Molecular Determinants of MG53 in Heart Structure and Function

项目摘要 心力衰竭，在亚细胞水平上测量，是受损的心肌细胞兴奋-收缩的结果 (E-C)偶合器. E-C偶联的一个关键结构成分是肌细胞横（T）小管系统。T型 小管在协调膜兴奋与肌肉收缩中起着重要作用， 肌浆网中钙离子的同步释放。我们的小组，以及其他小组， 提供了强有力的证据表明，来自患者和动物模型的衰竭肌细胞的特征在于： T-小管系统变性和紊乱，导致细胞内Ca 2+动力学受损， 肌丝收缩我们研究的长期目标是识别和利用 心脏病T-小管重塑过程的基本机制 完整性和减缓（如果不能逆转）心力衰竭进展。我们已经确认Mitsugumin 53 (MG53/TRIM 72）作为潜在的T-小管修复酶。我们发现MG 53，已知参与损伤诱导的 横纹肌中的膜囊泡运输和修复，定位于T-小管，并在人类中上调 衰竭心脏和慢性心力衰竭动物模型。我们的初步数据显示MG 53拥有 在心脏中的明显发散功能中，MG 53的MG 53缺失加剧了心脏中的T-小管变性。 而外源性MG 53过表达促进进行性和严重的T-小管破坏。 有趣的是，我们已经将过表达效应映射到其很少研究的E3连接酶结构域。是我们 因此，假设MG 53的膜修复与E3-泛素连接酶活性决定了 心肌细胞T-小管完整性和E-C耦合功能在健康和疾病。我们最近 产生了两种新的敲入动物，用于分别检测内源性MG 53 E3连接酶和膜 心脏应激反应中的修复功能。我们将通过首先确定 MG 53 E3-泛素连接酶及其膜的生理（目的1）和分子（目的2）作用 修复（目标3），T-小管结构域和Ca 2+处理在基线和响应心脏应激。 我们将归因于明显不一致的泛素依赖的蛋白水解和膜愈合功能， MG 53对整体T-小管完整性、E-C偶联和心脏重塑过程的影响。了解 MG 53促进T-小管重塑与修复的机制将使我们能够设计高度靶向的 以及心力衰竭治疗的有效治疗剂。

PROJECT SUMMARY Heart failure, measured at the subcellular level, is the result of impaired cardiomyocyte excitation-contraction (E-C) coupling. One key structural component of E–C coupling is the myocyte transverse (T)-tubule system. T- tubules play an essential role in coordinating membrane excitation with muscle contraction by facilitating the synchronized release of Ca2+ from the sarcoplasmic reticulum. Our group, as well as those of others, have provided strong evidence that failing myocytes from patients and animal models are characterized by a degenerated and disorganized T-tubule system resulting in impaired intracellular Ca2+ dynamics and myofilament contraction. The long-term goal of our research is to discern and take advantage of the fundamental mechanisms underlying T-tubule remodeling processes in heart disease toward restoring T-tubule integrity and slowing, if not reversing, heart failure progression. We have identified Mitsugumin 53 (MG53/TRIM72) as a potential T-tubule repair enzyme. We find MG53, known to be involved in injury-induced membrane vesicle trafficking and repair in striated muscle, localizes to T-tubules and is upregulated in human failing hearts and animal models of chronic heart failure. Our preliminary data indicate MG53 possesses apparent divergent functions in the heart, MG53 deletion of MG53 exacerbates T-tubule degeneration in stressed hearts while exogenous MG53 overexpression promotes progressive and severe T-tubule disruption. Interestingly, we have mapped overexpression effects to its little-studied E3-ligase domain. It is our hypothesis, therefore, that the membrane repair versus E3-ubiquitin ligase activities of MG53 determine cardiomyocyte T-tubule integrity and E-C coupling function during health and in disease. We have recently generated two novel knockin animals for separately examining endogenous MG53 E3-ligase and membrane repair functions during cardiac stress responses. We will test our hypothesis by first determining the physiological (Aim 1) and molecular (Aim 2) actions of the MG53 E3-ubiquitin ligase, as well as its membrane repair (Aim 3), domains in T-tubule structure and Ca2+ handling at baseline and in response to cardiac stress. We will attribute apparently discordant ubiquitin-dependent proteolysis and membrane healing functions of MG53 to overall T-tubule integrity, E-C coupling and cardiac remodeling processes. Understanding the mechanisms by which MG53 facilitates T-tubule remodeling versus repair will allow us design highly targeted and efficacious therapeutics for heart failure treatment.