Myocardial damage during ischemia and reperfusion is increased in the elderly heart. Two tandem defects are present in complex III at the time of reperfusion in the aging heart: damage to the Rieske iron-sulfur protein (ISP) and a aging-related defect in the cytochrome c binding site. Our preliminary evidence has identified the ISP as a prominent site of damage associated with mitochondrial functional deficits following ischemia. A hierarchical approach is developed to identify the chemical nature of oxidative damage suffered by the ISP during reperfusion, the probably chemical nature of the reactive species responsible for the protein modification, and the correlation of the observed modification with reperfusion induced myocardial injury.
Four specific aims are described to accomplish these goals. The first is to develop and demonstrate biochemical and mass spectrometric methods for efficient structural identification of oxidative-damage. The second specific aim investigates the spectrum of oxidative damage that can be inflicted on the soluble catalytic domain of the ISP, referred to as the iron-sulfur fragment, and then at the level of integration of the ISP within complex III. The third specific aim examines how these oxidants alter the function of the ISP when it functions within intact mitochondria.
This specific aim permits the characterization of the anticipated products from potentially physiologically relevant oxidants and reactive species generated by lipid peroxidation. The pattern of oxidative modifications will be examined when the electron flow is varied in response to well characterized inhibitors of electron transport. The fourth specific aim is to determine the oxidative modifications arising during ischemia-reperfusion. The use of /180/2 during reperfusion will permit the oxidative modifications detected in the ISP isolated following reperfusion to be unequivocally correlated with the reperfusion injury. Comparison of the identified protein modifications with the data accumulated from the first 3 aims will identify the probably reactive species generated by reperfusion, and suggest and mitochondrial a mitochondrial site for its generation and mechanism of protein damage. This information will be used in the fourth aim to design and evaluate mechanistic-based therapeutic interventions to ameliorate damage during ischemia and reperfusion in the aging heart.

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National Institute on Aging (NIA)
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Case Western Reserve University
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