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.

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.

230 Signal transduction via the mitogen activated protein kinase (MAPK) has been implicated in cardiac myocyte hyopertrophy, but the nature of its role is controversial. Our purpose was to determine whether basal levels or elevated levels of MAPK activity play a role in cardiac myocyte hypertrophy. We examined the relative roles of agonist-stimulated increases in MAPK activity and basal levels of MAPK activity in the subsequent hypertrophic growth of cultured neonatal rat cardiac myocytes. Serum-free myocyte cultures were stimulated with phenylephrine (PE) in the presence or absence of selected doses of PD98059, a specific inhibitor of the immediate upstream activator of MAPK, MEK. PE stimulated a transient (3-5-fold after 5 min) and a sustained(1.5-fold after 18 hours) elevation in MAPK activity, both of which were completely prevented by 10 mM PD98059. 50 mM (but not 10 mM) PD98059 reduced MAPK activity in PE-treated myocytes to levels below those in vehicle-treated cells. These actions of PD98059 were sustained for at least 72 hours. PE stimulated increases in the total protein/DNA ratio (1.3-fold), RNA content(1.4-fold), incorporation of labeled phenylalanine (14C-Phe; 2-fold), endogenous ANF mRNA (2-fold) and sarcomere organization. 10 mM PD98059 had no significant effects on the PE-induced increases in protein/DNA ratio, RNA content, incorporation of 14C-Phe, or sarcomere organization, but did suppress the increase in ANF mRNA level. In contrast, 50 mM PD98059 markedly inhibited the PE-stimulated increases in each of the aforementioned measures except sarcomere organization. It is concluded that neither the early transient, nor the sustained elevation in MAPK activity is required for PE-stimulated hypertrophy of cardiac myocytes, but that the basal level of MAPK activity plays a permissive role in hypertrophic growth. In contrast, the induction of ANF gene expression by PE requires an elevation in MAPK activity in neonatal rat cardiac myocytes.

Objectives: The aims of this study were to determine (1) whether neonatal rat cardiac fibroblasts (CAFB) express P2Y receptors; (2) whether CAFE respond to extracellular ATP by inducing expression of c-Sos mRNA; and (3) whether extracellular ATP modulates norepinephrine (NE)-stimulated cell growth in CAFE. Methods: Expression of P2Y(1) and P2Y(2) receptors and induction of c-Sos were examined by Northern blot analysis. CAFB growth was assessed by measuring [H-3]thymidine incorporation and DNA content. P2Y receptor pharmacology was studied using various ATP analogues. Results: Northern blot analysis of polyA enriched RNA confirmed that at least 2 subtypes of P2Y receptors (P2Y(1) and P2Y(2)) an expressed in cultured CAFE. Extracellular ATP induced the expression of c-foss mRNA through a pathway that was sensitive to inhibitors of protein kinase C (PKC), but not to inhibitors of intracellular Ca2+ signaling. Extracellular ATP inhibited the NE-stimulated increases in DNA content and in [3H]thymidine incorporation into DNA. Whereas the potency order for stimulation of c-fos expression was ATP = UTP > ADP > adenosine, the potency order to inhibit the NE-induced increase of [H-3]thymidine incorporation into DNA was ATP > ADP > UTP > adenosine. Conclusions: These data demonstrate that CAFE express both P2Y(1) and P2Y(2) receptor mRNA and that CAFE respond to P2Y receptor stimulation by induction of c-fos and inhibition of DNA synthesis. These findings suggest that the effects of ATP on [H-3]thymidine incorporation into DNA and on expression of c-Sos mRNA are exerted via distinct P2Y receptor subtypes. (C) 1998 Elsevier Science B.V.

The spontaneously hypertensive rat (SHR) exhibits a transition from stable compensated left ventricular (LV) hypertrophy to heart failure (HF) at a mean age of 21 months that is characterized by a decrease in alpha-myosin heavy chain (alpha-MHC) gene expression and increases in the expression of the atrial natriuretic factor (ANF), pro-alpha1(III) collagen, and transforming growth factor beta1 (TGF-beta1) genes. We tested the hypotheses that angiotensin-converting enzyme inhibition (ACEI) in SHR would prevent and reverse HF-associated changes in gene expression when administered prior to and after the onset of HF, respectively. We also investigated the effect of ACEI on circulating and cardiac components of the renin-angiotensin system. ACEI (captopril 2 g/L in the drinking water) was initiated at 12, 18, and 21 months of age in SHR without HF and in SHR with HF. Results were compared with those of age-matched normotensive Wistar-Kyoto (WKY) rats, and to untreated SHR with and without evidence of HF. ACEI initiated prior to failure prevented the changes in alpha-MHC, ANF, pro-alpha1(III) collagen, and TGF-beta1 gene expression that are associated with the transition to HF. ACEI initiated after the onset of HF lowered levels of TGF-beta1 mRNA by 50% (P<.05) and elevated levels of alpha-MHC mRNA two- to threefold (P<.05). Circulating levels of renin and angiotensin I were elevated four- to sixfold by ACEI, but surprisingly, plasma levels of angiotensin II were not reduced. ACEI increased LV renin mRNA levels in WKY and SHR by two- to threefold but did not influence LV levels of angiotensinogen mRNA. The results suggest that the anti-HF benefits of ACEI in SHR may be mediated, at least in part, by effects on the expression of specific genes, including those encoding alpha-MHC, ANF, TGF-beta1, pro-alpha1(III) collagen, and renin-angiotensin system components.

Boluyt, M. O.; Zheng, J-S.; Younes, A.; O'Neill, L.; Lakatta, E. G.; Crow, M. T. Author Information