work, we aimed to evaluate the potential use of CDP-choline as a cardioprotector in an in vitro model of ischaemia/reperfusion injury. Methods. Neonatal rat cardiac myocytes were isolated and subjected to hypoxia/reperfusion using the coverslip hypoxia model. To evaluate the effect of CDP-choline on oxidative stress-induced reperfusion injury, the cells were incubated with H2O2 during reperfusion. The effect of CDP-choline pre- and postconditioning was evaluated using the cell viability MTT assay, and the proportion of apoptotic and necrotic cells was analyzed using the Annexin V determination by flow cytometry. Results. Pre- and postconditioning with 50 mg/mL of CDP-choline induced a significant reduction of cells undergoing apoptosis after hypoxia/reperfusion. Preconditioning with CDP-choline attenuated postreperfusion cell death induced by oxidative stress. Conclusion. CDP-choline administration reduces cell apoptosis induced by oxidative stress after hypoxia/reperfusion of cardiac myocytes. Thus, it has a potential as cardioprotector in ischaemia/reperfusion-injured cardiomyocytes. PMID:24578622

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

PubMed Central

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

2013-01-01

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

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

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.

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

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

PubMed

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

2014-11-15

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

Measuring ability to enhance and suppress emotional expression: The Flexible Regulation of Emotional Expression (FREE) Scale.

PubMed

Burton, Charles L; Bonanno, George A

2016-08-01

Flexibility in self-regulatory behaviors has proved to be an important quality for adjusting to stressful life events and requires individuals to have a diverse repertoire of emotion regulation abilities. However, the most commonly used emotion regulation questionnaires assess frequency of behavior rather than ability, with little evidence linking these measures to observable capacity to enact a behavior. The aim of the current investigation was to develop and validate a Flexible Regulation of Emotional Expression (FREE) Scale that measures a person's ability to enhance and suppress displayed emotion across an array of hypothetical contexts. In Studies 1 and 2, a series of confirmatory factor analyses revealed that the FREE Scale consists of 4 first-order factors divided by regulation and emotional valence type that can contribute to 2 higher order factors: expressive enhancement ability and suppression ability. In Study 1, we also compared the FREE Scale to other commonly used emotion regulation measures, which revealed that suppression ability is conceptually distinct from suppression frequency. In Study 3, we compared the FREE Scale with a composite of traditional frequency-based indices of expressive regulation to predict performance in a previously validated emotional modulation paradigm. Participants' enhancement and suppression ability scores on the FREE Scale predicted their corresponding performance on the laboratory task, even when controlling for baseline expressiveness. These studies suggest that the FREE Scale is a valid and flexible measure of expressive regulation ability. (PsycINFO Database Record (c) 2016 APA, all rights reserved).

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.

78 FR 59851 - Defense Federal Acquisition Regulation Supplement: Enhancement of Contractor Employee...

Federal Register 2010, 2011, 2012, 2013, 2014

2013-09-30

...-AH97 Defense Federal Acquisition Regulation Supplement: Enhancement of Contractor Employee... contractor and subcontractor employees. DATES: Effective date: September 30, 2013. In accordance with FAR 1... Whistleblower Protections for Contractor Employees) made extensive changes to 10 U.S.C. 2409, entitled...

AMPKα2 regulates expression of estrogen-related receptor alpha, a metabolic transcription factor related to heart failure development

PubMed Central

Hu, Xinli; Xu, Xin; Lu, Zhongbing; Zhang, Ping; Fassett, John; Zhang, Ying; Xin, Yi; Hall, Jennifer L.; Viollet, Benoit; Bache, Robert J.; Huang, Yimin; Chen, Yingjie

2011-01-01

The normal expression of myocardial mitochondrial enzymes is essential to maintain the cardiac energy reserve and facilitate responses to stress, but the molecular mechanisms to maintain myocardial mitochondrial enzyme expression have been elusive. Here we report that congestive heart failure is associated with a significant decrease of myocardial Estrogen-Related Receptor alpha (ERRα), but not PPAR gamma coactivator-1 alpha (PGC1α), in human heart failure samples. In addition, chronic pressure overload in mice caused a decrease of ERRα expression that was significantly correlated to the degree of LV dysfunction, pulmonary congestion and decreases of a group of myocardial energy metabolism related genes. We found that the metabolic sensor AMP activated protein kinase (AMPK) regulates ERRα expression in vivo and in vitro. AMPKα2 KO decreased myocardial ERRα (both mRNA and protein) and its downstream targets under basal conditions, with no change in myocardial PGC1α expression. Using cultured rat neonatal cardiac myocytes, we found that overexpression of constitutively active AMPKα significantly induced ERRα mRNA, protein and promoter activity. Conversely, selective gene silencing of AMPKα2 repressed ERRα and its target gene levels, indicating that AMPKα2 is involved in the regulation of ERRα expression. In addition, over-expression of ERRα in AMPKα2 KO neonatal cardiac myocytes partially rescued the repressed expression of some energy metabolism related genes. These data support an important role for AMPKα2 in regulating the expression of myocardial ERRα and its downstream mitochondrial enzymes. PMID:21825219

Effects of angiotensin II type 1 receptor antagonist and temperature on prolonged cardioplegic arrest in neonatal rat myocytes.

PubMed

Lucchese, Gianluca; Cambi, Giulia Elisa; De Rita, Fabrizio; Franzoi, Mauro; Faggian, Giuseppe; Mazzucco, Alessandro; Modesti, Pietro Amedeo; Luciani, Giovanni Battista

2013-08-01

Cardioplegic arrest is a model of ischemia/reperfusion injury and results in the death of irreplaceable cardiac myocytes by a programmed cell death or apoptosis. Signal transducers and activators of transcription (STAT) signaling pathways play an important role in the modulation of apoptosis after ischemia and reperfusion. Angiotensin II type 1 (AT1) receptor antagonist added to cardioplegia could represent an additional modality for enhancing myocardial protection during cardioplegic arrest. To test that hypothesis, we studied the effect of AT1 receptor antagonism and cardioplegia temperature perfusion on STATs modulation during cardioplegic arrest in neonatal rat hearts. Isolated, nonworking hearts (n = 4 per group) from neonatal rats were perfused aerobically in the Langendorff mode according to the following scheme: Dulbecco's Modified Eagle's Medium solution (Group 1); cold (4°C) modified St. Thomas' Hospital no. 2 (MSTH2) cardioplegic solution (Group 2); cold (4°C) MSTH2 cardioplegic solution plus AT1 antagonist (Valsartan) (Group 3); and warm (34°C) MSTH2 cardioplegic solution (Group 4). Thus, myocytes were isolated by enzymatic digestion, and STAT1, STAT2, STAT3, and STAT5 were investigated in Western blot studies. Times to arrest after cardioplegia were 6-10 s for all groups with the exception of Group 1 (spontaneous arrest after 12-16 s). Total cardioplegia delivery volume was about 300 mL in 15 min. Perfusion with cold MSTH2 supplemented with AT1 receptor antagonist (Group 3) induced a significant reduction in STAT1, STAT2, and STAT5 tyrosine phosphorylation versus other groups (P < 0.05). The decreased activation of STAT1, STAT2, and STAT5 observed in Group 3 was accompanied by reduction of interleukin-1β (P < 0.05). On the other hand, STAT3 activation was significantly reduced in Groups 1 and 4 (P < 0.05). Only perfusion with AT1 receptor antagonist supplemented with cold MSTH2 significantly decreases the inflammatory

Influence of infrasound exposure on the whole L-type calcium currents in rat ventricular myocytes.

PubMed

Pei, Zhaohui; Zhuang, Zhiqiang; Xiao, Pingxi; Chen, Jingzao; Sang, Hanfei; Ren, Jun; Wu, Zhenbiao; Yan, Guangmei

2009-06-01

This study was designed to examine the effect of infrasound exposure (5 Hz at 130 dB) on whole-cell L-type Ca2+ currents (WLCC) in rat ventricular myocytes and the underlying mechanism(s) involved. Thirty-two adult Sprague-Dawley rats were randomly assigned to infrasound exposure and control groups. [Ca2+](i), WLCC, mRNA expression of the a(1c) subunit of L-type Ca2+ channels (LCC), and SERCA2 protein were examined on day 1, 7, and 14 after initiation of infrasound exposure. Fluo-3/AM fluorescence and the laser scanning confocal microscope techniques were used to measure [Ca2+](i) in freshly isolated ventricular myocytes. The Ca2+ fluorescence intensity (FI), denoting [Ca2+](i) in cardiomyocytes, was significantly elevated in a time-dependent manner in the exposure groups. There was a significant increase in WLCC in the 1-day group and a further significant increase in the 7- and 14-day groups. LCC mRNA expression measured by RT-PCR revealed a significant rise in the 1-day group and a significant additional rise in the 7- and 14-day groups compared with control group. SERCA2 expression was significantly upregulated in the 1-day group followed by an overt decrease in the 7- and 14-day groups. Prolonged exposure of infrasound altered WLCC in rat cardiomyocytes by shifting the steady-state inactivation curves to the right (more depolarized direction) without altering the slope and biophysical properties of I (Ca,L). Taken together, our data suggest that changes in [Ca2+](I) levels as well as expression of LCC and SERCA2 may contribute to the infrasound exposure-elicited cardiac response.

Sex differences in repolarization and slow delayed rectifier potassium current and their regulation by sympathetic stimulation in rabbits.

PubMed

Zhu, Yujie; Ai, Xun; Oster, Robert A; Bers, Donald M; Pogwizd, Steven M

2013-06-01

Slow delayed rectifier potassium current (IKs) is important in action potential (AP) repolarization and repolarization reserve. We tested the hypothesis that there are sex-specific differences in IKs, AP, and their regulation by β-adrenergic receptors (β-AR's) using whole-cell patch-clamp. AP duration (APD90) was significantly longer in control female (F) than in control male (M) myocytes. Isoproterenol (ISO, 500 nM) shortened APD90 comparably in M and F, and was largely reversed by β1-AR blocker CGP 20712A (CGP, 300 nM). Inhibition of IKs with chromanol 293B (10 μM) resulted in less APD prolongation in F at baseline (3.0 vs 8.9 %, p < 0.05 vs M) and even in the presence of ISO (5.4 vs 20.9 %, p < 0.05). This suggests that much of the ISO-induced APD abbreviation in F is independent of IKs. In F, baseline IKs was 42 % less and was more weakly activated by ISO (19 vs 68 % in M, p < 0.01). ISO enhancement of IKs was comparably attenuated by CGP in M and F. After ovariectomy, IKs in F had greater enhancement by ISO (72 %), now comparable to control M. After orchiectomy, IKs in M was only slightly enhanced by ISO (23 %), comparable to control F. Pretreatment with thapsigargin (to block SR Ca release) had bigger impact on ISO-induced APD shortening in F than that in M (p < 0.01). In conclusion, we found that there are sex differences in IKs, AP, and their regulation by β-AR's that are modulated by sex hormones, suggesting the potential for sex-specific antiarrhythmic therapy.

MED12 Regulates HSC-Specific Enhancers Independently of Mediator Kinase Activity to Control Hematopoiesis.

PubMed

Aranda-Orgilles, Beatriz; Saldaña-Meyer, Ricardo; Wang, Eric; Trompouki, Eirini; Fassl, Anne; Lau, Stephanie; Mullenders, Jasper; Rocha, Pedro P; Raviram, Ramya; Guillamot, María; Sánchez-Díaz, María; Wang, Kun; Kayembe, Clarisse; Zhang, Nan; Amoasii, Leonela; Choudhuri, Avik; Skok, Jane A; Schober, Markus; Reinberg, Danny; Sicinski, Piotr; Schrewe, Heinrich; Tsirigos, Aristotelis; Zon, Leonard I; Aifantis, Iannis

2016-12-01

Hematopoietic-specific transcription factors require coactivators to communicate with the general transcription machinery and establish transcriptional programs that maintain hematopoietic stem cell (HSC) self-renewal, promote differentiation, and prevent malignant transformation. Mediator is a large coactivator complex that bridges enhancer-localized transcription factors with promoters, but little is known about Mediator function in adult stem cell self-renewal and differentiation. We show that MED12, a member of the Mediator kinase module, is an essential regulator of HSC homeostasis, as in vivo deletion of Med12 causes rapid bone marrow aplasia leading to acute lethality. Deleting other members of the Mediator kinase module does not affect HSC function, suggesting kinase-independent roles of MED12. MED12 deletion destabilizes P300 binding at lineage-specific enhancers, resulting in H3K27Ac depletion, enhancer de-activation, and consequent loss of HSC stemness signatures. As MED12 mutations have been described recently in blood malignancies, alterations in MED12-dependent enhancer regulation may control both physiological and malignant hematopoiesis. Copyright © 2016 Elsevier Inc. All rights reserved.

A beta1-adrenergic receptor CaM kinase II-dependent pathway mediates cardiac myocyte fetal gene induction.

PubMed

Sucharov, Carmen C; Mariner, Peter D; Nunley, Karin R; Long, Carlin; Leinwand, Leslie; Bristow, Michael R

2006-09-01

Beta-adrenergic signaling plays an important role in the natural history of dilated cardiomyopathies. Chronic activation of beta-adrenergic receptors (beta1-AR and beta2-AR) during periods of cardiac stress ultimately harms the failing heart by mechanisms that include alterations in gene expression. Here, we show that stimulation of beta-ARs with isoproterenol in neonate rat ventricular myocytes causes a "fetal" response in the relative activities of the human cardiac fetal and/or adult gene promoters that includes repression of the human and rat alpha-myosin heavy chain (alpha-MyHC) promoters with simultaneous activation of the human atrial natriuretic peptide (ANP) and rat beta-MyHC promoters. We also show that the promoter changes correlate with changes in endogenous gene expression as measured by mRNA expression. Furthermore, we show that these changes are specifically mediated by the beta1-AR, but not the beta2-AR, and are independent of alpha1-AR stimulation. We also demonstrate that the fetal gene response is independent of cAMP and protein kinase A, whereas inhibition of Ca2+/calmodulin-dependent protein kinase (CaMK) pathway blocks isoproterenol-mediated fetal gene program induction. Finally, we show that induction of the fetal program is dependent on activation of the L-type Ca2+ channel. We conclude that in neonatal rat cardiac myocytes, agonist-occupied beta1-AR mobilizes Ca2+ stores to activate fetal gene induction through cAMP independent pathways that involve CaMK.

The heart as a self-regulating system: integration of homeodynamic mechanisms.

PubMed

Kresh, J Y; Armour, J A

1997-04-01

In the past the study of mechanical and electrical properties of the heart has been disjointed with minimal overlap and unification. The fact remains that these features are tightly coupled and central to the functioning heart. The maintenance of adequate cardiac output relies upon the highly integrated autoregulatory mechanisms and modulation of cardiac myocyte function. Regional ventricular mechanics and energetics are dependent upon muscle fiber stress-strain rate, the passive properties of myocardial collagen matrix, adequate vascular perfusion, transcapillary transport and electrical activation pattern. Intramural hydraulic "loading" is regulated by coronary arterial and venous dynamics. All of these components are under the constant influence of intrinsic cardiac and extracardiac autonomic neurons, as well as circulating hormones. A brief overview of the putative regulation of these various components is presented in this paper.

Enhancing well-being at work: The role of emotion regulation skills as personal resources.

PubMed

Buruck, Gabriele; Dörfel, Denise; Kugler, Joachim; Brom, Sarah Susanne

2016-10-01

Dealing with negative emotions is a crucial work demand, particularly for employees in health care. Job resources (e.g., autonomy, social support, or reward) but also personal resources (such as emotion regulation strategies) might reduce job stress and support well-being. Following this, the present study focused on strengthening emotion regulation as 1 way of dealing with high job demands. The aim of this study was to evaluate the impact of a standardized emotion regulation training (Affect Regulation Training [ART]; Berking, 2010) to improve emotion regulation skills and well-being of employees in elderly health care. Therefore, 96 elderly care workers filled out an established questionnaire of emotion regulation skills as well as a measure of well-being at pretreatment, posttreatment and at 6-month follow-up. The findings show that the ART fosters emotion regulation skills. In particularly, acceptance, tolerance, and modification of negative emotions was enhanced in the training groups in comparison to a control-group. Modification, meaning the ability to actively change emotions, improved even more over the follow-up-period. Simultaneously, well-being of participants increased over all measurement time points in the ART-group compared with the control-group. Additionally, the improvement in emotion regulation skills from pre to posttreatment was related to well-being at follow-up. In summary, our results support the ART as an effective intervention for dealing with negative emotions and to enhance well-being among employees in elderly care. (PsycINFO Database Record (c) 2016 APA, all rights reserved).

Endoplasmic reticulum chaperone GRP78 regulates macrophage function and insulin resistance in diet-induced obesity.

PubMed

Kim, Jong Hun; Lee, Eunjung; Friedline, Randall H; Suk, Sujin; Jung, Dae Young; Dagdeviren, Sezin; Hu, Xiaodi; Inashima, Kunikazu; Noh, Hye Lim; Kwon, Jung Yeon; Nambu, Aya; Huh, Jun R; Han, Myoung Sook; Davis, Roger J; Lee, Amy S; Lee, Ki Won; Kim, Jason K

2018-04-01

Obesity-mediated inflammation is a major cause of insulin resistance, and macrophages play an important role in this process. The 78-kDa glucose-regulated protein (GRP78) is a major endoplasmic reticulum chaperone that modulates unfolded protein response (UPR), and mice with GRP78 heterozygosity were resistant to diet-induced obesity. Here, we show that mice with macrophage-selective ablation of GRP78 (Lyz- GRP78 -/- ) are protected from skeletal muscle insulin resistance without changes in obesity compared with wild-type mice after 9 wk of high-fat diet. GRP78-deficient macrophages demonstrated adapted UPR with up-regulation of activating transcription factor (ATF)-4 and M2-polarization markers. Diet-induced adipose tissue inflammation was reduced, and bone marrow-derived macrophages from Lyz- GRP78 -/- mice demonstrated a selective increase in IL-6 expression. Serum IL-13 levels were elevated by >4-fold in Lyz- GRP78 -/- mice, and IL-6 stimulated the myocyte expression of IL-13 and IL-13 receptor. Lastly, recombinant IL-13 acutely increased glucose metabolism in Lyz- GRP78 -/- mice. Taken together, our data indicate that GRP78 deficiency activates UPR by increasing ATF-4, and promotes M2-polarization of macrophages with a selective increase in IL-6 secretion. Macrophage-derived IL-6 stimulates the myocyte expression of IL-13 and regulates muscle glucose metabolism in a paracrine manner. Thus, our findings identify a novel crosstalk between macrophages and skeletal muscle in the modulation of obesity-mediated insulin resistance.-Kim, J. H., Lee, E., Friedline, R. H., Suk, S., Jung, D. Y., Dagdeviren, S., Hu, X., Inashima, K., Noh, H. L., Kwon, J. Y., Nambu, A., Huh, J. R., Han, M. S., Davis, R. J., Lee, A. S., Lee, K. W., Kim, J. K. Endoplasmic reticulum chaperone GRP78 regulates macrophage function and insulin resistance in diet-induced obesity.

TEAD and YAP regulate the enhancer network of human embryonic pancreatic progenitors.

PubMed

Cebola, Inês; Rodríguez-Seguí, Santiago A; Cho, Candy H-H; Bessa, José; Rovira, Meritxell; Luengo, Mario; Chhatriwala, Mariya; Berry, Andrew; Ponsa-Cobas, Joan; Maestro, Miguel Angel; Jennings, Rachel E; Pasquali, Lorenzo; Morán, Ignasi; Castro, Natalia; Hanley, Neil A; Gomez-Skarmeta, Jose Luis; Vallier, Ludovic; Ferrer, Jorge

2015-05-01

The genomic regulatory programmes that underlie human organogenesis are poorly understood. Pancreas development, in particular, has pivotal implications for pancreatic regeneration, cancer and diabetes. We have now characterized the regulatory landscape of embryonic multipotent progenitor cells that give rise to all pancreatic epithelial lineages. Using human embryonic pancreas and embryonic-stem-cell-derived progenitors we identify stage-specific transcripts and associated enhancers, many of which are co-occupied by transcription factors that are essential for pancreas development. We further show that TEAD1, a Hippo signalling effector, is an integral component of the transcription factor combinatorial code of pancreatic progenitor enhancers. TEAD and its coactivator YAP activate key pancreatic signalling mediators and transcription factors, and regulate the expansion of pancreatic progenitors. This work therefore uncovers a central role for TEAD and YAP as signal-responsive regulators of multipotent pancreatic progenitors, and provides a resource for the study of embryonic development of the human pancreas.

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

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

Overexpression of human Hsp27 inhibits serum-induced proliferation in airway smooth muscle myocytes and confers resistance to hydrogen peroxide cytotoxicity.

PubMed

Salinthone, Sonemany; Ba, Mariam; Hanson, Lisa; Martin, Jody L; Halayko, Andrew J; Gerthoffer, William T

2007-11-01

Airway smooth muscle (ASM) hypertrophy and hyperplasia are characteristics of asthma that lead to thickening of the airway wall and obstruction of airflow. Very little is known about mechanisms underlying ASM remodeling, but in vascular smooth muscle, it is known that progression of atherosclerosis depends on the balance of myocyte proliferation and cell death. Small heat shock protein 27 (Hsp27) is antiapoptotic in nonmuscle cells, but its role in ASM cell survival is unknown. Our hypothesis was that phosphorylation of Hsp27 may regulate airway remodeling by modifying proliferation, cell survival, or both. To test this hypothesis, adenoviral vectors were used to overexpress human Hsp27 in ASM cells. Cells were infected with empty vector (Ad5) or wild-type Hsp27 (AdHsp27 WT), and proliferation and death were assessed. Overexpressing Hsp27 WT caused a 50% reduction in serum-induced proliferation and increased cell survival after exposure to 100 microM hydrogen peroxide (H(2)O(2)) compared with mock-infected controls. Overexpression studies utilizing an S15A, S78A, and S82A non-phosphorylation mutant (AdHsp27 3A) and an S15D, S78D, and S82D pseudo-phosphorylation mutant (AdHsp27 3D) showed phosphorylation of Hsp27 was necessary for regulation of ASM proliferation, but not survival. Hsp27 provided protection against H(2)O(2)-induced cytotoxicity by upregulating cellular glutathione levels and preventing necrotic cell death, but not apoptotic cell death. The results support the notion that ASM cells can be stimulated to undergo proliferation and death and that Hsp27 may regulate these processes, thereby contributing to airway remodeling in asthmatics.

NF-κB (p65) negatively regulates myocardin-induced cardiomyocyte hypertrophy through multiple mechanisms.

PubMed

Liao, Xing-Hua; Wang, Nan; Zhao, Dong-Wei; Zheng, De-Liang; Zheng, Li; Xing, Wen-Jing; Zhou, Hao; Cao, Dong-Sun; Zhang, Tong-Cun

2014-12-01

Myocardin is well known to play a key role in the development of cardiomyocyte hypertrophy. But the exact molecular mechanism regulating myocardin stability and transactivity to affect cardiomyocyte hypertrophy has not been studied clearly. We now report that NF-κB (p65) can inhibit myocardin-induced cardiomyocyte hypertrophy. Then we explore the molecular mechanism of this response. First, we show that p65 can functionally repress myocardin transcriptional activity and also reduce the protein expression of myocardin. Second, the function of myocardin can be regulated by epigenetic modifications. Myocardin sumoylation is known to transactivate cardiac genes, but whether p65 can inhibit SUMO modification of myocardin is still not clear. Our data show that p65 weakens myocardin transcriptional activity through attenuating SUMO modification of myocardin by SUMO1/PIAS1, thereby impairing myocardin-mediated cardiomyocyte hypertrophy. Furthermore, the expression of myocardin can be regulated by several microRNAs, which play important roles in the development and function of the heart and muscle. We next investigated potential role of miR-1 in cardiac hypotrophy. Our results show that p65 can upregulate the level of miR-1 and miR-1 can decrease protein expression of myocardin in cardiac myocytes. Notably, miR-1 expression is also controlled by myocardin, leading to a feedback loop. These data thus provide important and novel insights into the function that p65 inhibits myocardin-mediated cardiomyocyte hypertrophy by downregulating the expression and SUMO modification of myocardin and enhancing the expression of miR-1. Copyright © 2014 Elsevier Inc. All rights reserved.

Differential Sarcomere and Electrophysiological Maturation of Human iPSC-Derived Cardiac Myocytes in Monolayer vs. Aggregation-Based Differentiation Protocols

PubMed Central

Jeziorowska, Dorota; Fontaine, Vincent; Jouve, Charlène; Villard, Eric; Dussaud, Sébastien; Akbar, David; Letang, Valérie; Cervello, Pauline; Itier, Jean-Michiel; Pruniaux, Marie-Pierre; Hulot, Jean-Sébastien

2017-01-01

Human induced pluripotent stem cells (iPSCs) represent a powerful human model to study cardiac disease in vitro, notably channelopathies and sarcomeric cardiomyopathies. Different protocols for cardiac differentiation of iPSCs have been proposed either based on embroid body formation (3D) or, more recently, on monolayer culture (2D). We performed a direct comparison of the characteristics of the derived cardiomyocytes (iPSC-CMs) on day 27 ± 2 of differentiation between 3D and 2D differentiation protocols with two different Wnt-inhibitors were compared: IWR1 (inhibitor of Wnt response) or IWP2 (inhibitor of Wnt production). We firstly found that the level of Troponin T (TNNT2) expression measured by FACS was significantly higher for both 2D protocols as compared to the 3D protocol. In the three methods, iPSC-CM show sarcomeric structures. However, iPSC-CM generated in 2D protocols constantly displayed larger sarcomere lengths as compared to the 3D protocol. In addition, mRNA and protein analyses reveal higher cTNi to ssTNi ratios in the 2D protocol using IWP2 as compared to both other protocols, indicating a higher sarcomeric maturation. Differentiation of cardiac myocytes with 2D monolayer-based protocols and the use of IWP2 allows the production of higher yield of cardiac myocytes that have more suitable characteristics to study sarcomeric cardiomyopathies. PMID:28587156

Arabidopsis thaliana G2-LIKE FLAVONOID REGULATOR and BRASSINOSTEROID ENHANCED EXPRESSION1 are low-temperature regulators of flavonoid accumulation.

PubMed

Petridis, Antonios; Döll, Stefanie; Nichelmann, Lars; Bilger, Wolfgang; Mock, Hans-Peter

2016-08-01

Flavonoid synthesis is predominantly regulated at the transcriptional level through the MYB-basic helix-loop-helix (bHLH)-WD40 (MBW) (MYB: transcription factor of the myeloblastosis protein family, WD40: tanscription factor with a short structural motif of 40 amino acids which terminates in an aspartic acid-tryptophan dipeptide) complex, and responds to both environmental and developmental stimuli. Although the developmental regulation of flavonoid accumulation in Arabidopsis thaliana has been examined in great detail, the response of the flavonoid synthesis pathway to abiotic stress (particularly low temperature) remains unclear. A screen of a Dissociation element (Ds) transposon-induced mutation collection identified two lines which exhibited an altered profile of phenylpropanoid accumulation following exposure to low-temperature stress. One of the mutated genes (BRASSINOSTEROID ENHANCED EXPRESSION1 (BEE1)) encoded a brassinosteroid enhanced expression transcription factor, while the other (G2-LIKE FLAVONOID REGULATOR (GFR)) encoded a G2-like flavonoid regulator. Phenylpropanoid-targeted analysis was performed using high-performance LC-MS, and gene expression analysis using quantitative reverse transcription-PCR. In both mutants, the accumulation of quercetins and scopolin was reduced under low-temperature growing conditions, whereas that of anthocyanin was increased. BEE1 and GFR were both shown to negatively regulate anthocyanin accumulation by inhibiting anthocyanin synthesis genes via the suppression of the bHLH (TRANSPARENT TESTA8 (TT8) and GLABROUS3 (GL3)) and/or the MYB (PRODUCTION OF ANTHOCYANIN PIGMENTS2 (PAP2)) components of the MBW complex. Our results provide new insight into the regulatory control of phenylpropanoid metabolism at low temperatures, and reveal that BEE1 and GFR act as important components of the signal transduction chain. © 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.

Dialogue and Structure: Enabling Learner Self-Regulation in Technology-Enhanced Learning Environments

ERIC Educational Resources Information Center

Andrade, Maureen Snow

2014-01-01

Distance learning that incorporates technology-enhanced learning environments provides a solution to the ever-increasing global demand for higher education. To be successful in these contexts, learners must be self-regulated, or have the ability to control the factors affecting their learning. Based on the theories of transactional distance,…

75 FR 26916 - Federal Acquisition Regulation: FAR Case 2009-004, Enhancing Contract Transparency

Federal Register 2010, 2011, 2012, 2013, 2014

2010-05-13

... Regulation: FAR Case 2009-004, Enhancing Contract Transparency AGENCY: Department of Defense (DoD), General... commercial or financial information. This transparency effort is intended to promote efficiency in Government... the Administration's memorandum entitled Transparency and Open Government (January 21, 2009...

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.

Addiction-Related Gene Regulation: Risks of Exposure to Cognitive Enhancers vs. Other Psychostimulants

PubMed Central

Steiner, Heinz; Van Waes, Vincent

2012-01-01

The psychostimulants methylphenidate (Ritalin, Concerta), amphetamine (Adderall), and modafinil (Provigil) are widely used in the treatment of medical conditions such as attention-deficit hyperactivity disorder and narcolepsy and, increasingly, as “cognitive enhancers” by healthy people. The long-term neuronal effects of these drugs, however, are poorly understood. A substantial amount of research over the past 2 decades has investigated the effects of psychostimulants such as cocaine and amphetamines on gene regulation in the brain because these molecular changes are considered critical for psychostimulant addiction. This work has determined in some detail the neurochemical and cellular mechanisms that mediate psychostimulant-induced gene regulation and has also identified the neuronal systems altered by these drugs. Among the most affected brain systems are corticostriatal circuits, which are part of cortico-basal ganglia-cortical loops that mediate motivated behavior. The neurotransmitters critical for such gene regulation are dopamine in interaction with glutamate, while other neurotransmitters (e.g., serotonin) play modulatory roles. This review presents (1) an overview of the main findings on cocaine- and amphetamine-induced gene regulation in corticostriatal circuits in an effort to provide a cellular framework for (2) an assessment of the molecular changes produced by methylphenidate, medical amphetamine (Adderall), and modafinil. The findings lead to the conclusion that protracted exposure to these cognitive enhancers can induce gene regulation effects in corticostriatal circuits that are qualitatively similar to those of cocaine and other amphetamines. These neuronal changes may contribute to the addiction liability of the psychostimulant cognitive enhancers. PMID:23085425

PC4/Tis7/IFRD1 Stimulates Skeletal Muscle Regeneration and Is Involved in Myoblast Differentiation as a Regulator of MyoD and NF-κB*

PubMed Central

Micheli, Laura; Leonardi, Luca; Conti, Filippo; Maresca, Giovanna; Colazingari, Sandra; Mattei, Elisabetta; Lira, Sergio A.; Farioli-Vecchioli, Stefano; Caruso, Maurizia; Tirone, Felice

2011-01-01

In skeletal muscle cells, the PC4 (Tis7/Ifrd1) protein is known to function as a coactivator of MyoD by promoting the transcriptional activity of myocyte enhancer factor 2C (MEF2C). In this study, we show that up-regulation of PC4 in vivo in adult muscle significantly potentiates injury-induced regeneration by enhancing myogenesis. Conversely, we observe that PC4 silencing in myoblasts causes delayed exit from the cell cycle, accompanied by delayed differentiation, and we show that such an effect is MyoD-dependent. We provide evidence revealing a novel mechanism underlying the promyogenic actions of PC4, by which PC4 functions as a negative regulator of NF-κB, known to inhibit MyoD expression post-transcriptionally. In fact, up-regulation of PC4 in primary myoblasts induces the deacetylation, and hence the inactivation and nuclear export of NF-κB p65, in concomitance with induction of MyoD expression. On the contrary, PC4 silencing in myoblasts induces the acetylation and nuclear import of p65, in parallel with a decrease of MyoD levels. We also observe that PC4 potentiates the inhibition of NF-κB transcriptional activity mediated by histone deacetylases and that PC4 is able to form trimolecular complexes with p65 and HDAC3. This suggests that PC4 stimulates deacetylation of p65 by favoring the recruitment of HDAC3 to p65. As a whole, these results indicate that PC4 plays a role in muscle differentiation by controlling the MyoD pathway through multiple mechanisms, and as such, it positively regulates regenerative myogenesis. PMID:21127072

Insulin Activates RSK (p90 Ribosomal S6 Kinase) to Trigger a New Negative Feedback Loop That Regulates Insulin Signaling for Glucose Metabolism*

PubMed Central

Smadja-Lamère, Nicolas; Shum, Michael; Déléris, Paul; Roux, Philippe P.; Abe, Jun-Ichi; Marette, André

2013-01-01

We previously demonstrated that the mTORC1/S6K1 pathway is activated by insulin and nutrient overload (e.g. amino acids (AA)), which leads to the inhibition of the PI3K/Akt pathway via the inhibitory serine phosphorylation of IRS-1, notably on serine 1101 (Ser-1101). However, even in the absence of AA, insulin can still promote IRS-1 Ser-1101 phosphorylation by other kinases that remain to be fully characterized. Here, we describe a new negative regulator of IRS-1, the p90 ribosomal S6 kinase (RSK). Computational analyses revealed that Ser-1101 within IRS-1 falls into the consensus motif of RSK. Moreover, recombinant RSK phosphorylated IRS-1 C-terminal fragment on Ser-1101, which was prevented by mutations of this site or when a kinase-inactive mutant of RSK was used. Using antibodies directed toward the phosphorylation sites located in the activation segment of RSK (Ser-221 or Ser-380), we found that insulin activates RSK in L6 myocytes in the absence of AA overload. Inhibition of RSK using either the pharmacological inhibitor BI-D1870 or after adenoviral expression of a dominant negative RSK1 mutant (RSK1-DN) showed that RSK selectively phosphorylates IRS-1 on Ser-1101. Accordingly, expression of the RSK1-DN mutant in L6 myocytes and FAO hepatic cells improved insulin action on glucose uptake and glucose production, respectively. Furthermore, RSK1 inhibition prevented insulin resistance in L6 myocytes chronically exposed to high glucose and high insulin. These results show that RSK is a novel regulator of insulin signaling and glucose metabolism and a potential mediator of insulin resistance, notably through the negative phosphorylation of IRS-1 on Ser-1101. PMID:24036112

Stimulus-dependent regulation of nuclear Ca2+ signaling in cardiomyocytes: a role of neuronal calcium sensor-1.

PubMed

Nakao, Shu; Wakabayashi, Shigeo; Nakamura, Tomoe Y

2015-01-01

In cardiomyocytes, intracellular calcium (Ca2+) transients are elicited by electrical and receptor stimulations, leading to muscle contraction and gene expression, respectively. Although such elevations of Ca2+levels ([Ca2+]) also occur in the nucleus, the precise mechanism of nuclear [Ca2+] regulation during different kinds of stimuli, and its relationship with cytoplasmic [Ca2+] regulation are not fully understood. To address these issues, we used a new region-specific fluorescent protein-based Ca2+ indicator, GECO, together with the conventional probe Fluo-4 AM. We confirmed that nuclear Ca2+ transients were elicited by both electrical and receptor stimulations in neonatal mouse ventricular myocytes. Kinetic analysis revealed that electrical stimulation-elicited nuclear Ca2+ transients are slower than cytoplasmic Ca2+ transients, and chelating cytoplasmic Ca2+ abolished nuclear Ca2+ transients, suggesting that nuclear Ca2+ are mainly derived from the cytoplasm during electrical stimulation. On the other hand, receptor stimulation such as with insulin-like growth factor-1 (IGF-1) preferentially increased nuclear [Ca2+] compared to cytoplasmic [Ca2+]. Experiments using inhibitors revealed that electrical and receptor stimulation-elicited Ca2+ transients were mainly mediated by ryanodine receptors and inositol 1,4,5-trisphosphate receptors (IP3Rs), respectively, suggesting different mechanisms for the two signals. Furthermore, IGF-1-elicited nuclear Ca2+ transient amplitude was significantly lower in myocytes lacking neuronal Ca2+ sensor-1 (NCS-1), a Ca2+ binding protein implicated in IP3R-mediated pathway in the heart. Moreover, IGF-1 strengthened the interaction between NCS-1 and IP3R. These results suggest a novel mechanism for receptor stimulation-induced nuclear [Ca2+] regulation mediated by IP3R and NCS-1 that may further fine-tune cardiac Ca2+ signal regulation.

Endoplasmic Reticulum-Plasma Membrane Contacts Regulate Cellular Excitability.

PubMed

Dickson, Eamonn J

2017-01-01

Cells that have intrinsic electrical excitability utilize changes in membrane potential to communicate with neighboring cells and initiate cellular cascades. Excitable cells like neurons and myocytes have evolved highly specialized subcellular architectures to translate these electrical signals into cellular events. One such structural specialization is sarco-/endoplasmic reticulum-plasma membrane contact sites. These membrane contact sites are positioned by specific membrane-membrane tethering proteins and contain an ever-expanding list of additional proteins that organize information transfer across the junctional space (~ 15-25 nm distance) to shape membrane identity and control cellular excitability. In this chapter we discuss how contacts between the sarco-/endoplasmic reticulum and plasma membrane are essential for regulated excitation-contraction coupling in striated muscle and control of lipid-dependent ion channels.

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.

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

PubMed

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

2017-11-01

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

Phosphoinositide 3-kinase (PI3K(p110alpha)) directly regulates key components of the Z-disc and cardiac structure.

PubMed

Waardenberg, Ashley J; Bernardo, Bianca C; Ng, Dominic C H; Shepherd, Peter R; Cemerlang, Nelly; Sbroggiò, Mauro; Wells, Christine A; Dalrymple, Brian P; Brancaccio, Mara; Lin, Ruby C Y; McMullen, Julie R

2011-09-02

Maintenance of cardiac structure and Z-disc signaling are key factors responsible for protecting the heart in a setting of stress, but how these processes are regulated is not well defined. We recently demonstrated that PI3K(p110α) protects the heart against myocardial infarction. The aim of this study was to determine whether PI3K(p110α) directly regulates components of the Z-disc and cardiac structure. To address this question, a unique three-dimensional virtual muscle model was applied to gene expression data from transgenic mice with increased or decreased PI3K(p110α) activity under basal conditions (sham) and in a setting of myocardial infarction to display the location of structural proteins. Key findings from this analysis were then validated experimentally. The three-dimensional virtual muscle model visually highlighted reciprocally regulated transcripts associated with PI3K activation that encoded key components of the Z-disc and costamere, including melusin. Studies were performed to assess whether PI3K and melusin interact in the heart. Here, we identify a novel melusin-PI3K interaction that generates lipid kinase activity. The direct impact of PI3K(p110α) on myocyte structure was assessed by treating neonatal rat ventricular myocytes with PI3K(p110α) inhibitors and examining the myofiber morphology of hearts from PI3K transgenic mice. Results demonstrate that PI3K is critical for myofiber maturation and Z-disc alignment. In summary, PI3K regulates the expression of genes essential for cardiac structure and Z-disc signaling, interacts with melusin, and is critical for Z-disc alignment.

Phosphoinositide 3-Kinase (PI3K(p110α)) Directly Regulates Key Components of the Z-disc and Cardiac Structure*

PubMed Central

Waardenberg, Ashley J.; Bernardo, Bianca C.; Ng, Dominic C. H.; Shepherd, Peter R.; Cemerlang, Nelly; Sbroggiò, Mauro; Wells, Christine A.; Dalrymple, Brian P.; Brancaccio, Mara; Lin, Ruby C. Y.; McMullen, Julie R.

2011-01-01

Maintenance of cardiac structure and Z-disc signaling are key factors responsible for protecting the heart in a setting of stress, but how these processes are regulated is not well defined. We recently demonstrated that PI3K(p110α) protects the heart against myocardial infarction. The aim of this study was to determine whether PI3K(p110α) directly regulates components of the Z-disc and cardiac structure. To address this question, a unique three-dimensional virtual muscle model was applied to gene expression data from transgenic mice with increased or decreased PI3K(p110α) activity under basal conditions (sham) and in a setting of myocardial infarction to display the location of structural proteins. Key findings from this analysis were then validated experimentally. The three-dimensional virtual muscle model visually highlighted reciprocally regulated transcripts associated with PI3K activation that encoded key components of the Z-disc and costamere, including melusin. Studies were performed to assess whether PI3K and melusin interact in the heart. Here, we identify a novel melusin-PI3K interaction that generates lipid kinase activity. The direct impact of PI3K(p110α) on myocyte structure was assessed by treating neonatal rat ventricular myocytes with PI3K(p110α) inhibitors and examining the myofiber morphology of hearts from PI3K transgenic mice. Results demonstrate that PI3K is critical for myofiber maturation and Z-disc alignment. In summary, PI3K regulates the expression of genes essential for cardiac structure and Z-disc signaling, interacts with melusin, and is critical for Z-disc alignment. PMID:21757757

Networking Senescence-Regulating Pathways by Using Arabidopsis Enhancer Trap Lines1

PubMed Central

He, Yuehui; Tang, Weining; Swain, Johnnie D.; Green, Anthony L.; Jack, Thomas P.; Gan, Susheng

2001-01-01

The last phase of leaf development, generally referred to as leaf senescence, is an integral part of plant development that involves massive programmed cell death. Due to a sharp decline of photosynthetic capacity in a leaf, senescence limits crop yield and forest plant biomass production. However, the biochemical components and regulatory mechanisms underlying leaf senescence are poorly characterized. Although several approaches such as differential cDNA screening, differential display, and cDNA subtraction have been employed to isolate senescence-associated genes (SAGs), only a limited number of SAGs have been identified, and information regarding the regulation of these genes is fragmentary. Here we report on the utilization of enhancer trap approach toward the identification and analysis of SAGs. We have developed a sensitive large-scale screening method and have screened 1,300 Arabidopsis enhancer trap lines and have identified 147 lines in which the reporter gene GUS (β-glucuronidase) is expressed in senescing leaves but not in non-senescing ones. We have systematically analyzed the regulation of β-glucuronidase expression in 125 lines (genetically, each contains single T-DNA insertion) by six senescence-promoting factors, namely abscisic acid, ethylene, jasmonic acid, brassinosteroid, darkness, and dehydration. This analysis not only reveals the complexity of the regulatory circuitry but also allows us to postulate the existence of a network of senescence-promoting pathways. We have also cloned three SAGs from randomly selected enhancer trap lines, demonstrating that reporter expression pattern reflects the expression pattern of the endogenous gene. PMID:11402199

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.

Mechanisms whereby insulin increases diacylglycerol in BC3H-1 myocytes.

PubMed Central

Farese, R V; Cooper, D R; Konda, T S; Nair, G; Standaert, M L; Davis, J S; Pollet, R J

1988-01-01

We previously suggested that insulin increases diacylglycerol (DAG) in BC3H-1 myocytes, both by increases in synthesis de novo of phosphatidic acid (PA) and by hydrolysis of non-inositol-containing phospholipids, such as phosphatidylcholine (PC) and phosphatidylethanolamine (PE). We have now evaluated these insulin effects more thoroughly, and several potential mechanisms for their induction. In studies of the effect on PA synthesis de novo, insulin stimulated [2-3H]glycerol incorporation into PA, DAG, PC/PE and total glycerolipids of BC3H-1 myocytes, regardless of whether insulin was added simultaneously with, or after 2 h or 3 or 10 days of prelabelling with, [2-3H]glycerol. In prelabelled cells, time-related changes in [2-3H]glycerol labelling of DAG correlated well with increases in DAG content: both were maximal in 30-60 s and persisted for 20-30 min. [2-3H]Glycerol labelling of glycerol 3-phosphate, on the other hand, was decreased by insulin, presumably reflecting increased utilization for PA synthesis. Glycerol 3-phosphate concentrations were 0.36 and 0.38 mM before and 1 min after insulin treatment, and insulin effects could not be explained by increases in glycerol 3-phosphate specific radioactivity. In addition to that of [2-3H]glycerol, insulin increased [U-14C]glucose and [1,2,3-3H]glycerol incorporation into DAG and other glycerolipids. Effects of insulin on [2-3H]glycerol incorporation into DAG and other glycerolipids were half-maximal and maximal at 2 nM- and 20 nM-insulin respectively, and were not dependent on glucose concentration in the medium, extracellular Ca2+ or protein synthesis. Despite good correlation between [3H]DAG and DAG content, calculated increases in DAG content from glycerol 3-phosphate specific radioactivity (i.e. via the pathway of PA synthesis de novo) could account for only 15-30% of the observed increases in DAG content. In addition to increases in [3H]glycerol labelling of PC/PE, insulin rapidly (within 30 s) increased PC

Acute Fasting Regulates Retrograde Synaptic Enhancement through a 4E-BP-Dependent Mechanism.

PubMed

Kauwe, Grant; Tsurudome, Kazuya; Penney, Jay; Mori, Megumi; Gray, Lindsay; Calderon, Mario R; Elazouzzi, Fatima; Chicoine, Nicole; Sonenberg, Nahum; Haghighi, A Pejmun

2016-12-21

While beneficial effects of fasting on organismal function and health are well appreciated, we know little about the molecular details of how fasting influences synaptic function and plasticity. Our genetic and electrophysiological experiments demonstrate that acute fasting blocks retrograde synaptic enhancement that is normally triggered as a result of reduction in postsynaptic receptor function at the Drosophila larval neuromuscular junction (NMJ). This negative regulation critically depends on transcriptional enhancement of eukaryotic initiation factor 4E binding protein (4E-BP) under the control of the transcription factor Forkhead box O (Foxo). Furthermore, our findings indicate that postsynaptic 4E-BP exerts a constitutive negative input, which is counteracted by a positive regulatory input from the Target of Rapamycin (TOR). This combinatorial retrograde signaling plays a key role in regulating synaptic strength. Our results provide a mechanistic insight into how cellular stress and nutritional scarcity could acutely influence synaptic homeostasis and functional stability in neural circuits. Copyright © 2016 Elsevier Inc. All rights reserved.

Thyroid Hormone Signaling in Male Mouse Skeletal Muscle Is Largely Independent of D2 in Myocytes

PubMed Central

Werneck-de-Castro, Joao P.; Fonseca, Tatiana L.; Ignacio, Daniele L.; Fernandes, Gustavo W.; Andrade-Feraud, Cristina M.; Lartey, Lattoya J.; Ribeiro, Marcelo B.; Ribeiro, Miriam O.; Gereben, Balazs

2015-01-01

The type 2 deiodinase (D2) activates the prohormone T4 to T3. D2 is expressed in skeletal muscle (SKM), and its global inactivation (GLOB-D2KO mice) reportedly leads to skeletal muscle hypothyroidism and impaired differentiation. Here floxed Dio2 mice were crossed with mice expressing Cre-recombinase under the myosin light chain 1f (cre-MLC) to disrupt D2 expression in the late developmental stages of skeletal myocytes (SKM-D2KO). This led to a loss of approximately 50% in D2 activity in neonatal and adult SKM-D2KO skeletal muscle and about 75% in isolated SKM-D2KO myocytes. To test the impact of Dio2 disruption, we measured soleus T3 content and found it to be normal. We also looked at the expression of T3-responsive genes in skeletal muscle, ie, myosin heavy chain I, α-actin, myosin light chain, tropomyosin, and serca 1 and 2, which was preserved in neonatal SKM-D2KO hindlimb muscles, at a time that coincides with a peak of D2 activity in control animals. In adult soleus the baseline level of D2 activity was about 6-fold lower, and in the SKM-D2KO soleus, the expression of only one of five T3-responsive genes was reduced. Despite this, adult SKM-D2KO animals performed indistinguishably from controls on a treadmill test, running for approximately 16 minutes and reached a speed of about 23 m/min; muscle strength was about 0.3 mN/m·g body weight in SKM-D2KO and control ankle muscles. In conclusion, there are multiple sources of D2 in the mouse SKM, and its role is limited in postnatal skeletal muscle fibers. PMID:26214036

Spatial diversification of agroecosystems to enhance biological control and other regulating services: An agroecological perspective.

PubMed

Hatt, Séverin; Boeraeve, Fanny; Artru, Sidonie; Dufrêne, Marc; Francis, Frédéric

2018-04-15

Spatial diversification of crop and non-crop habitats in farming systems is promising for enhancing natural regulation of insect pests. Nevertheless, results from recent syntheses show variable effects. One explanation is that the abundance and diversity of pests and natural enemies are affected by the composition, design and management of crop and non-crop habitats. Moreover, interactions between both local and landscape elements and practices carried out at different spatial scales may affect the regulation of insect pests. Hence, research is being conducted to understand these interdependencies. However, insects are not the only pests and pests are not the only elements to regulate in agroecosystems. Broadening the scope could allow addressing multiple issues simultaneously, but also solving them together by enhancing synergies. Indeed, spatial diversification of crop and non-crop habitats can allow addressing the issues of weeds and pathogens, along with being beneficial to several other regulating services like pollination, soil conservation and nutrient cycling. Although calls rise to develop multifunctional landscapes that optimize the delivery of multiple ecosystem services, it still represents a scientific challenge today. Enhancing interdisciplinarity in research institutions and building interrelations between scientists and stakeholders may help reach this goal. Despite obstacles, positive results from research based on such innovative approaches are encouraging for engaging science in this path. Hence, the aim of the present paper is to offer an update on these issues by exploring the most recent findings and discussing these results to highlight needs for future research. Copyright © 2017 Elsevier B.V. All rights reserved.

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.

Calmodulin kinase II and protein kinase C mediate the effect of increased intracellular calcium to augment late sodium current in rabbit ventricular myocytes.

PubMed

Ma, Jihua; Luo, Antao; Wu, Lin; Wan, Wei; Zhang, Peihua; Ren, Zhiqiang; Zhang, Shuo; Qian, Chunping; Shryock, John C; Belardinelli, Luiz

2012-04-15

An increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) augments late sodium current (I(Na.L)) in cardiomyocytes. This study tests the hypothesis that both Ca(2+)-calmodulin-dependent protein kinase II (CaMKII) and protein kinase C (PKC) mediate the effect of increased [Ca(2+)](i) to increase I(Na.L). Whole cell and open cell-attached patch clamp techniques were used to record I(Na.L) in rabbit ventricular myocytes dialyzed with solutions containing various concentrations of [Ca(2+)](i). Dialysis of cells with [Ca(2+)](i) from 0.1 to 0.3, 0.6, and 1.0 μM increased I(Na.L) in a concentration-dependent manner from 0.221 ± 0.038 to 0.554 ± 0.045 pA/pF (n = 10, P < 0.01) and was associated with an increase in mean Na(+) channel open probability and prolongation of channel mean open-time (n = 7, P < 0.01). In the presence of 0.6 μM [Ca(2+)](i), KN-93 (10 μM) and bisindolylmaleimide (BIM, 2 μM) decreased I(Na.L) by 45.2 and 54.8%, respectively. The effects of KN-93 and autocamtide-2-related inhibitory peptide II (2 μM) were not different. A combination of KN-93 and BIM completely reversed the increase in I(Na.L) as well as the Ca(2+)-induced changes in Na(+) channel mean open probability and mean open-time induced by 0.6 μM [Ca(2+)](i). Phorbol myristoyl acetate increased I(Na.L) in myocytes dialyzed with 0.1 μM [Ca(2+)](i); the effect was abolished by Gö-6976. In summary, both CaMKII and PKC are involved in [Ca(2+)](i)-mediated augmentation of I(Na.L) in ventricular myocytes. Inhibition of CaMKII and/or PKC pathways may be a therapeutic target to reduce myocardial dysfunction and cardiac arrhythmias caused by calcium overload.

Modulation of K(ATP) currents in rat ventricular myocytes by hypoxia and a redox reaction.

PubMed

Yan, Xi-Sheng; Ma, Ji-Hua; Zhang, Pei-Hua

2009-10-01

The present study investigated the possible regulatory mechanisms of redox agents and hypoxia on the K(ATP) current (I(KATP)) in acutely isolated rat ventricular myocytes. Single-channel and whole-cell patch-clamp techniques were used to record the K(ATP) current (I(KATP)) in acutely isolated rat ventricular myocytes. Oxidized glutathione (GSSG, 1 mmol/L) increased the I(KATP), while reduced glutathione (GSH, 1 mmol/L) could reverse the increased I(KATP) during normoxia. To further corroborate the effect of the redox agent on the K(ATP) channel, we employed the redox couple DTT (1 mmol/L)/H2O2 (0.3, 0.6, and 1 mmol/L) and repeated the previous processes, which produced results similar to the previous redox couple GSH/GSSG during normoxia. H2O2 increased the I(KATP) in a concentration dependent manner, which was reversed by DTT (1 mmol/L). In addition, our results have shown that 15 min of hypoxia increased the I(KATP), while GSH (1 mmol/L) could reverse the increased I(KATP). Furthermore, in order to study the signaling pathways of the I(KATP) augmented by hypoxia and the redox agent, we applied a protein kinase C(PKC) inhibitor bisindolylmaleimide VI (BIM), a protein kinase G(PKG) inhibitor KT5823, a protein kinase A (PKA) inhibitor H-89, and Ca2+/calmodulin-dependent protein kinase II (CaMKII) inhibitors KN-62 and KN-93. The results indicated that BIM, KT5823, KN-62, and KN-93, but not H-89, inhibited the I(KATP) augmented by hypoxia and GSSG; in addition, these results suggest that the effects of both GSSG and hypoxia on K(ATP) channels involve the activation of the PKC, PKG, and CaMK II pathways, but not the PKA pathway. The present study provides electrophysiological evidence that hypoxia and the oxidizing reaction are closely related to the modulation of I(KATP).

Comparison of Voltage Gated K+ Currents in Arterial Myocytes with Heterologously Expressed K v Subunits.

PubMed

Cox, Robert H; Fromme, Samantha

2016-12-01

We have shown that three components contribute to functional voltage gated K + (K v ) currents in rat small mesenteric artery myocytes: (1) Kv1.2 plus Kv1.5 with Kvβ1.2 subunits, (2) Kv2.1 probably associated with Kv9.3 subunits, and (3) Kv7.4 subunits. To confirm and address subunit stoichiometry of the first two, we have compared the biophysical properties of K v currents in small mesenteric artery myocytes with those of K v subunits heterologously expressed in HEK293 cells using whole cell voltage clamp methods. Selective inhibitors of Kv1 (correolide, COR) and Kv2 (stromatoxin, ScTx) channels were used to separate these K v current components. Conductance-voltage and steady state inactivation data along with time constants of activation, inactivation, and deactivation of native K v components were generally well represented by those of Kv1.2-1.5-β1.2 and Kv2.1-9.3 channels. The slope of the steady state inactivation-voltage curve (availability slope) proved to be the most sensitive measure of accessory subunit presence. The availability slope curves exhibited a single peak for both native K v components. Availability slope curves for Kv1.2-1.5-β1.2 and Kv2.1-9.3 channels expressed in human embryonic kidney cells also exhibited a single peak that shifted to more depolarized voltages with increasing accessory to α subunit transfection ratio. Availability slope curves for SxTc-insensitive currents were similar to those of Kv1.2-1.5 expressed with Kvβ1.2 at a 1:5 molar ratio while curves for COR-insensitive currents closely resembled those of Kv2.1 expressed with Kv9.3 at a 1:1 molar ratio. These results support the suggested K v subunit combinations in small mesenteric artery, and further suggest that Kv1 α and Kvβ1.2 but not Kv2.1 and Kv9.3 subunits are present in a saturated (4:4) stoichiometry.

Self-Regulated Learning in Technology Enhanced Learning Environments: An Investigation with University Students

ERIC Educational Resources Information Center

Lenne, Dominique; Abel, Marie-Helene; Trigano, Philippe; Leblanc, Adeline

2008-01-01

In Technology Enhanced Learning Environments, self-regulated learning (SRL) partly relies on the features of the technological tools. The authors present two environments they designed in order to facilitate SRL: the first one (e-Dalgo) is a website dedicated to the learning of algorithms and computer programming. It is structured as a classical…

The Multi-Domain Fibroblast/Myocyte Coupling in the Cardiac Tissue: A Theoretical Study.

PubMed

Greisas, Ariel; Zlochiver, Sharon

2016-09-01

Cardiac fibroblast proliferation and concomitant collagenous matrix accumulation (fibrosis) develop during multiple cardiac pathologies. Recent studies have demonstrated direct electrical coupling between myocytes and fibroblasts in vitro, and assessed the electrophysiological implications of such coupling. However, in the living tissues, such coupling has not been demonstrated, and only indirect coupling via the extracellular space is likely to exist. In this study we employed a multi-domain model to assess the modulation of the cardiac electrophysiological properties by neighboring fibroblasts assuming only indirect coupling. Numerical simulations in 1D and 2D human atrial models showed that extracellular coupling sustains a significant impact on conduction velocity (CV) and a less significant effect on the action potential duration. Both CV and the slope of the CV restitution increased with increasing fibroblast density. This effect was more substantial for lower extracellular conductance. In 2D, spiral waves exhibited reduced frequency with increasing fibroblast density, and the propensity of wavebreaks and complex dynamics at high pacing rates significantly increased.

The CGTCA sequence motif is essential for biological activity of the vasoactive intestinal peptide gene cAMP-regulated enhancer.

PubMed Central

Fink, J S; Verhave, M; Kasper, S; Tsukada, T; Mandel, G; Goodman, R H

1988-01-01

cAMP-regulated transcription of the human vasoactive intestinal peptide gene is dependent upon a 17-base-pair DNA element located 70 base pairs upstream from the transcriptional initiation site. This element is similar to sequences in other genes known to be regulated by cAMP and to sequences in several viral enhancers. We have demonstrated that the vasoactive intestinal peptide regulatory element is an enhancer that depends upon the integrity of two CGTCA sequence motifs for biological activity. Mutations in either of the CGTCA motifs diminish the ability of the element to respond to cAMP. Enhancers containing the CGTCA motif from the somatostatin and adenovirus genes compete for binding of nuclear proteins from C6 glioma and PC12 cells to the vasoactive intestinal peptide enhancer, suggesting that CGTCA-containing enhancers interact with similar transacting factors. Images PMID:2842787

Transcription regulation by distal enhancers

PubMed Central

Stadhouders, Ralph; van den Heuvel, Anita; Kolovos, Petros; Jorna, Ruud; Leslie, Kris; Grosveld, Frank; Soler, Eric

2012-01-01

Genome-wide chromatin profiling efforts have shown that enhancers are often located at large distances from gene promoters within the noncoding genome. Whereas enhancers can stimulate transcription initiation by communicating with promoters via chromatin looping mechanisms, we propose that enhancers may also stimulate transcription elongation by physical interactions with intronic elements. We review here recent findings derived from the study of the hematopoietic system. PMID:22771987

Coordinated regulation of IFITM1, 2 and 3 genes by an IFN-responsive enhancer through long-range chromatin interactions.

PubMed

Li, Ping; Shi, Ming-Lei; Shen, Wen-Long; Zhang, Zhang; Xie, De-Jian; Zhang, Xiang-Yuan; He, Chao; Zhang, Yan; Zhao, Zhi-Hu

2017-08-01

Interferon-induced transmembrane protein (IFITM) 1, 2 and 3 genes encode a family of interferon (IFN)-induced transmembrane proteins that block entry of a broad spectrum of pathogens. However, the transcriptional regulation of these genes, especially whether there exist any enhancers and their roles during the IFN induction process remain elusive. Here, through public data mining, episomal luciferase reporter assay and in vivo CRISPR-Cas9 genome editing, we identified an IFN-responsive enhancer located 35kb upstream of IFITM3 gene promoter upregulating the IFN-induced expression of IFITM1, 2 and 3 genes. Chromatin immunoprecipitation (ChIP), electrophoretic mobility shift assay (EMSA) and luciferase reporter assay demonstrated that signal transducers and activators of transcription (STAT) 1 bound to the enhancer with the treatment of IFN and was indispensable for the enhancer activity. Furthermore, using chromosome conformation capture technique, we revealed that the IFITM1, 2 and 3 genes physically clustered together and constitutively looped to the distal enhancer through long-range interactions in both HEK293 and A549 cells, providing structural basis for coordinated regulation of IFITM1, 2 and 3 by the enhancer. Finally, we showed that in vivo truncation of the enhancer impaired IFN-induced resistance to influenza A virus (IAV) infection. These findings expand our understanding of the mechanisms underlying the transcriptional regulation of IFITM1, 2 and 3 expression and its ability to mediate IFN signaling. Copyright © 2017 Elsevier B.V. All rights reserved.

Endothelial cell SHP-2 negatively regulates neutrophil adhesion and promotes transmigration by enhancing ICAM-1-VE-cadherin interaction.

PubMed

Yan, Meiping; Zhang, Xinhua; Chen, Ao; Gu, Wei; Liu, Jie; Ren, Xiaojiao; Zhang, Jianping; Wu, Xiaoxiong; Place, Aaron T; Minshall, Richard D; Liu, Guoquan

2017-11-01

Intercellular adhesion molecule-1 (ICAM-1) mediates the firm adhesion of leukocytes to endothelial cells and initiates subsequent signaling that promotes their transendothelial migration (TEM). Vascular endothelial (VE)-cadherin plays a critical role in endothelial cell-cell adhesion, thereby controlling endothelial permeability and leukocyte transmigration. This study aimed to determine the molecular signaling events that originate from the ICAM-1-mediated firm adhesion of neutrophils that regulate VE-cadherin's role as a negative regulator of leukocyte transmigration. We observed that ICAM-1 interacts with Src homology domain 2-containing phosphatase-2 (SHP-2), and SHP-2 down-regulation via silencing of small interfering RNA in endothelial cells enhanced neutrophil adhesion to endothelial cells but inhibited neutrophil transmigration. We also found that VE-cadherin associated with the ICAM-1-SHP-2 complex. Moreover, whereas the activation of ICAM-1 leads to VE-cadherin dissociation from ICAM-1 and VE-cadherin association with actin, SHP-2 down-regulation prevented ICAM-1-VE-cadherin association and promoted VE-cadherin-actin association. Furthermore, SHP-2 down-regulation in vivo promoted LPS-induced neutrophil recruitment in mouse lung but delayed neutrophil extravasation. These results suggest that SHP-2- via association with ICAM-1-mediates ICAM-1-induced Src activation and modulates VE-cadherin switching association with ICAM-1 or actin, thereby negatively regulating neutrophil adhesion to endothelial cells and enhancing their TEM.-Yan, M., Zhang, X., Chen, A., Gu, W., Liu, J., Ren, X., Zhang, J., Wu, X., Place, A. T., Minshall, R. D., Liu, G. Endothelial cell SHP-2 negatively regulates neutrophil adhesion and promotes transmigration by enhancing ICAM-1-VE-cadherin interaction. © FASEB.

Fenofibrate inhibits aldosterone-induced apoptosis in adult rat ventricular myocytes via stress-activated kinase-dependent mechanisms

PubMed Central

De Silva, Deepa S.; Wilson, Richard M.; Hutchinson, Christoph; Ip, Peter C.; Garcia, Anthony G.; Lancel, Steve; Ito, Masa; Pimentel, David R.; Sam, Flora

2009-01-01

Aldosterone induces extracellular signal-regulated kinase (ERK)-dependent cardiac remodeling. Fenofibrate improves cardiac remodeling in adult rat ventricular myocytes (ARVM) partly via inhibition of aldosterone-induced ERK1/2 phosphorylation and inhibition of matrix metalloproteinases. We sought to determine whether aldosterone caused apoptosis in cultured ARVM and whether fenofibrate ameliorated the apoptosis. Aldosterone (1 μM) induced apoptosis by increasing terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL)-positive nuclei in ARVM. Spironolactone (100 nM), an aldosterone receptor antagonist, but not RU-486, a glucocorticoid receptor, inhibited aldosterone-mediated apoptosis, indicating that the mineralocorticoid receptor (MR) plays a role. SP-600125 (3 μM)—a selective inhibitor of c-Jun NH2-terminal kinase (JNK)—inhibited aldosterone-induced apoptosis in ARVM. Although aldosterone increased the expression of both stress-activated protein kinases, pretreatment with fenofibrate (10 μM) decreased aldosterone-mediated apoptosis by inhibiting only JNK phosphorylation and the aldosterone-induced increases in Bax, p53, and cleaved caspase-3 and decreases in Bcl-2 protein expression in ARVM. In vivo studies demonstrated that chronic fenofibrate (100 mg·kg body wt−1·day−1) inhibited myocardial Bax and increased Bcl-2 expression in aldosterone-induced cardiac hypertrophy. Similarly, eplerenone, a selective MR inhibitor, used in chronic pressure-overload ascending aortic constriction inhibited myocardial Bax expression but had no effect on Bcl-2 expression. Therefore, involvement of JNK MAPK-dependent mitochondrial death pathway mediates ARVM aldosterone-induced apoptosis and is inhibited by fenofibrate, a peroxisome proliferator-activated receptor (PPAR)α ligand. Fenofibrate mediates beneficial effects in cardiac remodeling by inhibiting programmed cell death and the stress-activated kinases. PMID:19395558

Estradiol up-regulates L-type Ca2+ channels via membrane-bound estrogen receptor/phosphoinositide-3-kinase/Akt/cAMP response element-binding protein signaling pathway.

PubMed

Yang, Xiaoyan; Mao, Xiaofang; Xu, Gao; Xing, Shasha; Chattopadhyay, Ansuman; Jin, Si; Salama, Guy

2018-05-01

In long QT syndrome type 2, women are more prone than men to the lethal arrhythmia torsades de pointes. We previously reported that 17β-estradiol (E2) up-regulates L-type Ca 2+ channels and current (I Ca,L ) (∼30%) in rabbit ventricular myocytes by a classic genomic mechanism mediated by estrogen receptor-α (ERα). In long QT syndrome type 2 (I Kr blockade or bradycardia), the higher Ca 2+ influx via I Ca,L causes Ca 2+ overload, spontaneous sarcoplasmic reticulum Ca 2+ release, and reactivation of I Ca,L that triggers early afterdepolarizations and torsades de pointes. The purpose of this study was to investigate the molecular mechanisms whereby E2 up-regulates I Ca,L , which are poorly understood. H9C2 and rat myocytes were incubated with E2 ± ER antagonist, or inhibitors of downstream transcription factors, for 24 hours, followed by western blots of Cav1.2α1C and voltage-clamp measurements of I Ca,L . Incubation of H9C2 cells with E2 (10-100 nM) increased I Ca,L density and Cav1.2α1C expression, which were suppressed by the ER antagonist ICI182,780 (1 μM). Enhanced I Ca,L and Cav1.2α1C expression by E2 was suppressed by inhibitors of phosphoinositide-3-kinase (Pi3K) (30 μM LY294002; P <.05) and Akt (5 μM MK2206) but not of mitogen-activated protein kinase (5 μM U0126) or protein kinase A (1 μM KT5720). E2 incubation increased p-CREB via the Pi3K/Akt pathway, reached a peak in 20 minutes (3-fold), and leveled off to 1.5-fold 24 hours later. Furthermore, a CREB decoy oligonucleotide inhibited E2-induced Cav1.2α1C expression, whereas membrane-impermeable E2 (E2-bovine serum albumin) was equally effective at Cav1.2α1C up-regulation as E2. Estradiol up-regulates Cav1.2α1C and I Ca,L via plasma membrane ER and by activating Pi3K, Akt, and CREB signaling. The promoter regions of the CACNA1C gene (human-rabbit-rat) contain adjacent/overlapping binding sites for p-CREB and ERα, which suggests a synergistic regulation by these pathways. Copyright © 2018

Characterization of two distinct depolarization-activated K+ currents in isolated adult rat ventricular myocytes

PubMed Central

1991-01-01

Depolarization-activated outward K+ currents in isolated adult rat ventricular myocytes were characterized using the whole-cell variation of the patch-clamp recording technique. During brief depolarizations to potentials positive to -40 mV, Ca(2+)-independent outward K+ currents in these cells rise to a transient peak, followed by a slower decay to an apparent plateau. The analyses completed here reveal that the observed outward current waveforms result from the activation of two kinetically distinct voltage-dependent K+ currents: one that activates and inactivates rapidly, and one that activates and inactivates slowly, on membrane depolarization. These currents are referred to here as Ito (transient outward) and IK (delayed rectifier), respectively, because their properties are similar (although not identical) to these K+ current types in other cells. Although the voltage dependences of Ito and IK activation are similar, Ito activates approximately 10-fold and inactivates approximately 30-fold more rapidly than IK at all test potentials. In the composite current waveforms measured during brief depolarizations, therefore, the peak current predominantly reflects Ito, whereas IK is the primary determinant of the plateau. There are also marked differences in the voltage dependences of steady-state inactivation of these two K+ currents: IK undergoes steady-state inactivation at all potentials positive to -120 mV, and is 50% inactivated at -69 mV; Ito, in contrast, is insensitive to steady-state inactivation at membrane potentials negative to -50 mV. In addition, Ito recovers from steady-state inactivation faster than IK: at -90 mV, for example, approximately 70% recovery from the inactivation produced at -20 mV is observed within 20 ms for Ito; IK recovers approximately 25-fold more slowly. The pharmacological properties of Ito and IK are also distinct: 4-aminopyridine preferentially attenuates Ito, and tetraethylammonium suppresses predominantly IK. The voltage- and

Facilitation of cytosolic calcium wave propagation by local calcium uptake into the sarcoplasmic reticulum in cardiac myocytes.

PubMed

Maxwell, Joshua T; Blatter, Lothar A

2012-12-01

The widely accepted paradigm for cytosolic Ca(2+) wave propagation postulates a 'fire-diffuse-fire' mechanism where local Ca(2+)-induced Ca(2+) release (CICR) from the sarcoplasmic reticulum (SR) via ryanodine receptor (RyR) Ca(2+) release channels diffuses towards and activates neighbouring release sites, resulting in a propagating Ca(2+) wave. A recent challenge to this paradigm proposed the requirement for an intra-SR 'sensitization' Ca(2+) wave that precedes the cytosolic Ca(2+) wave and primes RyRs from the luminal side to CICR. Here, we tested this hypothesis experimentally with direct simultaneous measurements of cytosolic ([Ca(2+)](i); rhod-2) and intra-SR ([Ca(2+)](SR); fluo-5N) calcium signals during wave propagation in rabbit ventricular myocytes, using high resolution fluorescence confocal imaging. The increase in [Ca(2+)](i) at the wave front preceded depletion of the SR at each point along the calcium wave front, while during this latency period a transient increase of [Ca(2+)](SR) was observed. This transient elevation of [Ca(2+)](SR) could be identified at individual release junctions and depended on the activity of the sarco-endoplasmic reticulum Ca(2+)-ATPase (SERCA). Increased SERCA activity (β-adrenergic stimulation with 1 μM isoproterenol (isoprenaline)) decreased the latency period and increased the amplitude of the transient elevation of [Ca(2+)](SR), whereas inhibition of SERCA (3 μM cyclopiazonic acid) had the opposite effect. In conclusion, the data provide experimental evidence that local Ca(2+) uptake by SERCA into the SR facilitates the propagation of cytosolic Ca(2+) waves via luminal sensitization of the RyR, and supports a novel paradigm of a 'fire-diffuse-uptake-fire' mechanism for Ca(2+) wave propagation in cardiac myocytes.

Facilitation of cytosolic calcium wave propagation by local calcium uptake into the sarcoplasmic reticulum in cardiac myocytes

PubMed Central

Maxwell, Joshua T; Blatter, Lothar A

2012-01-01

The widely accepted paradigm for cytosolic Ca2+ wave propagation postulates a ‘fire-diffuse-fire’ mechanism where local Ca2+-induced Ca2+ release (CICR) from the sarcoplasmic reticulum (SR) via ryanodine receptor (RyR) Ca2+ release channels diffuses towards and activates neighbouring release sites, resulting in a propagating Ca2+ wave. A recent challenge to this paradigm proposed the requirement for an intra-SR ‘sensitization’ Ca2+ wave that precedes the cytosolic Ca2+ wave and primes RyRs from the luminal side to CICR. Here, we tested this hypothesis experimentally with direct simultaneous measurements of cytosolic ([Ca2+]i; rhod-2) and intra-SR ([Ca2+]SR; fluo-5N) calcium signals during wave propagation in rabbit ventricular myocytes, using high resolution fluorescence confocal imaging. The increase in [Ca2+]i at the wave front preceded depletion of the SR at each point along the calcium wave front, while during this latency period a transient increase of [Ca2+]SR was observed. This transient elevation of [Ca2+]SR could be identified at individual release junctions and depended on the activity of the sarco-endoplasmic reticulum Ca2+-ATPase (SERCA). Increased SERCA activity (β-adrenergic stimulation with 1 μm isoproterenol (isoprenaline)) decreased the latency period and increased the amplitude of the transient elevation of [Ca2+]SR, whereas inhibition of SERCA (3 μm cyclopiazonic acid) had the opposite effect. In conclusion, the data provide experimental evidence that local Ca2+ uptake by SERCA into the SR facilitates the propagation of cytosolic Ca2+ waves via luminal sensitization of the RyR, and supports a novel paradigm of a ‘fire-diffuse-uptake-fire’ mechanism for Ca2+ wave propagation in cardiac myocytes. PMID:22988145

Active and Inactive Enhancers Cooperate to Exert Localized and Long-Range Control of Gene Regulation.

PubMed

Proudhon, Charlotte; Snetkova, Valentina; Raviram, Ramya; Lobry, Camille; Badri, Sana; Jiang, Tingting; Hao, Bingtao; Trimarchi, Thomas; Kluger, Yuval; Aifantis, Iannis; Bonneau, Richard; Skok, Jane A

2016-06-07

V(D)J recombination relies on the presence of proximal enhancers that activate the antigen receptor (AgR) loci in a lineage- and stage-specific manner. Unexpectedly, we find that both active and inactive AgR enhancers cooperate to disseminate their effects in a localized and long-range manner. Here, we demonstrate the importance of short-range contacts between active enhancers that constitute an Igk super-enhancer in B cells. Deletion of one element reduces the interaction frequency between other enhancers in the hub, which compromises the transcriptional output of each component. Furthermore, we establish that, in T cells, long-range contact and cooperation between the inactive Igk enhancer MiEκ and the active Tcrb enhancer Eβ alters enrichment of CBFβ binding in a manner that impacts Tcrb recombination. These findings underline the complexities of enhancer regulation and point to a role for localized and long-range enhancer-sharing between active and inactive elements in lineage- and stage-specific control. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

PH domain leucine-rich repeat protein phosphatase 2 (PHLPP2) regulates G-protein-coupled receptor kinase 5 (GRK5)-induced cardiac hypertrophy in vitro.

PubMed

Yeh, Szu-Tsen; Zambrano, Cristina M; Koch, Walter J; Purcell, Nicole H

2018-05-25

PH domain leucine-rich repeat protein phosphatase (PHLPP) is a serine/threonine phosphatase that has been shown to regulate cell growth and survival through dephosphorylation of several members of the AGC family of kinases. G-protein-coupled receptor kinase 5 (GRK5) is an AGC kinase that regulates phenylephrine (PE)-induced cardiac hypertrophy through its noncanonical function of directly targeting proteins to the nucleus to regulate transcription. Here we investigated the possibility that the PHLPP2 isoform can regulate GRK5-induced cardiomyocyte hypertrophy in neonatal rat ventricular myocytes (NRVMs). We show that removal of PHLPP2 by siRNA induces hypertrophic growth of NRVMs as measured by cell size changes at baseline, potentiated PE-induced cell size changes, and re-expression of fetal genes atrial natriuretic factor and brain natriuretic peptide. Endogenous GRK5 and PHLPP2 were found to interact in NRVMs, and PE-induced nuclear accumulation of GRK5 was enhanced upon down-regulation of PHLPP2. Conversely, overexpression of PHLPP2 blocked PE-induced hypertrophic growth, re-expression of fetal genes, and nuclear accumulation of GRK5, which depended on its phosphatase activity. Finally, using siRNA against GRK5, we found that GRK5 was necessary for the hypertrophic response induced by PHLPP2 knockdown. Our findings demonstrate for the first time a novel regulation of GRK5 by the phosphatase PHLPP2, which modulates hypertrophic growth. Understanding the signaling pathways affected by PHLPP2 has potential for new therapeutic targets in the treatment of cardiac hypertrophy and failure. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Regulating the dorsal neural tube expression of Ptf1a through a distal 3' enhancer.

PubMed

Mona, Bishakha; Avila, John M; Meredith, David M; Kollipara, Rahul K; Johnson, Jane E

2016-10-01

Generating the correct balance of inhibitory and excitatory neurons in a neural network is essential for normal functioning of a nervous system. The neural network in the dorsal spinal cord functions in somatosensation where it modulates and relays sensory information from the periphery. PTF1A is a key transcriptional regulator present in a specific subset of neural progenitor cells in the dorsal spinal cord, cerebellum and retina that functions to specify an inhibitory neuronal fate while suppressing excitatory neuronal fates. Thus, the regulation of Ptf1a expression is critical for determining mechanisms controlling neuronal diversity in these regions of the nervous system. Here we identify a sequence conserved, tissue-specific enhancer located 10.8kb 3' of the Ptf1a coding region that is sufficient to direct expression to dorsal neural tube progenitors that give rise to neurons in the dorsal spinal cord in chick and mouse. DNA binding motifs for Paired homeodomain (Pd-HD) and zinc finger (ZF) transcription factors are required for enhancer activity. Mutations in these sequences implicate the Pd-HD motif for activator function and the ZF motif for repressor function. Although no repressor transcription factor was identified, both PAX6 and SOX3 can increase enhancer activity in reporter assays. Thus, Ptf1a is regulated by active and repressive inputs integrated through multiple sequence elements within a highly conserved sequence downstream of the Ptf1a gene. Copyright © 2016 Elsevier Inc. All rights reserved.

Palmitoylation regulates intracellular trafficking of β2 adrenergic receptor/arrestin/phosphodiesterase 4D complexes in cardiomyocytes.

PubMed

Liu, Ruijie; Wang, Dayong; Shi, Qian; Fu, Qin; Hizon, Steven; Xiang, Yang K

2012-01-01

β(2) adrenergic receptor (β(2)AR) is a prototypical G-protein coupled receptor that stimulates the classic cAMP-protein kinase A (PKA) signaling pathway. Recent studies indicate that the cAMP-PKA activities are spatiotemporally regulated in part due to dynamic association of β(2)AR with phosphodiesterase 4D (PDE4D), a group of cAMP degradation enzymes. Here, we demonstrate that in cardiomyocytes, palmitoylation of β(2)AR, the covalent acylation of cysteine residue 341, plays a critical role in shaping subcellular cAMP-PKA activities in cardiomyocytes via regulating β(2)AR association with arrestin/PDE4D. Replacing cysteine 341 on β(2)AR with alanine (C341A) leads to an impaired binding to β arrestin 2. Surprisingly, the C341A mutant is able to internalize via an arrestin-independent pathway at saturated concentration of agonist stimulation; the internalization becomes caveolae-dependent and requires dynamin GTPase. However, the impaired binding to β arrestin 2 also leads to an impaired recruitment of PDE4D to the C341A mutant. Thus, the mutant C341A β(2)AR is transported alone from the plasma membrane to the endosome without recruiting PDE4D. This alteration leads to an enhanced cytoplasmic cAMP signal for PKA activation under β(2)AR stimulation. Functionally, Mutation of the C341 residue or inhibition of palmitoylation modification of β(2)AR enhances the receptor-induced PKA activities in the cytoplasm and increases in myocyte contraction rate. Our data reveal a novel function of palmitoylation in shaping subcellular cAMP-PKA signaling in cardiomyocytes via modulating the recruitment of β arrestin 2-PDE4D complexes to the agonist-stimulated β(2)AR.

Palmitoylation Regulates Intracellular Trafficking of β2 Adrenergic Receptor/Arrestin/Phosphodiesterase 4D Complexes in Cardiomyocytes

PubMed Central

Liu, Ruijie; Wang, Dayong; Shi, Qian; Fu, Qin; Hizon, Steven; Xiang, Yang K.

2012-01-01

β2 adrenergic receptor (β2AR) is a prototypical G-protein coupled receptor that stimulates the classic cAMP-protein kinase A (PKA) signaling pathway. Recent studies indicate that the cAMP-PKA activities are spatiotemporally regulated in part due to dynamic association of β2AR with phosphodiesterase 4D (PDE4D), a group of cAMP degradation enzymes. Here, we demonstrate that in cardiomyocytes, palmitoylation of β2AR, the covalent acylation of cysteine residue 341, plays a critical role in shaping subcellular cAMP-PKA activities in cardiomyocytes via regulating β2AR association with arrestin/PDE4D. Replacing cysteine 341 on β2AR with alanine (C341A) leads to an impaired binding to β arrestin 2. Surprisingly, the C341A mutant is able to internalize via an arrestin-independent pathway at saturated concentration of agonist stimulation; the internalization becomes caveolae-dependent and requires dynamin GTPase. However, the impaired binding to β arrestin 2 also leads to an impaired recruitment of PDE4D to the C341A mutant. Thus, the mutant C341A β2AR is transported alone from the plasma membrane to the endosome without recruiting PDE4D. This alteration leads to an enhanced cytoplasmic cAMP signal for PKA activation under β2AR stimulation. Functionally, Mutation of the C341 residue or inhibition of palmitoylation modification of β2AR enhances the receptor-induced PKA activities in the cytoplasm and increases in myocyte contraction rate. Our data reveal a novel function of palmitoylation in shaping subcellular cAMP-PKA signaling in cardiomyocytes via modulating the recruitment of β arrestin 2-PDE4D complexes to the agonist-stimulated β2AR. PMID:22912718

Expression, Regulation and Putative Nutrient-Sensing Function of Taste GPCRs in the Heart

PubMed Central

Foster, Simon R.; Porrello, Enzo R.; Purdue, Brooke; Chan, Hsiu-Wen; Voigt, Anja; Frenzel, Sabine; Hannan, Ross D.; Moritz, Karen M.; Simmons, David G.; Molenaar, Peter; Roura, Eugeni; Boehm, Ulrich; Meyerhof, Wolfgang; Thomas, Walter G.

2013-01-01

G protein-coupled receptors (GPCRs) are critical for cardiovascular physiology. Cardiac cells express >100 nonchemosensory GPCRs, indicating that important physiological and potential therapeutic targets remain to be discovered. Moreover, there is a growing appreciation that members of the large, distinct taste and odorant GPCR families have specific functions in tissues beyond the oronasal cavity, including in the brain, gastrointestinal tract and respiratory system. To date, these chemosensory GPCRs have not been systematically studied in the heart. We performed RT-qPCR taste receptor screens in rodent and human heart tissues that revealed discrete subsets of type 2 taste receptors (TAS2/Tas2) as well as Tas1r1 and Tas1r3 (comprising the umami receptor) are expressed. These taste GPCRs are present in cultured cardiac myocytes and fibroblasts, and by in situ hybridization can be visualized across the myocardium in isolated cardiac cells. Tas1r1 gene-targeted mice (Tas1r1Cre/Rosa26tdRFP) strikingly recapitulated these data. In vivo taste receptor expression levels were developmentally regulated in the postnatal period. Intriguingly, several Tas2rs were upregulated in cultured rat myocytes and in mouse heart in vivo following starvation. The discovery of taste GPCRs in the heart opens an exciting new field of cardiac research. We predict that these taste receptors may function as nutrient sensors in the heart. PMID:23696900

Active and inactive enhancers co-operate to exert localized and long-range control of gene regulation

PubMed Central

Proudhon, Charlotte; Snetkova, Valentina; Raviram, Ramya; Lobry, Camille; Badri, Sana; Jiang, Tingting; Hao, Bingtao; Trimarchi, Thomas; Kluger, Yuval; Aifantis, Iannis; Bonneau, Richard; Skok, Jane A

2016-01-01

V(D)J recombination relies on the presence of proximal enhancers that activate the antigen receptor (AgR) loci in a lineage and stage specific manner. Unexpectedly we find that both active and inactive AgR enhancers co-operate to disseminate their effects in a localized and long-range manner. Here we demonstrate the importance of short-range contacts between active enhancers that constitute an Igk super-enhancer in B cells. Deletion of one element reduces the interaction frequency between other enhancers in the hub, which compromises the transcriptional output of each component. We further establish that in T cells long-range contact and co-operation between the inactive Igk enhancer, MiEκ and the active Tcrb enhancer, Eβ, alters enrichment of CBFβ binding in a manner that impacts Tcrb recombination. These findings underline the complexities of enhancer regulation and point to a role for localized and long-range enhancer-sharing between active and inactive elements in lineage and stage specific control. PMID:27239026

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.

Does Using E-Portfolios for Reflective Writing Enhance High School Students' Self-Regulated Learning?

ERIC Educational Resources Information Center

Chang, Chi-Cheng; Liang, Chaoyun; Shu, Kuen-Ming; Tseng, Kuo-Hung; Lin, Chun-Yu

2016-01-01

The study aims to examine whether reflective writing using e-portfolios enhances high school students' self-regulated learning. Participants included two classes of eighth-graders majoring in Information Processing and taking a course called "Website Design" at a vocational high school in Taiwan. There were 41 students, with 18 males and…

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

PubMed

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

2004-12-10

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

Biodegradable Hollow Mesoporous Silica Nanoparticles for Regulating Tumor Microenvironment and Enhancing Antitumor Efficiency

PubMed Central

Kong, Miao; Tang, Jiamin; Qiao, Qi; Wu, Tingting; Qi, Yan; Tan, Songwei; Gao, Xueqin; Zhang, Zhiping

2017-01-01

There is accumulating evidence that regulating tumor microenvironment plays a vital role in improving antitumor efficiency. Herein, to remodel tumor immune microenvironment and elicit synergistic antitumor effects, lipid-coated biodegradable hollow mesoporous silica nanoparticle (dHMLB) was constructed with co-encapsulation of all-trans retinoic acid (ATRA), doxorubicin (DOX) and interleukin-2 (IL-2) for chemo-immunotherapy. The nanoparticle-mediated combinational therapy provided a benign regulation on tumor microenvironment through activation of tumor infiltrating T lymphocytes and natural killer cells, promotion of cytokines secretion of IFN-γ and IL-12, and down-regulation of immunosuppressive myeloid-derived suppressor cells, cytokine IL-10 and TGF-β. ATRA/DOX/IL-2 co-loaded dHMLB demonstrated significant tumor growth and metastasis inhibition, and also exhibited favorable biodegradability and safety. This nanoplatform has great potential in developing a feasible strategy to remodel tumor immune microenvironment and achieve enhanced antitumor effect. PMID:28900509

Novel approaches to determine contractile function of the isolated adult zebrafish ventricular cardiac myocyte.

PubMed

Dvornikov, Alexey V; Dewan, Sukriti; Alekhina, Olga V; Pickett, F Bryan; de Tombe, Pieter P

2014-05-01

The zebrafish (Danio rerio) has been used extensively in cardiovascular biology, but mainly in the study of heart development. The relative ease of its genetic manipulation may indicate the suitability of this species as a cost-effective model system for the study of cardiac contractile biology. However, whether the zebrafish heart is an appropriate model system for investigations pertaining to mammalian cardiac contractile structure-function relationships remains to be resolved. Myocytes were isolated from adult zebrafish hearts by enzymatic digestion, attached to carbon rods, and twitch force and intracellular Ca(2+) were measured. We observed the modulation of twitch force, but not of intracellular Ca(2+), by both extracellular [Ca(2+)] and sarcomere length. In permeabilized cells/myofibrils, we found robust myofilament length-dependent activation. Moreover, modulation of myofilament activation-relaxation and force redevelopment kinetics by varied Ca(2+) activation levels resembled that found previously in mammalian myofilaments. We conclude that the zebrafish is a valid model system for the study of cardiac contractile structure-function relationships.

Enzymological mechanism for the regulation of lanthanum chloride on flavonoid synthesis of soybean seedlings under enhanced ultraviolet-B radiation.

PubMed

Fan, Caixia; Hu, Huiqing; Wang, Lihong; Zhou, Qing; Huang, Xiaohua

2014-01-01

In order to probe into the enzymological mechanism for the regulation of lanthanum chloride (LaCl3) on flavonoid synthesis in plants under enhanced ultraviolet-B (UV-B) radiation, the effects of LaCl₃ (20 and 60 mg l(-1)) on the content of flavonoids as well as the activities of phenylalanine ammonia-lyase (PAL), cinnamate-4-hydroxylase (C4H), 4-coumarate : coenzyme A ligase (4CL), and chalcone synthase (CHS) in soybean seedlings under enhanced UV-B radiation (2.6 and 6.2 kJ m(-2) day(-1)) were investigated. Enhanced UV-B radiation (2.6 and 6.2 kJ m(-2) day(-1)) caused the increase in the content of flavonoids as well as the activities of PAL, C4H, 4CL, and CHS in soybean seedlings. The treatment of 20 mg l(-1) LaCl₃ also efficiently increased these indices, which promoted the flavonoid synthesis and provided protective effects for resisting enhanced UV-B radiation. On the contrary, the treatment of 60 mg l(-1) LaCl₃ decreased the content of flavonoids as well as the activities of C4H, 4CL, and CHS in soybean seedlings except increasing the activity of PAL, which were not beneficial to the flavonoid synthesis and provided negative effects for resisting enhanced UV-B radiation. In conclusion, enhanced UV-B radiation caused the increase in the flavonoid synthesis by promoting the activities of PAL, C4H, 4CL, and CHS in soybean seedlings. The treatment of LaCl₃ could change flavonoid synthesis in soybean seedlings under enhanced UV-B radiation by regulating the activities of PAL, C4H, 4CL, and CHS, which is an enzymological mechanism for the regulation of LaCl₃ on flavonoid synthesis in plants under enhanced UV-B radiation.

Voltage sensitivity of M2 muscarinic receptors underlies the delayed rectifier-like activation of ACh-gated K(+) current by choline in feline atrial myocytes.

PubMed

Navarro-Polanco, Ricardo A; Aréchiga-Figueroa, Iván A; Salazar-Fajardo, Pedro D; Benavides-Haro, Dora E; Rodríguez-Elías, Julio C; Sachse, Frank B; Tristani-Firouzi, Martin; Sánchez-Chapula, José A; Moreno-Galindo, Eloy G

2013-09-01

Choline (Ch) is a precursor and metabolite of the neurotransmitter acetylcholine (ACh). In canine and guinea pig atrial myocytes, Ch was shown to activate an outward K(+) current in a delayed rectifier fashion. This current has been suggested to modulate cardiac electrical activity and to play a role in atrial fibrillation pathophysiology. However, the exact nature and identity of this current has not been convincingly established. We recently described the unique ligand- and voltage-dependent properties of muscarinic activation of ACh-activated K(+) current (IKACh) and showed that, in contrast to ACh, pilocarpine induces a current with delayed rectifier-like properties with membrane depolarization. Here, we tested the hypothesis that Ch activates IKACh in feline atrial myocytes in a voltage-dependent manner similar to pilocarpine. Single-channel recordings, biophysical profiles, specific pharmacological inhibition and computational data indicate that the current activated by Ch is IKACh. Moreover, we show that membrane depolarization increases the potency and efficacy of IKACh activation by Ch and thus gives the appearance of a delayed rectifier activating K(+) current at depolarized potentials. Our findings support the emerging concept that IKACh modulation is both voltage- and ligand-specific and reinforce the importance of these properties in understanding cardiac physiology.

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

An ultra-low power output capacitor-less low-dropout regulator with slew-rate-enhanced circuit

NASA Astrophysics Data System (ADS)

Cheng, Xin; Zhang, Yu; Xie, Guangjun; Yang, Yizhong; Zhang, Zhang

2018-03-01

An ultra-low power output-capacitorless low-dropout (LDO) regulator with a slew-rate-enhanced (SRE) circuit is introduced. The increased slew rate is achieved by sensing the transient output voltage of the LDO and then charging (or discharging) the gate capacitor quickly. In addition, a buffer with ultra-low output impedance is presented to improve line and load regulations. This design is fabricated by SMIC 0.18 μm CMOS technology. Experimental results show that, the proposed LDO regulator only consumes an ultra-low quiescent current of 1.2 μA. The output current range is from 10 μA to 200 mA and the corresponding variation of output voltage is less than 40 mV. Moreover, the measured line regulation and load regulation are 15.38 mV/V and 0.4 mV/mA respectively. Project supported by the National Natural Science Foundation of China (Nos. 61401137, 61404043, 61674049).

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

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

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.

Spontaneous Ca2+ sparks and Ca2+ homeostasis in a minimal model of permeabilized ventricular myocytes

PubMed Central

Hartman, Jana M.; Sobie, Eric A.

2010-01-01

Many issues remain unresolved concerning how local, subcellular Ca2+ signals interact with bulk cellular concentrations to maintain homeostasis in health and disease. To aid in the interpretation of data obtained in quiescent ventricular myocytes, we present here a minimal whole cell model that accounts for both localized (subcellular) and global (cellular) aspects of Ca2+ signaling. Using a minimal formulation of the distribution of local [Ca2+] associated with a large number of Ca2+-release sites, the model simulates both random spontaneous Ca2+ sparks and the changes in myoplasmic and sarcoplasmic reticulum (SR) [Ca2+] that result from the balance between stochastic release and reuptake into the SR. Ca2+-release sites are composed of clusters of two-state ryanodine receptors (RyRs) that exhibit activation by local cytosolic [Ca2+] but no inactivation or regulation by luminal Ca2+. Decreasing RyR open probability in the model causes a decrease in aggregate release flux and an increase in SR [Ca2+], regardless of whether RyR inhibition is mediated by a decrease in RyR open dwell time or an increase in RyR closed dwell time. The same balance of stochastic release and reuptake can be achieved, however, by either high-frequency/short-duration or low-frequency/long-duration Ca2+ sparks. The results are well correlated with recent experimental observations using pharmacological RyR inhibitors and clarify those aspects of the release-reuptake balance that are inherent to the coupling between local and global Ca2+ signals and those aspects that depend on molecular-level details. The model of Ca2+ sparks and homeostasis presented here can be a useful tool for understanding changes in cardiac Ca2+ release resulting from drugs, mutations, or acquired diseases. PMID:20852058

L-type Ca2+ channels in the heart: structure and regulation.

PubMed

Treinys, Rimantas; Jurevicius, Jonas

2008-01-01

This review analyzes the structure and regulation mechanisms of voltage-dependent L-type Ca(2+) channel in the heart. L-type Ca(2+) channels in the heart are composed of four different polypeptide subunits, and the pore-forming subunit alpha(1) is the most important part of the channel. In cardiac myocytes, Ca(2+) enter cell cytoplasm from extracellular space mainly through L-type Ca(2+) channels; these channels are very important system in heart Ca(2+) uptake regulation. L-type Ca(2+) channels are responsible for the activation of sarcoplasmic reticulum channels (RyR2) and force of muscle contraction generation in heart; hence, activity of the heart depends on L-type Ca(2+) channels. Phosphorylation of channel-forming subunits by different kinases is one of the most important ways to change the activity of L-type Ca(2+) channel. Additionally, the activity of L-type Ca(2+) channels depends on Ca(2+) concentration in cytoplasm. Ca(2+) current in cardiac cells can facilitate, and this process is regulated by phosphorylation of L-type Ca(2+) channels and intracellular Ca(2+) concentration. Disturbances in cellular Ca(2+) transport and regulation of L-type Ca(2+) channels are directly related to heart diseases, life quality, and life span.

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

Combinatorial regulation of a Blimp1 (Prdm1) enhancer in the mouse retina

PubMed Central

Mills, Taylor S.; Eliseeva, Tatiana; Bersie, Stephanie M.; Randazzo, Grace; Nahreini, Jhenya; Park, Ko Uoon

2017-01-01

The mouse retina comprises seven major cell types that exist in differing proportions. They are generated from multipotent progenitors in a stochastic manner, such that the relative frequency of any given type generated changes over time. The mechanisms determining the proportions of each cell type are only partially understood. Photoreceptors and bipolar interneurons are derived from cells that express Otx2. Within this population, Blimp1 (Prdm1) helps set the balance between photoreceptors and bipolar cells by suppressing bipolar identity in most of the cells. How only a subset of these Otx2+ cells decides to upregulate Blimp1 and adopt photoreceptor fate is unknown. To understand this, we investigated how Blimp1 transcription is regulated. We identified several potential Blimp1 retinal enhancer elements using DNase hypersensitivity sequencing. Only one of the elements recapitulated Blimp1 spatial and temporal expression in cultured explant assays and within the retinas of transgenic mice. Mutagenesis of this retinal Blimp1 enhancer element revealed four discrete sequences that were each required for its activity. These included highly conserved Otx2 and ROR (retinoic acid receptor related orphan receptor) binding sites. The other required sequences do not appear to be controlled by Otx2 or ROR factors, increasing the complexity of the Blimp1 gene regulatory network. Our results show that the intersection of three or more transcription factors is required to correctly regulate the spatial and temporal features of Blimp1 enhancer expression. This explains how Blimp1 expression can diverge from Otx2 and set the balance between photoreceptor and bipolar fates. PMID:28829770

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

PubMed

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

2000-04-05

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

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

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

Liang, B.T.

1989-06-01

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

Higenamine protects ischemia/reperfusion induced cardiac injury and myocyte apoptosis through activation of β2-AR/PI3K/AKT signaling pathway

PubMed Central

Wu, Mei-ping; Zhang, Yi-shuai; Zhou, Qian-mei; Xiong, Jian; Dong, Yao-rong; Yan, Chen

2016-01-01

Cardiomyocyte apoptosis contributes to ischemic cardiac injury and the development of heart failure. Higenamine is a key component of the Chinese herb aconite root that has been prescribed for treating symptoms of heart failure for thousands of years in the oriental Asian countries. It has been shown that higenamine has anti-apoptotic effects in a few cell types including cardiomyocytes. However, the pharmacological target and molecular mechanism of higenamine in the heart are still not fully illustrated. Herein, we report that higenamine protected myocyte apoptosis and ischemia/reperfusion (I/R) injury through selective activation of beta2-adrenergic receptor (β2-AR). In particular, we show that higenamine significantly reduced I/R-induced myocardial infarction in mice. In both primary neonatal rat and adult mouse ventricular myocytes, we show higenamine inhibited cell apoptosis and also reduced biochemical markers of apoptosis such as cleaved caspase 3 and 9. More importantly, we show that the anti-apoptotic effects of higenamine in cardiomyocytes were completely abolished by β2-AR but not β1-AR antagonism. Furthermore, we confirmed that higenamine attenuated I/R-induced myocardial injury and reduced cleaved caspases in a β2-AR dependent manner in intact mouse hearts. Higenamine stimulated AKT phosphorylation and required PI3K activation for the anti-apoptotic effect in cardiomyocytes. These findings together suggest that anti-apoptotic and cardiac protective effects of higenamine are mediated by the β2-AR/PI3K/AKT cascade. PMID:26746354

Lsd1 regulates skeletal muscle regeneration and directs the fate of satellite cells.

PubMed

Tosic, Milica; Allen, Anita; Willmann, Dominica; Lepper, Christoph; Kim, Johnny; Duteil, Delphine; Schüle, Roland

2018-01-25

Satellite cells are muscle stem cells required for muscle regeneration upon damage. Of note, satellite cells are bipotent and have the capacity to differentiate not only into skeletal myocytes, but also into brown adipocytes. Epigenetic mechanisms regulating fate decision and differentiation of satellite cells during muscle regeneration are not yet fully understood. Here, we show that elevated levels of lysine-specific demethylase 1 (Kdm1a, also known as Lsd1) have a beneficial effect on muscle regeneration and recovery after injury, since Lsd1 directly regulates key myogenic transcription factor genes. Importantly, selective Lsd1 ablation or inhibition in Pax7-positive satellite cells, not only delays muscle regeneration, but changes cell fate towards brown adipocytes. Lsd1 prevents brown adipocyte differentiation of satellite cells by repressing expression of the novel pro-adipogenic transcription factor Glis1. Together, downregulation of Glis1 and upregulation of the muscle-specific transcription program ensure physiological muscle regeneration.

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.

Regulation of the neuropathy-associated Pmp22 gene by a distal super-enhancer.

PubMed

Pantera, Harrison; Moran, John J; Hung, Holly A; Pak, Evgenia; Dutra, Amalia; Svaren, John

2018-05-16

Peripheral nerve myelination is adversely affected in the most common form of the hereditary peripheral neuropathy called Charcot-Marie-Tooth Disease. This form, classified as CMT1A, is caused by a 1.4 Mb duplication on chromosome 17, which includes the abundantly expressed Schwann cell myelin gene, Peripheral Myelin Protein 22 (PMP22). This is one of the most common copy number variants causing neurological disease. Overexpression of Pmp22 in rodent models recapitulates several aspects of neuropathy, and reduction of Pmp22 in such models results in amelioration of the neuropathy phenotype. Recently we identified a potential super-enhancer approximately 90-130 kb upstream of the Pmp22 transcription start sites. This super-enhancer encompasses a cluster of individual enhancers that have the acetylated histone H3K27 active enhancer mark, and coincides with smaller duplications identified in patients with milder CMT1A-like symptoms, where the PMP22 coding region itself was not part of the duplication. In this study, we have utilized genome editing to create a deletion of this super-enhancer to determine its role in Pmp22 regulation. Our data show a significant decrease in Pmp22 transcript expression using allele-specific internal controls. Moreover, the P2 promoter of the Pmp22 gene, which is used in other cell types, is affected, but we find that the Schwann cell-specific P1 promoter is disproportionately more sensitive to loss of the super-enhancer. These data show for the first time the requirement of these upstream enhancers for full Pmp22 expression.

Cell Surface Expression of Human Ether-a-go-go-related Gene (hERG) Channels Is Regulated by Caveolin-3 Protein via the Ubiquitin Ligase Nedd4-2*