Molecular Regulation of Mitochondrial Permeability Transition and its Role in Regulated Necrosis

项目摘要/摘要 心肌梗死(MI)及其长期恢复是心脏病的主要原因。 美国1.虽然通过再灌流使血液回流到心脏可以挽救生命，但它也会导致 额外的心肌细胞死亡。2，3目前，没有作为预防措施的治疗性干预 抗缺血-再灌注(I/R)损伤中的心肌细胞死亡。17因此，新的药物 需要确认身份。这项建议的长期目标是减少I/R期间的心肌细胞损失 损伤可降低总死亡率，并减少心肌梗塞后心力衰竭的发生。要做到这一点 目的，我们正在研究两种形式的细胞死亡：线粒体通透性转换孔(MPTP)- 依赖性坏死和铁性下垂，导致缺血再灌注(I/R)损伤。5，6 依赖于坏死，致命数量的自由基氧物种(ROS)和钙(Ca+)被认为是开放的 线粒体内部的一个孔，称为MPTP，其机制未知，它会导致 线粒体膜电位导致心肌细胞坏死性死亡。7，8，9，10交替，在铁性下垂，过量 线粒体内的铁(Fe2+)会产生ROS，从而在一个被称为 脂质过氧化(LIPOX)。11、12、13在先前的研究中，两种机制都有单独的药物抑制作用 14，15不幸的是，针对MPTP的临床试验- 依赖通路在人类中不成功，提示在I/R损伤过程中细胞死亡更多 16因此，我们的研究战略将解决是否双重针对这些机制 同时可进一步缩小I/R损伤后的梗塞面积。我们的初步数据表明这条路径 启动取决于ROS浓度和Ca~(2+)有效性，因为低浓度的ROS能够 使钙依赖的MPTP开放敏感，而高ROS水平可通过以下途径导致线粒体功能障碍 LIPOX。我们还确定，在体外靶向这两条途径对ROS具有额外的保护作用。 诱导细胞死亡，通过处理铁下垂抑制剂铁抑素-1在基因敲除细胞系 MPTP调节因子亲环素D(CypD)。因此，我们假设MPTP的双重抑制依赖于 坏死和铁性下垂途径将对心肌I/R损伤具有额外的保护作用。在目标1中， 我们将用与I/R相关的侮辱，如钙、铁或ROS来挑战分离的心脏线粒体 从机械的角度考察通路诱导。在目标2中，我们将在治疗上针对MPTP依赖的两个 使用药物和遗传策略的坏死症和铁下垂途径。我们将使用MPTP 环孢菌素A抑制剂和LIPOX抑制剂铁蛋白-1联合抗I/R，以及执行腺病毒感染。 CypD缺失遗传模型中抗铁链抗氧化剂Gpx4的相关病毒(AAV)过表达 重述小鼠的药物策略。通过这种方法，我们的目标是完成以下任务 国家心脏、肺和血液(HLBS)研究所防止心肌细胞(CM)在I/R损伤期间死亡。

Project Summary/Abstract Myocardial infarction (MI) and its consequent long term recovery constitutes a majority of heart disease in the United States.1 While the return of blood flow to the heart by reperfusion can be lifesaving, it also induces additional cardiomyocyte death.2,3 Currently, there is no therapeutic intervention that serves as a preventative against cardiomyocyte cell death during ischemia-reperfusion (I/R) injury.17 Thus, new pharmacological agents need to be identified. The long-term objective of this proposal is to reduce cardiomyocyte loss during I/R injury to reduce overall mortality and diminish the onset of heart failure following MI. To accomplish this goal, we are investigating two forms of cell death: mitochondrial permeability transition pore (MPTP)- dependent necrosis and ferroptosis, which contribute to ischemia reperfusion (I/R) injury.5,6 During MPTP- dependent necrosis, lethal amounts of radical oxygen species (ROS) and calcium (Ca2+) are thought to open an inner mitochondrial pore known as the MPTP in an unknown mechanism, which causes a loss of mitochondrial membrane potential resulting in myocyte necrotic death.7,8,9,10 Alternatively, in ferroptosis, excess iron (Fe2+) within mitochondria generates ROS which dysregulates lipid membranes in a process known as lipid peroxidation (LIPOX).11,12,13 In previous studies, individual pharmacological inhibition of both mechanisms demonstrated a promising reduction of infarct size in mice.14,15 Unfortunately, clinical trials targeting the MPTP- dependent pathway were not successful in humans, suggestive that cell death during I/R injury is more complex than anticipated.16 Therefore, our research strategy will address if dually targeting these mechanisms simultaneously can further reduce infarct size post I/R injury. Our preliminary data suggests that pathway initiation depends on ROS concentration and Ca2+ availability, as low concentrations of ROS are able to sensitize Ca2+-dependent MPTP opening, whereas high ROS levels can lead to mitochondrial dysfunction via LIPOX. We also determined that targeting both pathways in vitro was additively protective against ROS- induced cell death, achieved by treating ferroptosis inhibitor ferrostatin-1 on a genetic knock-out cell line of MPTP regulator cyclophilin D (CypD). Therefore, we hypothesize that dual inhibition of MPTP-dependent necrosis and ferroptosis pathways will be additively protective against myocardial I/R injury. In Aim 1, we will challenge isolated heart mitochondria with I/R-relevant insults such as Ca2+, Fe2+, or ROS to mechanistically examine pathway induction. In Aim 2, we will therapeutically target both MPTP-dependent necrosis and ferroptosis pathways using both pharmaceutical and genetic strategies. We will use MPTP inhibitor cyclosporine A and LIPOX inhibitor ferrostatin-1 in combination against I/R, as well as perform adeno- associated viral (AAV) overexpression of anti-ferroptotic antioxidant GPX4 on genetic models of CypD null mice to recapitulate the pharmaceutical strategy. With this approach, we aim to accomplish the mission of the National heart, lung, and blood (HLBS) institute to prevent cardiomyocyte (CM) death during I/R injury.