IRON-MEDIATED CARDIOVASCULAR INJURY

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We propose that substantial reductions in the morbidity and mortality of acute myocardial infarction can be achieved by new approaches aimed at preventing the iron-mediated damage that occurs during reperfusion of ischemic myocardium. Our major hypothesis is: Production of reactive oxygen species during reperfusion causes injury due to .OH production or other processes dependent upon availability of iron, which is determined by transferrin and ferritin regulation of the cellular labile iron pool. We will investigate, using molecular biological methods in cultured cardiac myocytes, the cellular regulation of free iron. We will then ascertain the degree to which free iron is involved in oxidant injury in cultured myocytes, and apply our findings to potential therapeutic interventions in isolated, perfused rabbit hearts. In AIM 1, we will infect cultured adult rat cardiac myocytes with recombinant adenoviruses encoding constitutively expressed, unregulated, or inducible, iron- regulated, human transferrin receptors. We will examine the effect of infection with these adenoviruses on iron-transferrin uptake and cellular free iron and ferritin levels. Under conditions simulating oxidant exposure during reperfusion, we will measure cell injury, lipid peroxidation and glutathione oxidation, all of which we postulate will be increased in the genetically altered myocytes with unregulated transferrin receptors. In AIM 2 we will study a group of unique, newly discovered iron chelators, the exochelins of Mycobacterium tuberculosis, which prevent .OH production and are both water and lipid soluble. These agents enter cells more rapidly than other chelators and have a very high binding affinity for iron. We anticipate that the exochelins will reduce intracellular iron levels and susceptibility to oxidant injury. In AIM 3 we will examine in cultured myocytes and isolated perfused rabbit hearts several measures to reduce intracellular iron. In cultured myocytes we will study a receptor- dependent iron chelator, lactoferrin, and the effect of agents (chloroquine and ammonium chloride) that increase pH in endocytic vesicles and lysosomes, thereby inhibiting release of iron from transferrin and ferritin. In conjunction with these studies we will employ a new highly sensitive assay for .OH, which employs gas chromatography and mass spectrometry detection of salicylate isomers of this radical. Further studies of iron-directed potential therapies will measure ventricular function, myocardial enzyme release and lipid peroxidation in isolated hearts exposed to hypoxia and reoxygenation, using the chelators and other methods for reducing intracellular free iron which were previously examined in the experiments with cultured cardiac myocytes.