Foetal nicotine exposure causes PKC epsilon gene repression by promoter methylation in rat hearts

Aims Foetal nicotine exposure results in decreased protein kinase C epsilon (PKC epsilon) expression and increased cardiac vulnerability to ischaemia and reperfusion injury in adult rat offspring. The present study tested the hypothesis that maternal nicotine administration causes increased promoter methylation of the PKC epsilon gene resulting in PKC epsilon repression in the heart. Methods and results Nicotine treatment of pregnant rats starting at day 4 of gestation increased the methylation of the Egr-1 binding site at the PKC epsilon gene promoter and decreased PKC epsilon protein and mRNA abundance in near-term foetal hearts. Methylation of the Egr-1 binding site reduced Egr-1 binding to the PKC epsilon promoter in the heart. Site-specific deletion of the Egr-1 binding site significantly decreased PKC epsilon promoter activity. The effects of nicotine were sustained in the heart of adult offspring. Ex vivo studies found no direct effect of nicotine on PKC epsilon gene expression. However, maternal nicotine administration increased norepinephrine content in the foetal heart. Treatment of isolated foetal hearts with norepinephrine resulted in the same effects of increased methylation of the Egr-1 binding site and PKC epsilon gene repression in the heart. 5-Aza-2'-deoxycytidine inhibited the norepinephrine-induced increase in methylation of the Egr-1 binding site and restored Egr-1 binding and PKC epsilon gene expression to the control levels. Conclusion This study demonstrates that prolonged nicotine exposure increases the sympathetic neurotransmitter release in the foetal heart and causes programming of PKC epsilon gene repression through promoter methylation, linking maternal smoking to pathophysiological consequences in the offspring heart.