ABSTRACT Heart disease is the most common cause of death in industrialized nations. The presence of underlying diabetes is the greatest risk factor for the progression of heart disease. During the current grant interval, we have discovered previously unknown lipid metabolic pathways and signaling molecules which lead to the generation of eicosanoid-lysophospholipids. Remarkably, the vast majority of eicosanoids in myocardium were found to be esterified to the glycerol backbone of lysophospholipids. In addition, induction of Type I diabetes in wild-type mice or ischemic damage in isolated wild-type mouse hearts resulted in dramatic increases in pro-inflammatory eicosanoid-lysophospholipids. This new class of phospholipids serve as inflammatory mediators by inducing the release of TNFa from monocytes or macrophages. Importantly, genetic ablation of iPLA2g (PNPLA8) substantially decreased the levels of eicosanoid-lysophospholipids in myocardium in the diabetic state, during myocardial ischemia and synergistically decreased their synthesis in diabetic myocardium rendered ischemic. Accordingly, we propose that iPLA2g plays a central role in the pathophysiologic development of diabetic heart disease and promotes the lethal sequelae of diabetic cardiomyopathy. In Specific Aim 1, we will utilize stable isotope labeling of isolated perfused mouse hearts from genetically engineered cardiac myocyte-specific conditional iPLA2g knockout mice we have generated. These studies will investigate the roles of iPLA2g in the metabolic flux of: 1) non-esterified and esterified eicosanoids; 2) eicosanoid-lysophospholipids; and 3) other salient oxidized phospholipids. Stable isotope pulse-chase experiments followed by penetrating bioinformatic analyses will determine rates of metabolic flux through these newly discovered pathways. Translationally, we will explore the impact of Type 2 diabetes on myocardial ischemic damage and the potential salvage of ischemic myocardium in cardiac myocyte-specific iPLA2g KO mice we engineered. Endpoints of analysis include infarct size, hemodynamic performance, and post-translational modifications of iPLA2g. In Specific Aim 2, we will utilize cardiac myocyte-specific iPLA2b KO mice we have generated to explore the role of iPLA2b in promoting myocardial ischemic damage and arrhythmias in WT vs. iPLA2b KO mice in the context of Type II diabetes. Next, we will determine the ability of iPLA2b to catalyze acyltransferase or transacylase mediated re-esterification of eicosanoid-lysophospholipids to generate oxidized phospholipids which have been implicated in damage associated molecular patterns. In Specific Aim 3, the mechanisms through which a high fat diet induces eicosanoid-lysolipid synthesis accompanied by inflammation and mitochondrial dysfunction will be studied. The roles of lysophospholipases in modulating eicosanoid-lysophospholipid levels and activation mechanisms for iPLA2g will be examined. Collectively, the proposed studies wil...