Intracellular Sorting of Connexin 43 by the Ubiquitin Ligase Wwp1

描述(申请人提供)：与肥厚性肌病(HCM，最常见的遗传性心肌病)相关的心律失常是运动员和年轻人猝死的主要原因。大体上，肥厚性心肌病的特征是在没有可识别的临床原因的情况下出现左心室肥厚(LVH)。组织学上，患者的心肌表现为心肌细胞肥大和紊乱，间盘排列紊乱，连接蛋白43(Cx43)定位改变，通常间质纤维化增加。尽管预后、左心室肥厚程度、纤维化量和致病突变之间存在相关性，但这种风险分层方法的范围有限。特别是，尽管Cx43的调节可能起到关键作用，但对轻至中度LVH患者的室性心律失常的确切机制尚不清楚，特别是在儿童肥厚性心肌病患者中。我们最近建立了一种可诱导的转基因小鼠模型，当泛素连接酶Wwp1在全球过表达时，该模型会产生类似HCM的表型。有趣的是，这些小鼠在6-12周龄(相当于人类的青春期或成年期早期)非常突然地死亡，并且这种表型是100%穿透的。与人类HCM相似，转基因动物心脏重量/体重比增加，中度LVH伴随着肥大标志物的转录激活，心肌细胞紊乱，间盘破坏，Cx43蛋白水平显著降低，心率不规则。重要的是，我们已经能够证明WWP1在人心肌细胞内的囊泡中正常表达，WWP1在HCM中上调和错误定位，Wwp1可以泛素化Cx43。基于这些观察，我们假设以下中心假设：Wwp1介导的Cx43泛素化增加导致Cx43溶酶体降解，随后发生心律失常和猝死。为了验证这一假说，我们的目标是1)确定Wwp1在心肌细胞中过表达的作用及其促进HCM表型的能力；2)确定Wwp1介导的Cx43的细胞内转运机制；3)确定Wwp1介导的Cx43上的泛素化位点(S)，以确定该位点(S)泛素化的功能意义。完成这些目标将有助于阐明缝隙连接重塑的分子机制。然后，这些信息可以应用于肥厚性心肌病，这是一种常见的破坏性疾病，已知表现为Cx43错位和WWP1表达增加，以识别可能面临更大致命性心律失常风险的患者群体。 公共卫生相关性：与肥厚性心肌病相关的心律失常是运动员和年轻人猝死的主要原因。目前，我们对为什么一些患者更容易发生这些心律失常缺乏了解，尽管似乎一种名为连接蛋白43的蛋白质是这一过程中的关键。这个应用定义了一个新的参与者Wwp1是如何控制连接蛋白43的数量的，因此可以作为一种诊断方法来识别哪些肥厚型心肌病患者最有可能遭受心脏性猝死。

DESCRIPTION (provided by applicant): Cardiac arrhythmias associated with hypertrophic myopathy (HCM, the most common genetic myocardial disease) are the leading cause of sudden death in athletes and young people. Grossly, HCM is characterized by left ventricular hypertrophy (LVH) in the absence of identifiable clinical causes. Histologically, the myocardium of affected individuals displays myocyte hypertrophy and disarray, disorganization of intercalated discs with altered localization of Connexin 43 (Cx43), and, usually, increased amounts of interstitial fibrosis. Although there is a correlation among prognosis, the degree of LVH, amount of fibrosis, and causative mutation, this risk stratification methodology is limited in scope. In particular, the precise mechanisms underlying ventricular arrhythmias in patients with little-to-moderate LVH and are poorly defined, especially in pediatric HCM patients, although the regulation of Cx43 likely plays a critical role. We have recently created an inducible transgenic mouse model which develops a HCM-like phenotype when the ubiquitin ligase Wwp1 is globally overexpressed. Interestingly, these mice die very suddenly at 6-12 weeks of age (equivalent to adolescence or early adulthood in humans), and this phenotype is 100% penetrant. Similar to human HCM, transgenic animals display an increase in heart weight-to-body weight ratio, moderate LVH with an accompanying transcriptional activation of hypertrophic markers, myocyte disarray, a disruption of intercalated discs, dramatically decreased Cx43 protein levels, and an irregular heart rate. Importantly, we have been able to show that WWP1 is normally expressed in vesicles within human cardiomyocytes, WWP1 is upregulated and mislocalized in HCM, and Wwp1 can ubiquitinate Cx43. Based on these observations, we posit the following central hypothesis: increased Wwp1-mediated ubiquitination of Cx43 causes lysosomal degradation of Cx43, ensuing arrhythmia, and sudden death. In order to test this hypothesis, we aim to 1) define the role of Wwp1 overexpression in cardiomyocytes and its ability to promote the HCM phenotype; 2) determine the mechanism of Wwp1-mediated intracellular trafficking of Cx43; and 3) identify the Wwp1-mediated ubiquitination site(s) on Cx43 to determine the functional significance of ubiquitination of this/these site(s). Accomplishing the Specific Aims outlined here will elucidate the molecular mechanism underlying gap junction remodeling. This information can then be applied to HCM, a frequent and devastating disease known to display mislocalized Cx43 and increased WWP1 expression, in order to identify patient populations that might be at greater risk for fatal arrhythmias. PUBLIC HEALTH RELEVANCE: Cardiac arrhythmias associated with hypertrophic cardiomyopathy are the leading cause of sudden death in athletes and young people. Currently, our understanding of why some patients are more prone to these arrhythmias is lacking, although it seems that a protein called Connexin43 is the lynchpin in this process. This application defines how a novel player, Wwp1, controls the amount of Connexin43, and as such might serve as a diagnostic to identify those hypertrophic cardiomyopathy patients most likely to suffer sudden cardiac death.