Project Abstract The goals of this project are two-fold: 1) to evaluate the role of lysosomal dysfunction in lipotoxic cardiomyopathy and 2) to provide mentorship and training to allow the PI’s transition to independence. The prevalence of diabetes and obesity are increasing in the population, and both conditions are independent risk factors for the development of heart failure. Cardiac lipotoxicity has been identified as a potential pathway through which diabetes causes cardiomyopathy, but the understanding of how lipid overload disrupts cardiac function remains limited. Preliminary data acquired by the PI demonstrate that lipid overload-induced cardiac dysfunction is rescued by intermittent fasting. Intermittent fasting does not reduce cardiac triglyceride levels, but protein aggregate pathology is attenuated. Also, the levels of specific cardiac ceramide species are altered, suggesting that the effect of intermittent fasting is medicated by changing the level of individual lipid species, rather than overall reduction in lipids. Based on this data, this project hypothesizes that myocardial lipid overload impairs lysosome function via ceramide accumulation to induce cardiomyopathy and that stimulation of lysosomal biogenesis program by TFEB activation is sufficient to rescue this outcome. This hypothesis will be tested by assessing autophagic flux and lysosomal function in mice models of cardiac lipid overload and in cultured cardiomyocytes under lipid stress (Aim 1), examining cardiac lysosomal ceramide accumulation under lipid stress and the requirement for ceramide synthesis in cardiac lipotoxicity (Aim 2), and evaluating the role of TFEB in stimulating lysosomal biogenesis to improve lipotoxic cardiomyopathy (Aim 3). Completion of these aims will address knowledge gaps in the field of cardiac lipotoxicity and also provide training to facilitate the career development of the PI. The PI has previously obtained PhD training in molecular and cellular biology and has undertaken postdoctoral training in the lab of Dr. Abhinav Diwan, following completion of clinical training in medicine and cardiology. The PI seeks additional scientific training in a mentored setting to address the knowledge gaps described. Specifically, the proposed career development plan will provide additional training in 4 areas: 1) autophagy and lysosomal biology, 2) lipid biology and lipidomics, with a focus on sphingolipid biology, 3) echocardiographic analysis of animal models of cardiac disease, and 4) training in grant writing, presentation skills, and leadership. This plan will be guided by mentorship and research support from Dr. Abhinav Diwan and Dr. Douglas Mann, experts in lysosomal biology and myocardial biology, respectively, as well as by additional mentorship from the advisory committee with renowned expertise in lysosomal biology (Dr. Stuart Kornfeld), lipid biology (Dr. Brian Finck and Dr. Clay Semenkovich), and sphingolipid biology and lipidomics (Dr. Ashley ...