Project Summary The goals of this proposal are to further train Sasha Prisco, MD, PhD as a physician-scientist in Cardiology and to advance the understanding of the mechanisms of right ventricular dysfunction in pulmonary hypertension. Pulmonary arterial hypertension is caused by pathological remodeling of the pulmonary vasculature which subsequently increases right ventricular afterload, leading to right ventricular failure. Unfortunately, there are no current therapies that directly target the right ventricle. Despite the fact that females outnumber males by 3- 4:1 in pulmonary arterial hypertension, males develop more severe right ventricular dysfunction and subsequently die sooner. We have previously shown that the microtubule-junctophilin-2 (MT-JPH2) pathway is dysregulated in a preclinical model of pulmonary arterial hypertension. While there are data linking sex hormones to microtubules, a clear mechanism by which sex hormones regulate microtubules has not been established. This research project will investigate the role of sex hormones on the microtubule cytoskeleton and right ventricular function in pulmonary arterial hypertension. Aim 1 tests the hypothesis that estrogen has anti-microtubule properties, which augment right ventricular function by limiting microtubule-dependent junctophilin-2 dysregulation (MT-JPH2 pathway) and t-tubule remodeling in preclinical right ventricular overload. Aim 2 examines the hypothesis that testosterone stabilizes microtubules, which exerts a negative effect on right ventricular function by promoting microtubule-mediated junctophilin-2 downregulation and t- tubule remodeling in experimental models of right ventricular pressure overload. In both aims, we will evaluate how the sex hormones, estrogen and testosterone, affect microtubule polymerization and microtubule stability and whether sex hormones directly interact with microtubules with advanced microscopy. Furthermore, we will investigate how estrogen and testosterone regulate expression of the MT-JPH2 pathway in isolated right ventricular cardiomyocytes. Finally, we will assess how alterations of estrogen and testosterone affect the MT- JPH2 pathway and right ventricular function in two rat models of right ventricular pressure overload (monocrotaline and pulmonary artery banded), right ventricle pressure overloaded rats that have either undergone oophorectomy or castration, and oophorectomized/castrated right ventricle pressure overloaded rats given estrogen or testosterone replacement, respectively. This project has potential to provide molecular mechanistic insight into the divergent response of the right ventricle in pulmonary arterial hypertension based on sex. The training of Dr. Prisco as a physician-scientist will advance the mission of the NIH by helping support the training of a scientist who will conduct clinically relevant research.