Direct effect of cocaine on epigenetic regulation of PKCepsilon gene repression in the fetal rat heart.

Maternal cocaine administration during gestation caused a down-regulation of PKCepsilon expression in the heart of adult offspring resulting in an increased sensitivity to ischemia and reperfusion injury. The present study investigated the direct effect of cocaine in epigenetic modification of PKCepsilon gene repression in the fetal heart. Hearts were isolated from gestational day 17 fetal rats and treated with cocaine in an ex vivo organ culture system. Cocaine treatment for 48 h resulted in significant decreases in PKCepsilon protein and mRNA abundance and increases in CpG methylation at two SP1 binding sites in the PKCepsilon promoter region (-346 and -268). Electrophoretic mobility shift assays demonstrated that CpG methylation of both SP1 sites inhibited SP1 binding. Consistently, chromatin immunoprecipitation assays showed that cocaine treatment significantly decreased binding of SP1 to the SP1 sites in the intact fetal heart. Reporter gene assays revealed that site-directed mutations of CpG methylation at both SP1 sites significantly reduced the PKCepsilon promoter activity while methylation of a single site at either -346 or -268 did not have a significant effect. The causal effect of increased methylation in the cocaine-induced down-regulation of PKCepsilon was demonstrated with the use of DNA methylation inhibitors. The presence of either 5-aza-2'-deoxycytodine or procainamide blocked the cocaine-induced increase in SP1 sites methylation and decrease in PKCepsilon mRNA. The results demonstrate a direct effect of cocaine in epigenetic modification of DNA methylation and programming of cardiac PKCepsilon gene repression linking prenatal cocaine exposure and pathophysiological consequences in the heart of adult offspring.