Sarco/endoplasmic reticulum calcium ATPase-2 expression is regulated by ATF6 during the endoplasmic reticulum stress response: intracellular signaling of calcium stress in a cardiac myocyte model system.

The recently described transcription factor, ATF6, mediates the expression of proteins that compensate for potentially stressful changes in the endoplasmic reticulum (ER), such as reduced ER calcium. In cardiac myocytes the maintenance of optimal calcium levels in the sarcoplasmic reticulum (SR), a specialized form of the ER, is required for proper contractility. The present study investigated the hypothesis that ATF6 serves as a regulator of the expression of sarco/endoplasmic reticulum calcium ATPase-2 (SERCA2), a protein that transports calcium into the SR from the cytoplasm. Depletion of SR calcium in cultured cardiac myocytes fostered the translocation of ATF6 from the ER to the nucleus, activated the promoter for rat SERCA2, and led to increased levels of SERCA2 protein. SERCA2 promoter induction by calcium depletion was partially blocked by dominant-negative ATF6, whereas constitutively activated ATF6 led to SERCA2 promoter activation. Mutation analyses identified a promoter-proximal ER stress-response element in the rat SERCA2 gene that was required for maximal induction by ATF6 and calcium depletion. Although this element was shown to be responsible for all of the effects of ATF6 on SERCA2 promoter activation, it was responsible for only a portion of the effects of calcium depletion. Thus, SERCA2 induction in response to calcium depletion appears to be a potentially physiologically important compensatory response to this stress that involves intracellular signaling pathways that are both dependent and independent of ATF6.

Ras reduces L-type calcium channel current in cardiac myocytes. Corrective effects of L-channels and SERCA2 on [Ca(2+)](i) regulation and cell morphology.

Heart failure is associated with dysregulation of intracellular calcium ([Ca(2+)](i)), reduction in myofibrils, and increased activation of Ras, a regulator of signal-transduction pathways. To evaluate the potential effects of Ras on [Ca(2+)](i), we expressed constitutively active Ras (Ha-Ras(V12)) in cardiac myocytes and monitored [Ca(2+)](i) via fluorescence and electrophysiological techniques. Ha-Ras(V12) reduced the magnitude of the contractile calcium transients. Unexpectedly, however, calcium loading of the sarcoplasmic reticulum was increased, suggesting that Ha-Ras(V12) introduces a defect in excitation-calcium release coupling. Consistent with this idea, L-channel calcium currents were reduced by Ha-Ras(V12), which also downregulated the activity of the L-channel gene promoter. Coexpression of L-channels and SERCA2 largely corrected Ha-Ras(V12)-induced dysregulation of [Ca(2+)](i). Furthermore, whereas Ha-Ras(V12) downregulated myofibrils, this effect was blocked by coexpression of L-channels. These results suggest that Ras downregulates L-channel expression, which may play a pathophysiological role in cardiac disease.

Expression and characterization of Edg-1 receptors in rat cardiomyocytes: calcium deregulation in response to sphingosine 1-phosphate.

Recent evidence indicates that sphingolipids are produced by the heart during hypoxic stress and by blood platelets during thrombus formation. It is therefore possible that sphingolipids may influence heart cell function by interacting with G-protein-coupled receptors of the Edg family. In the present study, it was found that sphingosine 1-phosphate (Sph1P), the prototypical ligand for Edg receptors, produced calcium overload in rat cardiomyocytes. The cDNA for Edg-1 was cloned from rat cardiomyocytes and, when transfected in an antisense orientation, effectively blocked Edg-1 protein expression and reduced the Sph1P-mediated calcium deregulation. Taken together, these results demonstrate that cardiomyocytes express an extracellular lipid-sensitive receptorsystem that can respond to sphingolipid mediators. Because the major source of Sph1P is from blood platelets, we speculate that Edg-mediated Sph1P negative inotropic and cardiotoxic effects may play important roles in acute myocardial ischemia where Sph1P levels are probably elevated in response to thrombus.

p38 MAPK and NF-kappa B collaborate to induce interleukin-6 gene expression and release. Evidence for a cytoprotective autocrine signaling pathway in a cardiac myocyte model system.

In cardiac myocytes, the stimulation of p38 MAPK by the MAPKK, MKK6, activates the transcription factor, NF-kappaB, and protects cells from apoptosis. In the present study in primary neonatal rat cardiac myocytes, constitutively active MKK6, MKK6(Glu), bound to IkappaB kinase (IKK)-beta and stimulated its abilities to phosphorylate IkappaB and to activate NF-kappaB. MKK6(Glu) induced NF-kappaB-dependent interleukin (IL)-6 transcription and IL-6 release in a p38-dependent manner. IL-6 protected myocardial cells against apoptosis. Like IL-6, TNF-alpha, which activates both NF-kappaB and p38, also induced p38-dependent IL-6 expression and release and protected myocytes from apoptotis. While TNF-alpha was relatively ineffective, IL-6 activated myocardial cell STAT3 by about 8-fold, indicating a probable role for this transcription factor in IL-6-mediated protection from apoptosis. TNF-alpha-mediated IL-6 induction was inhibited by a kinase-inactive form of the MAPKKK, TGF-beta activated protein kinase (Tak1), which is known to activate p38 and NF-kappaB in other cell types. Thus, by stimulating both p38 and NF-kappaB, Tak1-activating cytokines, like TNF-alpha, can induce IL-6 expression and release. Moreover, the myocyte-derived IL-6 may then function in an autocrine and/or paracrine fashion to augment myocardial cell survival during stresses that activate p38.

Effects of mutant and antisense RNA of phospholamban on SR Ca(2+)-ATPase activity and cardiac myocyte contractility.

BACKGROUND: The delayed cardiac relaxation in failing hearts has been attributed to a reduced activity of sarcoplasmic reticulum Ca(2+)-ATPase (SERCA2). Phospholamban (PLB) inhibits SERCA2 activity and is therefore a potential target to improve the cardiac performance in heart failure. METHODS AND RESULTS: Mutants of PLB (Adv/mPLB) or antisense RNA of PLB (Adv/asPLB) was expressed in cardiac myocytes by recombinant adenovirus, and their effects on SERCA2 activity and myocyte contractility were studied. One mPLB, K3E/R14E, pentamerized with endogenous PLB in neonatal myocytes and resulted in a 45% increase in the affinity of SERCA2 for Ca(2+) and 27% faster diastolic Ca(2+) decline as determined by SR (45)Ca uptake assays and by indo 1-facilitated Ca(2+) transient measurement, respectively. Edge-detection analysis of adult myocyte contractility showed a 74% increase in fractional shortening, accompanied by 115% increase in velocity of relengthening and 25% decrease in time to half-maximal relengthening. In parallel, infection of neonatal cardiac myocytes by Adv/asPLB decreased the endogenous PLB level by 54%, which was associated with a 35% increase in Ca(2+) affinity of SERCA2 and 21% faster diastolic Ca(2+) decline. However, in adult cardiac myocytes, Adv/asPLB failed to significantly alter the endogenous PLB level, the SERCA2 activity, or most of the contractile parameters. CONCLUSIONS: K3E/R14E is a dominant negative mutant of PLB that disrupts the structural integrity and function of the endogenous PLB and consequently enhances SERCA2 activity and myocyte contractility. In neonatal myocytes, the decrease in steady-state abundance of PLB by asPLB also leads to increased SERCA2 activity.

p38 Mitogen-activated protein kinase mediates the transcriptional induction of the atrial natriuretic factor gene through a serum response element. A potential role for the transcription factor ATF6.

In various cell types certain stresses can stimulate p38 mitogen-activated protein kinase (p38 MAPK), leading to the transcriptional activation of genes that contribute to appropriate compensatory responses. In this report the mechanism of p38-activated transcription was studied in cardiac myocytes where this MAPK is a key regulator of the cell growth and the cardiac-specific gene induction that occurs in response to potentially stressful stimuli. In the cardiac atrial natriuretic factor (ANF) gene, a promoter-proximal serum response element (SRE), which binds serum response factor (SRF), was shown to be critical for ANF induction in primary cardiac myocytes transfected with the selective p38 MAPK activator, MKK6 (Glu). This ANF SRE does not possess sequences typically required for the binding of the Ets-related ternary complex factors (TCFs), such as Elk-1, indicating that p38-mediated induction through this element may take place independently of such TCFs. Although p38 did not phosphorylate SRF in vitro, it efficiently phosphorylated ATF6, a newly discovered SRF-binding protein that is believed to serve as a co-activator of SRF-inducible transcription at SREs. Expression of an ATF6 antisense RNA blocked p38-mediated ANF induction through the ANF SRE. Moreover, when fused to the Gal4 DNA-binding domain, an N-terminal 273-amino acid fragment of ATF6 was sufficient to support trans-activation of Gal4/luciferase expression in response to p38 but not the other stress kinase, N-terminal Jun kinase (JNK); p38-activating cardiac growth promoters also stimulated ATF6 trans-activation. These results indicate that through ATF6, p38 can augment SRE-mediated transcription independently of Ets-related TCFs, representing a novel mechanism of SRF-dependent transcription by MAP kinases.

The Raf-MEK-ERK cascade represents a common pathway for alteration of intracellular calcium by Ras and protein kinase C in cardiac myocytes.

Ras and protein kinase C (PKC), which regulate the Raf-MEK-ERK cascade, may participate in the development of cardiac hypertrophy, a condition characterized by diminished and prolonged contractile calcium transients. To directly examine the influence of this pathway on intracellular calcium ([Ca2+]i), cardiac myocytes were cotransfected with effectors of this pathway and with green fluorescent protein, which allowed the living transfected myocytes to be identified and examined for [Ca2+]i via indo-1. Transfection with constitutively active Ras (Ha-RasV12) increased cell size, decreased expression of the myofibrils and the calcium-regulatory enzyme SERCA2, and reduced the magnitude and prolonged the decay phase of the contractile [Ca2+]i transients. Similar effects on [Ca2+]i were obtained with Ha-RasV12S35, a Ras mutant that selectively couples to Raf, and with constitutively active Raf. In contrast, Ha-RasV12C40, a Ras mutant that activates the phosphatidylinositol 3-kinase pathway, had a lesser effect. The PKC-activating phorbol ester, phorbol 12-myristate 13-acetate, also prolonged the contractile [Ca2+]i transients. Cotransfection with dnMEK inhibited the effects of Ha-RasV12, Raf, and phorbol 12-myristate 13-acetate on [Ca2+]i. The effects of Ha-RasV12 and Raf on [Ca2+]i were also counteracted by SERCA2 overexpression. Both Ras and PKC may thus regulate cardiac [Ca2+]i via the Raf-MEK-ERK cascade, and this pathway may represent a critical determinant of cardiac physiological function.

MKK6 activates myocardial cell NF-kappaB and inhibits apoptosis in a p38 mitogen-activated protein kinase-dependent manner.

In cardiac myocytes the stimulation of p38 mitogen-activated protein kinase activates a hypertrophic growth program and the induction of the cardiac-specific genes associated with this program. This study focused on determining whether these novel growth-promoting effects are accompanied by the p38-mediated inhibition of apoptosis, and if so, what signaling pathways might be responsible. Primary neonatal rat ventricular myocytes were driven into apoptosis by treatments known to induce apoptosis in other cell types, e.g. incubation with anisomycin or overexpression constitutively active MEKK-1 (MEKK-1COOH), a protein that strongly activates extracellular signal-regulated kinase and N-terminal c-Jun kinase, but not p38. Overexpression of constitutively active MKK6, MKK6 (Glu), which selectively activates p38 in cardiac myocytes, protected cells from either anisomycin- or MEKK-1COOH-induced apoptosis. This protection was blocked by SB 203580, a selective p38 inhibitor. MKK6 (Glu) also activated transcription mediated by NF-kappaB, a factor which protects other cell types from apoptosis. The activation of NF-kappaB and the protection from apoptosis mediated by MKK6 (Glu) were both blocked by SB 203580. Interestingly, overexpression of a mutant form of I-kappaBalpha, which inhibits nuclear translocation of NF-kappaB, completely blocked MKK6 (Glu)-activated NF-kappaB but had little effect on MKK6s anti-apoptotic effects. These findings suggest that, in part, the overexpression of MKK6 (Glu) may foster growth and survival of cardiac myocytes by protecting them from apoptosis in a p38-dependent manner. Additionally, while NF-kappaB is activated in myocardial cells by p38, this does not appear to be the major mechanism by which MKK6 (Glu) exerts its anti-apoptotic effects in this cell type, suggesting a novel pathway for p38-mediated protection from apoptosis.

A role for the p38 mitogen-activated protein kinase pathway in myocardial cell growth, sarcomeric organization, and cardiac-specific gene expression.

Three hallmark features of the cardiac hypertrophic growth program are increases in cell size, sarcomeric organization, and the induction of certain cardiac-specific genes. All three features of hypertrophy are induced in cultured myocardial cells by alpha1- adrenergic receptor agonists, such as phenylephrine (PE) and other growth factors that activate mitogen- activated protein kinases (MAPKs). In this study the MAPK family members extracellular signal-regulated kinase (ERK), c-jun NH2-terminal kinase (JNK), and p38 were activated by transfecting cultured cardiac myocytes with constructs encoding the appropriate kinases possessing gain-of-function mutations. Transfected cells were then analyzed for changes in cell size, sarcomeric organization, and induction of the genes for the A- and B-type natriuretic peptides (NPs), as well as the alpha-skeletal actin (alpha-SkA) gene. While activation of JNK and/or ERK with MEKK1COOH or Raf-1 BXB, respectively, augmented cell size and effected relatively modest increases in NP and alpha-SkA promoter activities, neither upstream kinase conferred sarcomeric organization. However, transfection with MKK6 (Glu), which specifically activated p38, augmented cell size, induced NP and alpha-Ska promoter activities by up to 130-fold, and elicited sarcomeric organization in a manner similar to PE. Moreover, all three growth features induced by MKK6 (Glu) or PE were blocked with the p38-specific inhibitor, SB 203580. These results demonstrate novel and potentially central roles for MKK6 and p38 in the regulation of myocardial cell hypertrophy.

Collaborative roles for c-Jun N-terminal kinase, c-Jun, serum response factor, and Sp1 in calcium-regulated myocardial gene expression.

Electrical stimulation of contractions (pacing) of primary neonatal rat ventricular myocytes increases intracellular calcium and activates a hypertrophic growth program that includes expression of the cardiac-specific gene, atrial natriuretic factor (ANF). To investigate the mechanism whereby pacing increases ANF, pacing was tested for its ability to regulate mitogen-activated protein kinase family members, ANF promoter activity, and the trans-activation domain of the transcription factor, Sp1. Pacing and the calcium channel agonist BAYK 8644 activated c-Jun N-terminal kinase (JNK) but not extracellular signal-regulated kinase. Pacing stimulated ANF-promoter activity approximately 10-fold. Furthermore, transfection with an expression vector for c-Jun, a substrate for JNK, also activated the ANF promoter, and the combination of pacing and c-Jun was synergystic, consistent with roles for JNK and c-Jun in calcium-activated ANF expression. Proximal serum response factor and Sp1 binding sites were required for the effects of pacing or c-Jun on the ANF promoter. Pacing and c-Jun activated a GAL4-Sp1 fusion protein by 3- and 12-fold, respectively, whereas the two stimuli together activated GAL4-Sp1 synergistically, similar to their effect on the ANF promoter. Transfection with an expression vector for c-Fos inhibited the effects of c-Jun, suggesting that c-Jun acts independently of AP-1. These results demonstrate an interaction between c-Jun and Sp1 and are consistent with a novel mechanism of calcium-mediated transcriptional activation involving the collaborative actions of JNK, c-Jun, serum response factor, and Sp1.

Overexpression of the rat sarcoplasmic reticulum Ca2+ ATPase gene in the heart of transgenic mice accelerates calcium transients and cardiac relaxation.

The Ca2+ ATPase of the sarcoplasmic reticulum (SERCA2) plays a dominant role in lowering cytoplasmic calcium levels during cardiac relaxation and reduction of its activity has been linked to delayed diastolic relaxation in hypothyroid and failing hearts. To determine the contractile alterations resulting from increased SERCA2 expression, we generated transgenic mice overexpressing a rat SERCA2 transgene. Characterization of a heterozygous transgenic mouse line (CJ5) showed that the amount of SERCA2 mRNA and protein increased 2. 6-fold and 1.2-fold, respectively, relative to control mice. Determination of the relative synthesis rate of SERCA2 protein showed an 82% increase. The mRNA levels of some of the other genes involved in calcium handling, such as the ryanodine receptor and calsequestrin, remained unchanged, but the mRNA levels of phospholamban and Na+/Ca2+ exchanger increased 1.4-fold and 1.8-fold, respectively. The increase in phospholamban or Na+/Ca2+ exchanger mRNAs did not, however, result in changes in protein levels. Functional analysis of calcium handling and contractile parameters in isolated cardiac myocytes indicated that the intracellular calcium decline (t1/2) and myocyte relengthening (t1/2) were accelerated by 23 and 22%, respectively. In addition, the rate of myocyte shortening was also significantly faster. In isolated papillary muscle from SERCA2 transgenic mice, the time to half maximum postrest potentiation was significantly shorter than in negative littermates. Furthermore, cardiac function measured in vivo, demonstrated significantly accelerated contraction and relaxation in SERCA2 transgenic mice that were further augmented in both groups with isoproterenol administration. Similar results were obtained for the contractile performance of myocytes isolated from a separate line (CJ2) of homozygous SERCA2 transgenic mice. Our findings suggest, for the first time, that increased SERCA2 expression is feasible in vivo and results in enhanced calcium transients, myocardial contractility, and relaxation that may have further therapeutic implications.

Phorbol myristate acetate-induced hypertrophy of neonatal rat cardiac myocytes is associated with decreased sarcoplasmic reticulum Ca2+ ATPase (SERCA2) gene expression and calcium reuptake.

The decreased expression of the sarcoplasmic reticulum Ca(2+)-ATPase associated with cardiac hypertrophy was investigated in cultured neonatal rat cardiac myocytes. Northern blot analysis indicated a significant 55-60% decrease in Ca(2+)-ATPase mRNA levels and after 12 and 24 h of treatment with the phorbol ester phorbol myristate acetate (PMA). Myocytes treated with the phorbol ester for 80 h showed a significant 34% decrease (relative to vehicle-treated control cells) in the levels of Ca(2+)-ATPase protein, and a significant 38% increase in the levels of alpha-sarcomeric actin, as assessed by Western blot analysis using specific antibodies. Immunocytochemistry of myocytes treated for 72 h with the phorbol ester revealed a hypertrophied cell morphology, and showed a marked decrease in Ca(2+)-ATPase staining intensity. Contractile calcium transients were evaluated through the use of indo-1. It was found that the t1/2 for the decline of calcium transient was significantly prolonged by PMA treatment (0.51 +/- 0.15) when compared to controls (0.38 +/- 0.17, P < 0.001). Treatment of myocytes with endothelin-1 also led to a 35% decrease in sarcoplasmic reticulum Ca(2+)-ATPase mRNA levels. It is concluded that phorbol ester treatment of neonatal rat cardiac myocytes induces similar changes in Ca(2+)-ATPase mRNA levels. It is concluded that phorbol ester treatment of neonatal rat cardiac myocytes induces similar changes in Ca(2+)-ATPase gene expression as observed in vivo in the hypertrophied and failing heart. The observed prolongation in t1/2 for [Ca2+]i decline might be due to the observed depressed levels for sarcoplasmic reticulum Ca(2+)-ATPase in PMA treated cells.

TNF alpha receptor expression in rat cardiac myocytes: TNF alpha inhibition of L-type Ca2+ current and Ca2+ transients.

Tumor necrosis factor-alpha (TNF alpha) is a potentially powerful anti-neoplastic agent; however, its therapeutic usefulness is limited by its cardiotoxic and negative inotropic effects. Accordingly, studies were undertaken to gain a better understanding of the mechanisms of TNF alpha-mediated cardiodepression. Single cell RT-PCR, [125I]TNF alpha ligand binding and Western immunoblotting experiments demonstrated that rat cardiac cells predominantly express type I TNF alpha receptors (TNFRI or p60). TNF alpha inhibited cardiac L-type Ca2+ channel current (ICa) and contractile Ca2+ transients. Thus, it is possible that the negative inotropic effects of TNF alpha are the result of TNFRI-mediated blockade of cardiac excitation-contraction coupling.

Control of cardiac Ca2+ levels. Inhibitory actions of sphingosine on Ca2+ transients and L-type Ca2+ channel conductance.

The naturally occurring second messenger sphingosine (SPH) was examined for its ability to influence cardiac myocyte Ca2+ regulation. SPH inhibited intracellular Ca2+ transients in adult and neonatal rat ventricular myocytes. The inhibition was steeply dose dependent, with complete blockage of the Ca2+ transients occurring in the 20- to 25-mumol/L range. Whole-cell patch clamping revealed substantial inhibition of the L-type Ca2+ channel current (ICa) by SPH. The ability of SPH to block both the Ca2+ transients and ICa was not dependent on protein kinases, since the general protein kinase inhibitor H7 failed to prevent the actions of SPH. The specificity of the effect of SPH was determined in experiments showing that SPH analogues did not produce comparable effects. Neither the naturally occurring ceramide, N-stearoyl SPH, nor the cell-permeant ceramide, N-acetyl SPH, had SPH-like actions on the Ca2+ transients or L-type channel conductances. Caffeine-induced Ca2+ transients were also inhibited by the actions of SPH on cardiac sarcoplamic reticulum Ca2+ release, and the threshold for caffeine-induced Ca2+ release was raised. We conclude that SPH inhibits excitation-contraction coupling in cardiac myocytes by reducing the amount of entering "trigger Ca2+" for Ca(2+)-induced Ca2+ release and by simultaneously raising the threshold of the ryanodine receptor for Ca(2+)-induced Ca2+ release. Consequently, we propose that sphingolipids produced by the sphingomyelin signal transduction pathway could be physiologically relevant regulators of cardiac [Ca2+]i and therefore cardiac contractility.

Involvement of cytoplasmic calcium and protein kinases in the regulation of atrial natriuretic factor secretion by contraction rate and endothelin.

To characterize the effects of the cellular events associated with contraction on atrial natriuretic factor (ANF) secretion, primary neonatal rat atrial myocytes were electrically paced to contract while being monitored for ANF release, cytoplasmic calcium, phosphoinositide hydrolysis, and protein kinase C activation. Similar measurements were also carried out in the presence of endothelin-1 (ET) for comparison of contraction-related and hormone-stimulated ANF secretion. Pacing (6-8 Hz) immediately increased ANF secretion by 3-5-fold and the time-averaged cytoplasmic calcium concentration (as monitored with indo-1 fluorescence) varied with pace frequency in a similar manner, suggesting that cytoplasmic calcium may play a key role in pace-induced ANF secretion. Furthermore, nifedipine and ryanodine, which inhibited the contractile calcium transients, inhibited pace-induced ANF release, whereas Bay K 8644 increased both the calcium transients and ANF secretion. Pace-induced ANF release was also completely inhibited by KN-62, a specific inhibitor of Ca2+/calmodulin-dependent protein kinase II (CaMK) but was not inhibited by chelerythrine, a protein kinase C-selective inhibitor. Pace-induced ANF release averaged 40% of that elicited by ET which is known to require both PKC and CaMK for maximal effects on ANF secretion. The effects of pacing and ET on ANF secretion were approximately additive. In contrast to pacing, ET strongly stimulated phosphoinositide hydrolysis, activated PKC, and did not increase cytoplasmic calcium. Thus, regulation of ANF secretion by contraction rate depends primarily on the contractile calcium transients and CaMK and is independent of PKC.

Phenylephrine and endothelin differentially stimulate cardiac PI hydrolysis and ANF expression.

In ventricular myocytes, phenylephrine (PE) and endothelin (ET) stimulate phosphoinositide (PI) hydrolysis, cell growth, and expression of several genes [e.g., atrial natriuretic factor (ANF)] often associated with cardiac hypertrophy. In this study the production of inositol monophosphate (InsP) and diglyceride (DG), both products of PI hydrolysis, and ANF were monitored during long-term exposure (up to 72 h) to PE or ET. For PE, InsP production increased and continued unabated throughout the time course; DG levels also increased and remained elevated, and similar rates of ANF production were observed from 24 to 72 h. For ET, the initial InsP and DG responses equaled those to PE, but diminished by 24 h. ET-stimulated ANF production equaled the PE response at 24 h but subsided by 72 h. Adding PE to cells previously desensitized to ET elicited maximal InsP formation, indicating the desensitization was ET specific. These data emphasize that while phenylephrine and endothelin have similar initial effects on cardiac second messenger production and ANF expression, hormonally specific patterns develop over extended periods of exposure.

Induction of atrial natriuretic factor and myosin light chain-2 gene expression in cultured ventricular myocytes by electrical stimulation of contraction.

While hormonal stimuli and mechanical stretch can induced cardiac-specific gene expression and in some cases cellular hypertrophy, the relationship between myocyte contraction frequency, gene expression, and myocyte growth has not been well characterized. In this study a new model system was developed in which cultures of neonatal rat ventricular myocytes were subjected to long term pacing of contractions with pulsatile electrical stimulation. Myocytes submitted to electrical stimulation for 3 days displayed dramatic increases in cellular size and myofibrillar organization, and a 5-10-fold increase in the expression of the cardiac genes atrial natriuretic factor and myosin light chain-2. Atrial natriuretic factor expression induced by electrical stimulation of contractions was inhibited by nifedipine or W7, indicating a dependence on calcium influx and calmodulin activity. Phosphoinositide hydrolysis and cAMP formation were not affected by electrical stimulation suggesting that gene induction occurred independently of the activation of protein kinases C or A above basal levels. These findings show that the cellular events associated with contraction, such as changes in cytoplasmic free calcium levels and/or cellular stretch, may serve as important determinants of myocyte growth and cardiac gene expression.

The alpha-adrenergic stimulation of atrial natriuretic factor expression in cardiac myocytes requires calcium influx, protein kinase C, and calmodulin-regulated pathways.

It has been shown recently that alpha-adrenergic agonists can stimulate atrial natriuretic factor (ANF) expression in ventricular cardiac myocytes; however, little is known about the intracellular signals mediating this activation. The present study focused on the potential roles of calcium-regulated kinases and calcium influx in the alpha-adrenergic stimulation of ANF gene expression in ventricular myocardial cell cultures. Myocardial cells maintained for 48 h in serum-free medium supplemented with phenylephrine (PE) possessed up to 15-fold higher levels of ANF peptide and ANF mRNA than control cells. The removal of PE, or the addition of nifedipine, resulted in a rapid decline in ANF expression, suggesting that the sustained elevation of some intracellular messenger (e.g. calcium and/or phospholipid hydrolysis products) was required for the adrenergic response. The calcium channel agonist BAY K 8644 was capable of increasing ANF expression in a nifedipine-sensitive manner; however, unlike PE, it did not stimulate phosphoinositide hydrolysis. The protein kinase C inhibitor, H7, caused an approximate 75% reduction in PE-stimulated ANF expression, but had no effect on BAY K-stimulated expression. W7, a calcium/calmodulin inhibitor, completely blocked the effects of both PE and BAY K 8644. The addition of either H7 or W7 24 h after the PE addition resulted in a decline of ANF expression. These results indicate that alpha-adrenergic agonists augment ANF gene expression through at least two pathways, one that is H7-sensitive, perhaps involving the sustained activation of protein kinase C, and the other that is W7-sensitive, perhaps involving the sustained activation of calmodulin-regulated kinases. Further, it appears that BAY K 8644-mediated increases in ANF expression are independent of protein kinase C activation and dependent on calmodulin-regulated events.

Dissociation of protein kinase C redistribution from the phosphorylation of its substrates.

Increases in cytoplasmic [Ca2+] caused by receptor activation are thought to stimulate the redistribution of loosely associated protein kinase C (PKC) to a tightly membrane-bound form that is activated by diacylglycerol. The precise role of Ca2(+)-dependent redistribution of PKC in the activation of this enzyme has not been critically assessed. We examined the relationship between PKC redistribution and substrate phosphorylation by comparing the kinetics and the Ca2+ dependence of the two events. Using immunoblotting with specific PKC antibodies, we find that 1321N1 cells express the alpha form of PKC, approximately 10-20% of which is membrane-associated in unstimulated cells. This fraction is increased to 60% in response to muscarinic receptor stimulation. Agonist-induced redistribution of PKC is rapid and transient, peaking at 30 s and returning to control levels by 2-5 min. Stimulation of muscarinic receptors also rapidly increases phosphorylation of both an endogenous 80-kDa protein and the peptide substrate, VRKRTLRRL. However, unlike the time course of PKC redistribution, PKC-mediated phosphorylation of these substrates is sustained for up to 30 min. To compare the Ca2+ dependence of PKC redistribution and substrate phosphorylation, we buffered muscarinic receptor-induced increases in cytoplasmic [Ca2+] with the divalent cation chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid. Under these conditions, redistribution of PKC and phosphorylation of the exogenous peptide substrate are inhibited by about 80%. In contrast, muscarinic receptor-stimulated phosphorylation of the 80-kDa protein occurs even when increases in cytoplasmic [Ca2+] are prevented. Taken together, these data demonstrate that the redistribution of PKC does not correlate in extent or duration with phosphorylation of PKC substrates.

Endothelin induction of inositol phospholipid hydrolysis, sarcomere assembly, and cardiac gene expression in ventricular myocytes. A paracrine mechanism for myocardial cell hypertrophy.

The present study examined the effects of endothelin-1 on phosphoinositide hydrolysis, diacylglycerol formation, and the induction of myocardial cell hypertrophy utilizing a well characterized cultured neonatal rat myocardial cell model. In this system, a hypertrophic response can be assessed by increases in myocardial cell size, an increase in the assembly of an individual contractile protein (myosin light chain-2) into organized contractile units, accumulation of contractile proteins, the activation of a program of immediate early gene expression, and the induction of genes encoding contractile and embryonic proteins (Iwaki, K., Sukhatme, V., Shubeita, H.E., Chien, K.R., (1990) J. Biol. Chem. 265, 13809-13817). Utilizing these criteria, the present study documents that stimulation with endothelin-1 can produce myocardial cell hypertrophy, induce the expression and release of ANF in ventricular cells, and can activate the transcription of cardiac-specific genes. In addition, endothelin-1 stimulates phosphoinositide hydrolysis and the accumulation of diacylglycerol. It is proposed that endothelin-1 stimulation may represent an important paracrine mechanism for the in vivo regulation of cardiac growth and hypertrophy.