Mechanisms of cardiac ischemia-reperfusion injury and cardioprotection

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的变化。有人提出，缺血预处理（PC）启动信号向线粒体聚集并导致心脏保护。已知PC与一氧化氮信号有关。我们验证了一个假设，即小泡可能作为一个信号模块，将质膜上g蛋白偶联受体的信号传递到线粒体。一氧化氮（NO）和蛋白质s -亚硝基化（SNO）已被证明在缺血预处理（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，IPC导致SNO增加的大多数蛋白质都是线粒体。然而，目前尚不清楚IPC如何将NO/SNO信号转导到线粒体。在这项使用Langendorff灌注小鼠心脏的研究中，我们发现，通过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，一种破坏小泡的胆固醇隔离剂）处理，ipc诱导的心脏保护作用被阻断。IPC不仅激活AKT/eNOS信号通路，还导致eNOS向线粒体易位。CD处理破坏了小泡结构，导致eNOS与caveolin-3分离，阻断了ipc诱导的AKT/eNOS信号通路的激活。与灌注对照组相比，IPC心脏中线粒体SNO显著增加，M&#61538；CD治疗对小泡的破坏不仅可以消除IPC诱导的心脏保护，还可以阻断IPC诱导的SNO增加。综上所述，这些结果表明，小泡介导了ipc诱导的eNOS/NO/SNO急性心脏保护信号。我们还测试了一种新的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，我们证实了CaSR在小鼠心肌细胞中表达，并表明它主要定位于小泡中。我们研究了CaSR是否在缺血预处理（IPC）中发挥心脏保护作用。采用Langendorff模式灌注C57BL/6J小鼠心脏，并进行以下处理：(1)对照灌注；(2)灌注特异性CaSR拮抗剂NPS2143；(3) IPC（全脑缺血5 min再灌注5 min 4个周期）；或(4)在IPC之前和期间灌注NPS2143。在这些治疗之后，心脏进行20分钟的无血流全脑缺血和120分钟的再灌注。与对照组相比，IPC可显著改善缺血后左心室功能恢复，缩小梗死面积。虽然单独灌注NPS2143没有改变血流动力学功能，也没有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，我们发现IPC显著提高了磷酸化的ERK1/2、AKT和GSK3&#946；的水平，而NPS2143也可以阻止这些磷酸化。综上所述，CaSR在小泡中的分布以及nps2143可阻断的IPC诱导的心脏保护信号表明，在IPC过程中，CaSR的激活具有心脏保护作用，这一过程涉及小泡。另一个项目是研究亲环蛋白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。亲环蛋白是唯一确定的mPTP成分。线粒体通透性过渡孔（mPTP）开放在缺血/再融合（I/R）损伤中介导细胞死亡中起关键作用。我们之前的研究表明，蛋白质s -亚硝基化（SNO）在I/R损伤中起保护作用，而亲环蛋白D （CypD）的SNO是一种关键的mPTP介质，可能是协调细胞保护的功能靶点。为了研究CypD的SNO是否会减弱mPTP的激活，我们将CypD的SNO位点半胱氨酸203突变为丝氨酸残基（C203S），并确定其对mPTP开放的影响。H2O2处理野生型（WT）小鼠胚胎成纤维细胞（mef）导致线粒体钙黄蛋白荧光丢失50%，表明mPTP被大量激活。与报道的CypD在mPTP激活中的作用一致，CypD无效（CypD-/-） MEFs表现出明显较少的mPTP开放。在H2O2之前，将一氧化氮供体GSNO添加到WT中，而不是CypD-/- MEFs中，可以减弱mPTP的开放。为了测试C203是否需要这种保护，我们用C203S-CypD载体感染了CypD-/- mef。令人惊讶的是，C203S-CypD重组的mef在GSNO存在或不存在的情况下都能抵抗mPTP的开放，这表明C203在mPTP激活中起着至关重要的作用。为了确定C203S-CypD突变是否会改变体内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-/-小鼠体内。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+诱导的肿胀具有抗性。我们的研究结果表明CypD的半胱氨酸203残基是氧化还原应激诱导的mPTP激活所必需的。我们还对研究亲环蛋白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对Ca2+诱导的线粒体通透性转变（MPT）的开放不太敏感。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+，但不一定导致死亡。如果CypD-/-小鼠的MPT被阻断，我们假设与WT相比，CypD-/-小鼠的基质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氧气消耗。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亚基的表达水平发生了变化。为了评估代谢的差异，我们向心脏灌注13c -葡萄糖和13c -棕榈酸酯，并通过测量谷氨酸C4的标签掺入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酯进入克雷布斯循环并标记与谷氨酸平衡的α - kg池，谷氨酸通常以较高的水平存在。CypD-/-心脏中葡萄糖与棕榈酸盐代谢的比率比WT高1.5倍，这可能表明PDH活性增加。与谷氨酸相比，在CypD-/-心脏中，13c标记为琥珀酸盐也显著增加，这一结果与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，以评估琥珀酸盐上游的克雷布斯循环通量是否在CypD-/-心脏中升高，结果发现α - kgdh活性增加了1.4倍。因此，这些结果表明，MPT成分CypD的缺失导致克雷布斯循环和氧化代谢的生理通量变化，这与Ca2+m的增加是一致的。

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.