Role of the mitochondrial LonP1 in myocardial ischemia and reperfusion injury protection

项目摘要 缺血-再灌注（IR）损伤是治疗心肌梗死（MI）的重大挑战， 美国的死因。线粒体活性氧（mtROS）由电子 转运链（ETC）复合物-I是IR损伤的主要介质。过量的mtROS产生于 早期IR触发自由基产生的恶性循环，促进心肌细胞死亡。因此，我们认为， 了解再灌注的早期分子事件将为开发新的药物提供新的靶点。 限制心脏损伤的干预措施。我们发表的研究结果表明，LonP 1-一种主要的线粒体应激， 反应蛋白酶减轻了早期IR期间氧化应激诱导的损伤;因此，LonP 1可能是一种 有望成为减轻再灌注损伤的靶点。我们的长期目标是利用线粒体蛋白 LonP 1的质量控制机制是开发减轻IR损伤的治疗策略的关键点 和心梗后心力衰竭我们已发表的研究结果表明，心脏中LonP 1表达的增加诱导了 通过缺血预处理（IPC）或转基因过表达（LonTg）减少IR损伤， 心脏保护。而LonP 1下调（LONP 1 +/-）则取消了IPC介导的心脏保护作用。 重要的是，LonTg心脏显示复合物-I亚基（但不是复合物II-V亚基）和复合物-I亚基（但不是复合物II-V亚基）水平降低。 与NTg对照相比，IR早期（再灌注30分钟内）的氧化损伤。相反，我们的 另外的发现表明心肌细胞中LonP 1的下调上调了复合物-I的活性， 增加超氧化物水平，并显示早期再灌注诱导的细胞死亡激活。另外我们有 鉴定了LonP 1的小分子激活剂，其显著降低了由LNP诱导的缺氧-复氧（H/R）。 在体外以剂量依赖性方式引起心肌细胞死亡。通过IR诱导的乙酰化的额外数据， 复合物-I基质亚单位和LonP 1依赖的复合物-I重构在IR期间，我们假设LonP 1 通过严格调节复合物I抑制过量mtROS产生来减轻心肌损伤 我们将通过以下具体目标来测试我们的假设：目标1将描述 LonP 1调节复合物-I水平、活性和降低IR期间氧化应激的机制。 目的2将测试LonP 1通过降解IR诱导的复合物来重塑复合物-I及其相关的超复合物。 后修饰（PTM）复合物-I基质亚基，从而减少早期IR期间的mtROS。 将确定LonP 1激活剂在体内治疗心肌IR损伤中的治疗潜力。通过 确定LonP 1介导的心脏保护作用的分子机制和 LonP 1激活剂，我们将确定LonP 1在心脏保护中的作用，并开发新的治疗工具， 减轻IR损伤的策略。

PROJECT SUMMARY Ischemia-reperfusion (IR) injury is a significant challenge in treating myocardial infarction (MI), the leading cause of death in the United States. Mitochondrial reactive oxygen species (mtROS) generated by electron transport chain (ETC) Complex-I are the principal mediators of IR injury. Excess mtROS generated during early IR triggers vicious cycles of free radical production promoting cardiomyocyte death. Therefore, understanding the early molecular events of reperfusion will provide new targets for developing novel interventions for limiting cardiac injury. Our published findings show that LonP1- a major mitochondrial stress response protease mitigates oxidative stress-induced damage during early IR; therefore, LonP1 could be a promising target for attenuating reperfusion injury. Our long-term goal is to leverage the mitochondrial protein quality control mechanisms of LonP1 as a pivotal point to develop therapeutic strategies for mitigating IR injury and post MI- heart failure. Our published findings show that increased LonP1 expression in the heart induced by ischemic preconditioning (IPC) or transgenic overexpression (LonTg) reduced IR injury and favors cardioprotection. Whereas, LonP1 downregulation (LONP1+/-) abrogated IPC-mediated cardioprotection. Importantly, LonTg hearts showed reduced levels of Complex-I subunits (but not Complex II-V subunit) and oxidative damage during early IR (within 30 min reperfusion) compared to NTg controls. Conversely, our additional findings show that LonP1 downregulation in cardiomyocytes upregulated Complex-I activity, increased superoxide levels, and showed early reperfusion-induced cell death activation. In addition, we have identified a small molecule activator of LonP1 that significantly reduced hypoxia-reoxygenation (H/R) induced myocyte death in a dose-dependent manner in vitro. With additional data on IR-induced acetylation of Complex-I matrix subunits and LonP1 dependent Complex-I remodeling during IR, we hypothesize that LonP1 mitigates myocardial injury by suppressing excess mtROS generation through tight regulation of Complex-I during early IR. We will test our hypothesis by the following specific aims: Aim 1 will delineate the mechanism(s) by which LonP1 modulates Complex-I levels, activity and reduces oxidative stress during IR. Aim 2 will test that LonP1 remodels Complex-I and its associated supercomplexes by degrading IR-induced post-translationally modified (PTM) Complex-I matrix subunits, thereby reduce mtROS during early IR. Aim 3 will determine the therapeutic potential of LonP1 activators in treating myocardial IR injury in vivo. By determining the molecular mechanisms of LonP1-mediated cardioprotection and the therapeutic potential of LonP1 activators, we will define the role of LonP1 in cardioprotection and develop novel therapeutic tools and strategies to mitigate IR injury.