This proposal focuses on development of a new treatment for patients with heart failure and preserved ejection fraction (HFpEF). Among symptomatic patients with HF, half have HFpEF, and their mortality is similar to those with HFrEF. However, there are currently no treatments for HFpEF that prolong life, and few (if any) that reliably reduce HF hospitalization rates. We need new therapies to address this unmet medical need. Cardiovascular gene transfer is conceptually an attractive method for treating HF, but difficulty in obtaining high yield transgene expression in the heart in a manner that can be easily and safely applied has been challenging. However, we recently have demonstrated the effectiveness of intravenous (i.v.) delivery of a long-term expression vector (AAV8) encoding a peptide, urocortin 2 (Ucn2), which possesses favorable cardiovascular effects through its paracrine/endocrine actions. In our current VA Merit grant we have established the safety and efficacy of a 1-time i.v. administration of AAV8.Ucn2 in two murine models of HFpEF—aging and pressure overload, two risk factors for clinical HFpEF. However, there are several important issues that must be resolved prior to clinical translation of this much-needed therapy. Hypothesis: Ucn2/Ucn3 gene transfer, through paracrine/endocrine effects, will improve cardiac function and survival in two models of HFpEF (in different species) via corticotropin releasing hormone receptor 2 (CRHR2)-dependent enhancement of Ca2+ handling in cardiac myocytes Aim 1: To determine the effectiveness and survival advantage of Ucn2 vs Ucn3 gene transfer in a model of HFpEF due to hypertension (Dahl Salt-Sensitive Rat) Aim 2a: To determine if the beneficial effects of Ucn2 gene transfer on LV function in aged mice are abrogated by cardiac-targeted deletion of CRHR2 Aim 2b: To determine whether the beneficial effects of Ucn2 gene transfer on the aged LV result from enhanced Ca2+ handling and increased rates of sarcomere shortening and lengthening Aim 3: To determine the intracellular mechanisms linking Ucn2 and Ucn3 peptide activation of CRHR2 with enhanced Ca2+ handling in cultured cardiac myocytes These Aims are designed to answer key questions: 1) Will the benefits of Ucn2 gene transfer be seen in a second species in a second model of HFpEF (hypertensive Dahl Salt-Sensitive rats), will it confer a survival advantage, and will Ucn3 gene transfer have similar beneficial effects? These questions will be addressed in Aim 1; 2) To what degree are the beneficial effects of Ucn2 gene transfer due to cardiac CRHR2 activation vs systemic vasodilation due to activation of CRHR2 in smooth muscle and attendant systemic vasodilation? We will address this question by using cardiac-targeted deletion of CRHR2 (Aim 2); 3) Finally, we propose to map the intracellular pathways (mechanisms) from CM CRHR2 stimulation by Ucn2/Ucn3 to the predicted increase in Ca2+ handling in a series of studies conducted in isolated CM...