Chronic Prenatal Hypoxia Induces Epigenetic Programming of PKCε Gene Repression in Rat Hearts

Abstract

Rationale: Epidemiological studies demonstrate a clear association of adverse intrauterine environment with an increased risk of ischemic heart disease in adulthood. Hypoxia is a common stress to the fetus and results in decreased protein kinase C epsilon (PKCε) expression in the heart and increased cardiac vulnerability to ischemia and reperfusion injury in adult offspring in rats. Objectives: The present study tested the hypothesis that fetal hypoxia-induced methylation of cytosine-phosphate-guanine dinucleotides at the PKCε promoter is repressive and contributes to PKCε gene repression in the heart of adult offspring. Methods and Results: Hypoxic treatment of pregnant rats from days 15 to 21 of gestation resulted in significant decreases in PKCε protein and mRNA in fetal hearts. Similar results were obtained in ex vivo hypoxic treatment of isolated fetal hearts and rat embryonic ventricular myocyte cell line H9c2. Increased methylation of PKCε promoter at SP1 binding sites, −346 and −268, were demonstrated in both fetal hearts of maternal hypoxia and H9c2 cells treated with 1% O 2 for 24 hours. Whereas hypoxia had no significant effect on the binding affinity of SP1 to the unmethylated sites in H9c2 cells, hearts of fetuses and adult offspring, methylation of both SP1 sites reduced SP1 binding. The addition of 5-aza-2′-deoxycytidine blocked the hypoxia-induced increase in methylation of both SP1 binding sites and restored PKCε mRNA and protein to the control levels. In hearts of both fetuses and adult offspring, hypoxia-induced methylation of SP1 sites was significantly greater in males than in females, and decreased PKCε mRNA was seen only in males. In fetal hearts, there was significantly higher abundance of estrogen receptor α and β isoforms in females than in males. Both estrogen receptor α and β interacted with the SP1 binding sites in the fetal heart, which may explain the sex differences in SP1 methylation in the fetal heart. Additionally, selective activation of PKCε restored the hypoxia-induced cardiac vulnerability to ischemic injury in offspring. Conclusions: The findings demonstrate a direct effect of hypoxia on epigenetic modification of DNA methylation and programming of cardiac PKCε gene repression in a sex-dependent manner, linking fetal hypoxia and pathophysiological consequences in the hearts of adult offspring.