Metabolic enzyme activity mediating myocardial stunning

描述(由申请人提供)：已知通过激活丙酮酸脱氢酶(PDH)来刺激碳水化合物氧化可以促进缺血后心肌的收缩恢复。我们已经确定，激活PDH的好处既不依赖于糖酵解，也不依赖于线粒体能量的产生。相反，胞浆氧化还原状态的PDH依赖性改变影响缺血后心脏的恢复。我们对清醒猪在体心脏再灌流后1小时内PDH活性的研究结果表明，早期再灌流损伤可以逆转，从而导致心脏收缩能力的持续改善。令人振奋的新数据显示，刺激复氧心肌细胞丙酮酸氧化可改善对钙的收缩反应，消除钙超载，提高收缩松弛速度。因此，本研究探讨了PDH激活通过细胞内氧化还原平衡的有利改变来消除早期再灌注损伤的假说，这种改变改善了钙稳态和线粒体功能，从而消除了钙超载，并潜在地改善了肌丝的完整性。这一假设将在单个成年大鼠心肌细胞和分离的灌流大鼠心脏中进行验证，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。具体目标是：1)确定细胞内氧化还原状态对复氧心肌细胞内钙水平和对钙的收缩反应的影响；2)评估SERCA2a活性对加强碳水化合物氧化的反应作为改善再灌流心脏钙处理的一个机制；b)如果再灌流心脏中的SERCA2a活性通过PDH激活而提高，那么确定在活体大鼠心脏中过表达SERCA2a是否对收缩恢复有类似的益处；3)确定钙与细胞内氧化还原状态的改变在再灌流心肌增强碳水化合物氧化影响收缩功能中的关系；4)阐明钙激活的线粒体脱氢酶、α-酮戊二酸脱氢酶与胞浆还原等量转运(苹果酸-天冬氨酸穿梭)活性之间的关系/竞争，以响应再灌流心肌中的钙负荷；5)研究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顿抑的可能性。

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.