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肌保护中的作用；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)了解参与心肌保护和细胞死亡的胞浆和线粒体信号的变化。有人认为，缺血预适应(PC)启动了汇聚到线粒体上的信号，从而导致心脏保护。众所周知，PC涉及一氧化氮信号转导。 最近的数据表明，心脏保护可以在涉及热休克蛋白90(HSP90)的过程中导致特定蛋白质进入线粒体，并被HSP90抑制剂格尔达那霉素(GD)阻断。为了验证线粒体输入的改变是更广泛的心脏保护特征的假设，在本研究中，我们使用广泛的蛋白质组学方法研究了糖原合成酶激酶(GSK)-3抑制诱导的心肌保护后线粒体蛋白质组的变化。从对照心脏中分离线粒体，在分离线粒体之前，用GSK抑制剂SB 216763(SB)灌流心脏15min。用同位素标记的相对和绝对定量(ITRAQ)标记对照和SB灌流心脏的线粒体提取液，并用质谱仪测定线粒体蛋白水平的差异。为了检测HSP90介导的蛋白导入的作用，在有或没有GD的情况下，心脏灌流15min，然后用SB灌流，然后进行线粒体分离和iTRAQ标记。我们证实，在缺血20分钟和再灌流40分钟的方案中，GD治疗阻断了SB治疗所提供的保护。我们发现有16种蛋白质在SB处理后线粒体比例明显增加。GD处理显著抑制了SB对其中5种蛋白线粒体结合的影响，这些蛋白包括膜联蛋白A6、纽蛋白和丙酮酸激酶。我们还发现，SB处理导致了8种蛋白质的线粒体含量下降，除2种外，其余都是已建立的线粒体蛋白质。为了确认线粒体输入与蛋白质合成和/或降解的变化之间的作用，我们测量了整个细胞提取物中这些蛋白质的变化。综上所述，这些数据表明，SB导致部分对GD敏感的线粒体蛋白质组重塑。 我们还对亲环素D的生理作用感兴趣，从亲环素D缺陷小鼠分离的线粒体(CypD-/-)对钙离子诱导的体外线粒体通透性转变(MPT)的开放不那么敏感。因此，缺乏CypD使心脏线粒体在接受MPT之前能够吸收更多的钙离子。我们假设MPT是一个钙安全阀，可以打开以释放过量的钙，但不一定会导致死亡。如果MPT在CypD-/-小鼠中被阻断，我们假设CypD-/-小鼠的基质钙(Ca~(2+)m)将比WT更高，这将激活对钙敏感的NADH脱氢酶(例如，丙酮酸脱氢酶(PDH)和α-酮戊二酸脱氢酶(α-KGDH))，这将反过来改变氧化代谢和增加氧气消耗。与此一致，我们利用2D DGE蛋白质组学发现，PDH E1亚基和α-KGDHE2亚基在CypD/-心脏中的表达水平发生了变化。因此，这些结果表明，MPT组分CypD的丢失导致了Krebs循环和氧化代谢的生理通量变化，这与Ca~(2+)m的增加是一致的。 如上所述。缺乏线粒体伴侣蛋白亲环素D(CypD-/-)的小鼠已经改变了心脏代谢。由于乙酰化已被证明可以调节新陈代谢，我们测试了蛋白质乙酰化的变化是否可能在CypD-/-心脏的这些代谢变化中发挥作用。为了确定CypD消融后赖氨酸乙酰化蛋白的变化和MAP乙酰化位点，我们对WT和CypD-/-小鼠分离的心肌线粒体进行胰酶消化，使用偶联抗乙酰赖氨酸抗体的琼脂糖珠进行免疫沉淀，然后进行质谱分析。我们使用无标记分析对WT和CypD-/-样品中乙酰化的875个常见多肽进行了相对定量，发现CypD-/-样品中11个多肽(10个蛋白质)减少，96个多肽(48个蛋白质)增加。我们发现，在脂肪酸氧化和支链氨基酸代谢中，蛋白质的乙酰化程度增加。为了评估这种乙酰化增加是否可能在抑制CypD-/-心脏的脂肪酸氧化中发挥作用，我们测量了CypD-/-心脏中乙酰化的L-3-羟基酰辅酶A脱氢酶(LCHAD)的活性。与假设一致，与WT线粒体相比，LCHAD活性被抑制了大约50%。这些结果暗示了CypD在调节蛋白质乙酰化中的作用。综上所述，这些结果表明，CypD的消融导致线粒体乙酰组的改变，这可能是导致CypD-/-小鼠线粒体代谢改变的原因之一。

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. 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 proteomics. 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. As described above. mice lacking cyclophilin D (CypD-/-), a mitochondrial chaperone protein, have altered cardiac metabolism. As acetylation has been shown to regulate metabolism, we tested whether changes in protein acetylation might play a role in these metabolic changes in CypD-/- hearts. To identify changes in lysine-acetylated proteins and map acetylation sites following ablation of CypD, we subjected tryptic digests of isolated cardiac mitochondria from WT and CypD-/- mice to immunoprecipitation using agarose beads coupled to anti-acetyl lysine antibodies followed by mass spectrometry. We used label-free analysis for the relative quantification of the 875 common peptides that were acetylated in WT and CypD-/- samples and found 11 peptides (10 proteins) decreased and 96 peptides (48 proteins) increased in the CypD-/- samples. We found increased acetylation of proteins in fatty acid oxidation and branched-chain amino acid metabolism. To evaluate whether this increase in acetylation might play a role in the inhibition of fatty acid oxidation that was previously reported in CypD-/- hearts, we measured the activity of L-3-hydroxyacyl-CoA dehydrogenase (LCHAD), which was acetylated in the CypD-/- hearts. Consistent with the hypothesis, LCHAD activity was inhibited by approximately 50% compared to the WT mitochondria. These results implicate a role for CypD in modulating protein acetylation. Taken together, these results suggest that ablation of CypD leads to changes in the mitochondrial acetylome, which may contribute to altered mitochondrial metabolism in CypD-/- mice.