Received for publication October 20, 2006.
Revised January 2, 2007.
Accepted for publication January 3, 2007.

Maternal Cocaine Administration Causes an Epigenetic Modification of PKC Gene Expression in Fetal Rat Heart

Abstract

PKC plays a pivotal role in cardioprotection during cardiac ischemia and reperfusion injury. Recent studies demonstrated that prenatal cocaine exposure caused a decrease in PKC expression and increased heart susceptibility to ischemic injury in adult offspring, suggesting an in utero programming of PKC gene expression pattern in the heart. The present investigation aimed to elucidate whether an epigenetic mechanism, DNA methylation, accounts for cocaine-mediated repression of the PKC gene in the heart. Pregnant rats were administered either saline or cocaine intraperitoneally (15 mg/kg) twice daily from day 15 to day 20 of gestational age, and term fetal hearts were studied. Cocaine treatment significantly decreased PKC mRNA and protein levels in the heart. CpG dinucleotides found in CREB, CREB/c-Jun1 and CREB/c-Jun2 binding sites at the proximal promoter region of the PKC gene were densely methylated and were not affected by cocaine. In contrast, methylation of CpGs in the AP-1 binding sites was low, but was significantly increased by cocaine. Reporter gene assays showed that the AP-1 binding site played a strong stimulatory role of PKC gene transcription. Methylation of the AP-1 binding sites significantly decreased AP-1 binding to the PKC promoter. Super-shift analyses implicated c-Jun homodimers binding to the AP-1 binding sites. Cocaine did not affect nuclear c-Jun levels or the binding of c-Jun to the unmethylated AP-1 binding sites. The results indicate a role for DNA methylation in cocaine-mediated PKC gene repression in the developing heart, and suggest an epigenetic mechanism affecting this gene linked with vulnerability of ischemic injury in the heart of adult offspring.

Key words: AP-1, CREB, NFkappaB, DNA binding sites, Promoter analysis, Cocaine, Ischemia/Reperfusion

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