Project Summary Ischemic cardiomyopathy is the leading cause of death in the world and affects approximately 1% to 2% of the general population. Sphingolipids including ceramides and sphingosine-1-phosphate have been demonstrated to play roles in myocardial injury. Previous research from the lab showed that SPTLC3-derived sphingolipids comprise greater than 1/3rd the myocardial sphingolipid pool, a previously underappreciated group of sphingolipids. SPTLC3, a recently identified subunit of the serine palmitoyltransferase (SPT) enzyme, along with SPTLC1, synthesizes the SPTLC3-derived or non-canonical sphingolipids, whereas SPTLC2 with SPTLC1 synthesizes canonical sphingolipids. Over-abundance of SPTLC3-derived sphingolipids has been associated with increased apoptosis in cardiomyocytes and other heart pathologies. Little is known of the induction of the programmed cell death pathways by these particular sphingolipids. Interestingly, I found that SPTLC3 and derived sphingolipids showed robust induction in human and mouse ventricle ischemic tissue as compared to complete absence of SPTLC3 in the non-ischemic control tissues. Based on my preliminary results, I hypothesize that induction of SPTLC3 increases d16-DHS1P which in turn promotes formation of an ATG7/P53/Parkin complex by direct binding. This leads to inhibition of Parkin- mediated mitophagy thereby promoting mitochondria-dependent cell death. Our preliminary data support that SPTLC3-derived sphingolipids induce formation of a novel complex between ATG7, P53, and Parkin, and we propose complex formation of this complex mediates apoptosis. Therefore, to test this hypothesis we will overexpress and/or knockdown SPTLC3 and determine protein-lipid and protein-protein interactions and whether this complex regulates mitophagy and/or apoptotic pathways. Second, We hypothesize that cardiomyocyte specific Sptlc3 knockout mice will show improved cardiovascular health and subsequent increased longevity in ischemia as compared to their control cohorts. To test our hypothesis, we recently developed a cardiomyocyte specific Sptlc3 null mouse (cSptlc3 KO) line, which to our knowledge is the first of its kind. We will be performing permanent left anterior descending (LAD) coronary artery ligation in the cardiomyocyte specific Sptlc3 null mouse (cSptlc3 KO). We will then determine the impact of SPTLC3 depletion on cardiac sphingolipid profiles, mitophagy/apoptosis, and cardiac structure/function in ischemia including adverse remodeling. Understanding the biological activities of the SPTLC3-derived sphingolipids will provide insights and establish the role of these sphingolipids in cardiac ischemia. This proposal will lay the foundation for further research on potential targeting of the pathway as an innovative therapeutic option to circumvent ischemia leading to heart failure and improve patient outcome.