Isoenzyme-selective regulation of SERCA2 gene expression by protein kinase C in neonatal rat ventricular myocytes

Patients with cardiac hypertrophy and heart failure display abnormally slowed myocardial relaxation, which is associated with downregulation of sarco(endo)plasmic reticulum [Ca.sup.2+]-ATPase (SERCA2) gene expression. We previously showed that SERCA2 downregulation can be simulated in cultured neonatal rat ventricular myocytes (NRVM) by treatment with the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA). However, NRVM express three different PMA-sensitive PKC isoenzymes (PKC[alpha], PKC[epsilon], and PKC[delta]), which may be differentially regulated and have specific functions in the cardiomyocyte. Therefore, in this study we used adenoviral vectors encoding wild-type (wt) and kinase-defective, dominant negative (dn) mutant forms of PKC[alpha], PKC[epsilon], and PKC[delta] to analyze their individual effects in regulating SERCA2 gene expression in NRVM. Overexpression of wtPKC[epsilon] and wtPKC[delta], but not wtPKC[alpha], was sufficient to downregulate SERCA2 mRNA levels, as assessed by Northern blotting and quantitative, real-time RTPCR (69 [+ or -] 7 and 61 [+ or -] 9% of control levels for wtPKC[epsilon] and wtPKC[delta], respectively; P < 0.05 for each adenovirus; n = 8 experiments). Conversely, overexpression of all three dnPKCs appeared to significantly increase SERCA2 mRNA levels (dnPKC[delta] > dnPKC[epsilon] > dnPKC[alpha]). dnPKC[delta] overexpression produced the largest increase (2.8 [+ or -] 1.0-fold; n = 11 experiments). However, PMA treatment was still sufficient to downregulate SERCA2 mRNA levels despite overexpression of each dominant negative mutant. These data indicate that the novel PKC isoenzymes PKC[epsilon] and PKC[delta] selectively regulate SERCA2 gene expression in cardiomyocytes but that neither PKC alone is necessary for this effect if the other novel PKC can be activated. heart; signal transduction; hypertrophy; transcription; mRNA stability; sarco(endo)plasmic reticulum [Ca.sup.2+]-ATPase