Project Summary This proposal examines the previously unaddressed role of altered retinoid metabolism in heart failure (HF). We have recently shown that in both idiopathic dilated cardiomyopathy (IDCM) and experimental heart failure (HF) there is up to a 40% decline in the cardiac levels of the vitamin A metabolite and potent hormone, all-trans retinoic acid (ATRA), despite adequate vitamin A. We have also shown that direct administration of ATRA prevents HF, in response to pressure-overload. However, implementing ATRA therapy for HF could face headwinds, given the pleiotropy of ATRA signaling and the limited half-life of ATRA in the circulation. Alternatively, therapeutic specificity might best be achieved by targeting the enzymes of cardiac retinoid metabolism. This proposal addresses 3 critical knowledge gaps in our understanding of the mechanism of ATRA metabolism and its impact on the post-natal heart. 1. The enzymes that are responsible for the metabolism of retinol to retinoic acid in the adult mammalian heart are unknown. We have begun to identify the pertinent enzymes in human stem cell-derived cardiomyocytes (hSC-CMs). We show that Dhrs4 regulates the retinaldehyde pool in human stem-cell-derived cardiomyocytes hSC-CMs. We detail a workflow to validate other candidates, leveraging advances hSC-CMs and human engineered heart tissue (hEHT). 2. The pathophysiological mechanisms of a clinically-pertinent cardiac ATRA insufficiency have not been addressed. Knockout of cellular retinol-binding protein 1 (Crbp1) recapitulates the ATRA decline seen in human IDCM. Herein, we show the ATRA decline is sufficient to cause diastolic dysfunction and slow myofibrillar relaxation kinetics. We evaluate the role of the retinaldehyde reductase Dhrs4 by conditional cardiac knockout in the mouse heart. We test the hypothesis that boosting cardiomyocyte ATRA can prevent or even rescue HF and ameliorate cross-bridge cycling. We propose to manipulate DHRS4 and ATRA levels to identify ATRA-sensitive transcriptional programs in mouse hearts, and hEHT. 3. How pervasive the ATRA decline is across HF etiologies is unknown, though proteomic biosignatures of ischemic cardiomyopathy (ICM) and HF with preserved ejection fraction (HFpEF) are consistent with ATRA decline. Even so, another study has shown increased cardiac ATRA in the setting of advanced coronary heart disease. Identifying suitable cohorts for ATRA homeostasis therapy requires that the magnitude and direction of ATRA changes in HF be quantified across HF etiologies. We will quantify retinoids and retinoid-associated protein multiple etiologies including HF with reduced ejection fraction (HFrEF), HFpEF, ICM, and hypertrophic cardiomyopathy (HCM), using state-of-the-art targeted mass spectrometry assays (LC-MS3, isPRM) and examine their distribution within the heart using MALDI-MS Imaging. Restated, this proposal addresses fundamentally novel cardiobiology of a transcriptional master regulator with trans...