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.

项目摘要 心肌梗塞后心肌细胞死亡（MI）是心力衰竭的主要原因之一 和患者死亡率。我们的小组以前鉴定出MG53是Trim家族蛋白（TRIM72）的成员，为 细胞膜修复机械的重要组成部分。没有MG53基因发育的小鼠 心脏中的病理学，容易受到心脏损伤的影响，而转基因小鼠的水平升高 MG53（CTPA-MG53）对应力诱导的MI有抵抗力，从而支持MG53在心脏保护中的功能。 使用CRISPR-CAS9介导的基因敲除和活细胞生物素接近标记测定法，我们确定了RIPK1 作为I/R诱导心脏损伤后MG53的新分子伴侣。 RIPK1是坏死性的关键因素， 一个编程的坏死，在MI中尚未广泛研究。人类的体外细胞培养研究 诱导多能干细胞（HIPSC）和体内鼠MI研究表明坏死性发挥作用 在损伤引起的心脏细胞死亡中的重要作用。更重要的是，遵循MI，MG53 - / - 心的较高 坏死性水平，而CTPA-MG53心脏的坏死性水平低于野生型。 同窝官，表明MG53可能可能调节坏死性。确实，生化实验揭示了 MI之后，野生型MG53用作RIPK1的E3连接酶，而没有E3连接酶的突变体Mg53 功能无法靶向RIPK1的介质降解。这项研究集中于测试 假设MG53在抑制坏死作用方面起着至关重要的作用，直接与和破坏稳定 RIPK1，针对这种功能相互作用对于维持心肌稳态和 为心脏病开发有效的治疗方法。 通过调节RIPK1剖析MG53介导的心肌保护的分子机制 或其他坏死因素。 AIM 2：定义MG53介导的坏死抑制的功能以保护 心肌受伤。活细胞成像，hipsC中的CRISPR-CAS9介导的基因编辑，AAV介导的基因 递送和诱变方法将用于剖析Mg53在上的细胞和分子功能 坏死作用的调节。总体而言，从该项目中获得的知识将扩展我们当前对 MG53作为其功能在MI后调节细胞死亡功能的膜修复因子，并且可能具有 潜在的转化含义，以制定治疗MI的新治疗策略。