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

线粒体通透性转变的分子调节及其在调节性坏死中的作用

• 批准号: 10630723
• 负责人: Jason Michael Karch
• 金额: $ 6.06万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-01 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10630723
• 关键词: Address; Antioxidants; Blood flow; Calcium; Cardiac Myocytes; Cardiac health; Cell Death; Cell Line; Cessation of life; Clinical Trials; Complex; Cyclosporine; Data; Genetic; Genetic Models; Goals; Heart; Heart Diseases; Heart Mitochondria; Heart failure; Human; In Vitro; Individual; Infarction; Iron; Ischemia; Knock-out; Knockout Mice; Knowledge; Lead; Life; Lipid Peroxidation; Membrane Lipids; Membrane Potentials; Mission; Mitochondria; Molecular; Mus; Muscle Cells; Myocardial Infarction; Myocardial Ischemia; National Heart, Lung, and Blood Institute; Necrosis; Pathway interactions; Permeability; Pharmacologic Substance; Pharmacology; Play; Process; Reactive Oxygen Species; Recovery; Regulation; Reperfusion Injury; Reperfusion Therapy; Research; Role; Suggestion; Therapeutic Intervention; United States; Viral; cyclophilin D; genetic approach; improved; inhibitor; lipid peroxidation inhibitor; mitochondrial dysfunction; mitochondrial membrane; mitochondrial permeability transition pore; mortality; new therapeutic target; overexpression; prevent; therapeutic target

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.

项目总结/摘要 心肌梗死（MI）及其随后的长期恢复构成了大多数心脏病， 美国。1虽然通过再灌注使血液回流到心脏可以挽救生命，但它也会诱导 额外的心肌细胞死亡。2，3目前，没有治疗干预，作为预防 抗缺血-再灌注（I/R）损伤期间心肌细胞死亡。17因此， 需要被识别。该提案的长期目标是减少I/R期间的心肌细胞损失 损伤以降低总体死亡率并减少MI后心力衰竭的发作。为了实现这一 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）- 依赖性坏死和铁凋亡，这有助于缺血再灌注（I/R）损伤。 依赖性坏死，致死量的自由基氧（ROS）和钙（Ca 2+）被认为是开放的 一个内部的线粒体孔被称为MPTP在一个未知的机制，这导致损失的 线粒体膜电位导致肌细胞坏死死亡。7，8，9，10或者，在铁凋亡中，过量 线粒体内的铁（Fe 2+）产生ROS，ROS在称为 脂质过氧化（LIPOX）。11，12，13在先前的研究中，两种机制的个体药理学抑制 在小鼠中证明了有希望的梗死面积减少。14，15不幸的是，针对MPTP的临床试验- 依赖性途径在人类中不成功，这表明I/R损伤期间的细胞死亡更多 16因此，我们的研究战略将解决，如果双重针对这些机制， 同时可以进一步减少I/R损伤后的梗死面积。我们的初步数据表明， 启动取决于ROS浓度和Ca 2+的可用性，因为低浓度的ROS能够 敏感的Ca 2+依赖的MPTP开放，而高ROS水平可导致线粒体功能障碍， LIPOX。我们还确定，体外靶向这两种途径对ROS具有相加保护作用。 诱导的细胞死亡，通过处理铁凋亡抑制剂ferrostatin-1对基因敲除的细胞系， MPTP调节剂亲环素D（CypD）。因此，我们假设MPTP依赖性的双重抑制 坏死和铁凋亡途径将对心肌I/R损伤具有额外的保护作用。在目标1中， 我们将用I/R相关的损伤如Ca 2+、Fe 2+或ROS挑战分离的心脏线粒体， 机械地检查通路诱导。在目标2中，我们将在治疗上靶向MPTP依赖性 坏死和铁凋亡途径使用药物和遗传策略。我们将使用MPTP 抑制剂环孢菌素A和LIPOX抑制剂ferrostatin-1联合抗I/R，以及进行腺 在CypD缺失的遗传模型上，抗铁促凋亡抗氧化剂GPX 4的相关病毒（AAV）过表达 小鼠来概括药物策略。通过这种方法，我们的目标是完成以下使命 国家心脏、肺和血液（HLBS）研究所，以防止I/R损伤期间心肌细胞（CM）死亡。