Project Summary Pregnancy is associated with a striking increase of uterine blood flow that is essential for normal fetal development as well as for cardiovascular well-being of the mother. Hypoxia during pregnancy has profound adverse effects on uterine artery hemodynamics adaptation, increasing incidence of pregnancy complications including preeclampsia and fetal intrauterine growth restriction. Previous studies in an animal model of pregnant sheep acclimatized to high altitude hypoxia demonstrated that pregnant ewes were similar to pregnant women in that they both showed an increase in uterine vascular resistance and elevation in maternal systemic blood pressure in response to gestational hypoxia. Yet, much remains unknown of the mechanisms underlying maternal cardiovascular maladaptation to chronic hypoxia during pregnancy. Our preliminary study in sheep suggests a highly novel mechanism of a monomeric G protein, Rad in inhibition of L-type CaV1.2 calcium channel currents in the uterine artery. The L-type CaV1.2 calcium channel, as the major pathway of Ca2+ influx, is essential for vascular smooth muscle contractions and plays a central role in regulating organ blood flow and arterial pressure. We identify that both ovine and human Rad gene promoters have multiple estrogen response elements (EREs), suggesting a robust mechanism of sex steroid hormones in the regulation of Rad gene expression in the uterine artery. In addition, the approach of RNA-seq analysis revealed a downregulation of Rad gene expression in uterine arteries of pregnant ewes acclimatized to high altitude hypoxia. Of importance, we demonstrated that chronic hypoxia during gestation abrogated pregnancy-induced upregulation of Rad protein expression and increased CaV1.2 channel currents in ovine uterine arteries. These exciting findings and many highly novel leads provide a strong scientific premise for us to move the field forward significantly by launching a new focus of research aimed at understanding the molecular and epigenetic mechanisms of Rad in regulating CaV1.2 channel currents and phenotypic programming of uterine vascular adaptation to pregnancy and gestational hypoxia. The proposed study will be conducted in a unique animal model of pregnant sheep exposed to high altitude (3801 m/12,470 ft) hypoxia during gestation. The overall hypothesis of the proposed study is that Rad is a novel regulatory mechanism and plays an essential role in the regulation of L-type CaV1.2 calcium channel currents and uterine vascular adaptation to pregnancy and gestational hypoxia. The proposed study has the strong scientific premise with a novel conceptual framework and mechanistic approach. It will provide new insights into fundamental mechanisms in uterine vascular adaptation to pregnancy, and will have a major impact on our understanding of pathophysiologic mechanisms underlying pregnancy complications including preeclampsia caused by gestational hypoxia. Of importance, the similarit...