Isoenzyme-specific protein kinase C and c-jun N-terminal kinase activationby electrically stimulated contraction of neonatal rat ventricular myocytes

Previous studies from our laboratory and others indicate that contraction-i nduced mechanical loading of cultured neonatal rat ventricular myocytes pro duces many of the phenotypic changes associated with cardiomyocyte hypertro phy in vivo, and that these changes occur via the activation of serine-thre onine protein kinases. These may include the extracellular regulated protei n kinases (ERK1 and ERK2), the c-Tun N-terminal kinases (JNK1, JNK2, and JN K3), and one or more isoenzymes of protein kinase C. In this study, we asse ssed whether one or more of these kinases are activated by stimulated contr action, and whether activation was isoenzyme-specific. Low-density, quiesce nt cultures of neonatal rat ventricular myocytes were maintained in serum-f ree medium, or electrically stimulated to contract (3 Hz) for up to 48 h. E RK and JNK activation was assessed by Western blotting with polyclonal anti bodies specific for the phosphorylated forms of both kinases, PKC activatio n was analysed by subcellular fractionation, detergent extraction and Weste rn blotting using isoenzyme-specific monoclonal antibodies. Stimulated cont ractile activity produced myocyte hypertrophy, as indicated by increased ce ll size, a 15 +/- 5% increase in total protein/DNA ratio, and induction of ANF and beta MHC gene transcription. Electrical pacing did not cause ERK1/2 or JNK1 activation, but increased JNK2 and JNK3 phosphorylation by similar to two-fold. Subcellular fractionation revealed a lime-dependent increase in PKC delta, and to a much lesser extent PKC epsilon, in a Triton X-100-so luble membrane fraction within 5 min of the onset of stimulated contraction . PKC alpha was not activated by electrical pacing. These results indicate that contraction-induced mechanical loading acutely activates some but not all of the specific isoenzymes of JNKs and PKCs in cardiomyocytes. (C) 2000 Academic Press.