Myocyte cell loss is a prominent and important pathogenic feature of cardiac ischemia. We have used cultured neonatal rat cardiac myocytes exposed to prolonged hypoxia as an experimental system to identify critical factors involved in cardiomyocyte death. Exposure of myocytes to hypoxia for 48 h resulted in intranucleosomal cleavage of genomic DNA characteristic of apoptosis and was accompanied by increased p53 transactivating activity and protein accumulation. Expression of p21/WAF-1/CIP-1, a well-characterized target of p53 transactivation, also increased in response to hypoxia. Hypoxia did not cause DNA laddering or cell loss in cardiac fibroblasts. To determine whether the increase in p53 expression in myocytes was sufficient to induce apoptosis, normoxic cultures were infected with a replication-defective adenovirus expressing wild-type human p53 (AdCMV.p53). Infected cells expressed high intracellular levels of p53 protein and exhibited the morphological changes and genomic DNA fragmentation characteristic of apoptosis. In contrast, no genomic DNA fragmentation was observed in myocytes infected with the control virus lacking an insert (AdCMV.null) or in cardiac fibroblasts infected with AdCMV.p53. These results suggest that the intracellular signaling pathways activated by p53 might play a critical role in the regulation of hypoxia-induced apoptosis of cardiomyocytes. ( J. Clin. Invest. 1997. 99:2635-2643.)

The failing heart is characterized by impaired cardiac muscle function and increased interstitial fibrosis. Our purpose was to determine whether the functional impairment of the failing heart is associated with changes in levels of mRNA encoding proteins that modulate parameters of contraction and relaxation and whether the increased fibrosis observed in the failing heart is related to elevated expression of genes encoding extracellular matrix components. We studied hearts of 18- to 24-month-old spontaneously hypertensive rats with signs and symptoms of heart failure (SHR-F) or without evidence of failure (SHR-NF) and of age-matched normotensive Wistar-Kyoto (WKY) rats. Compared with WKY rats, SHR-NF exhibited left ventricular (LV) hypertrophy (2.2-fold) and right ventricular (RV) hypertrophy (1.5-fold), whereas SHR-F were characterized by comparable LV hypertrophy (2.1-fold) and augmented RV hypertrophy (2.4-fold; all P < .01). Total RNA was isolated from ventricles and subjected to Northern blot analysis. In SHR-F hearts, the level of alpha-myosin heavy chain mRNA was decreased in both ventricles to 1/3 and 1/5 of the SHR-NF and WKY values, respectively (both P < .01). Levels of beta-myosin heavy chain, alpha-cardiac actin, and myosin light chain-2 mRNAs were not significantly altered in hearts of SHR-NF or SHR-F. Levels of alpha-skeletal actin were twofold greater in SHR-NF hearts compared with WKY hearts and were intermediate in SHR-F hearts. Levels of atrial natriuretic factor (ANF) mRNA were elevated threefold in the LV of SHR-NF (P < .05) but were not significantly increased in the RV of SHR-NF compared with WKY rats. During the transition to failure (SHR-F versus SHR-NF), ANF mRNA levels increased an additional 1.6-fold in the LV and were elevated 4.7-fold in the RV (both P < .05). Levels of sarcoplasmic reticulum Ca(2+)-ATPase (SRCA) mRNA were maintained in the LV of hypertensive and failing hearts at levels not significantly different from WKY values. In contrast, the level of RV SRCA mRNA was 24% less in SHR-NF compared with WKY rats, and during the transition to failure, this difference was not significantly exacerbated (29% less than the WKY value). The levels of fibronectin and pro-alpha 1(I) and pro-alpha 1(III) collagen mRNAs were not significantly elevated in either ventricle of the SHR-NF group but were fourfold to fivefold higher in both ventricles of SHR-F (all P < .05).(ABSTRACT TRUNCATED AT 400 WORDS)

The 70-kD S6 kinase (p70S6K) has been implicated in the regulation of protein synthesis in many cell types and in the angiotensin II-stimulated hypertrophy of cardiac myocytes. Our purpose was to determine whether p70S6K plays a role in cardiomyocyte hypertrophy induced by the alpha 1-adrenergic receptor (alpha 1-AR) agonist phenylephrine (PE). PE stimulated the activity of p70S6K > 3-fold, and this increase was blocked by rapamycin, an immunosuppressant macrolide that selectively inhibits p70S6K. When administered for 3 days, PE stimulated a 30% increase in total protein content, a 2-fold increase in the incorporation of [14C]phenylalanine (14C-Phe) into protein, and a 50% increase in two-dimensional myocyte area. Rapamycin pretreatment (> or = 500 pg/mL) significantly inhibited each of these PE-stimulated changes. Two days of PE treatment resulted in a 1.6-fold increase in total RNA yield per dish, a 2-fold increase in incorporation of [14C]uridine into myocyte RNA, and increases in relative mRNA levels of the hypertrophy-associated atrial natriuretic factor (ANF, 2.1-fold) and skeletal alpha-actin (SK, 2.2-fold) genes. Although rapamycin abolished the PE-stimulated increases in total RNA and incorporation of [14C]uridine, it had no effect on the induction of the ANF and SK genes. LY294002, a specific inhibitor of phosphatidylinositol 3-kinase (PI3-K) activity, inhibited PE-stimulated increases in p70S6K activity and the incorporation of labeled precursors into myocyte protein and RNA. These results demonstrate that p70S6K is activated by the hypertrophic agent PE and that a PI3-K or PI3-K-like activity is required for p70S6K activation and myocyte hypertrophy. The data suggest that p70S6K activation may be required for PE-stimulated hypertrophy of cardiac myocytes. Our results demonstrate that intracellular signaling pathways responsible for transcriptional and translational responses diverge early after alpha 1-AR stimulation in cardiac myocytes.

Activation of the vacuolar proton ATPase (VPATPase) has been implicated in the prevention of apoptosis in neutrophils and adult cardiac myocytes. To determine the role of the VPATPase in apoptosis of cardiac myocytes, we used a potent and specific inhibitor of the VPATPase, bafilomycin A1. Bafilomycin A1 alone caused increased DNA laddering of genomic DNA and increased nuclear staining for fragmented DNA in neonatal cardiomyocyte apoptosis in a dose- and time-dependent manner. Intracellular acidification in cardiac myocytes was also observed after 18 h of bafilomycin A1 treatment. Accordingly, bafilomycin A1-treated myocytes also showed increased accumulation of p53 protein and p53-dependent transactivation of gene expression, including a persistent upregulation of p21/wild-type p53 activated fragment 1/cyclin kinase inhibitor protein-1 mRNA. The bafilomycin A1-induced increase in p53 protein levels was accompanied by a marked increase in p53 mRNA accumulation. In contrast, cardiac fibroblasts treated with bafilomycin A1 showed no change in p53 protein expression or pHi and did not undergo apoptosis even after 24 h of treatment. Our data suggest that blockade of the VPATPase induces apoptotic cell death of cardiac myocytes and that this may occur through a p53-mediated apoptotic pathway.

We have previously shown that extracellular ATP, like norepinephrine (NE) and many other hypertrophy-inducing agents, increases expression of the immediate-early genes c-fos and junB in cultured neonatal cardiac myocytes but that the intracellular signaling pathways activated by ATP and responsible for these changes differ from those stimulated by NE. Furthermore, whereas NE increases incorporation of [14C]phenylalanine (14C-Phe) and cell size in neonatal cardiomyocytes, ATP does not. Since ATP is coreleased with NE from sympathetic nerve endings in the heart, we investigated whether ATP could modulate cardiac hypertrophy induced by adrenergic agonists, such as NE. We report in the present study that extracellular ATP inhibited the increase in incorporation of 14C-Phe into cellular protein and the increase in cell size in neonatal rat cardiac myocytes that was induced by NE, phenylephrine (PE), basic fibroblast growth factor, or endothelin-1. This inhibition was dose dependent, occurred predominantly through P2 purinergic receptors, and was observed even when cells were treated with ATP for as little as 1 hour before the addition of the hypertrophy-inducing agent. ATP also selectively affected changes in gene expression associated with hypertrophy. It prevented PE-stimulated increases in atrial natriuretic factor and myosin light chain-2 mRNA levels, while appearing to augment basal and PE-stimulated skeletal alpha-actin mRNA levels. ATP alone increased sarcoplasmic reticulum Ca2+-ATPase mRNA levels but had no effect when added with PE. ATP did not significantly affect the level of the constitutively expressed mRNA for GAPDH. Neither the PE-stimulated increase in immediate-early gene expression nor the initial induction of mitogen-activated protein kinase activity by PE was inhibited by ATP. These results demonstrate that extracellular ATP can inhibit hypertrophic growth of neonatal cardiac myocytes and differentially alter the changes in gene expression that accompany hypertrophy.

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Although previous studies have shown that cardiac myosin heavy chain (MHC) composition undergoes a switch from the alpha- to beta-isoform in the heart during adult aging, the underlying mechanisms responsible for this switch are unknown. Cardiac MHC gene expression is regulated, in part, by thyroid hormone responsive elements present in the regulatory control regions of the alpha- and beta-MHC genes. Age-associated changes in the expression of thyroid hormone receptors (THRs) and/or retinoid X receptors (RXRs), the heterodimeric partner for THRs, could explain the age-associated changes in MHC expression. Accordingly, we measured mRNA levels for the cardiac muscle MHCs and the rat THR and RXR genes in the left ventricles of Wistar rats at 2, 6, and 24 months of age. Although there were no significant changes in RXR alpha or RXR beta mRNA levels with age, both alpha 1 and alpha 2 THR mRNA levels decreased significantly between 2 and 6 months of age. During this same time period, the mRNA levels for alpha-MHC declined by more than half, whereas beta-MHC mRNA levels remained low and unchanged. On the other hand, between 6 and 24 months, when mRNA levels for beta-MHC increased and alpha-MHC continued to decrease, there was a significant decline in THR beta 1 and RXR gamma mRNA levels accompanied by a reduction in the THR beta 1 and RXR gamma protein levels. These data show a pattern of change that suggests that the decline in alpha-MHC gene expression may be biphasic and due to a decline in alpha 1 (and possibly alpha 2) THR levels between 2 and 6 months of age and a decline in THR beta 1 and RXR gamma levels at later stages. In contrast, the increase in beta-MHC gene expression was associated only with the changes in THR beta 1 and RXR gamma mRNA and protein levels.

It is well-documented that norepinephrine (NE) induces the expression of immediate-early genes (IEGs), such as c-fos, c-jun, and jun-B, in cultured neonatal heart cells and leads to cell growth without cell division (ie, hypertrophy). Although purinergic receptors activated by ATP are present on cardiac myocytes and ATP is coreleased with NE from sympathetic nerve endings within the heart, the potential role of the purinergic system in the cascade of events that leads to cardiac hypertrophy is unknown. We report in the present study that stimulation of purinergic receptors by micromolar concentrations of extracellular ATP increased the levels of c-fos and jun-B mRNA as well as FOS and JUN-B proteins in neonatal cardiac myocytes. The magnitude of response to micromolar ATP was comparable to that elicited by NE. The increase in IEG expression induced by ATP was preceded by a rapid transient increase in cytosolic Ca2+. Pretreatment of myocytes with the intracellular Ca2+ chelator BAPTA-AM prevented the ATP-stimulated increase in cytosolic Ca2+ and attenuated the ATP-stimulated increase in c-fos expression. In contrast, NE did not increase cytosolic Ca2+ in quiescent myocytes, and pretreatment with BAPTA-AM did not inhibit the NE-stimulated increase in c-fos gene expression. Furthermore, although NE markedly increased [14C]phenylalanine incorporation into protein and myocyte hypertrophy measured by cell size, ATP did not. These results demonstrate that stimulation of purinergic receptors by ATP activates IEGs via a Ca(2+)-dependent pathway in cardiac myocytes that differs from the NE stimulated activation of these genes.(ABSTRACT TRUNCATED AT 250 WORDS)