The long term objective is to elucidate the mechanism(s) of ischemic preconditioning in order to develop a useful clinical tool for myocardial protection against ischemia/reperfusion injury. Ischemic preconditioning is the paradoxical phenomenon whereby a brief (non-lethal), transient episode of ischemia enhances the tolerance of the heart to a subsequent prolonged (lethal) ischemic insult. One of the reported benefits of ischemic preconditioning is attenuation of postischemic contractile dysfunction. Ischemic preconditioning also attenuates acidosis during prolonged ischemia. Ischemia/reperfusion injury may result from hydroxyl radical formation, which is catalyzed by the ischemia-induced release of protein-bound iron to a "free", low molecular weight pool. This project will test the hypothesis that preconditioning attenuates contractile dysfunction by limiting H+ accumulation during ischemia, which attenuates accumulation of "free" iron during ischemia, and which then attenuates the formation of injurious hydroxyl radicals. The isolated perfused rat heart will be used in this project. Aim l is to determine if preconditioning attenuates accumulation of "free" iron during ischemia, and attenuates hydroxyl radical formation during reperfusion. Aim 2 is to determine if varying the availability of "free" iron (by perfusion with an iron chelator and by dietary supplementation and restriction) will cause proportional changes in the amount of hydroxyl radicals produced during reperfusion, and in postischemic contractile dysfunction. Aim 3 is to determine if release of bound iron is pH-sensitive in homogenates of nonconditioned and preconditioned hearts. Aim 4 is to determine if accumulation of "free" iron during ischemia; hydroxyl radical formation; and postischemic contractile function correlate with myocardial pH during ischemia. The myocardial pH during ischemia will be modulated by l) perfusion with different glucose concentrations to vary the preischemic glycogen levels, and 2) perfusion with highly buffered perfusates prior to ischemia. Aim 5 is to test the alternative possibility that preconditioning may limit the accumulation of "free" iron by attenuating accumulation of reductants (NADH, NADPH, and xanthine) that are known to enhance the release of protein-bound iron.