Oxidants and Nitric Oxide in Post-Ischemic Heart Injury

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Oxygen radical generation is increased in the postischemic heart and leads to altered nitric oxide (NO) production and postischemic injury. Using Electron Paramagnetic Resonance (EPR) and other techniques, we directly measured the mechanisms of oxygen radical and NO generation in the postischemic heart. From these and other recent studies, it has been shown that oxidants alter NO generation from nitric oxide synthase (NOS). However, the precise role of oxidants in the regulation of NOS structure and function remains unknown. Recently, it has been shown that under ischemic conditions oxygen radical generating enzymes, such as xanthine oxidase (XO), also form NO through reduction of nitrite or nitrate. However, the nature and role of NOS-independent pathways of NO formation in the modulation of postischemic injury is unclear. Therefore, this project will characterize these processes of NO formation and their oxidant interactions. Studies will be performed first at the enzyme level; then in endothelial cells, followed by studies in isolated heart models and finally in vivo models of coronary occlusion and reflow. This project has the following 5 specific aims. 1) To characterize mechanisms by which 'O2~ and 'O2~ -derived oxidants affect the structure and function of human endothelial NOS (eNOS). 2) To determine the mechanism by which -O2" and 'O2 -derived oxidants alter eNOS structure and function in the isolated postischemic heart and evaluate approaches to restore eNOS function. 3) To characterize fundamental mechanisms of nitrite, nitrate or organic nitrate mediated NO generation. 4) To characterize the role of nitrite or nitrate mediated pathways of NO generation in the isolated postischemic heart and the mechanisms that regulate this process. 5) In vivo testing and EPR/NMR coimaging of novel therapeutics to inhibit oxidant injury, restore NOS function and confer myocardial protection. For these aims; EPR, electrochemical, and chemiluminescence measurements of oxygen radicals, NO, and NO derived species will be performed along with characterization of the function, structure and modification of the critical NO generating enzymes. Overall, this project will determine the interactions between oxygen radicals and the pathways of NO generation that occur in the process of postischemic injury, and lead to the development of optimal strategies to salvage heart muscle at risk.