Role of protein kinase C-epsilon in hypertrophy of cultured neonatal rat ventricular myocytes

Using adenovirus (Adv)-mediated overexpression of constitutively active (ca) and dominant-negative (dn) mutants, we examined whether protein kinase C (PKC)-epsilon, the major novel PKC isoenzyme expressed in the adult heart, was necessary and/or sufficient to induce specific aspects of the hypertrophic phenotype in low-density, neonatal rat ventricular myocytes (NRVM) in serum-free culture. Adv-caPKC-epsilon did not increase cell surface area or the total protein-to-DNA ratio. However, cell shape was markedly affected, as evidenced by a 67% increase in the cell length-to-width ratio and a 17% increase in the perimeter-to-area ratio. Adv-caPKC-epsilon also increased atrial natriuretic factor (ANF) and beta-myosin heavy chain (MHC) mRNA levels 2.5 +/- 0.3- and 2.1 +/- 0.2-fold, respectively, compared with NRVM infected with an empty, parent vector (P < 0.05 for both). Conversely, Adv-dnPKC-epsilon did not block endothelin-induced increases in cell surface area, the total protein-to-DNA ratio, or upregulation of beta-MHC and ANF gene expression. However, the dominant-negative inhibitor markedly suppressed endothelin-induced extracellular signal-regulated kinase (ERK) 1/2 activation. Taken together, these results indicate that caPKC-epsilon overexpression alters cell geometry, producing cellular elongation and remodeling without a significant, overall increase in cell surface area or total protein accumulation. Furthermore, PKC-epsilon activation and downstream signaling via the ERK cascade may not be necessary for cell growth, protein accumulation, and gene expression changes induced by endothelin.     

Chronotropic response of cultured neonatal rat ventricular myocytes to short-term fluid shear

Ventricular myocytes are continuously exposed to fluid shear in vivo by relative movement of laminar sheets and adjacent cells. Preliminary observations have shown that neonatal myocytes respond to fluid shear by increasing their beating rate, which could have an arrhythmogenic effect under elevated shear conditions. The objective of this study is to investigate the characteristics of the fluid shear response in cultured myocytes and to study selected potential mechanisms. Cultured neonatal rat ventricular myocytes that were spontaneously beating were subjected to low shear rates (5-50/s) in a fluid flow chamber using standard culture medium. The beating rate was measured from digital microscopic recordings. The myocytes reacted to low shear rates by a graded and reversible increase in their spontaneous beating rate of up to 500%. The response to shear was substantially attenuated in the presence of the beta-adrenergic agonist isoproterenol (by 86+/-8%), as well as after incubation with integrin-blocking RGD peptides (by 92+/-8%). The results suggest that the beta-adrenergic signaling pathway and integrin activation, which are known to interact, may play an important role in the response mechanism.     

Nitric oxide-induced cardioprotection in cultured rat ventricular myocytes

The aim of this study was to investigate the role of nitric oxide (NO) in a cellular model of early preconditioning (PC) in cultured neonatal rat ventricular myocytes. Cardiomyocytes "preconditioned" with 90 min of stimulated ischemia (SI) followed by 30 min reoxygenation in normal culture conditions were protected against subsequent 6 h of SI. PC was blocked by N(G)-monomethyl-L-arginine monoacetate but not by dexamethasone pretreatment. Inducible nitric oxide synthase (NOS) protein expression was not detected during PC ischemia. Pretreatment (90 min) with the NO donor S-nitroso-N-acetyl-L,L-penicillamine (SNAP) mimicked PC, resulting in significant protection. SNAP-triggered protection was completely abolished by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) but was unaffected by chelerythrine or the presence of glibenclamide and 5-hydroxydecanoate. With the use of RIA, SNAP treatment increased cGMP levels, which were blocked by ODQ. Hence, NO is implicated as a trigger in this model of early PC via activation of a constitutive NOS isoform. After exposure to SNAP, the mechanism of cardioprotection is cGMP dependent but independent of protein kinase C or ATP-sensitive K(+) channels. This differs from the proposed mechanism of NO-induced cardioprotection in late PC.     

收缩活动促进新生大鼠培养心室肌细胞的^3H—亮氨酸...

To determine whether contraction could influence cell growth, the rate of protein synthesis (3H-leucine incorporation) and cell diameter and volume were measured in cultured neonatal rat cardiac myocytes beating spontaneously or arrested by high potassium. In medium supplemented with 10% calf serum, the 3H-leucine incorporation for 24 h in contracting myocytes (CMC) was significantly higher by 14.2% than that in quiescent myocytes (QMC), i.e. 1,229 +/- 29 cpm/10(5) cells vs. 1,076 +/- 60 cpm/10(5) cells (P < 0.01, n = 5 for each group). The cell diameter and cell volume in QMC group were respectively 15.14 +/- 0.42 microns and 1,842 +/- 123 microns3, while in the CMC group the corresponding figures reached to 16.82 +/- 0.64 microns3 and 2,495 +/- 210 microns3, increased by 11.1% and 35.5% respectively (P < 0.01, n = 6 for each group). With prolongation of culture time, the differences in these parameters between CMC and QMC became even more significant. In all these experiments, there was no significant difference in cell number between the two groups (P > 0.05). It is concluded that contraction per se can accelerate protein synthesis and cell growth in neonatal rat ventricular myocardium.     

Plasmalogen content and distribution in the sarcolemma of cultured neonatal rat myocytes

Phospholipids are believed to play an important role in pathology and physiology of the myocardium. Because of the distinct physico-chemical properties of plasmalogens we studied the plasmalogen content and distribution in the sarcolemma of cultured rat myocytes. Treatment with phospholipase A2 degraded all glycerophospholipids in the outer monolayer. The hydrolysis products were analyzed for plasmalogen content. It is shown that the inner sarcolemmal leaflet is highly enriched in phosphatidylcholine and ethanolamine plasmalogen. This distribution of the plasmalogens might affect bilayer stability and thereby be involved in the destruction of the sarcolemma upon ischemia and reperfusion.     

血管紧张素II诱导培养的成年大鼠心肌细胞凋亡

研究血管紧张素Ⅱ（ＡｎｇⅡ）诱导培养的成年大鼠心肌细胞（ＡＲＶＭｓ）凋亡。酶灌流消化法分离培养ＡＲＶＭｓ,不同处理后,光镜观察形态改变,琼脂糖凝胶电泳定性分析ＤＮＡ降解程度。结果发现培养的ＡＲＶＭｓ经ＡｎｇⅡ１０μｍｏｌ／Ｌ处理４８ｈ后,大部分细胞变圆,胞浆浓缩;电泳显示核酸断裂片段“梯形”结构,上述改变在７２ｈ更为明显。上述作用可被氯沙坦、维拉帕米和ｓｔａｕｒｏｓｐｏｒｉｎｅ所取消。这表明Ａｎｇ     

Aldosterone modulates I(f) current through gene expression in cultured neonatal rat ventricular myocytes

Mineralocorticoid receptor (MR) antagonists decrease the incidence of sudden cardiac death in patients with heart failure, as has been reported in two clinical trials (Randomized Aldactone Evaluation Study and Eplerenone Post-Acute Myocardial Infarction Heart Failure Efficacy and Survival Study). Aldosterone has been shown to increase the propensity to arrhythmias by changing the expression or function of various ion channels. In this study, we investigate the effect of aldosterone on the expression of hyperpolarization-activated current (I(f)) channels in cultured neonatal rat ventricular myocytes, using the whole cell patch-clamp technique, real-time PCR, and Western blotting. Incubation with 10 nM aldosterone for 17-24 h significantly accelerates the rate of spontaneous beating by increasing diastolic depolarization. I(f) current elicited by hyperpolarization from -50 to -130 mV significantly increases aldosterone by 10 nM (by 1.9-fold). Exposure to aldosterone for 1.5 h increases hyperpolarization-activated cyclic nucleotide-gated (HCN) 2 mRNA by 26.3% and HCN4 mRNA by 47.2%, whereas HCN1 mRNA expression remains unaffected. Aldosterone (24-h incubation) increases the expression of HCN2 protein (by 60.0%) and HCN4 protein (by 84.8%), but not HCN1 protein. MR antagonists (1 microM eplerenone or 0.1 microM spironolactone) abolish the increase of I(f) channel expression (currents, mRNA, and protein levels) by 10 nM aldosterone. In contrast, 1 microM aldosterone downregulated I(f) channel gene expression. Glucocorticoid receptor antagonist (100 nM RU-38486) did not affect the increase of I(f) current by 10 nM aldosterone. These findings suggest that aldosterone in physiological concentrations upregulates I(f) channel gene expression by MR activation in cardiac myocytes and may increase excitability, which may have a potential proarrhythmic bearing under pathophysiological conditions.     

Protein kinase C-alpha-induced hypertrophy of neonatal rat ventricular myocytes

Protein kinase C (PKC) isoenzymes play a critical role in cardiomyocyte hypertrophy. At least three different phorbol ester-sensitive PKC isoenzymes are expressed in neonatal rat ventricular myocytes (NRVMs): PKC-alpha, -delta, and -epsilon. Using replication-defective adenoviruses (AdVs) that express wild-type (WT) and dominant-negative (DN) PKC-alpha together with phorbol myristate acetate (PMA), which is a hypertrophic agonist and activator of all three PKC isoenzymes, we studied the role of PKC-alpha in signaling-specific aspects of the hypertrophic phenotype. PMA induced nuclear translocation of endogenous and AdV-WT PKC-alpha in NRVMs. WT PKC-alpha overexpression increased protein synthesis and the protein-to-DNA (P/D) ratio but did not affect cell surface area (CSA) or cell shape compared with uninfected or control AdV beta-galactosidase (AdV betagal)-infected cells. PMA-treated uninfected cells displayed increased protein synthesis, P/D ratio, and CSA and elongated morphology. PMA did not further enhance protein synthesis or P/D ratio in AdV-WT PKC-alpha-infected cells. To assess the requirement of PKC-alpha for these PMA-induced changes, AdV-DN PKC-alpha or AdV betagal-infected NRVMs were stimulated with PMA. Without PMA, AdV-DN PKC-alpha had no effects on protein synthesis, P/D ratio, CSA, or shape vs. AdV betagal-infected NRVMs. PMA increased protein synthesis, P/D ratio, and CSA in AdV betagal-infected cells, but these parameters were significantly reduced in PMA-stimulated AdV-DN PKC-alpha-infected NRVMs. Overexpression of DN PKC-alpha enhanced PMA-induced cell elongation. Neither WT PKC-alpha nor DN PKC-alpha affected atrial natriuretic factor gene expression. Insulin-like growth factor-1 also induced nuclear translocation of endogenous PKC-alpha. PMA but not WT PKC-alpha overexpression induced ERK1/2 activation. However, AdV-DN PKC-alpha partially blocked PMA-induced ERK activation. Thus PKC-alpha is necessary for certain aspects of PMA-induced NRVM hypertrophy.     

Activation of focal adhesion kinase by protein kinase C epsilon in neonatal rat ventricular myocytes

Focal adhesion kinase (FAK) is a nonreceptor protein tyrosine kinase critical for both cardiomyocyte survival and sarcomeric assembly during endothelin (ET)-induced cardiomyocyte hypertrophy. ET-induced FAK activation requires upstream activation of one or more isoenzymes of protein kinase C (PKC). Therefore, with the use of replication-defective adenoviruses (Adv) to overexpress constitutively active (ca) and dominant negative (dn) mutants of PKCs, we examined which PKC isoenzymes are necessary for FAK activation and which downstream signaling components are involved. FAK activation was assessed by Western blot analysis with an antibody specific for FAK autophosphorylated at Y397 (Y397pFAK). ET (10 nmol/l; 2-30 min) resulted in the time-dependent activation of FAK which was inhibited by chelerythrine (5 micromol/l; 1 h pretreatment). Adv-caPKC epsilon, but not Adv-caPKC delta, activated FAK compared with a control Adv encoding beta-galactosidase. Conversely, Adv-dnPKC epsilon inhibited ET-induced FAK activation. Y-27632 (10 micromol/l; 1 h pretreatment), an inhibitor of Rho-associated coiled-coil-containing protein kinases (ROCK), prevented ET- and caPKC epsilon-induced FAK activation as well as cofilin phosphorylation. Pretreatment with cytochalasin D (1 micromol/l, 1 h pretreatment) also inhibited ET-induced Y397pFAK and cofilin phosphorylation and caPKC epsilon-induced Y397pFAK. Neither inhibitor, however, interfered with ET-induced ERK1/2 activation. Finally, PP2 (50 micromol/l; 1 h pretreatment), a highly selective Src inhibitor, did not alter basal or ET-induced Y397pFAK. PP2 did, however, reduce basal and ET-induced phosphorylation of other sites on FAK, namely, Y576, Y577, Y861, and Y925. We conclude that the ET-induced signal transduction pathway resulting in downstream Y397pFAK is partially dependent on PKC epsilon, ROCK, cofilin, and assembled actin filaments, but not ERK1/2 or Src.     

Substrate stiffness affects the functional maturation of neonatal rat ventricular myocytes

Cardiac cells mature in the first postnatal week, concurrent with altered extracellular mechanical properties. To investigate the effects of extracellular stiffness on cardiomyocyte maturation, we plated neonatal rat ventricular myocytes for 7 days on collagen-coated polyacrylamide gels with varying elastic moduli. Cells on 10 kPa substrates developed aligned sarcomeres, whereas cells on stiffer substrates had unaligned sarcomeres and stress fibers, which are not observed in vivo. We found that cells generated greater mechanical force on gels with stiffness similar to the native myocardium, 10 kPa, than on stiffer or softer substrates. Cardiomyocytes on 10 kPa gels also had the largest calcium transients, sarcoplasmic calcium stores, and sarcoplasmic/endoplasmic reticular calcium ATPase2a expression, but no difference in contractile protein. We hypothesized that inhibition of stress fiber formation might allow myocyte maturation on stiffer substrates. Treatment of maturing cardiomyocytes with hydroxyfasudil, an inhibitor of RhoA kinase and stress fiber-formation, resulted in enhanced force generation on the stiffest gels. We conclude that extracellular stiffness near that of native myocardium significantly enhances neonatal rat ventricular myocytes maturation. Deviations from ideal stiffness result in lower expression of sarcoplasmic/endoplasmic reticular calcium ATPase, less stored calcium, smaller calcium transients, and lower force. On very stiff substrates, this adaptation seems to involve RhoA kinase.     

Hydrogen peroxide predisposes neonatal rat ventricular myocytes to Fas-mediated apoptosis

Neonatal rat ventricular myocytes (NRVM) grown in normoxic environment are not susceptible to Fas-induced apoptosis. In the present work, we tested the hypothesis that free radical injury represented by transient exposure to H2O2 sensitizes NRVM to Fas-mediated apoptosis. NRVM were treated with H2O2 (0.5 mM) for 2-4 h and thereafter exposed for 7 h to recombinant Fas ligand (rFasL, 10 ng/ml) plus an enhancing antibody (1 microg/ml). Apoptotic cardiomyocytes were counted and apoptosis-related proteins were measured by Western blot. H2O2 alone induced apoptosis (9.4+/-1.0%) that was preceded by activation of caspases-8 and -3, and PARP degradation. Incubation of NRVM with H2O2, followed by exposure to rFasL, increased the apoptotic index to 13.8+/-2.0%, but did not change caspase-8 or PARP activation. To investigate the mechanism underlying the sensitizing affect of H2O2 towards Fas-induced apoptosis, we studied the effects of H2O2 on the expression of key apoptosis signaling proteins. Incubation with H2O2 for 2-4 h decreased Fas expression and the expression of the Fas-related antiapoptotic proteins FLIP(L) and ARC, and increased the expression of the antiapoptotic proteins bcl-2 and xIAP. FADD expression was unchanged. Next, we tested the effect of H2O2 on the apoptosis-inducing, Fas-dependent Daxx-ASK-1-JUN kinase pathway. H2O2 dramatically increased ASK-1 expression and JUN kinase activation, but did not effect Daxx expression. Based on these findings we concluded that H2O2 sensitizes NRVM to Fas-mediated apoptosis by activating the Daxx-ASK-1-JUN kinase pathway, and by shifting the balance between proapoptotic and antiapoptotic proteins towards the former.     

Angiotensin II stimulates hypertrophic growth of cultured neonatal rat ventricular myocytes: roles of PKC and PGF2alpha

Angiotensin II (Ang II) has been shown to regulate growth in smooth muscle cells. Protein kinase C (PKC), which mediates Ang II action, has been implicated in myocardial cell hypertrophy. Acute pressure overload in the left ventricles has been demonstrated to produce prostaglandin F2 alpha (PGF2alpha) release. Therefore, we used cultured neonatal rat ventricular myocytes to study Ang II, PKC and PGF2alpha and their relationship to hypertrophy. The amount of PGF2alpha produced was determined by radioimmunoassay, Ang II-induced hypertrophy and PGF2alpha release. Pretreatment with 10(-6) M of PKC inhibitor, 1-(5-isoquinolinesulfonyl-methyl) piperazine (H7), blocked Ang II-induced hypertrophy and PGF2alpha release. In neonatal rat ventricular myocytes that were treated with either Ang II or PKC activator (Phorbol 12, 13, dibutyrate; PDBu), PKC enzyme assay showed PKC was translocated from the cytosol to the membrane which indicates activation. This suggests that PKC mediates, in part, Ang II-induced PGF2alpha release and hypertrophy. In summary, Ang II activates PKC, which causes PGF2alpha release and hypertrophy, and this PGF2alpha release and hypertrophy can be overcome by pretreatment with PKC inhibitor.     

Glucose uptake and glycolysis reduce hypoxia-induced apoptosis in cultured neonatal rat cardiac myocytes

Myocardial ischemia/reperfusion is well recognized as a major cause of apoptotic or necrotic cell death. Neonatal rat cardiac myocytes are intrinsically resistant to hypoxia-induced apoptosis, suggesting a protective role of energy-generating substrates. In the present report, a model of sustained hypoxia of primary cultures of Percoll-enriched neonatal rat cardiac myocytes was used to study specifically the modulatory role of extracellular glucose and other intermediary substrates of energy metabolism (pyruvate, lactate, propionate) as well as glycolytic inhibitors (2-deoxyglucose and iodoacetate) on the induction and maintenance of apoptosis. In the absence of glucose and other substrates, hypoxia (5% CO2 and 95% N2) caused apoptosis in 14% of cardiac myocytes at 3 h and in 22% of cells at 6-8 h of hypoxia, as revealed by sarcolemmal membrane blebbing, nuclear fragmentation, and chromatin condensation (Hoechst staining), terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining, and DNA laddering. This was accompanied by translocation of cytochrome c from the mitochondria to the cytosol and cleavage of the death substrate poly(ADP-ribose) polymerase. Cleavage of poly(ADP-ribose) polymerase and DNA laddering were prevented by preincubation with the caspase inhibitors benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (zVAD-fmk) and benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethyl ketone (zDEVD-fmk), indicating activation of caspases in the apoptotic process. The caspase inhibitor zDEVD-fmk also partially inhibited cytochrome c translocation. The presence of as little as 1 mM glucose, but not pyruvate, lactate, or propionate, before hypoxia prevented apoptosis. Inhibiting glycolysis by 2-deoxyglucose or iodoacetate, in the presence of glucose, reversed the protective effect of glucose. This study demonstrates that glycolysis of extracellular glucose, and not other metabolic pathways, protects cardiac myocytes from hypoxic injury and subsequent apoptosis.     

Myofibrillar and cytoskeletal assembly in neonatal rat cardiac myocytes cultured on laminin and collagen

Summary Neonatal rat cardiomyocytes were cultured on extracellular matrix components laminin and collagens I+III to examine effects of extracellular matrix on the assembly of cytoskeletal proteins during myofibrillogenesis. Myofibril assembly was visualized by immunofluorescence of marker proteins for myofibrils (f-actin for I bands and -actinin for Z bands), focal adhesions (vinculin), and transmembrane extracellular matrix receptors (₁ integrin) as cells spread for various times in culture. By 4 h in culture, f-actin appeared organized into nonstriated stress-fiber-like structures while -actinin, vinculin and ₁ integrin were localized in small streaks and beads. Subsequently, striated patterns were observed sequentially in the intracellular cytoskeletal components -actinin, vinculin, f-actin, and then in the transmembrane ₁ integrin receptor. These data support an earlier model for sarcomerogenesis in which stress-fiber-like structures serve as initial scaffolds upon which -actinin and then vinculin-containing costameres are assembled. This sequential and temporal assembly was the same on both laminin and collagens I+III. A quantitative difference, however, was apparent on the 2 matrices. There was an increased appearance on collagens I+III of rosettes (also called podosomes or cortical actin-containing bodies in other cells) which consisted of an f-actin core surrounded by -actinin, vinculin and ₁ integrin rims. The increased incidence of rosettes in neonatal myocytes on collagens I+III suggests that these cytoskeletal complexes are involved in recognition and interaction with extracellular matrix components.     

Protective effects of the potassium channel opener-diazoxide against injury in neonatal rat ventricular myocytes

The mitochondrial ATP-regulated potassium channel is present in the inner membrane of heart mitochondria. Similarly to plasma membrane K(ATP), the mitochondrial channel is inhibited by antidiabetic sulfonylureas and activated by potassium channel openers, such as diazoxide. In the present work, the cytoprotective properties of diazoxide on the H9c2 cardiac myoblast cell line and neonatal rat ventricular cardiomyocytes were analysed. It was observed that 100 micromol/l diazoxide protected neonatal rat ventricular cardiomyocytes, but not H9c2 myoblasts, against injury induced by hydrogen peroxide or simulated ischemia. Moreover, diazoxide prevented hydrogen peroxide-induced mitochondrial potential depolarisation in neonatal rat ventricular cardiomyocytes. Diazoxide, at the same time, did not affect the expression level of the anti-apoptotic protein bcl-2 in these cells. The protective effects of diazoxide were suppressed by 5-hydroxydecanoic acid, a potassium channel blocker. These observations suggest that activation of the mitochondrial ATP-regulated potassium channel plays an important role in protection of neonatal cardiomyocytes against injury.     

Chemical hypoxia triggers apoptosis of cultured neonatal rat cardiac myocytes: Modulation by calcium-regulated proteases and protein kinases

Myocardial infarctions and stroke arise primarily as a result of hypoxia/ischemia-induced cell injury. However, the molecular mechanism of cardiac cell death due to hypoxia has not been elucidated. We showed here that chemical hypoxia induced by 1 mM azide triggered apoptosis of isolated neonatal rat ventricular cardiac myocytes but had no effect on cardiac fibroblasts. The azide-induced cardiomyocyte apoptosis could be characterized by a reversible initiation phase (0-6 h after azide exposure) during which cytosolic ATP levels remained little affected. This was followed by an irreversible execution phase (12-18 h) exhibiting prominent internucleosomal DNA fragmentation, cell membrane leakage, mitochondrial dysfunction, and increased calpain messenger RNA. Blocking extracellular calcium influx or intracellular calcium release was each effective in suppressing myocyte apoptosis. Cell death was also found to be mediated by calcium sensitive signal transduction events based on the use of specific antagonists. Consistent with the induction of calpain expression during apoptosis, blocking de novo protein synthesis and calpain activity inhibited cell death. These regulatory features coupled with the ease of the cell system suggest that the myocyte apoptosis model described here should be useful in the study of events leading to the demise of the myocardium.     

Angiotensin II stimulates PGF(2 alpha)release in cultured neonatal rat ventricular myocytes via L-type calcium channels.

Angiotensin II (Ang II) has been shown to cause Prostaglandin F(2 alpha)(PGF(2 alpha)) release in neonatal rat ventricular myocytes and smooth muscle cells. In these cells, Ang II has also been shown to regulate growth. We used neonatal rat ventricular myocytes to investigate the role of calcium in maintenance of Ang II-induced PGF(2 alpha)release. The amount of PGF(2 alpha)produced was determined by radioimmunoassay. Ang II-induced PGF(2 alpha)release. Pretreatment of neonatal rat ventricular myocytes with different doses (10(-8)M, 10(-7)M, 10(-6)M and 10(-5)M) of diltiazm (voltage-sensitive L-type calcium channel blocker) produced significant inhibition in Ang II-induced PGF(2 alpha)release. Inhibition was first noted at 10(-8)M and was complete at 10(-6)M. Conversely, pretreatment of neonatal rat ventricular myocytes with different doses (10(-8)M, 10(-7)M, 10(-6)M and 10(-5)M) of calcium channel blockers (conotoxin; voltage-sensitive N-type calcium channel blocker or thapsigargin; intracellular calcium channel blocker) produced no changes in Ang II-induced PGF(2 alpha)release. These results strongly suggest that Ang II-induced PGF(2 alpha)release in neonatal rat ventricular myocytes is maintained, at least in part, via increase in extracellular calcium influx.     

Quantification of t-tubule area and protein distribution in rat cardiac ventricular myocytes

The transverse (t-) tubules of cardiac ventricular myocytes are invaginations of the surface membrane that form a complex network within the cell. Many of the key proteins involved in excitation–contraction coupling appear to be located predominantly at the t-tubule membrane. Despite their importance, the fraction of cell membrane within the t-tubules remains unclear: measurement of cell capacitance following detubulation suggests 32%, whereas optical measurements suggest up to 65%. We have, therefore, investigated the factors that may account for this discrepancy. Calculation of the combinations of t-tubule radius, length and density that produce t-tubular membrane fractions of 32% or 56% suggest that the true fraction is at the upper end of this range. Assessment of detubulation using confocal and electron microscopy suggests that incomplete detubulation can account for some, but not all of the difference. High cholesterol, and a consequent decrease in specific capacitance, in the t-tubule membrane, may also cause the t-tubule fraction calculated from the loss of capacitance following detubulation to be underestimated. Correcting for both of these factors results in an estimate that is still lower than that obtained from optical measurements suggesting either that optical methods overestimate the fraction of membrane in the t-tubules, or that other, unknown, factors, reduce the apparent fraction obtained by detubulation. A biophysically realistic computer model of a rat ventricular myocyte, incorporating a t-tubule network, is used to assess the effect of the altered estimates of t-tubular membrane fraction on the calculated distribution of ion flux pathways.     

Nitric oxide-mediated inhibition of DNA synthesis was attenuated in hypertrophied neonatal rat ventricular myocytes

The antiproliferative action of nitric oxide (NO) has been well established and increased production was reported in the infarcted rat heart. Concomitantly, increased DNA synthesis and hyperplasia of cardiac myocytes were documented in the hypertrophied myocardium. Despite these observations, the effect of NO on DNA synthesis in hypertrophied cardiac myocytes remains unexamined. Hypertrophy of the non-infarcted left ventricle (NILV) in 1-week post-MI rats was characterized by the increased prepro-ANP and reduction of alpha-myosin heavy chain protein expression. Inducible NO synthase was expressed in the NILV and associated with a concomitant attenuation of MnSuperoxide dismutase protein content. The latter data suggest that an antiproliferative action of NO in the hypertrophied NILV may proceed via either a cyclic GMP-dependent pathway and/or facilitated by a peroxynitrite-dependent mechanism. In neonatal rat ventricular myocytes (NNVM), the NO donor S-nitroso-N-acetyl-penicillamine (SNAP) promoted a dose-dependent attenuation of DNA synthesis via a cyclic GMP-independent pathway. The permeable superoxide dismutase mimetic and peroxynitrite scavenger MnTBAP abrogated SNAP-dependent attenuation of DNA synthesis in NNVM. MnTBAP failed to inhibit SNAP-mediated recruitment of extracellular signal regulated kinase 1/2 (ERK1/2) but partially attenuated p38 phosphorylation. In hypertrophied NNVM induced by norepinephrine, SNAP-mediated peroxynitrite-dependent inhibition of DNA synthesis, ERK1/2 and p38 phosphorylation were significantly attenuated. Collectively, these data suggest that despite a favourable environment for NO and subsequent peroxynitrite generation in the NILV, hypertrophied cardiac myocytes may be partially refractory to their biological actions.     

Extracellular signal-regulated protein kinase activation is required for the anti-hypertrophic effect of atrial natriuretic factor in neonatal rat ventricular myocytes.

Atrial natriuretic factor (ANF) inhibits proliferation in non-myocardial cells and is thought to be anti-hypertrophic in cardiomyocytes. We investigated the possibility that the anti-hypertrophic actions of ANF involved the mitogen-activated protein kinase signal transduction cascade. Cultured neonatal rat ventricular myocytes treated for 48 h with the alpha(1)-adrenergic agonist phenylephrine (PE) had an 80% increase in cross-sectional area (CSA). ANF alone had no effect but inhibited PE-induced increases in CSA by approximately 50%. The mitogen-activated protein kinase/ERK kinase (MEK) inhibitor PD098059 minimally inhibited PE-induced increases in CSA, but it completely abolished ANF-induced inhibition of PE-induced increases. ANF-induced extracellular signal-regulated protein kinase (ERK) nuclear translocation was also eliminated by PD098059. ANF treatment caused MEK phosphorylation and activation but failed to activate any of the Raf isoforms. ANF induced a rapid increase in ERK phosphorylation and in vitro kinase activity. PE also increased ERK activity, and the combined effect of ANF and PE appeared to be additive. ANF-induced ERK phosphorylation was eliminated by PD098059. ANF induced minimal phosphorylation of JNK or p38, indicating that its effect on ERK was specific. ANF-induced activation of ERK was mimicked by cGMP analogs, suggesting that ANF-induced ERK activation involves the guanylyl cyclase activity of the ANF receptor. These data suggest that there is an important linkage between cGMP signaling and the mitogen-activated protein kinase cascade and that selective ANF activation of ERK is required for the anti-hypertrophic action of ANF. Thus, ANF expression might function as the natural defense of the heart against maladaptive hypertrophy through its ability to activate ERK.