The main aim of this proposal is to elucidate the role of oxygen- derived radicals (ODR) or cardiac injury during the calcium paradox. A major hypothesis of this project is that during Ca++ depletion, the damage to myocytes is initiated by superoxide radicals (O2-) and hydrogen peroxide (H2O2) generated in the vascular endothelium. This damage is potentiated by hydroxyl radical (OH-) production after reintroduction of Ca++. The injury induced by O2-/H2O2 and OH- is not well known. Therefore, the focus of the proposal will be investigate the roles of H2O2 alone, or in combination with O2-, in causing injury to myocyte and endothelial cells in culture, intact hearts, and cocultures of myocytes and endothelial cells. Secondly, the roles of O2-/H2O2 and OH- in the induction of the CA++ paradox utilizing cultures of myocytes and endothelial cells will be investigated. In this manner, the potential contribution of endothelial cells in the generation of O2-/H2O2 as initiating species in the pathogenesis of Ca++ paradox will be investigated. The above aims will be accomplished as follows: The cell injury due to O2-/H2O2/OH- will be correlated with functional studies including measuring contractility, enzyme release, lipid peroxidation, altered distribution of Fe++ and quantitative cell damage before and after specific interventions. Experiments proposed utilizing endothelial cell and myocyte cultures will permit us to study the direct effect of the individual O2-derived species generated exogenously or during Ca++ depletion and Ca++ repletion, and the progressive stages in the development of Ca++ paradox. Superoxide and OH- generated during Ca++ depletion and Ca++ repletion of cultured endothelial cells myocytes will be identified and measured by cytochrome C assay and high pressure liquid chromatography (HPLC). The use of HPLC for direct measurement of OH- will be a unique and novel technique which will enable us to define the role of OH- in myocyte injury. The information derived from these studies will increase our understanding of the fundamental mechanisms underlying the Ca++ paradox. Furthermore, the study will provide insight into the development of new strategies to prevent the myocardial injury which occurs during calcium-free cardioplegia, Ca++ paradox and post-ischemic reperfusion.
- National Heart, Lung, and Blood Institute
- National Institutes of Health
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