Metabolic enzyme activity mediating myocardial stunning

描述（由申请人提供）：已知通过丙酮酸脱氢酶（PDH）激活刺激碳水化合物氧化可改善缺血后心肌的收缩恢复。我们已经确定激活PDH的益处既不依赖于糖酵解也不依赖于线粒体能量的产生。相反，胞质氧化还原状态的ph依赖性变化影响缺血后心脏的恢复。我们在清醒猪体内心脏再灌注的第一个小时内PDH激活的结果表明，早期再灌注损伤的逆转导致收缩力的持续改善。令人兴奋的新数据表明，刺激再氧化心肌细胞中的丙酮酸氧化可改善对钙的收缩反应，消除钙超载，并提高收缩松弛率。因此，本研究探讨了PDH激活消除早期再灌注损伤的假设，通过细胞质氧化还原平衡的有利改变，通过消除钙超载和潜在的肌丝完整性，改善钙稳态和线粒体功能。这一假设将在单个成年大鼠心肌细胞和分离的灌注大鼠心脏中进行测试，使用包括光学显微镜和13C核磁共振波谱在内的新技术组合。具体目的是：1)确定胞质氧化还原状态对细胞内钙水平和复氧心肌细胞对钙的收缩反应的影响；2) a)评估SERCA2a活性对碳水化合物氧化增强的反应，作为再灌注心脏中改善钙处理的机制；b)如果再灌注心脏中PDH的激活提高了SERCA2a的活性，那么确定SERCA2a在体内大鼠心脏中的过度表达是否对收缩恢复产生类似的益处；3)在再灌注心肌碳水化合物氧化增强过程中，确定钙与诱导胞质氧化还原状态变化在影响收缩功能中的关系；4)阐明钙激活的线粒体脱氢酶- α -酮戊二酸脱氢酶与再灌注心肌中响应钙负荷的细胞质还原等效转运（苹果酸-天冬氨酸穿梭）活性之间的关系/竞争；5)检查通过减少肌丝蛋白降解和提高钙敏感性来增强碳水化合物氧化以逆转休克的潜力。

DESCRIPTION (provided by applicant): Stimulating carbohydrate oxidation via pyruvate dehydrogenase (PDH) activation is known to irmprove 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 the 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 improvement in contractility. 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, this study explores the hypothesis that PDH activation eliminates early reperfusion injury via favorable shifts in cytosolic redox balance that improve calcium homeostasis and mitochondrial function due to elimination of calcium overload and potentially, the integrity of the myofilaments. This hypothesis will be tested in both the single adult rat cardiomyocyte and in the isolated, perfused rat heart using a novel combination of techniques that include optical microscopy and 13C NMR spectroscopy. Specific aims are: 1) Determine the effects of cytosolic redox state on intracellular calcium levels and contractile response to calcium in reoxygenated cardiomyocytes; 2) a) Evaluate the response of SERCA2a activity to augmented carbohydrate oxidation as a mechanism of improved calcium handling in reperfused hearts; b) if SERCA2a activity is improved by PDH activation in reperfused hearts then determine whether SERCA2a over expression in vivo rat hearts produces similar benefits to contractile recovery; 3) Determine the relationship between calcium and induced shifts in cytosolic redox state in affecting contractile function during augmented carbohydrate oxidation in reperfused myocardium; 4) Elucidate the relationship/competition between the calcium activated mitochondrial dehydrogenase, alpha-ketoglutarate dehydrogenase, versus activity of cytosolic reducing equivalent transport (malate-aspartate shuttle) in response to calcium load in the reperfused myocardium; 5) Examine the potential for augmented carbohydrate oxidation to reverse stunning via reduced myofilament protein degradation and improved calcium sensitivity.