Reduction of cardiac injury by targeting damaged mitochondria during reperfusion

描述(由申请人提供)： 心肌线粒体(MITO)是心肌缺血再灌注损伤的重要介质。在缺血期间，Mito遭受由线粒体电子传输链(ETC)介导的损伤。我们以前的工作发现了ETC在缺血时对Mito造成损伤的两种主要机制。首先，ETC本身在复合体I和复合体III处被破坏。其次，MITO中的抗凋亡多肽BCL-2被耗尽。我们发现，当在缺血期间使用抑制剂来阻断ETC时，这种对Mito的损害被防止。更值得注意的是，当Mito在缺血期间受到保护时，REP后测量的梗塞面积显著减少。由于缺血时ETC引起的损伤，Mito成为再灌流期间心肌细胞损伤的效应因子。我们假设ETC依赖性缺血损伤的两种机制在再灌流期间主导心脏损伤。在再灌流开始时，受损的Mito会产生氧化损伤，经历灾难性的通透性变化，并激活细胞死亡程序。确定了导致这些损伤性整体反应的机制，并将导致在再灌注早期和后期进行治疗干预的策略。在这项建议的前期工作中，我们发现，尽管ETC已经受到损害，但在再灌注期间进行干预，直接操纵线粒体功能可以减少心脏损伤。我们专注于通过药理学和遗传学对线粒体功能的调节来直接调节代谢的机制驱动的方法，包括使用对复合体I的瞬时和部分阻断。实验方法旨在绕过上游信号级联，而直接与心脏损伤的最终靶点和效应者Mito相互作用。我们认为，在再灌注开始时，受损的ETC氧化代谢的恢复与电子从复合体I到复合体III的传递产生氧化剂，导致线粒体通透性转换孔开放和细胞死亡。目的1识别和研究复合体I和III中在再灌流时产生损伤的部位。我们推测，在缺血过程中依赖于ETC的BCL-2从Mito中耗尽，导致线粒体外膜通透，并在再灌流过程中激活细胞程序性死亡。目的2研究bcl2缺失在再灌流早期通透性转换介导的心脏损伤中的作用，这可能有助于加强复杂的I驱动的激活。在再灌流初期经干预保护的心肌细胞在再灌流较长时间内仍易受有丝分裂引起的细胞死亡的影响。我们假设MITO持续存在缺血损伤的ETC和BCL-2将驱动包括自噬和破坏MITO分裂/融合平衡的非适应性线粒体重塑反应，如目标3中所研究的。这项工作关键地探索了ETC介导的缺血损伤的再灌注期间心脏损伤的机制，既包括ETC损伤的直接氧化损伤，也包括通过包括BCL-2在内的ETC依赖损伤的下游效应分子的损伤。

DESCRIPTION (provided by applicant): Cardiac mitochondria (MITO) are critical mediators of myocardial injury during ischemia and reperfusion. During ischemia, MITO sustain damage that is mediated by the mitochondrial electron transport chain (ETC). Our previous work discovered two major mechanisms of ETC-driven injury to MITO during ischemia. First, the ETC itself is damaged at complex I and complex III. Second, the anti-apoptotic peptide bcl-2 is depleted from MITO. We found that when inhibitors were used to block the ETC during ischemia, this damage to MITO was prevented. More remarkably, when MITO were protected during ischemia, infarct size measured after REP was substantially reduced. As a result of the ETC-driven damage during ischemia, MITO become effectors of cardiomyocyte injury during reperfusion. We hypothesize that the two mechanisms of ETC-dependent ischemic damage dominate cardiac injury during reperfusion. At the onset of reperfusion, damaged MITO generates oxidative injury, undergo catastrophic permeability changes and activate cell death programs. The mechanisms leading to these injurious global responses are identified and will lead to strategies for therapeutic intervention during early and later periods of reperfusion. In preliminary work for this proposal, we found tha although the ETC has already sustained damage, intervention during reperfusion to directly manipulate mitochondrial function can decrease cardiac injury. We have focused on mechanism-driven approaches to directly modulate metabolism using pharmacologic and genetic modulation of mitochondrial function, including the use transient and partial blockade of complex I. The experimental approaches are designed to bypass upstream signaling cascades and instead directly interact with the ultimate target and effector of the cardiac injury, the MITO We propose that at the onset of reperfusion, resumption of oxidative metabolism by the damaged ETC with electron transport from complex I into complex III generates oxidants that lead to opening of the mitochondrial permeability transition pore and cell death. Aim 1 identifies and studies sites within complexes I and III that generate the injury at reperfusion. We hypothesize that the ETC-dependent depletion of bcl-2 from MITO during ischemia leads to mitochondrial outer membrane permeation and the activation of programmed cell death during reperfusion. Aim 2 studies the role of bcl-2 depletion in permeability transition-mediated cardiac injury during early reperfusion that may serve to reinforce complex I-driven activation. Myocytes protected by intervention during the initial phase of reperfusion remain susceptible to MITO- driven cell death during longer periods of reperfusion. We hypothesize that the persistence of MITO with ischemia-damaged ETC and bcl-2 depletion will drive maladaptive mitochondrial remodeling responses including autophagy and the disruption of the MITO fission/fusion balance as studied in Aim 3. This work critically explores the mechanisms of cardiac injury during reperfusion of ETC-mediated injury to MITO from ischemia, both direct oxidative injury from the damaged ETC and injury via downstream effectors of ETC- dependent damage including bcl-2.