Inhibiting Cell Death for Protecting Cardiac Injury

抑制细胞死亡以保护心脏损伤

• 批准号: 10206269
• 负责人: Hua Zhu
• 金额: $ 39万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-01 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10206269
• 关键词: Acute myocardial infarction; Affinity; Animal Model; Apoptosis; Area; Autophagocytosis; Binding; Biochemical; Biochemistry; Biological Assay; Biology; Biotin; Blood Circulation; CRISPR/Cas technology; Cardiac; Cardiac Myocytes; Cell Culture Techniques; Cell Death; Cell membrane; Cell physiology; Cells; Cellular Structures; Cellular biology; Cessation of life; Cleaved cell; Co-Immunoprecipitations; Disease; Event; Extracellular Space; Gene Delivery; Genes; Goals; Heart; Heart Diseases; Heart Injuries; Heart failure; Homeostasis; Human; In Vitro; Infarction; Injury; Ischemia; Knock-out; Knowledge; Label; Lead; Mediating; Membrane; Molecular; Molecular Biology; Mus; Mutagenesis; Myocardial; Myocardial Infarction; Myocardial Ischemia; Myocardium; Necrosis; Operative Surgical Procedures; Oxidative Stress; Pathology; Pathway interactions; Patients; Periodicity; Pharmacology; Pilot Projects; Play; Protein Family; Protein-Serine-Threonine Kinases; Proteins; Public Health; Recombinants; Regulation; Relaxation; Repair Complex; Reperfusion Injury; Reperfusion Therapy; Research; Resistance; Rodent Model; Role; Stress; Syndrome; TRIM Family; Testing; Tissues; Transgenic Mice; Transgenic Model; Ubiquitination; cardiogenesis; cardioprotection; defined contribution; effective therapy; experimental study; genome editing; heart function; improved; in vivo; induced pluripotent stem cell; injured; injury and repair; insight; intravenous administration; knockout gene; live cell imaging; member; mortality; mouse model; mutant; myocardial injury; novel; novel therapeutic intervention; overexpression; porcine model; preconditioning; receptor; repaired; ubiquitin-protein ligase; uptake

PROJECT SUMMARY The death of cardiomyocyte following myocardial infarction (MI) is one of the main causes of heart failure and patient mortality. Our group previously identified MG53, a member of the TRIM family protein (TRIM72), as an essential component of the cell membrane repair machinery. Mice without the mg53 gene develop pathology in the heart and are susceptible to cardiac injury, while transgenic mice with increased levels of MG53 (ctPA-MG53) are resistant to stress-induced MI, supporting the function of MG53 in cardioprotection. Using CRISPR-Cas9 mediated gene knockout and live cell biotin proximity-labeling assay, we identified RIPK1 as a novel molecular partner of MG53 following I/R induced cardiac injury. RIPK1 is a key factor of necroptosis, a programmed necrosis, which hasn’t been extensively studied in MI. In vitro cell culture studies with human induced pluripotent stem cells (hiPSCs) and in vivo murine MI studies demonstrated that necroptosis plays an important role in injury induced cardiac cell death. More importantly, following MI, mg53-/- hearts had higher level of necroptosis, while ctPA-MG53 hearts displayed lower level of necroptosis than those of wild type littermates, indicating MG53 could potentially regulate necroptosis. Indeed, biochemical experiments revealed that wild type MG53 serves as an E3 ligase of RIPK1 following MI, while mutant MG53 without E3 ligase function failed to target and mediate degradation of RIPK1. This research is centered on testing the hypothesis that MG53 plays a critical role in inhibiting necroptosis via directly interacting with and destabilizing RIPK1, targeting this functional interaction could be important for maintaining myocardial homeostasis and developing effective treatments for cardiac diseases”. We will test the hypothesis with two specific aims. Aim 1: Dissect the molecular mechanisms underlying MG53-mediated myocardial protection through regulating RIPK1 or other necroptotic factors. Aim 2: Define the function of MG53-mediated necroptotic inhibition for protection of myocardial injury. Live cell imaging, CRISPR-Cas9 mediated gene editing in hiPSCs, AAV mediated gene delivery and mutagenesis approaches will be utilized to dissect cellular and molecular functions of MG53 on regulation of necroptosis. Overall, knowledge gained from this project will extend our current understanding of MG53 as a membrane repair factor to its function for regulation of cell death following MI, and may have potential translational implications for developing new therapeutic strategies for treating MI.

项目概要 心肌梗死（MI）后心肌细胞死亡是心力衰竭的主要原因之一 和患者死亡率。我们的小组之前鉴定出 MG53，TRIM 家族蛋白 (TRIM72) 的成员，作为 细胞膜修复机制的重要组成部分。没有 mg53 基因的小鼠发育 心脏病理学并且容易受到心脏损伤，而转基因小鼠的心脏功能水平增加 MG53 (ctPA-MG53) 可抵抗应激诱发的 MI，支持 MG53 的心脏保护功能。 使用 CRISPR-Cas9 介导的基因敲除和活细胞生物素邻近标记测定，我们鉴定了 RIPK1 作为 I/R 诱导的心脏损伤后 MG53 的新型分子伴侣。 RIPK1是坏死性凋亡的关键因子， 一种程序性坏死，尚未在心肌梗死中得到广泛研究。人类体外细胞培养研究 诱导多能干细胞 (hiPSC) 和小鼠体内 MI 研究表明，坏死性凋亡在 在损伤引起的心肌细胞死亡中起重要作用。更重要的是，心肌梗死后，mg53-/- 心脏具有更高的 坏死性凋亡水平，而 ctPA-MG53 心脏的坏死性凋亡水平低于野生型 同窝小鼠，表明 MG53 可能调节坏死性凋亡。事实上，生化实验表明 野生型 MG53 在 MI 后充当 RIPK1 的 E3 连接酶，而突变型 MG53 没有 E3 连接酶 功能未能靶向并介导 RIPK1 的降解。这项研究的重点是测试 假设 MG53 通过直接相互作用和破坏稳定而在抑制坏死性凋亡中发挥关键作用 RIPK1，针对这种功能相互作用对于维持心肌稳态和 开发心脏病的有效治疗方法”。我们将通过两个具体目标来检验该假设。目标 1： 解析 MG53 通过调节 RIPK1 介导心肌保护的分子机制 或其他坏死性凋亡因素。目标 2：定义 MG53 介导的坏死性凋亡抑制的功能，以保护 心肌损伤。活细胞成像、CRISPR-Cas9 介导的 hiPSC 基因编辑、AAV 介导的基因 将利用递送和诱变方法来剖析 MG53 的细胞和分子功能 坏死性凋亡的调节。总的来说，从这个项目中获得的知识将扩展我们目前对 MG53 作为一种膜修复因子，具有调节 MI 后细胞死亡的功能，并且可能具有 开发治疗心肌梗死的新治疗策略的潜在转化意义。