Mechanisms of cardiac ischemia-reperfusion injury and cardioprotection

本项目的目标是：1）了解线粒体在缺血-再灌注损伤和心脏保护中的作用; 2）了解离子稳态改变和代谢改变在缺血-再灌注和心脏保护中的作用; 3）了解参与心脏保护和细胞死亡的细胞溶质和线粒体信号转导的变化。 缺血预处理（PC）可能通过激活线粒体信号通路而发挥心肌保护作用。 我们还研究了抑制糖原合成酶激酶（GSK）通过作用于线粒体介导心脏保护的机制。 GSK-3的抑制通过涉及线粒体的机制减少缺血再灌注损伤。本研究的目的是确定这种心脏保护作用的分子靶点和机制基础。我们发现GSK抑制在这些条件下减缓ATP消耗，这可能是由于抑制ATP通过VDAC和/或ANT进入线粒体或抑制F1 F0 ATP酶。 因此，我们使用蛋白质组学的方法来测试的假设，PC和药理学预处理同样改变线粒体信号复合物。 Langendorff灌注的小鼠心脏用特异性GSK-3抑制剂AR-A014418（GSK InhibVIII）处理10分钟或在20分钟全缺血和120分钟再灌注之前进行4个循环的5分钟缺血/再灌注（PC）。 与时间匹配的对照组相比，PC和GSK InhibVIII均改善了缺血后左心室发展压（LVDP）恢复，缩小了梗死面积，并减少了缺血期间的乳酸产生。 我们使用蛋白质组学检查PC和GSK InhibVIII之间常见的线粒体蛋白水平/翻译后修饰。 细胞色素c氧化酶亚基Va和VIb、ATP酶偶联因子6和细胞色素b-c1复合物亚基6的水平升高，而细胞色素c在PC和GSK Inhib VIII中降低。 此外，发现细胞色素c氧化酶亚基VIb的量在PC和GSK InhibVIII线粒体超复合物中增加，所述超复合物由复合物I、III和IV组成。 这一结果表明，与心脏保护相关的复合物亚基的变化可能会影响超复合物的组成。 因此，PC和GSK抑制改变电子传递复合物的表达水平的能力将对线粒体功能具有重要意义。 从亲环素D（CypD-/-）缺陷的小鼠分离的线粒体在体外对Ca 2+诱导的线粒体通透性转换（MPT）开放不太敏感。 因此，CypD的缺乏使心脏线粒体在接受MPT之前能够吸收更多的Ca 2+。 我们假设MPT作为一个Ca 2+安全阀，可以打开释放过量的Ca 2+，但不一定导致死亡。 如果在CypD-/-小鼠中MPT被阻断，我们假设与WT相比，CypD-/-小鼠中的基质Ca 2+（Ca 2 +m）将更高，并且这将激活Ca 2+敏感性NADH脱氢酶（例如，丙酮酸脱氢酶（PDH）和α-酮戊二酸脱氢酶（α-KGDH）），这反过来会改变氧化代谢并增加氧消耗。 与此一致，我们发现改变表达水平的PDH E1亚基和α-KGDH E2亚基CypD-/-心脏使用二维DIGE蛋白质组学。 为了评估代谢的差异，我们用13 C-葡萄糖和13 C-棕榈酸灌注心脏，并通过测量标记掺入谷氨酸的C4来观察它们对乙酰辅酶A池的贡献。 13 C标记的葡萄糖或棕榈酸进入克雷布斯循环，并标记与谷氨酸平衡的α-KG池，谷氨酸通常以较高的水平存在。 CypD-/-心脏中葡萄糖与棕榈酸代谢的比率比WT高1.5倍，这表明PDH活性增加。 在CypD-/-心脏中，与谷氨酸盐相比，13 C标记到琥珀酸盐中也显著增加，并且该结果与相对于其他竞争性反应，α-KGDH的活性增加一致。 我们测量了α-KGDH活性以评估琥珀酸上游的克雷布斯循环通量是否在CypD-/-心脏中升高，并发现α-KGDH活性增加了1.4倍。 因此，这些结果表明，MPT组分CypD的损失导致三羧酸循环和氧化代谢中的生理通量变化，这与Ca 2 +m增加一致。

The goal of this project is to 1) understand the role of mitochondria in ischemia-reperfusion injury and cardioprotection ; 2) to understand the role of altered ion homeostasis and altered metabolism in ischemia-reperfusion and cardioprotection and 3) to understand changes in cytosolic and mitochondrial signaling involved in cardioprotection and cell death. It is proposed that ischemic preconditioning (PC) initiates signaling that converges on mitochondria and results in cardioprotection. We also examined mechanisms by which inhibition of glycogen synthase kinase (GSK) mediates cardioprotection by acting on the mitochondria. Inhibition of GSK-3 reduces ischemia-reperfusion injury by mechanisms that involve the mitochondria. The goal of this study was to determine the molecular targets and mechanistic basis of this cardioprotective effect. We found that GSK inhibition slowed ATP consumption under these conditions, which could be due to inhibition of ATP entry into the mitochondria through VDAC and/or ANT or to inhibition of the F1F0 ATPase. We therefore used proteomic methods to test the hypothesis that PC and pharmacological preconditioning similarly alter mitochondrial signaling complexes. Langendorff-perfused murine hearts were treated with a specific GSK-3 inhibitor AR-A014418 (GSK Inhib VIII) for 10 min or subjected to 4 cycles of 5-min ischemia/reperfusion (PC) prior to 20-min global ischemia and 120-min reperfusion. PC and GSK Inhib VIII both improved post-ischemic left ventricular developed pressure (LVDP) recovery, decreased infarct size, and reduced lactate production during ischemia compared to their time-matched controls. We used proteomics to examine mitochondrial protein levels/post-translational modifications that were common between PC and GSK Inhib VIII. Levels of cytochrome c oxidase subunits Va and VIb, ATP synthase-coupling factor 6, and cytochrome b-c1 complex subunit 6 were increased while cytochrome c was decreased in PC and GSK Inhib VIII. Furthermore, the amount of cytochrome c oxidase subunit VIb was found to be increased in PC and GSK Inhib VIII mitochondrial supercomplexes, which are comprised of complexes I, III, and IV. This result would suggest that changes in complex subunits associated with cardioprotection may affect supercomplex composition. Thus, the ability of PC and GSK inhibition to alter the expression levels of electron transport complexes will have important implications for mitochondrial function. Isolated mitochondria from mice deficient in cyclophilin D (CypD-/-) are less sensitive to Ca2+-induced opening of the mitochondrial permeability transition (MPT) in vitro. Thus, the lack of CypD enables heart mitochondria to take up more Ca2+ before undergoing the MPT. We hypothesize that the MPT serves as a Ca2+-safety valve that can open to release excess Ca2+, but not necessarily result in death. If the MPT is blocked in CypD-/- mice, we hypothesize that matrix Ca2+ (Ca2+m) would be higher in CypD-/- mice compared to WT and this would activate Ca2+-sensitive NADH dehydrogenases (e.g., pyruvate dehydrogenase (PDH) and alpha-ketoglutarate dehydrogenase (alpha-KGDH)), which would in turn, alter oxidative metabolism and increase oxygen consumption. Consistent with this, we found altered expression levels of PDH E1 subunit and the alpha-KGDH E2 subunit in CypD-/- hearts using 2D DIGE proteomics. To evaluate differences in metabolism, we perfused hearts with 13C-glucose and 13C-palmitate and looked at their contribution to the acetyl-CoA pool by measuring label incorporation into the C4 of glutamate. The 13C-labeled glucose or palmitate enters the Krebs cycle and labels the alpha-KG pool that is in equilibrium with glutamate, which is usually present at higher levels. The ratio of glucose to palmitate metabolism in CypD-/- hearts was 1.5-fold higher than in WT, which would suggest increased PDH activity. 13C-labeling into succinate compared to glutamate was also increased significantly in CypD-/- hearts, and this result would be consistent with increased activity of alpha-KGDH relative to other competing reactions. We measured alpha-KGDH activity to evaluate whether Krebs cycle flux upstream of succinate was elevated in CypD-/- hearts and found a 1.4 fold increase in alpha-KGDH activity. Therefore, these results demonstrate that the loss of a MPT component, CypD, results in physiological flux changes in the Krebs cycle and oxidative metabolism that are consistent with increased Ca2+m.