Inhibiting Cell Death for Protecting Cardiac Injury

项目摘要 心肌梗死（MI）后心肌细胞的死亡是导致心力衰竭的主要原因之一 和患者死亡率。我们的小组先前鉴定了TRIM家族蛋白（TRIM 72）的成员MG 53， 细胞膜修复机制的重要组成部分。没有mg 53基因的老鼠 转基因小鼠的心脏病理学水平升高，易受心脏损伤，而转基因小鼠的心脏病理学水平升高， MG 53（ctPA-MG 53）对应激诱导的MI具有抗性，支持MG 53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邻近标记试验，我们鉴定了RIPK 1 作为I/R诱导的心脏损伤后MG 53的新分子伴侣。RIPK 1是坏死性凋亡的关键因子， 程序性坏死，在心肌梗死中尚未得到广泛研究人的体外细胞培养研究 诱导性多能干细胞（hiPSC）和体内鼠MI研究表明，坏死性凋亡起着重要作用， 在损伤诱导的心肌细胞死亡中起重要作用。更重要的是，在MI后，mg 53-/-心脏具有更高的 而ctPA-MG 53心脏显示出比野生型心脏更低的坏死性凋亡水平 这表明MG 53可以潜在地调节坏死性凋亡。事实上，生化实验表明， 野生型MG 53在MI后充当RIPK 1的E3连接酶，而没有E3连接酶的突变型MG 53在MI后充当RIPK 1的E3连接酶， 功能未能靶向和介导RIPK 1的降解。这项研究的重点是测试 假设MG 53通过与细胞直接相互作用并使其不稳定而在抑制坏死性凋亡中起关键作用 RIPK 1，靶向这种功能性相互作用对于维持心肌稳态可能很重要， 为心脏病开发有效的治疗方法”。我们将以两个具体目标来检验这个假设。目标1： 探讨MG 53通过调节RIPK 1介导心肌保护的分子机制 或其他坏死性因素。目的2：确定MG 53介导的坏死性凋亡抑制对保护细胞凋亡的功能。 心肌损伤活细胞成像，hiPSC中CRISPR-Cas9介导的基因编辑，AAV介导的基因编辑 将利用递送和诱变方法来剖析MG 53的细胞和分子功能， 调节坏死性凋亡。总的来说，从这个项目中获得的知识将扩展我们目前对 MG 53作为膜修复因子调节MI后细胞死亡的功能，并且可能具有 潜在的翻译意义，开发新的治疗策略，治疗心肌梗死。

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.