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Principal Investigator: Nicole Colussi
Organization: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
Fiscal Year: 2024
Award: $48,974
Funding agency: National Heart Lung and Blood Institute
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 during IR injury.
Terms: <Active Oxygen><Amino Acids><Anti-Inflammatories><Anti-Inflammatory Agents><Anti-inflammatory><Asystole><Autoimmune><Biochemical><Biological><Blood Plasma><Blood flow><Body Tissues><Cardiac><Cardiac Arrest><Cardiac Muscle Cells><Cardiac Myocytes><Cardiac artery><Cardiocyte><Cell Body><Cell Communication and Signaling><Cell Culture Techniques><Cell Membrane Lipids><Cell Protection><Cell Signaling><Cell membrane><Cells><Chemicals><Chemistry><Clinical Research><Clinical Study><Closure by Ligation><Complex><Conjugated Linoleic Acids><Coronary artery><Coupled><Crowding><Cysteine><Cytoplasmic Membrane><Cytoprotection><Dietary Intervention><EDRF Synthase><Endogenous Nitrate Vasodilator><Endothelium-Derived Growth Factor Synthase><Endothelium-Derived Nitric Oxide><Esterification><Evaluation><Event><Fatty Acids><Free Radicals><Generations><Guanylyl Cyclase-Activating Factor Synthase><Half-Cystine><Heart><Heart Arrest><Heart Muscle Cells><Heart artery><Heart myocyte><Immunofluorescence><Immunofluorescence Immunologic><Immunoglobulin Enhancer-Binding Protein><Infarction><Inflammation><Inflammatory><Injury><Interruption><Intracellular Communication and Signaling><Intravenous><Ischemia><Ischemia-Reperfusion Injury><Ischemic Heart><Ischemic Heart Disease><Ischemic myocardium><L-Cysteine><Label><Lecithinases><Ligation><Lipid Bilayers><Lipid Peroxidation><Lipids><Liquid Chromatography><Mass Photometry/Spectrum Analysis><Mass Spectrometry><Mass Spectroscopy><Mass Spectrum><Mass Spectrum Analyses><Mass Spectrum Analysis><Measurement><Mediating><Membrane><Membrane Lipids><Mercaptans><Mercapto Compounds><Metabolic><Mice><Mice Mammals><Mitochondria><Modeling><Mononitrogen Monoxide><Murine><Mus><Myocardial><Myocardial Ischemia><Myocardial Ischemic Reperfusion Injury><Myocardial Reperfusion Injury><Myocardium><NF-kB><NF-kappa B><NF-kappaB><NFKB><NO Synthase><NO2><NO2-cLA><Nitric Oxide><Nitric Oxide Synthase><Nitric-Oxide Synthetase><Nitrites><Nitrogen Dioxide><Nitrogen Monoxide><Nitrogen Peroxide><Nitrogen Protoxide><Nuclear Factor kappa B><Nuclear Transcription Factor NF-kB><Nutrition Interventions><Nutritional Interventions><O element><O2 element><Oral><Outcome><Oxidative Stress><Oxygen><Oxygen Radicals><Patients><Phosphatides><Phospholipase><Phospholipids><Physiologic><Physiological><Plasma><Plasma Membrane><Plasma Serum><Post-Translational Modification Protein/Amino Acid Biochemistry><Post-Translational Modifications><Post-Translational Protein Modification><Post-Translational Protein Processing><Posttranslational Modifications><Posttranslational Protein Processing><Pre-Clinical Model><Preclinical Models><Pro-Oxidants><Production><Protein Modification><Proteins><Proteomics><Public Health><Reaction><Reactive Oxygen Species><Reperfusion Damage><Reperfusion Injury><Reporting><Resolution><Reticuloendothelial System, Serum, Plasma><Role><Scheme><Signal Pathway><Signal Transduction><Signal Transduction Systems><Signaling><Signaling Molecule><Source><Sulfhydryl Compounds><Superoxide Anion><Superoxide Radical><Superoxides><Testing><Therapeutic><Thiols><Tissues><Transcription Factor NF-kB><Triacylglycerol><Triglycerides><Unsaturated Fatty Acids><Vascular blood supply><Viral Diseases><Virus Diseases><Work><aminoacid><biologic><biological signal transduction><biological systems><blood supply><cardiac function><cardiac ischemia><cardiac muscle><cardiac preservation><cardiomyocyte><cell culture><cell cultures><coronary ischemia><cytoprotective><denitration><diet intervention><endothelial cell derived relaxing factor><function of the heart><heart function><heart ischemia><heart muscle><heart preservation><in vivo><infarct><injuries><innovate><innovation><innovative><insight><kappa B Enhancer Binding Protein><lipid bilayer membrane><membrane structure><mitochondrial><myocardial ischemia/hypoxia><myocardium ischemia><nitrated conjugated linoleic acid><nitration><nitro-CLA><nitro-conjugated linoleic acid><nitroalkene><novel><nuclear factor kappa beta><oxidative damage><oxidative injury><plasmalemma><pre-clinical><pre-clinical study><preclinical><preclinical study><preference><protective effect><resolutions><response><restoration><small molecule><social role><sulfhydryl group><systemic inflammation><systemic inflammatory response><tandem mass spectrometry><vascular supply><viral infection><virus infection><virus-induced disease>