Reduction of cardiac injury by targeting damaged mitochondria during reperfusion

描述（由申请人提供）： 心脏线粒体（MITO）是缺血和再灌注期间心肌损伤的关键介质。缺血期间，MITO 会受到线粒体电子传递链 (ETC) 介导的损伤。我们之前的工作发现了缺血期间 ETC 驱动的 MITO 损伤的两种主要机制。首先，ETC 本身在复合体 I 和复合体 III 处受损。其次，抗凋亡肽 bcl-2 从 MITO 中被耗尽。我们发现，当在缺血期间使用抑制剂阻断 ETC 时，可以防止对 MITO 的损害。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着减小。 由于缺血期间 ETC 驱动的损伤，MITO 成为再灌注期间心肌细胞损伤的效应器。我们假设 ETC 依赖性缺血损伤的两种机制主导再灌注期间的心脏损伤。再灌注开始时，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。导致这些有害的整体反应的机制已被确定，并将导致再灌注早期和后期的治疗干预策略。 在该提案的初步工作中，我们发现虽然 ETC 已经受到损伤，但在再灌注期间进行干预以直接操纵线粒体功能可以减少心脏损伤。我们重点关注机制驱动的方法，利用线粒体功能的药理和遗传调节来直接调节代谢，包括使用复合物 I 的瞬时和部分阻断。实验方法旨在绕过上游信号级联，直接与心脏损伤的最终目标和效应器 MITO 相互作用。我们提出，在再灌注开始时，受损的 ETC 通过从复合物 I 到复合物 III 的电子传输恢复氧化代谢，产生氧化剂，导致线粒体通透性过渡孔打开和细胞死亡。目标 1 识别并研究复合物 I 和 III 内产生再灌注损伤的位点。我们假设缺血期间 ETC 依赖性的 MITO 中 bcl-2 的消耗导致线粒体外膜渗透并在再灌注期间激活程序性细胞死亡。目标 2 研究 bcl-2 耗竭在早期再灌注期间通透性转变介导的心脏损伤中的作用，这可能有助于增强复合物 I 驱动的激活。 在再灌注初始阶段受到干预保护的肌细胞在较长的再灌注期间仍然容易受到 MITO 驱动的细胞死亡的影响。我们假设 MITO 与缺血损伤的 ETC 和 bcl-2 耗竭的持续存在将驱动适应不良的线粒体重塑反应，包括自噬和 MITO 裂变/融合平衡的破坏，如目标 3 中所研究的。这项工作批判性地探讨了 ETC 介导的缺血对 MITO 损伤再灌注过程中心脏损伤的机制，包括来自受损 ETC 的直接氧化损伤和通过 ETC 依赖性损伤的下游效应器造成的损伤，包括bcl-2。

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.