Mechanisms of cardiac ischemia-reperfusion injury and cardioprotection

这个项目的长期目标是：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心肌保护的作用。 缺血-再灌流和心脏保护中代谢的改变以及3)了解 胞浆和线粒体信号参与心脏保护和细胞死亡。有建议说 缺血预适应(PC)启动信号，汇聚到线粒体并导致 在心脏保护方面。 有研究认为，细胞在缺血再灌注后死亡，是由于线粒体钙离子升高，导致线粒体通透性转换孔(MPT)激活。线粒体钙的增加被认为是通过线粒体钙单转运体摄取钙而发生的。MPT的组成尚不清楚，但亲环素D已被证明是MPT的调节因子。 我们研究了线粒体钙单一转运体(MCU)在调节细胞死亡和代谢中的钙摄取作用。在与Toren Finkel博士的合作中，我们描述了一种缺乏最近发现的线粒体钙单转运体(MCU)表达的小鼠模型。来自MCU(-/-)小鼠的线粒体没有明显的快速吸收钙的能力。虽然基础代谢似乎没有受到影响，但MCU(-/-)小鼠的骨骼肌表现出丙酮酸脱氢酶的磷酸化和活性的变化。此外，MCU(-/-)小鼠在执行繁重工作的能力方面表现出明显的损害。我们进一步表明，来自MCU(-/-)小鼠的线粒体缺乏钙诱导的通透性转换孔(PTP)开放的证据。令人惊讶的是，缺乏PTP开放似乎并不能保护MCU(-/-)细胞和组织免于细胞死亡。在朗宁多夫灌流心脏模型中，在整体I/R后，WT心脏和MCU-/-小鼠的心脏之间无法区分梗死面积。使情况进一步复杂化的是，MPTP的抑制剂环孢菌素A可以减少WT心脏的梗塞面积，但不能缩小MCU-/-心脏的梗塞面积。我们一直在测试MCU的缺失是否会导致其他细胞死亡途径的改变。我们还检测了小鼠对添加异丙肾上腺素的反应。 我们还研究了琥珀酸化在调节心脏功能和对缺血和再灌流反应中的作用。琥珀酸化是指用琥珀酰辅酶A提供的琥珀酸基对赖氨酸残基进行修饰。Sirtuin5(SIRT5)是一种依赖于线粒体NAD+的脱酰基酶，催化蛋白质中琥珀酸基的去除。SIRT5和蛋白质琥珀酸化在物种中是保守的，这表明了修饰的功能重要性。SIRT5缺失会影响肝脏代谢，但琥珀酸化在心脏中的作用尚未被探索。我们将亲和富集与蛋白质组学和质谱学相结合，分析了WT和SIRT5-/-小鼠心脏线粒体的总琥珀酸化赖氨酸含量。我们在184个蛋白质中鉴定了887个琥珀酰化赖氨酸残基。44个多肽(5个蛋白质)在WT样本中独有，289个(46个蛋白质)在SIRT5-/-样本中出现，554个(133个蛋白质)在两组中共同存在。SIRT5-/-心脏中的46个独特蛋白质参与代谢过程，如脂肪酸氧化(ECI2)和支链氨基酸分解代谢，包括呼吸链蛋白(Ndufa7，12，13，Dhsa)。我们对WT和SIRT5-/-心脏共有的多肽进行了无标记分析。在SIRT5-/-中，来自9个蛋白的16个多肽显著增加了至少30%。SIRT5-/-中腺嘌呤核苷酸转运体1的琥珀酸化水平最高(108.4倍)。这些数据表明，琥珀酸化作用在心脏中广泛存在，并在代谢途径中丰富。我们研究了SIRT5的缺失是否会影响缺血再灌注(I/R)损伤，我们发现在缺血20分钟和再灌注90分钟后，SIRT5-/-心脏的梗塞面积比WT组增加(68.5+/-1.1%SIRT5-/-vs39.6+/-6.8%WT)。我们进一步证明，用琥珀酸脱氢酶(SDH)的竞争性抑制剂丙二酸二甲酯预处理，SIRT5/-心脏的I/R损伤程度可以恢复到WT水平，这表明SDH活性的变化是导致损伤的原因。

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. It has been proposed that cells die following ischemia and reperfusion because of a rise in mitochondrial calcium which leads to activation of the mitochondrial permeability transition pore (MPT). The increase in mitochondrial calcium is proposed to occur via calcium uptake by the mitochondrial calcium uniporter. The composition of the MPT is unknown, but cyclophilin D has been shown to be a regulator of the MPT. We examined the role of calcium uptake by mitochondrial calcium uniporter (MCU) in regulating cell death and metabolism. In collaboration with Dr. Toren Finkel, we characterize a mouse model that lacks expression of the recently discovered mitochondrial calcium uniporter (MCU). Mitochondria derived from MCU(-/-) mice have no apparent capacity to rapidly uptake calcium. Whereas basal metabolism seems unaffected, the skeletal muscle of MCU(-/-) mice exhibited alterations in the phosphorylation and activity of pyruvate dehydrogenase. In addition, MCU(-/-) mice exhibited marked impairment in their ability to perform strenuous work. We further show that mitochondria from MCU(-/-) mice lacked evidence for calcium-induced permeability transition pore (PTP) opening. Surprisingly, the lack of PTP opening does not seem to protect MCU(-/-) cells and tissues from cell death. Following global I/R in a Langendorff perfused heart model infarct size was indistinguishable between WT hearts and hearts from MCU-/- mice. To further complicate the picture, cyclosporine A, an inhibitor of the mPTP, reduced infarct size in WT hearts, but not in MCU-/- hearts. We have been testing whether loss of MCU leads to alterations in other cell death pathways. We also examined the response of the mouse to addition of isoproterenol. We also examined the role of succinylation in regulating cardiac function and response to ischemia and reperfusion. Succinylation refers to modification of lysine residues with succinyl groups donated by succinyl-CoA. Sirtuin5 (Sirt5) is a mitochondrial NAD+-dependent deacylase that catalyzes the removal of succinyl groups from proteins. Sirt5 and protein succinylation are conserved across species, suggesting functional importance of the modification. Sirt5 loss impacts liver metabolism but the role of succinylation in the heart has not been explored. We combined affinity enrichment with proteomics and mass spectrometry to analyze total succinylated lysine content of mitochondria isolated from WT and Sirt5-/- mouse hearts. We identified 887 succinylated lysine residues in 184 proteins. 44 peptides (5 proteins) occurred uniquely in WT samples, 289 (46 proteins) in Sirt5-/- samples, and 554 (133 proteins) were common to both groups. The 46 unique proteins in Sirt5-/- heart participate in metabolic processes such as fatty acid β-oxidation (Eci2) and branched chain amino acid catabolism, and include respiratory chain proteins (Ndufa7, 12, 13, Dhsa). We performed label-free analysis of the peptides common to WT and Sirt5-/- hearts. 16 peptides from 9 proteins were significantly increased in Sirt5-/- by at least 30%. The adenine nucleotide transporter 1 showed the highest increase in succinylation in Sirt5-/- (108.4 fold). The data indicates that succinylation is widespread in the heart and enriched in metabolic pathways. We examined whether the loss of Sirt5 would impact ischemia-reperfusion (I/R) injury and we found an increase in infarct size in Sirt5-/- hearts compared to WT littermates (68.5+/-1.1% Sirt5-/- vs 39.6+/- 6.8% WT) following 20 minutes of ischemia and 90 minutes reperfusion. We further demonstrate that the degree of I/R injury in Sirt5-/- heart is restored to WT levels by pretreatment with dimethyl malonate, a competitive inhibitor of succinate dehydrogenase (SDH), implicating alteration in SDH activity as causative of the injury.