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: 8557913
• 负责人: Elizabeth Murphy
• 金额: $ 54.77万
• 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/8557913
• 关键词: Acute; Annexin A6; Ascorbic Acid; Calcium; Cardiac; Caveolae; Cell Death; Cell Extracts; Cell Respiration; Cell membrane; Cessation of life; Cholesterol; Citric Acid Cycle; Cyclodextrins; Cytochrome c Reductase; Data; Dissociation; Electron Transport; G Protein-Coupled Receptor Signaling; Geldanamycin; Generations; Glycogen Synthase Kinase 3; Glycogen Synthase Kinases; Goals; Heart; Heart Mitochondria; Heat-Shock Proteins 90; Homeostasis; In Vitro; Infarction; Injury; Ion Pumps; Ions; Ischemia; Ischemic Preconditioning; Isotopes; Ketoglutarate Dehydrogenase Complex; Label; Location; Mass Spectrum Analysis; Measures; Mediating; Metabolism; Methods; Mitochondria; Mitochondrial Proteins; Modeling; Mus; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Synthase; Oxygen; Oxygen Consumption; Perfusion; Permeability; Physiological; Plasma Cells; Play; Post-Translational Protein Processing; Process; Protein Biosynthesis; Protein Import; Protein S; Proteins; Proteome; Proteomics; Proto-Oncogene Proteins c-akt; Protocols documentation; Pyruvate Dehydrogenase E1; Pyruvate Kinase; Reactive Oxygen Species; Reducing Agents; Relative (related person); Reperfusion Injury; Reperfusion Therapy; Reporting; Role; Rupture; Safety; Signal Pathway; Signal Transduction; Structure; Testing; Vinculin; arginine methyl ester; base; caveolin-3; cyclophilin D; 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. 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.

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。我们验证了小凹可能作为一个信号模块将信号从质膜上的G蛋白偶联受体传递到线粒体的假设。一氧化氮(NO)和蛋白S亚硝酸酯(SNO)在缺血预适应(IPC)诱导的心肌保护中起重要作用。线粒体是预适应的关键调节者，随着IPC的增加，SNO增加的大多数蛋白质都是线粒体。然而，目前还不清楚IPC是如何将NO/SNO信号转导到线粒体的。在采用朗宁多夫灌流小鼠心脏的研究中，我们发现，N-硝基-L-精氨酸甲酯(L-NAME，一种结构性NO合成酶抑制剂)、抗坏血酸(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隔离剂)均可阻断IPC的心脏保护作用。IPC不仅激活了AKT/eNOS信号转导通路，还导致eNOS易位到线粒体。M-β；Cd处理破坏了小窝结构，导致eNOS从小窝-3解离，并阻断了IPC诱导的AKT/eNOS信号通路的激活。与灌流对照组相比，IPC心肌线粒体SNO含量显著增加，Mbeta；Cd不仅可阻断IPC诱导的心肌保护作用，还可阻断IPC诱导的SNO增加。这些结果表明，小窝转导了IPC诱导的eNOS/NO/SNO心脏急性保护信号。 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。与此一致的是，我们利用2D DGE蛋白质组学发现，在CypD/-心脏中，PDH E1亚基和α-KGDHE2亚基的表达水平发生了变化。因此，这些结果表明，MPT组分CypD的缺失导致了Krebs循环和氧化代谢的生理性通量变化，这与Ca~(2+)m的增加是一致的。