Neonatal Ventricular Myocytes Research Articles

Calcium-sensing receptor activation contributed to apoptosis stimulates TRPC6 channel in rat neonatal ventricular myocytes

Capacitative calcium entry (CCE) refers to the influx of calcium through plasma membrane channels activated on depletion of endoplasmic sarcoplasmic/reticulum (ER/SR) Ca 2+ stores, which is performed mainly by the transient receptor potential (TRP) channels. TRP channels are expressed in cardiomyocytes. Calcium-sensing receptor (CaR) is also expressed in rat cardiac tissue and plays an important role in mediating cardiomyocyte apoptosis. However, there are no data regarding the link between CaR and TRP channels in rat heart. In this study, in rat neonatal myocytes, by Ca 2+ imaging, we found that the depletion of ER/SR Ca 2+ stores by thapsigargin (TG) elicited a transient rise in cytoplasmic Ca 2+ ([Ca 2+] i), followed by sustained increase depending on extracellular Ca 2+. But, TRP channels inhibitor (SKF96365), not L-type channels or the Na +/Ca 2+ exchanger inhibitors, inhibited [Ca 2+] i relatively high. Then, we found that the stimulation of CaR with its activator gadolinium chloride (GdCl 3) or by an increased extracellular Ca 2+([Ca 2+] o) increased the concentration of intracelluar Ca 2+, whereas, the sustained elevation of [Ca 2+] i was reduced in the presence of SKF96365. Similarly, the duration of [Ca 2+] i increase was also shortened in the absence of extracellular Ca 2+. Western blot analysis showed that GdCl 3 increased the expression of TRPC6, which was reversed by SKF96365. Additionally, SKF96365 reduced cardiomyocyte apoptosis induced by GdCl 3. Our results suggested that CCE exhibited in rat neonatal myocytes and CaR activation induced Ca 2+-permeable cationic channels TRPCs to gate the CCE, for which TRPC6 was one of the most likely candidates. TRPC6 channel was functionally coupled with CaR to enhance the cardiomyocyte apoptosis.

Effects of Continous Electrical Field Stimulation and Hypertrophic Stimulation on Micropatterned Cardiac Myocytes

Neonatal ventricular myocytes (CM) have long been used as an in vitro model for hypertrophy studies. In conventional 2D culture, CM lack axial orientation and rhythmic electrical stimulation. Micropatterned cultures can restrict cell attachment to narrow stripes, leading to enhanced axial orientation, particularly in spontaneously contracting CM (Rohr et. al., Circ Res, 1991). In this study, we investigated the effect of continous electrical field stimulation (CES) on micropatterned CM and examined their response to hypertrophic stimulation. Rat CM plated in serum-containing media selectively attached to stripes (100 μm x 10 mm) of fibronectin (FN) that were microcontact-printed onto coverslips. CM cultures were subjected to CES (1 Hz, 5 V/cm) for 48 hrs, with the current applied parallel or perpendicular to FN stripes. To induce a hypertrophic response, micropatterned CM were incubated for 48 hrs in serum-free medium with the α1 adrenoceptor agonist phenylephrine (PE, together with timolol). We determined that the size, minor/major axis ratio and angles relative to FN stripes of DAPI-stained nuclei can be used as surrogate measures of CM size, elongation and alignment, respectively. Compared to unpaced CM, parallel CES increased nuclear size (1028±121 vs. 798±87 μm2, P<0.001), elongation (minor/major axis: 0.76±0.10 vs. 0.84±0.08, P<0.001) and alignment (P<0.001, Mardia-Watson-Wheeler circular statistics). Perpendicular CES caused similar but significantly less pronounced changes. PE stimulation increased nuclear size (809±93 vs. 682±99 μm2, P<0.05), but did not increase elongation or alignment with or without CES. In conclusion, CES can be used to enhance the degree of differentiation of micropatterned CM due to continuous electrical activation and/or contractions and does not interfere with their hypertrophic response. Continuously paced micropatterned CM represent an advanced model for the investigation of hypertrophic responses and mechanisms and may be suitable for other applications.

PKA, Rap1, ERK1/2, and p90RSK mediate PGE2and EP4 signaling in neonatal ventricular myocytes

We have previously reported that 1) inhibition of cyclooxygenase-2 and PGE(2) production reduces hypertrophy after myocardial infarction in mice and 2) PGE(2) acting through its EP4 receptor causes hypertrophy of neonatal ventricular myocytes (NVMs) via ERK1/2. It is known that EP4 couples to adenylate cyclase, cAMP, and PKA. The present study was designed to determine interactions between the cAMP-PKA pathway and ERK1/2 and to further characterize events downstream of ERK1/2. We hypothesized that PKA and the small GTPase Rap are upstream of ERK1/2 and that 90-kDa ribosomal S6 kinase (p90RSK) is activated downstream. Treatment of NVMs with PGE(2) activated Rap, and this activation was inhibited in part by an EP4 antagonist and PKA inhibition. Transfection of a dominant negative mutant of Rap reduced PGE(2) activation of ERK1/2. PGE(2) activation of p90RSK was also dependent on EP4, PKA, and Rap. We also tested the involvement of Rap, ERK1/2, and p90RSK in PGE(2) regulation of gene expression. PGE(2) stimulation of brain natriuretic peptide promoter activity was blocked by either ERK1/2 inhibition or a dominant negative mutation of p90RSK. PGE(2) stimulation of c-Fos was dependent on EP4, PKA, ERK1/2, and p90RSK, whereas only the latter two kinases were involved in PGE(2) regulation of early growth response-1. Finally, we tested the involvement of EP4-dependent signaling in the NVM growth response and found that the overexpression of EP4 increased NVM cell size. We conclude that EP4-dependent signaling in NVMs in part involves PKA, Rap, ERK1/2, and p90RSK and results in the increased expression of brain natriuretic peptide and c-Fos.

Importance of the bioenergetic reserve capacity in response to cardiomyocyte stress induced by 4-hydroxynonenal

Mitochondria play a critical role in mediating the cellular response to oxidants formed during acute and chronic cardiac dysfunction. It is widely assumed that, as cells are subjected to stress, mitochondria are capable of drawing upon a 'reserve capacity' which is available to serve the increased energy demands for maintenance of organ function, cellular repair or detoxification of reactive species. This hypothesis further implies that impairment or depletion of this putative reserve capacity ultimately leads to excessive protein damage and cell death. However, it has been difficult to fully evaluate this hypothesis since much of our information about the response of the mitochondrion to oxidative stress derives from studies on mitochondria isolated from their cellular context. Therefore the goal of the present study was to determine whether 'bioenergetic reserve capacity' does indeed exist in the intact myocyte and whether it is utilized in response to stress induced by the pathologically relevant reactive lipid species HNE (4-hydroxynonenal). We found that intact rat neonatal ventricular myocytes exhibit a substantial bioenergetic reserve capacity under basal conditions; however, on exposure to pathologically relevant concentrations of HNE, oxygen consumption was increased until this reserve capacity was depleted. Exhaustion of the reserve capacity by HNE treatment resulted in inhibition of respiration concomitant with protein modification and cell death. These data suggest that oxidized lipids could contribute to myocyte injury by decreasing the bioenergetic reserve capacity. Furthermore, these studies demonstrate the utility of measuring the bioenergetic reserve capacity for assessing or predicting the response of cells to stress.

An Adenylyl Cyclase-mAKAPβ Signaling Complex Regulates cAMP Levels in Cardiac Myocytes

Protein kinase A-anchoring proteins (AKAPs) play important roles in the compartmentation of cAMP signaling, anchoring protein kinase A (PKA) to specific cellular organelles and serving as scaffolds that assemble localized signaling cascades. Although AKAPs have been recently shown to bind adenylyl cyclase (AC), the functional significance of this association has not been studied. In cardiac myocytes, the muscle protein kinase A-anchoring protein beta (mAKAPbeta) coordinates cAMP-dependent, calcium, and MAP kinase pathways and is important for cellular hypertrophy. We now show that mAKAPbeta selectively binds type 5 AC in the heart and that mAKAPbeta-associated AC activity is absent in AC5 knock-out hearts. Consistent with its known inhibition by PKA phosphorylation, AC5 is inhibited by association with mAKAPbeta-PKA complexes. AC5 binds to a unique N-terminal site on mAKAP-(245-340), and expression of this peptide disrupts endogenous mAKAPbeta-AC association. Accordingly, disruption of mAKAPbeta-AC5 complexes in neonatal cardiac myocytes results in increased cAMP and hypertrophy in the absence of agonist stimulation. Taken together, these results show that the association of AC5 with the mAKAPbeta complex is required for the regulation of cAMP second messenger controlling cardiac myocyte hypertrophy.

Calcium-Mediated Translocation of Fission Protein DLP1 to Mitochondria and Augmentation of Reactive Oxygen Species (ROS) Levels in Heart

Background: Mitochondrial fission/fusion/movement plays a critical role in bioenergetics, Ca2+ homeostasis, redox signaling, and apoptosis. We have recently shown that Ca2+ regulates mitochondrial fission, ROS production, and mitochondrial permeability transition (MPT) in several cell types. The adult heart mitochondria in vivo are continuously exposed to cytosolic Ca2+ transients. They situate orderly in the sarcomeres so that their movement is minimal. Here we test the hypothesis that cardiac muscle cells contain DLP1 and its translocation to mitochondria is regulated by Ca2+. The translocation of DLP1 is a critical step for Ca2+-mediated ROS increases.Results: Western blots showed fission proteins DLP1 and hFis were present in the mitochondria of adult and neonatal ventricular myocytes. To raise cytosolic Ca2+, we used: 1) 50 mM KCl to open L-type Ca2+ channels, 2) 1 μM thapsigargin to inhibit Ca2+ uptake into sarcoplasmic reticulum, and 3) combination of electrical and β-adrenergic receptor stimulation to increase the frequency and magnitude of Ca2+ transients. All three interventions increase translocation of DLP1 to mitochondria (p<0.01) and elevation of ROS levels (3 fold). Overexpression of dominant-negative mutant DLP1-K38A led to web-like interconnected long mitochondria in cultured neonatal rat ventricular myocytes that no longer respond to the Ca2+-mediated ROS increases. The translocation of DLP1 was also observed in mitochondria isolated from Langendorff hearts perfused with 10 nM isoproterenol (p<0.05) suggesting the Ca2+-mediated mitochondrial fission could occur in vivo. Current experiments are addressing whether the DLP1 translocation could promote MPT openings.Conclusion: Adult cardiac myocytes possess the key proteins involved in mitochondrial fission. Ca2+ increases DLP1 translocation to mitochondria and thus augments ROS generation. Therefore, the mitochondrial fission machinery could play a critical role in physiological regulation of cardiac energy metabolism and ROS homeostasis.

Model of Excitation-Contraction Coupling of Rat Neonatal Ventricular Myocytes

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 Ca 2+ 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 Ca 2+ signals, weaker sarcoplasmic reticulum Ca 2+ handling, and stronger sarcolemmal Ca 2+ handling, with a significant contribution by the Na +/Ca 2+ exchanger. The developed model reproduces faithfully the ECC of rat neonatal cardiomyocytes with a novel description of spatial cytosolic [Ca 2+] 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.

Abstract 1531: SCN5A Promoter and Enhancer Variants associated with Atrial Fibrillation

We have recently reported that 5.9% of patients with atrial fibrillation (AF) have rare variants in the coding region of the predominant cardiac sodium channel gene SCN5A . In this study, we tested the hypothesis that AF susceptibility is also influenced by variants in the SCN5A core promoter and short non-coding nucleotide sequences highly conserved across species (CNS) that we previously identified. We resequenced the SCN5A core promoter region (~3 kb) and CNS regions in intron 1 (&lt;1 kb for each) in 376 patients with AF (111 patients with lone AF and 234 patients with AF and cardiac disease) and 47 ethnically-defined controls. The promoter activity of variants showing different frequencies in patients with AF compared to controls was studied using promoter-reporter assays in CHO cells and in neonatal mouse ventricular myocytes. Identification of variants Four common variants were identified in the core promoter in controls, and one of the variants (c.−225– 834 T&gt;C) was significantly more common in patients with AF than in controls (minor allele frequency: lone AF, 17%; AF and heart disease, 17%; control, 7%; P&lt;0.05 vs. control for each). In addition, 10 novel rare promoter variants were identified in patients with AF and were absent in controls. In CNS28, a 406 bp region in intron 1 that includes three TEF-1 binding sites, one variant (c.−53±15307 C&gt;T) which is predicted to disrupt one of the TEF-1 binding sites was identified in 7 out of 376 patients with AF (1 with lone AF, 6 with AF and heart disease), but not in controls. Functional study There was no difference in promoter activity between wild-type and c.−225– 834 T&gt;C SCN5A core promoters expressed in CHO cells. Wild-type CNS28 coexpressed with the SCN5A core promoter increased reporter activity by 77 ± 14% (P=0.03) in CHO cells and 261 ± 8% (P=0.001) in cardiomyocytes, compared to SCN5A promoter alone. However, c.−53+15307 C&gt;T CNS28 showed smaller effects on promoter activity in both CHO cells (52 ± 2 % of wild-type, P&lt;0.05) and cardiomyocytes (62 ± 8%, P&lt;0.05) compared to wild-type CNS28. Variants in the SCN5A promoter and an enhancer region in intron 1 were associated with AF. Variability in SCN5A transcription may affect AF susceptibility.

Interaction between transcription factors Iroquois proteins 4 and 5 controls cardiac potassium channel Kv4.2 gene transcription

The homeobox transcription factor, Iroquois protein 5 (Irx5), plays an essential role in the generation of region-selective expression of Kv4.2 gene across the left ventricular wall of rodent hearts. Here, we analyse molecular mechanisms underlying the Irx5-induced regulation of the rat Kv4.2 promoter. The mRNA levels for Irx members in various heart regions were assessed by RT-PCR. A luciferase reporter gene with the rat Kv4.2 promoter was used to test the effects of Irx members on channel promoter activity. Irx3 and Irx5 mRNAs were differentially distributed across the left ventricular wall, whereas Irx4 message was equally abundant in various ventricular regions. Irx5, but not Irx3 or Irx4, increased Kv4.2 promoter activity in 10T1/2 fibroblasts, whereas the transcription factor decreased promoter activity in neonatal ventricular myocytes. These effects were mediated by the C-terminal portion of Irx5. Irx4 appeared to inhibit the Irx5-induced increase in channel promoter activity in 10T1/2 cells. The N-terminal region of Irx4 was necessary and sufficient for this inhibition. Furthermore, when endogenous Irx4 expression was suppressed with siRNA, Irx5 increased channel promoter activity in neonatal myocytes. These results indicate that Irx5 possesses the ability to activate the Kv4.2 promoter. The abundant Irx4 expression throughout the rat ventricle may play a role in the inverse relationship between Irx5 and Kv4.2 levels across the left ventricular wall.

κ-Opioid receptor stimulation inhibits augmentation of Ca 2+ transient and hypertrophy induced by isoprenaline in neonatal rat ventricular myocytes — Role of CaMKIIδ B

We aimed to further define the pathway mediating the inhibitory effects of κ-opioid receptor stimulation on Ca 2+ transients and hypertrophic responses to β 1-adrenoceptor stimulation. We determined the effects of Trans-(±)-3,4-dichloro- N-methyl- N-[2-(1-pyrrolidinyl)-cyclohexyl]-benzeneacetamid methanesulfonate salt (U50,488H), a selective κ-opioid receptor agonist, on the enhancement of spontaneous Ca 2+ transients and the induction of hypertrophy by isoprenaline, a β-adrenoceptor agonist, in cultured neonatal ventricular myocytes. The results were compared with those found with KN93, a selective Ca 2+/calmodulin-dependent kinase (CaMKII) inhibitor, propranolol, a β-adrenoceptor antagonist, and verapamil, a L-type Ca 2+ channel antagonist. Hypertrophy of cardiomyocytes was characterized by increases in (i) total protein content; (ii) cell size; and (iii) [ 3H]leucine incorporation. 10 μmol/l isoprenaline increased all three parameters. We also determined the expression of nuclear CaMKIIδ in response to U50,488H in the presence or absence of isoprenaline. To determine whether the effects of U50,488H were receptor-mediated, its effects were also measured following blockade of the κ-opioid receptor with nor-binaltorphimine. κ-Opioid receptor stimulation suppressed the stimulatory effect of isoprenaline on Ca 2+ transients and cardiac hypertrophy, as did KN93, propranalol and verapamil. U50,488H also suppressed the expression of nuclear CaMKIIδ B in the presence, but not in the absence of isoprenaline. These results suggest that the inhibitory effect of κ-opioid receptor stimulation on β 1-adrenoceptor stimulation may also involve CaMKIIδ.

Proteasome malfunction increases autophagosomes in cardiomyocytes and mouse hearts

Background:Autophagy and the ubiquitin‐proteasome system (UPS) are two main routes for intracellular protein degradation. The effect of proteasome malfunction on autophagy in cardiomyocytes and the heart remains to be demonstrated.Methods &amp; results:Proteasomal function in cultured neonatal ventricular myocytes (NRVMs) or mouse hearts is manipulated using pharmacological and/or genetic means. The resultant effects on autophagy are assessed by two methods: LC3‐II/LC‐I ratio measured by western blot analysis; autophagy index measured via confocal microscopic analysis of GFP‐LC3 distribution. Cardiac‐restricted expression of a 7‐amino‐acid deletion mutation of the desmin gene (D7‐des) has been linked to human desminopathy which develops aberrant desmin aggregation and severe proteasome malfunction in heart muscle cells. D7‐des increases LC3‐II/LC3‐I ratio and punctate GFP‐LC3 in mouse heart as early as 2 month. The increased autophagosomes is also detected in mouse heart 12 hours after an intravenous injection of proteasome inhibitor MG262 (5umol/kg). In NRVMs, proteasome inhibition by MG132 (1uM) increases the LC3‐II/LC3‐I ratio and autophagic flux measured as the difference of autophagosomes in the absence and presence of lysosomal protease inhibitors.Conclusions:Pharmacological and/or genetic proteasome inhibition increase autophagosomes in both NRVMs and mouse hearts.

The Cell Cycle Factor E2F-1 Activates Bnip3 and the Intrinsic Death Pathway in Ventricular Myocytes

The cell cycle factor E2F-1 is known to regulate a variety of cellular processes including apoptosis. Previously we showed that disruption of Rb-E2F-1 complexes provoked apoptosis of postmitotic adult and neonatal ventricular myocytes; however, the underlying mechanism was undetermined. In this report, we show that E2F-1 provokes cell death of ventricular myocytes through a mechanism that directly impinges on the intrinsic death pathway. Furthermore, we show mechanistically that the hypoxia-inducible death factor Bnip3 is a direct transcriptional target of E2F-1 that is necessary and sufficient for E2F-1-induced cell death. Expression of E2F-1 resulted in a 4.9-fold increase (P<0.001) in nucleosomal DNA fragmentation and cell death by Hoechst 33258 dye and vital staining. E2F-1 provoked mitochondrial perturbations that were consistent with permeability transition pore opening. As determined by quantitative real-time PCR analysis, a 6.2-fold increase (P<0.001) in endogenous Bnip3 gene transcription was observed in cells expressing wild-type E2F-1 but not in cells expressing a mutation of E2F-1 defective for DNA binding. Rb, the principle regulator of cellular E2F-1 activity, was proteolytically cleaved and inactivated in ventricular myocytes during hypoxia. Consistent with the proteolytic cleavage of Rb, chromatin immunoprecipitation analysis revealed increased binding of E2F-1 to the Bnip3 promoter during hypoxia, a finding concordant with the induction of Bnip3 gene transcription. The Bnip3 homolog Nix/Bnip3L was unaffected in ventricular myocytes by either E2F-1 or hypoxia. Genetic knockdown of E2F-1 or expression of a caspase-resistant form of Rb suppressed basal and hypoxia-inducible Bnip3 gene transcription. Loss-of-function mutations of Bnip3 defective for mitochondrial membrane insertion or small interference RNA directed against Bnip3 suppressed cell death signals elicited by E2F-1. To our knowledge, the data provide the first direct evidence that activation of the intrinsic mitochondrial death pathway by E2F-1 is mutually dependent on and obligatorily linked to the transcriptional activation of Bnip3.

Abstract 1067: CamKII Regulates NFAT Nuclear Translocation in a Calcineurin Independent Manner

Pathologic cardiac myocyte hypertrophy involves stress induced increases in intracellular Ca 2+ , which binds to calmodulin (CaM) and then activates both the phosphatase calcineurin (Cn) and CaM kinase (CaMKII). In hypertrophy, Cn dephosphorylates the cytoplasmic nuclear factor of activated T-cells (NFAT), inducing nuclear translocation where it activates hypertrophic target genes. While GSK3, p38, and JNK are kinases that have been shown to directly phosphorylate NFAT, resulting in nuclear export, the role of cytoplasmic CamKII in NFAT translocation is unknown. This study explored the hypothesis that cytoplasmic CamKII directly induces NFAT cytoplasmic localization which antagonizes Cn mediated nuclear NFAT translocation. Methods: Adenovirus gene delivery was used to express GFP-tagged NFATc3 (m.o.i - 100, 12h) in either cultured neonatal rat (NRVM) or in adult feline ventricular myocytes (AFVM). Myocytes were co-infected with either constitutively active (CamKII-CA) or dominant negative (CamKII-DN) mutants of cytoplasmic targeted CamKII δc (m.o.i - 100, 12 h). FK506 (2uM) or CsA (2uM) were used to inhibit Cn. The % of myocytes with nuclear translocated NFAT is reported. Results: In NRVM (and in AFVM), NFAT nuclear translocation (Control: 14.9±1%) was significantly increased (29±2%, p&lt;0.05 vs control) by raising the bath [Ca 2+ ] by 2X. This Ca 2+ mediated NFAT translocation was blocked by FK506 (3.8±1%) and CsA (7.3±2%, p&lt;0.05 vs control). CamKII-DN (CaMKII inhibition) increased (44.9±3%, P&lt;0.001 vs control) and CamKII-CA (CaMKII activation) decreased (6.8±2%, P&lt;0.01 vs control) NFAT translocation. FK506 and CsA did not block NFAT translocation in CamKII-DN NRVM (FK506: 50.7±2, CsA: 46.1±2%). CamKII-DN also increased NFAT translocation (50.5±3% vs 24.8±2% in control, P&lt;0.01) in AFVM. CsA treatment also failed to reduce CamKII-DN mediated NFAT translocation (59.6±9, P&lt;0.01 vs control) in adult myocytes. Conclusion: These data show for the first time that activation of cytoplasmic CaMKII inhibits NFAT nuclear translocation in both neonatal and adult ventricular myocytes, possibly by direct NFAT phosphorylation. These effects are in parallel with but independent of Ca 2+ dependent Cn signaling.

Activation of glycogen synthase kinase 3β participates in hypoxia‐induced cardiomyocyte apoptosis

Background: Hypoxia is a vital initiating factor in myocardial dysfunction, and apoptosis of myocytes contributes to hypoxia-induced heart failure. Glycogen synthase kinase 3β (GSK3β) has recently been identified as a key intermediate in several apoptotic signaling pathways. This study tested the hypothesis that GSK-3β activation results in hypoxia-induced cardiomyocyte apoptosis. Methods: Primary cultured neonatal ventricular myocytes from normal Wister rats (3d) were used. Cell apoptosis and viability were determined by Hoechst 33342 nuclear staining and MTT absorbance at 0, 3, 6, 12, 24 h after hypoxia, respectively. Expressions of GSK3β, caspases−9, −3 mRNA and phosphorylation of GSK3β protein were measured by RT-PCR and Western blot, respectively. Changes of caspase-9 enzymes activity were assessed by a colorimetric assay with the use of specific substrates. Results: Typical cell apoptosis was observed in cardiomyocytes and cell viability was significantly decreased 24 h after hypoxia. GSK3β mRNA and Caspase-9 mRNA expressions were increased 3–24 h after hypoxia, whereas caspase-3 mRNA expression was increased 12, 24 h after hypoxia. The phosphorylated form of GSK3β was increased 6-24 h after hypoxia, whereas caspase-9 activity was markedly elevated 3–24 h after hypoxia. Conclusion: Activation of GSK3β signal transduction pathway participates in hypoxia-induced cardiomyocytes apoptosis.

Innate Defense Mechanism Against Virus Infection Within the Cardiac Myocyte Requiring gp130-STAT3 Signaling

Little is known about innate immune mechanisms within the cardiac myocyte that determine susceptibility to enterovirus infection, an important cause of myocarditis and subsequent heart failure. Although interferon (IFN) generally plays a key role in innate immunity, ablation of IFN receptors has little or no effect on acute coxsackievirus B3 infection in the heart. Interestingly, gp130-cytokine-mediated stimulation of neonatal ventricular myocytes has a cytoprotective effect against virus infection in culture that can be inhibited by suppressors of cytokine signaling (SOCS)-3, a physiological inhibitor of gp130 signaling that does not affect IFN signaling. Therefore, we hypothesized that inhibition of gp130 signaling by SOCS3 would change cardiac myocyte susceptibility to virus infection without affecting IFN signaling. We generated cardiac-specific SOCS3 transgenic mice. Despite an intact IFN-mediated antiviral response in adult transgenic myocytes, there was a marked increase in susceptibility to viral infection in the SOCS3 transgenic mouse hearts. This indicated the presence of IFN-independent innate defense mechanisms within the cardiac myocyte. Subsequently, we demonstrated that cardiac-specific gp130-knockout mice also had increased susceptibility to viral infection. Furthermore, we demonstrated that the gp130-mediated increase in survival of infected myocytes occurred through a signal transducers and activators of transcription-3-dependent mechanism that did not affect viral replication. This was accompanied by a persistent expression of full-length dystrophin after coxsackievirus B3 infection. In addition, we found that both SOCS3 transgenic and gp130-deficient mice had a decrease in alpha-sarcoglycan. SOCS3-mediated regulation of gp130 signaling can affect susceptibility to viral infection in the heart. Increased cardiac cell survival through gp130-signal transducers and activators of transcription-3 signaling appears to play an important role in preserving nondividing cardiac myocytes until specific immune responses begin to clear the virus.

κ-opioid receptor stimulation inhibits cardiac hypertrophy induced by β 1-adrenoceptor stimulation in the rat

To test the hypothesis that κ-opioid receptor stimulation inhibits cardiac hypertrophy induced by β 1-adrenoceptor stimulation, we determined the effects of trans-(±)-3,4-dichloro- N-methyl- N-[2-(1-pyrrolidinyl)-cyclohexyl]-benzeneacetamide methanesulfonate salt (U50,488H), a selective κ-opioid receptor agonist, on cardiac hypertrophy induced by isoprenaline, a selective β-adrenoceptor agonist, in neonatal ventricular myocytes upon blockade of β 2-adrenoceptor. Hypertrophy of cardiomyocytes was determined by increases in (i) total protein content; (ii) [ 3H]leucine incorporation; and iii) cell size. 10 μmol/l isoprenaline increased all three parameters. The effects were abolished by 2 μmol/l propranolol, a β-adrenergic receptor antagonist, or 300 nmol/l CGP20712A, a β 1-adrenoceptor antagonist, but not by 100 nmol/l ICI118,551, a β 2-adrenoceptor antagonist. The effects were also abolished by Rp-cAMPs 100 μmol/l, a protein kinase A inhibitor and not by pertussis toxin 5 mg/l. The effects of isoprenaline in the presence or absence of ICI118,551 were also abolished by 1 μmol/l U50,488H. The inhibitory effects of U50,488H were abolished by 1 μmol/l nor-binaltorphimine, a selective κ-opioid receptor antagonist. U50,488H also abolished the increases in the amplitude and frequency of the spontaneous intracellular Ca 2+ transient induced by 10 μmol/l isoprenaline in the presence or absence of ICI118,551, an effect also abolished by nor-binaltorphimine. In conclusion the results show that κ-opioid receptor stimulation abolished both the cardiac hypertrophy and enhanced amplitude and frequency of the spontaneous intracellular Ca 2+ transient induced by β 1-adrenoceptor stimulation.

Calcium signalling during excitation-contraction coupling in mammalian atrial myocytes

Atrial cardiomyocytes make an important contribution to the refilling of ventricles with blood, which enhances the subsequent ejection of blood from the heart. The dependence of cardiac function on the contribution of atria becomes increasingly important with age and exercise. We know much less about the calcium signals that link electrical depolarisation to contraction within atrial myocytes in comparison with ventricular myocytes. Nevertheless, recent work has shed new light on calcium signalling in atrial cells. At an ultrastructural level, atrial and ventricular myocytes have many similarities. However, a few key structural differences, in particular the lack of transverse tubules (;T-tubules') in atrial myocytes, make these two cell types display vastly different calcium patterns in response to depolarisation. The lack of T-tubules in atrial myocytes means that depolarisation provokes calcium signals that largely originate around the periphery of the cells. To engage the contractile machinery, the calcium signal must propagate centripetally deeper into the cells. This inward movement of calcium is ultimately controlled by hormones that can promote or decrease calcium release within the myocytes. Enhanced centripetal movement of calcium in atrial myocytes leads to increased contraction and a more substantial contribution to blood pumping. The calcium signalling paradigm within atrial cells applies to other cardiac cell types that also do not express T-tubules, such as neonatal ventricular myocytes, and Purkinje cells that aid in the spread of electrical depolarisation. Furthermore, during heart failure ventricular myocytes progressively lose their regular T-tubule expression, and their pattern of response resembles that of atrial cells.

Essential role of Rho/ROCK-dependent processes and actin dynamics in mediating leptin-induced hypertrophy in rat neonatal ventricular myocytes

Obesity is associated with increased leptin production, which may contribute to cardiac hypertrophy. Although leptin has been shown to produce cardiomyocyte hypertrophy, its mechanism of action is far from clear. Rho proteins have been suggested as major contributors to cardiac hypertrophy, although their potential role in mediating the effect of leptin has not been studied. We determined the role of Rho and Rho-associated kinase (ROCK) as mediators of leptin-induced cell hypertrophy in cultured neonatal rat ventricular myocytes. Leptin (3.1 nmol/L) significantly increased cell surface area by 32+/-5% and leucine incorporation by 43 +/- 7%. These effects were associated with significant activation of RhoA to 450 +/- 40% of pre-leptin levels that was attenuated by pretreatment with an anti-leptin receptor (anti-OBR) antibody (166 ng/mL) to 120 +/- 20% of control values. Both the RhoA inhibitor C3 exoenzyme and ROCK inhibitor Y-27632 potently attenuated leptin-induced increased cell surface area and leucine incorporation. The hypertrophic effect of leptin was associated with an increase in phosphorylation of the actin binding protein cofilin to 290 +/- 20% of control values. In addition, the increase in polymerization of actin, as reflected by a decrease in the G/F-actin ratio, was significantly inhibited by both the anti-OBR antibody and Y-27632. Leptin-induced hypertrophy was also attenuated by disruption of actin filaments with 50 nmol/L latrunculin B. RhoA pathway inhibitors and latrunculin B also both attenuated leptin-induced ERK1/2 and p38 activation. Our results indicate that the activation of Rho and actin dynamics play a pivotal role in leptin signaling leading to the development of cardiomyocyte hypertrophy.

Myocyte Enhancer Factors 2A and 2C Induce Dilated Cardiomyopathy in Transgenic Mice

Cardiac hypertrophy and dilation are mediated by neuroendocrine factors and/or mitogens as well as through internal stretch- and stress-sensitive signaling pathways, which in turn transduce alterations in cardiac gene expression through specific signaling pathways. The transcription factor family known as myocyte enhancer factor 2 (MEF2) has been implicated as a signal-responsive mediator of the cardiac transcriptional program. For example, known hypertrophic signaling pathways that utilize calcineurin, calmodulin-dependent protein kinase, and MAPKs can each affect MEF2 activity. Here we demonstrate that MEF2 transcription factors induced dilated cardiomyopathy and lengthening of myocytes. Specifically, multiple transgenic mouse lines with cardiac-specific overexpression of MEF2A or MEF2C presented with cardiomyopathy at base line or were predisposed to more fulminant disease following pressure overload stimulation. The cardiomyopathic response associated with MEF2A and MEF2C was not further altered by activated calcineurin, suggesting that MEF2 functions independently of calcineurin in this response. In cultured cardiomyocytes, MEF2A, MEF2C, and MEF2-VP16 overexpression induced sarcomeric disorganization and focal elongation. Mechanistically, MEF2A and MEF2C each programmed similar profiles of altered gene expression in the heart that included extracellular matrix remodeling, ion handling, and metabolic genes. Indeed, adenoviral transfection of cultured cardiomyocytes with MEF2A or of myocytes from the hearts of MEF2A transgenic adult mice showed reduced transient outward K(+) currents, consistent with the alterations in gene expression observed in transgenic mice and partially suggesting a proximal mechanism underlying MEF2-dependent cardiomyopathy.

Selective Loss of Fine Tuning of Gq/11 Signaling by RGS2 Protein Exacerbates Cardiomyocyte Hypertrophy

Alterations in cardiac G protein-mediated signaling, most prominently G(q/11) signaling, are centrally involved in hypertrophy and heart failure development. Several RGS proteins that can act as negative regulators of G protein signaling are expressed in the heart, but their functional roles are still poorly understood. RGS expression changes have been described in hypertrophic and failing hearts. In this study, we report a marked decrease in RGS2 (but not other major cardiac RGS proteins (RGS3-RGS5)) that occurs prior to hypertrophy development in different models with enhanced G(q/11) signaling (transgenic expression of activated Galpha(q)(*) and pressure overload due to aortic constriction). To assess functional consequences of selective down-regulation of endogenous RGS2, we identified targeting sequences for effective RGS2 RNA interference and used lipid-based transfection to achieve uptake of fluorescently labeled RGS2 small interfering RNA in >90% of neonatal and adult ventricular myocytes. Endogenous RGS2 expression was dose-dependently suppressed (up to 90%) with no major change in RGS3-RGS5. RGS2 knockdown increased phenylephrine- and endothelin-1-induced phospholipase Cbeta stimulation in both cell types and exacerbated the hypertrophic effect (increase in cell size and radiolabeled protein) in neonatal myocytes, with no major change in G(q/11)-mediated ERK1/2, p38, or JNK activation. Taken together, this study demonstrates that endogenous RGS2 exerts functionally important inhibitory restraint on G(q/11)-mediated phospholipase Cbeta activation and hypertrophy in ventricular myocytes. Our findings point toward a potential pathophysiological role of loss of fine tuning due to selective RGS2 down-regulation in G(q/11)-mediated remodeling. Furthermore, this study shows the feasibility of effective RNA interference in cardiomyocytes using lipid-based small interfering RNA transfection.