PROJECT SUMMARY Myocardial ischemia-reperfusion injury is a multifactorial event characterized by oxidative stress and mitochondrial damage. Interrupted blood supply establishes ischemia, and restoration of flow introduces a second wave of damage caused by uncontrolled reduction of oxygen (i.e., superoxide). Superoxide and nitric oxide produced by nitric oxide synthase in the myocardium initiates both lipid peroxidation and nitration in cell membranes. Lipid peroxidation mechanisms and consequences have been the focus of many studies, yet efforts are not matched for lipid nitration. Mechanistically, lipid nitration proceeds by the addition of nitrogen dioxide, a product of NO and oxygen radical reactions, to conjugated fatty acids. Complex lipids (i.e., triglycerides and phospholipids) are recognized as the main substrates for lipid nitration. However, their chemical complexity amplifies the challenges of analytical characterization. Nitrated conjugated linoleic acid (NO2-CLA) is an anti- inflammatory fatty acid that forms during cardiac IR injury. NO2-CLA formation was first established in a model of coronary artery ligation, and later work identified the mitochondria as a source of the nitrated species. Due to its electrophilic character, NO2-CLA reversibly modifies cysteines in target proteins to modulate signaling cascades. The impacts of NO2-CLA-mediated signaling have been evaluated in inflammatory, autoimmune, and viral infection preclinical models. Through these studies, covalent targets were identified, including Nf-kB, Keap1, and STING. Complementary to preclinical efforts, in a small clinical study of cardiac arrest, plasma levels of NO2- CLA correlated with patient survival. Despite this evidence, the mechanism of NO2-CLA formation in IR remains to be established, as well as the target protective signaling pathways. Given that phospholipids represent a major class of lipids in the myocardium and accumulate CLA, it is hypothesized that during cardiac IR injury, CLA esterified to phospholipids acts as a scavenger for nitration radicals, forming NO2-CLA that is released by phospholipases to initiate protective signaling cascades by modification of protein thiol targets. This hypothesis will be tested by the following two Aims: Aim 1: Establish that CLA esterified to phospholipids is a target for lipid nitration. Aim 2: Determine the protein targets that are modified by NO2-CLA during IR injury. Using cardiac H9c2 cells and isolated hearts subject to IR conditions, we will evaluate lipid nitration products, covalent targets, and protection endpoints. Mass spectrometry and novel bio-orthogonal labeling will serve as the experimental pillars, as they bring analytical value, physiological relevance, and innovation to this project. Overall, the proposed work will be the first to demonstrate the role of myocardial phospholipid nitration in establishing levels of NO2-CLA for the activation of protective and anti-inflammatory signaling pathways ...