The decreased expression of the sarcoplasmic reticulum Ca 2 + -ATPase associated with cardiac hypertrophy was investigated in cultured neonatal rat cardiac myocytes. Northern blot analysis indicated a significant 55-60% decrease in Ca 2 + -ATPase mRNA levels and after 12 and 24 h of treatment with the phorbol ester phorbol myristate acetate (PMA). Myocytes treated with the phorbol ester for 80 h showed a significant 34% decrease (relative to vehicle-treated control cells) in the levels of Ca 2 + -ATPase protein, and a significant 38% increase in the levels ofα-sarcomeric actin, as assessed by Western blot analysis using specific antibodies. Immunocytochemistry of myocytes treated for 72 h with the phorbol ester revealed a hypertrophied cell morphology, and showed a marked decrease in Ca 2 + -ATPase staining intensity. Contractile calcium transients were evaluated through the use of indo-1. It was found that thet 1 / 2 for the decline of calcium transient was significantly prolonged by PMA treatment (0.51+/-0.15) when compared to controls (0.38+/-0.17,P<0.001). Treatment of myocytes with endothelin-1 also led to a 35% decrease in sarcoplasmic reticulum Ca 2 + -ATPase mRNA levels. It is concluded that phorbol ester treatment of neonatal rat cardiac myocytes induces similar changes in Ca 2 + -ATPase gene expression as observedin vivoin the hypertrophied and failing heart. The observed prolongation int 1 / 2 for [Ca 2 + ] i decline might be due to the observed depressed levels for sarcoplasmic reticulum Ca 2 + -ATPase in PMA treated cells.