PHORBOL 12-MYRISTATE 13-ACETATE ALTERS SR CA2-ATPASE GENE-EXPRESSION IN CULTURED NEONATAL RAT-HEART CELLS()

Primary cultures of neonatal rat ventricular myocytes were used to exa mine how the cardiac myocyte cytoplasmic Ca2+ ([Ca2+](i)) transient an d sarcoplasmic reticulum Ca2+-ATPase (SERCA2) gene expression change i n response to treatment with the protein kinase C activator phorbol 12 -myristate 13-acetate (PMA). Exposure of neonatal myocytes to PMA (200 nM, 48-72 h) produced myocyte growth and a 70% prolongation of the ha lf-time for [Ca2+](i) decline induced by potassium depolarization in t he absence of extracellular Na+ (in which the sarcoplasmic reticulum C a2+ pump is the main mechanism responsible for [Ca2+](i) decline). The reduced rate of [Ca2+](i) transient decline corresponded to a 53% red uction in SERCA2 protein levels and a 43% reduction in SERCA2 mRNA lev els as compared with control myocytes. Exposure to PMA for as little a s 30 min or for as long as 48 h produced a similar degree of SERCA2 mR NA downregulation over time. PMA-induced downregulation of SERCA2 mRNA levels was blocked by either 10 nM staurosporine or 4 mu M chelerythr ine, whereas treatment with either agent alone increased SERCA2 mRNA l evels as compared with control cells. Actinomycin D mRNA stability ass ays revealed that PMA treatment appeared to markedly destabilize the r elatively long-lived SERCA2 mRNA transcript. Taken together, these res ults indicate that downregulation of SERCA2 gene by PMA in cultured ne onatal myocytes occurs at least in part by alterations in mRNA stabili ty and results in functional alterations in [Ca2+](i) decline that-are similar to that observed in the hypertrophied and failing adult myoca rdium.