ABSTRACT Cardiovascular disease is the most common cause of death in industrialized nations. During the course of our studies, we have identified previously undiscovered mitochondrial pathways of lipid metabolism and signaling which lead to the generation of 2-arachidonoyl-lysophosphatidylcholine (2-AA-LPC) and 2-arachidonoyl- lysophosphatidylethanolamine (2-AA-LPE). These include: 1) the identification of iPLA2g (PNPLA8) as a phospholipase with sn-1 specificity; and 2) oxidized cardiolipin-activated cytochrome c serving as a plasmalogenase catalyzing the production of 2-AA LPC and 2-AA LPE. Further research demonstrated that 2-AA-LPC and 2-AA-LPE are excellent substrates for cyclooxygenase-2 resulting in a plethora of unanticipated metabolites. Remarkably, incubation of these substrates with either 12-lipoxygenase or 15-lipoxygenase resulted in their oxidation to 12- H(p)ETE- or 15-H(p)ETE-lysophospholipids. Building upon these discoveries we identified 2-AA-LPC and 2-AA-LPE as signaling and metabolic nodes in lipid synthesis and human heart mitochondrial function. Importantly, we have identified the ability of failing human heart mitochondria to generate increased amounts of HETE eicosanoids in response to Ca2+ challenge in comparison to non-failing control mitochondria. Recently, ferroptosis has been identified as a mechanism that leads to cell death through the accumulation of lipid hydroperoxides. During myocardial ischemia and heart failure, a substantial portion of mitochondrial iron (Fe+3) is released from its bound state to become free Fe+2 that initiates the formation of reactive oxygen species (ROS) through Haber-Weiss and Fenton-type chemistries. The present research is targeted to identifying the roles of prominent mechanisms responsible for the oxidized lipids and their roles in membrane dysfunction in failing myocardium. The proposed research will focus on mechanistically understanding the mechanisms leading to oxidized lipid production in the failing human heart. Specifically, we will identify the chiral enrichment in different classes of oxidized lipids to gain mechanistic insight into future translational targets for therapy of heart failure. If lipid oxidation is largely enzyme-mediated, then specific enzymes can be targeted that will be identified in the proposed research. Alternatively, if lipid oxidation is largely mediated/initiated by non-enzymatic Fe+2 mechanisms then Fe chelation approaches and intramembrane radical traps can be explored. In Specific Aim 3, we will investigate the roles of oxidized lipids in the activation of different cell types from control and from failing hearts from female and male subjects. The importance of inflammation in heart disease is now well documented, but the roles oxidized lipids play in activating different cells of the immune system is still at its earliest stages of understanding. To traverse this gap in our knowledge, we will examine the effects of different oxidized lipids on select...