Mechanisms of cardiac ischemia-reperfusion injury and cardioprotection

心脏缺血再灌注损伤机制及心脏保护作用

• 批准号: 8746558
• 负责人: Elizabeth Murphy
• 金额: $ 53.13万
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8746558
• 关键词: Ablation; Acetylation; Annexin A6; Antibodies; Branched-Chain Amino Acids; Calcium; Cardiac; Cell Death; Cell Extracts; Cell Respiration; Cell membrane; Cessation of life; Citric Acid Cycle; Coenzyme A; Coupled; Cytochrome c Reductase; Data; Electron Transport; Geldanamycin; Generations; Glycogen Synthase Kinase 3; Glycogen Synthase Kinases; Goals; Heart; Heart Mitochondria; Heat-Shock Proteins 90; Homeostasis; Immunoprecipitation; In Vitro; Infarction; Injury; Ion Pumps; Ions; Ischemia; Ischemic Preconditioning; Isotopes; Ketoglutarate Dehydrogenase Complex; L-3; Label; Location; Lysine; Maps; Mass Spectrum Analysis; Measures; Mediating; Metabolic; Metabolism; Methods; Mitochondria; Mitochondrial Proteins; Modeling; Molecular Chaperones; Mus; Nitric Oxide; Oxidoreductase; Oxygen; Oxygen Consumption; Peptides; Perfusion; Permeability; Physiological; Plasma Cells; Play; Post-Translational Protein Processing; Process; Protein Acetylation; Protein Biosynthesis; Protein Import; Proteins; Proteome; Proteomics; Protocols documentation; Pyruvate Dehydrogenase E1; Pyruvate Kinase; Reactive Oxygen Species; Relative (related person); Reperfusion Injury; Reperfusion Therapy; Reporting; Role; Rupture; Safety; Sampling; Sepharose; Signal Pathway; Signal Transduction; Site; Testing; Vinculin; base; cyclophilin D; fatty acid oxidation; heart metabolism; inhibitor/antagonist; interest; kinase inhibitor; mitochondrial permeability transition pore; preconditioning; pyruvate dehydrogenase

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.

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。 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16种蛋白质在SB处理后显示线粒体分数明显增加。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。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。 如上所述。缺乏亲环素D（CypD-/-）（一种线粒体伴侣蛋白）的小鼠心脏代谢发生改变。 由于乙酰化已被证明可以调节代谢，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。为了鉴定赖氨酸乙酰化蛋白的变化和CypD消融后的乙酰化位点，我们将WT和CypD-/-小鼠的分离心脏线粒体的胰蛋白酶消化物进行免疫沉淀，使用偶联抗乙酰赖氨酸抗体的琼脂糖珠，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种常见肽进行相对定量，发现CypD-/-样品中11种肽（10种蛋白质）减少，96种肽（48种蛋白质）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增加。 为了评估这种乙酰化的增加是否可能在先前报道的CypD-/-心脏中脂肪酸氧化的抑制中起作用，我们测量了L-3-羟酰辅酶A脱氢酶（LCHAD）的活性，该酶在CypD-/-心脏中被乙酰化。 与该假设一致，与WT线粒体相比，LCHAD活性被抑制约50%。 这些结果暗示CypD在调节蛋白质乙酰化中的作用。 总之，这些结果表明，CypD的消融导致线粒体乙酰组的变化，这可能有助于改变CypD-/-小鼠的线粒体代谢。