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: 8344761
• 负责人: Elizabeth Murphy
• 金额: $ 52.79万
• 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/8344761
• 关键词: Acetyl Coenzyme A; Acute; Adenoviruses; Ascorbic Acid; Attenuated; Calcium; Calcium-Sensing Receptors; Cardiac; Cardiac Myocytes; Cardiovascular system; Caveolae; Cell Death; Cell Respiration; Cell membrane; Cell physiology; Cessation of life; Cholesterol; Citric Acid Cycle; Cyclodextrins; Cyclophilins; Cysteine; Cytochrome c Reductase; Cytoprotection; Dissociation; Electron Transport; Embryo; Equilibrium; Exhibits; Fibroblasts; Fluorescence; G Protein-Coupled Receptor Signaling; G-Protein-Coupled Receptors; Generations; Glucose; Glutamates; Goals; Heart; Heart Mitochondria; Homeostasis; Hydrogen Peroxide; Immunoblot Analysis; In Vitro; Infarction; Injury; Ion Pumps; Ions; Ischemia; Ischemic Preconditioning; Ketoglutarate Dehydrogenase Complex; Knockout Mice; Label; Left; Ligands; Liver; Location; MAPK3 gene; Measures; Mediating; Mediator of activation protein; Metabolism; Methods; Mitochondria; Modeling; Mus; Mutate; Mutation; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase; Oxidation-Reduction; Oxygen; Oxygen Consumption; Palmitates; Pathology; Perfusion; Permeability; Physiological; Plasma Cells; Play; Post-Translational Protein Processing; Process; Protein S; Proteins; Proteomics; Proto-Oncogene Proteins c-akt; Pyruvate Dehydrogenase E1; Reaction; Reactive Oxygen Species; Recombinants; Recovery of Function; Reducing Agents; Relative (related person); Reperfusion Injury; Reperfusion Therapy; Reporting; Resistance; Role; Rupture; Safety; Serine; Signal Pathway; Signal Transduction; Site; Stress; Structure; Succinates; Swelling; Tail; Testing; Veins; Ventricular; arginine methyl ester; caveolin-3; cyclophilin D; extracellular; fluorexon; hemodynamics; improved; in vivo; inhibitor/antagonist; interest; mitochondrial permeability transition pore; novel; preconditioning; prevent; pyruvate dehydrogenase; reconstitution; vector

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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 MCD treatment not only abolished IPC-induced cardioprotection, but also blocked IPC-induced increase in SNO. In conclusion, these results suggest that caveolae transduce IPC-induced eNOS/NO/SNO acute cardioprotective signaling in the heart. We also test the role of a novel G-protein coupled receptor the extracellular Ca2+-sensing receptor (CaSR) in cardioprotection. The CaSR responds to changes not only in extracellular Ca2+ but also to many other ligands. CaSR has been found to be expressed in the hearts and cardiovascular system. In this study, we confirmed that CaSR is expressed in mouse cardiomyocytes, and showed that it is predominantly localized in caveolae. We investigated whether CaSR plays a cardioprotective role in ischemic preconditioning (IPC). Hearts from C57BL/6J mice were perfused in the Langendorff mode and subjected to the following treatments: (1) control perfusion; (2) perfusion with a specific CaSR antagonist, NPS2143; (3) IPC (four cycles of 5 min of global ischemia and 5 min of reperfusion); or (4) perfusion with NPS2143 prior to and during IPC. Following these treatments hearts were subjected to 20 min of no-flow global ischemia and 120 min of reperfusion. Compared with control, IPC significantly improved post-ischemic left ventricular functional recovery and reduced infarct size. Although NPS2143 perfusion alone did not change the hemodynamic function and did not change the extent of post-ischemic injury, NPS2143 treatment abolished cardioprotection of IPC. Through immunoblot analysis, it was demonstrated that IPC significantly increased the levels of phosphorylated ERK1/2, AKT, and GSK3β, which were also prevented by NPS2143 treatment. Taken together, the distribution of CaSR in caveolae along with NPS2143-blockable IPC-induced cardioprotective signaling suggest that the activation of CaSR during IPC is cardioprotective, a process involving caveolae. Another project involves examining the role of cyclophilin D in cell physiology and pathology. Following ischemia and reperfusion a mitochondrial pore, known as the mitochondrial transition pore (mPTP) opens and leads to cell death. Cyclophilin is the only identified component of mPTP. Mitochondrial permeability transition pore (mPTP) opening plays a critical role in mediating cell death during ischemia/ reper-fusion (I/R) injury. Our previous studies have shown that protein S-nitrosylation (SNO) plays a protective role in I/R injury and that the SNO of cyclophilin D (CypD), a critical mPTP mediator, may be a functional target in orchestrating cytoprotection. To investigate whether SNO of CypD might attenuate mPTP activation, we mutated cysteine 203 of CypD, the SNO site, to a serine residue (C203S) and determined its effects on mPTP opening. Treatment of wildtype (WT) mouse embryonic fibroblasts (MEFs) with H2O2 resulted in an ≈50% loss of the mitochondrial calcein fluorescence, suggesting substantial activation of the mPTP. Consistent with the reported role of CypD in mPTP activation, CypD null (CypD-/-) MEFs exhibited significantly less mPTP opening. Addition of a nitric oxide donor, GSNO, to WT but not CypD-/- MEFs prior to H2O2 attenuated mPTP opening. To test whether C203 is required for this protection, we infected CypD-/- MEFs with a C203S-CypD vector. Surprisingly, C203S-CypD re-constituted MEFs were resistant to mPTP opening in the presence or absence of GSNO, suggesting a crucial role for C203 in mPTP activation. To determine whether mutation of C203S-CypD would alter mPTP in vivo, we injected a recombinant adenovirus encoding C203S-CypD or WT CypD into CypD-/- mice via tail-vein. Mitochondria isolated from livers of CypD-/- mice or mice expressing C203S-CypD were resistant to Ca2+-induced swelling as compared to WT CypD reconstituted mice. Our results indicate that the cysteine 203 residue of CypD is necessary for redox stress-induced activation of mPTP. 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. 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.

该项目的长期目标是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质和线粒体信号传导的变化。 有人提出，缺血预适应（PC）启动信号汇聚于线粒体并产生心脏保护作用。 众所周知，PC 涉及一氧化氮信号传导。 我们测试了这样的假设：小窝可能作为信号模块，将信号从质膜上的 G 蛋白偶联受体传递到线粒体。 一氧化氮 (NO) 和蛋白质 S-亚硝基化 (SNO) 已被证明在缺血预适应 (IPC) 诱导的心脏保护中发挥重要作用。线粒体是预处理的关键调节因子，大多数显示 IPC 中 SNO 增加的蛋白质都是线粒体。然而，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，我们发现用 N-硝基-L-精氨酸甲酯（L-NAME，一种组成型 NO 合酶抑制剂）、抗坏血酸（一种分解 SNO 的还原剂）或甲基-b-环糊精（MbCD，一种胆固醇螯合剂，可破坏 SNO）治疗可阻断 IPC 诱导的心脏保护作用。 小穴）。 IPC不仅激活AKT/eNOS信号传导，还导致eNOS易位至线粒体。 MCD 治疗破坏了小凹结构，导致 eNOS 从 Caveolin-3 解离，并阻断 IPC 诱导的 AKT/eNOS 信号通路激活。与灌注对照相比，IPC 心脏中线粒体 SNO 显着增加，并且 MCD 治疗对小窝的破坏不仅消除了 IPC 诱导的心脏保护作用，而且还阻断了 IPC 诱导的 SNO 增加。总之，这些结果表明，小窝转导了 IPC 诱导的 eNOS/NO/SNO 心脏中的急性心脏保护信号。 我们还测试了一种新型 G 蛋白偶联受体细胞外 Ca2+ 感应受体 (CaSR) 在心脏保护中的作用。 CaSR 不仅响应细胞外 Ca2+ 的变化，还响应许多其他配体的变化。 CaSR 已被发现在心脏和心血管系统中表达。在这项研究中，我们证实 CaSR 在小鼠心肌细胞中表达，并表明它主要位于小凹中。我们研究了 CaSR 在缺血预处理 (IPC) 中是否发挥心脏保护作用。 C57BL/6J小鼠心脏采用Langendorff模式进行灌注，并进行以下处理：（1）对照灌注； (2)用特异性CaSR拮抗剂NPS2143进行灌注； (3) IPC(4个周期，5分钟的整体缺血和5分钟的再灌注)；或 (4) 在 IPC 之前和期间用 NPS2143 进行灌注。在这些治疗之后，心脏经历 20 分钟的无血流整体缺血和 120 分钟的再灌注。与对照组相比，IPC 显着改善了缺血后左心室功能恢复并减少了梗塞面积。虽然单独使用 NPS2143 灌注不会改变血流动力学功能，也不会改变缺血后损伤的程度，但 NPS2143 治疗消除了 IPC 的心脏保护作用。通过免疫印迹分析，表明 IPC 显着增加了磷酸化 ERK1/2、AKT 和 GSK3β 的水平，而 NPS2143 治疗也可以阻止这种情况。总而言之，CaSR 在小凹中的分布以及 NPS2143 可阻断的 IPC 诱导的心脏保护信号表明，IPC 期间 CaSR 的激活具有心脏保护作用，这是一个涉及小凹的过程。 另一个项目涉及检查亲环蛋白 D 在细胞生理学和病理学中的作用。缺血和再灌注后，线粒体孔（称为线粒体过渡孔（mPTP））打开并导致细胞死亡。 亲环蛋白是 mPTP 中唯一已确定的成分。线粒体通透性转换孔（mPTP）开放在介导缺血/再灌注（I/R）损伤期间的细胞死亡中发挥着关键作用。 我们之前的研究表明，蛋白质 S-亚硝基化 (SNO) 在 I/R 损伤中发挥保护作用，而亲环蛋白 D (CypD)（一种关键的 mPTP 介质）的 SNO 可能是协调细胞保护的功能靶点。 为了研究 CypD 的 SNO 是否可能减弱 mPTP 激活，我们将 CypD 的半胱氨酸 203（SNO 位点）突变为丝氨酸残基 (C203S)，并确定其对 mPTP 打开的影响。 用 H2O2 处理野生型 (WT) 小鼠胚胎成纤维细胞 (MEF) 导致线粒体钙黄绿素荧光损失约 50%，表明 mPTP 显着激活。 与报道的 CypD 在 mPTP 激活中的作用一致，CypD 无效 (CypD-/-) MEF 表现出明显较少的 mPTP 开放。 在 H2O2 减弱 mPTP 开放之前，向 WT 添加一氧化氮供体 GSNO，但不添加 CypD-/- MEF。 为了测试这种保护是否需要 C203，我们用 C203S-CypD 载体感染 CypD-/- MEF。令人惊讶的是，C203S-CypD 重建的 MEF 在存在或不存在 GSNO 的情况下都能抵抗 mPTP 打开，这表明 C203 在 mPTP 激活中发挥着至关重要的作用。 为了确定 C203S-CypD 的突变是否会改变体内的 mPTP，我们通过尾静脉将编码 C203S-CypD 或 WT CypD 的重组腺病毒注射到 CypD-/- 小鼠中。 与 WT CypD 重组小鼠相比，从 CypD-/- 小鼠或表达 C203S-CypD 的小鼠肝脏中分离的线粒体对 Ca2+ 诱导的肿胀具有抵抗力。我们的结果表明 CypD 的半胱氨酸 203 残基对于氧化还原应激诱导的 mPTP 激活是必需的。 我们还对检查亲环蛋白 D 的生理作用感兴趣。从缺乏亲环蛋白 D (CypD-/-) 的小鼠中分离出的线粒体在体外对 Ca2+ 诱导的线粒体通透性转变 (MPT) 打开不太敏感。 因此，CypD 的缺乏使心脏线粒体在进行 MPT 之前吸收更多的 Ca2+。 我们假设 MPT 作为 Ca2+ 安全阀，可以打开释放过量的 Ca2+，但不一定会导致死亡。 如果 CypD-/- 小鼠中的 MPT 被阻断，我们假设与 WT 相比，CypD-/- 小鼠中的基质 Ca2+ (Ca2+m) 会更高，这将激活 Ca2+ 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)）， 这反过来会改变氧化代谢并增加耗氧量。 与此一致的是，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 亚基的表达水平发生了变化。 为了评估代谢差异，我们用 13C-葡萄糖和 13C-棕榈酸酯灌注心脏，并通过测量谷氨酸 C4 中的标签掺入情况来观察它们对乙酰辅酶 A 库的贡献。 13C 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 库，谷氨酸通常以较高水平存在。 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。 与谷氨酸相比，在 CypD-/- 心脏中，琥珀酸中的 13C 标记也显着增加，并且该结果与 α-KGDH 相对于其他竞争反应的活性增加是一致的。 我们测量了 α-KGDH 活性，以评估 CypD-/- 心脏中琥珀酸上游的克雷布斯循环通量是否升高，并发现 α-KGDH 活性增加了 1.4 倍。 因此，这些结果表明，MPT 成分 CypD 的丢失会导致三羧酸循环和氧化代谢的生理通量变化，这与 Ca2+m 的增加一致。