Inhibiting Cell Death for Protecting Cardiac Injury

项目总结 心肌梗死(MI)后心肌细胞死亡是心力衰竭的主要原因之一 和病人死亡率。本课题组此前鉴定了TRIM家族蛋白(TRIM72)的成员MG53为 细胞膜修复机械的重要组成部分。没有MG53基因的小鼠会发生 在心脏中的病理，并对心脏损伤敏感，而转基因小鼠的水平增加 MG53(CTPA-MG53)能抵抗应激性心肌梗死，支持MG53的心脏保护作用。 通过CRISPR-Cas9介导的基因敲除和活细胞生物素邻近标记实验，我们鉴定了RIPK1 作为MG53在心肌I/R损伤后的一个新的分子伴侣。RIPK1是坏死性下垂的关键因素， 一种程序性坏死，在心肌梗死中尚未得到广泛研究。人的体外细胞培养研究 诱导多能干细胞(HiPSCs)和体内小鼠心肌梗死的研究表明，坏死性下垂在 在损伤所致的心肌细胞死亡中起重要作用。更重要的是，心肌梗死后，MG53-/-心脏的比例更高 CTPA-MG53心脏的坏死性下垂水平低于野生型 表明MG53对坏死性下垂有潜在的调节作用。事实上，生化实验揭示了 野生型MG53在心肌梗塞后作为RIPK1的E3连接酶，而突变体MG53没有E3连接酶 功能无法针对和调节RIPK1的降解。这项研究的中心是测试 假设MG53通过与坏死性下垂的直接相互作用和破坏稳定而在抑制坏死性下垂中起关键作用 RIPK1，靶向这种功能相互作用可能对维持心肌内稳态和 开发心脏疾病的有效治疗方法“。我们将用两个具体的目标来检验这一假设。目标1： MG53通过调节RIPK1介导心肌保护的分子机制剖析 或其他坏死性下巴因子。目的2：明确MG53介导的抗坏死性上皮病的作用 心肌损伤。活细胞成像，CRISPR-Cas9介导的HiPSCs基因编辑，AAV介导的基因 将利用递送和诱变的方法来剖析MG53的细胞和分子功能 对坏死性下垂的调节。总体而言，从这个项目中获得的知识将扩大我们目前对 MG53作为膜修复因子对心肌梗死后细胞死亡的调节作用，可能具有 开发治疗心肌梗死的新治疗策略的潜在翻译含义。

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.