Phorbol myristate acetate-induced hypertrophy of neonatal rat cardiac myocytes is associated with decreased sarcoplasmic reticulum Ca2+ ATPase (SERCA2) gene expression and calcium reuptake

The decreased expression of the sarcoplasmic reticulum Ca2+-ATPase associated with cardiac hypertrophy was investigated in cultured neonatal rat cardiac myocytes. Northern blot analysis indicated a significant 55-60% decrease in Ca2+-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 Ca2+-ATPase protein, and a significant 38% increase in the levels of alpha-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 Ca2+-ATPase staining intensity. Contractile calcium transients were evaluated through the use of indo-1. It was found that the t(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 Ca2+-ATPase mRNA levels. It is concluded that phorbol ester treatment of neonatal rat cardiac myocytes induces similar changes in Ca2+-ATPase gene expression as observed in vivo in the hypertrophied and failing heart. The observed prolongation in t(1/2) for [Ca2+](i) decline might be due to the observed depressed levels for sarcoplasmic reticulum Ca2+-ATPase in PMA treated cells. (C) 1996 Academic Press Limited