This supplemental application has two purposes: 1) to restore specific aims that were eliminated due to an administrative cut of 40% of the originally requested budget (Aims 2b, 4, and 5 shown below); 2) to support newly designed experiments, enabling an additional mechanistic level of investigation (Aim 1b) that will provide new insight into our hypothesis. Stimulating carbohydrate oxidation via pyruvate dehydrogenase (PDH) activation is known to improve contractile recovery of postischemic myocardium. We have determined that the benefits of activating PDH rely neither on glycolytic nor mitochondrial energy production. Instead, PDH-dependent changes in cytosolic redox state influence recovery of the postischemic heart. Our results on PDH activation during the first hour of reperfusion in the in vivo heart of conscious pigs demonstrate reversal of early reperfusion injury that results in sustained contractile improvement. Exciting new data show that stimulating pyruvate oxidation in reoxygenated cardiomyocytes improves contractile response to calcium, eliminates calcium overload, and improves contractile relaxation rate. Therefore, we offer the hypothesis that PDH activation counters early reperfusion injury via favorable shifts in cytosolic redox balance that improve calcium homeostasis and mitochondrial function or alternatively affect the integrity of the myofilaments. The hypothesis will be tested in both single adult rat cardiomyocytes and isolated, perfused rat hearts using a novel combination of techniques that include optical microscopy and 13C NMR. The supplemental Specific Aims are: 1) b) Elucidate relative contributions of the SR Ca ATPase and Na/Ca exchange to calcium removal and respective effects on cell shortening in response to glucose oxidation and cytosolic redox state in reoxygenated cells; 2) b) determine whether SERCA2a over expression in rat hearts produces similar benefits to contractile recovery as PDH activation; 4) Elucidate the relationship/competition between the mitochondrial, calcium-activated alpha-ketoglutarate dehydrogenase, versus activity of cytosolic reducing equivalent transport (malate-aspartate shuttle) in response to calcium load in the reperfused heart; 5) Examine the potential for augmented carbohydrate oxidation to reverse stunning via reduced myofilament protein degradation and improved calcium sensitivity. Together with existing aims, the supplemental studies combine an important new level of inquiry with a more comprehensive investigative approach. ? ?
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