Mechanisms of cardiac ischemia-reperfusion injury and cardioprotection

本项目的长期目标是：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。 已知PC涉及一氧化氮信号传导。 我们测试的假设，caveolea可能作为一个信号传导模块，从G蛋白偶联受体的质膜上的线粒体的信号。 一氧化氮（NO）和蛋白质S-亚硝基化（SNO）在缺血预处理（IPC）诱导的心肌保护中起重要作用。线粒体是预处理的关键调节物，并且显示SNO随着IPC增加的大多数蛋白质是线粒体的。然而，目前尚不清楚IPC如何将NO/SNO信号转导至线粒体。 在这项使用Langendorff灌注小鼠心脏的研究中，我们发现IPC诱导的心脏保护作用被N-硝基-L-精氨酸甲酯（L-NAME，一种组成型NO合成酶抑制剂），抗坏血酸（一种分解SNO的还原剂）或甲基-b-环糊精（MbCD，一种破坏小窝的胆固醇螯合剂）阻断。IPC不仅激活AKT/eNOS信号通路，而且使eNOS向线粒体转位。M-β; CD处理破坏了小窝结构，导致eNOS从小窝蛋白-3上解离，并阻断了IPC诱导的AKT/eNOS信号通路的激活。与灌注对照相比，IPC心脏中线粒体SNO显著增加，并且Mbeta的小窝破坏;CD治疗不仅废除了IPC诱导的心脏保护作用，而且还阻断了IPC诱导的SNO增加。这些结果表明，Caveolae EMPIC诱导的eNOS/NO/SNO急性心脏保护信号在心脏中。 最近的数据表明，心脏保护可以导致特定蛋白质进入线粒体的过程中，涉及热休克蛋白90（HSP 90），并被格尔德霉素（GD），HSP 90抑制剂阻断。为了验证线粒体输入的改变是心脏保护的一个更普遍的特征这一假设，在这项研究中，我们使用了一种广泛的蛋白质组学方法来研究糖原合成酶激酶（GSK）-3抑制诱导的心脏保护后线粒体蛋白质组的变化。从对照心脏分离线粒体，在分离线粒体之前，用GSK抑制剂SB 216763（SB）灌注心脏15分钟。对照组和SB灌注心脏的线粒体提取物用同位素标记进行相对和绝对定量（iTRAQ），并通过质谱法测定线粒体蛋白水平的差异。为了测试HSP 90介导的蛋白质输入的作用，在GD存在和不存在的情况下灌注心脏15分钟，然后用SB灌注，随后进行线粒体分离和iTRAQ标记。我们证实，在20分钟的缺血和40分钟的再灌注方案中，GD治疗阻断了SB治疗所提供的保护。我们发现了16种蛋白质，显示出明显的增加，在SB治疗后的线粒体分数。GD治疗显著阻断了SB介导的这些蛋白质中的五种的线粒体缔合的增加，所述蛋白质包括膜联蛋白A6、黏着斑蛋白和丙酮酸激酶。我们还发现SB处理导致8种蛋白质的线粒体含量减少，其中除了两种之外，其他都是已建立的线粒体蛋白质。为了证实线粒体输入相对于蛋白质合成和/或降解的变化的作用，我们测量了全细胞提取物中这些蛋白质的变化。总之，这些数据表明，SB导致部分GD敏感的线粒体蛋白质组的重塑。 我们也有兴趣研究亲环素D的生理作用。 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，我们使用2D DIGE蛋白质组学发现CypD-/-心脏中PDH E1亚基和α-KGDH E2亚基的表达水平改变。因此，这些结果表明，MPT组分CypD的丢失导致Krebs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 long-term goals of this project are 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. PC is known to involve nitric oxide signaling. We tested the hypothesis that caveolea might serve as a signaling module to transmit signals from G-protein coupled receptors on the plasma membrane to the mitochondria. Nitric oxide (NO) and protein S-nitrosylation (SNO) have been shown to play important roles in ischemic preconditioning (IPC)-induced cardioprotection. Mitochondria are key regulators of preconditioning and most proteins showing an increase in SNO with IPC are mitochondrial. However, it is not clear how IPC transduces NO/SNO signaling to mitochondria. In this study using Langendorff perfused mouse hearts, we found that IPC-induced cardioprotection was blocked by treatment with either N-nitro-L-arginine methyl ester (L-NAME, a constitutive NO synthase inhibitor), ascorbic acid (a reducing agent to decompose SNO), or methyl-b-cyclodextrin (MbCD, a cholesterol sequestering agent to disrupt caveolae). IPC not only activated AKT/eNOS signaling but also led to translocation of eNOS to mitochondria. M-beta;CD treatment disrupted caveolae structure, leading to dissociation of eNOS from caveolin-3 and blockade of IPC-induced activation of the AKT/eNOS signaling pathway. A significant increase in mitochondrial SNO was found in IPC hearts compared to perfusion control, and the disruption of caveolae by Mbeta;CD treatment not only abolished IPC-induced cardioprotection, but also blocked IPC-induced increase in SNO. These results suggest that caveolae transduce IPC-induced eNOS/NO/SNO acute cardioprotective signaling in the heart. Recent data have shown that cardioprotection can result in the import of specific proteins into the mitochondria in a process that involves heat shock protein 90 (HSP90) and is blocked by geldanamycin (GD), a HSP90 inhibitor. To test the hypothesis that an alteration in mitochondrial import is a more widespread feature of cardioprotection, in this study, we used a broad-based proteomics approach to investigate changes in the mitochondrial proteome following cardioprotection induced by inhibition of glycogen synthase kinase (GSK)-3. Mitochondria were isolated from control hearts, and hearts were perfused with the GSK inhibitor SB 216763 (SB) for 15 min before isolation of mitochondria. Mitochondrial extracts from control and SB-perfused hearts were labeled with isotope tags for relative and absolute quantification (iTRAQ), and differences in mitochondrial protein levels were determined by mass spectrometry. To test for the role of HSP90-mediated protein import, hearts were perfused in the presence and absence of GD for 15 min before perfusion with SB followed by mitochondrial isolation and iTRAQ labeling. We confirmed that treatment with GD blocked the protection afforded by SB treatment in a protocol of 20 min of ischemia and 40 min of reperfusion. We found 16 proteins that showed an apparent increase in the mitochondrial fraction following SB treatment. GD treatment significantly blocked the SB-mediated increase in mitochondrial association for five of these proteins, which included annexin A6, vinculin, and pyruvate kinase. We also found that SB treatment resulted in a decrease in mitochondrial content of eight proteins, of which all but two are established mitochondrial proteins. To confirm a role for mitochondrial import versus a change in protein synthesis and/or degradation, we measured changes in these proteins in whole cell extracts. Taken together, these data show that SB leads to a remodeling of the mitochondrial proteome that is partially GD sensitive. We were also interested in examining the physiological role of cyclophilin D. 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 proteomicsTherefore, 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.