Intracellular Sorting of Connexin 43 by the Ubiquitin Ligase Wwp1

描述（由申请人提供）：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，HCM的特征是在缺乏可识别的临床原因的情况下的左心室肥大（LVH）。在组织学上，受影响的个体的心肌显示肌细胞肥大和紊乱，闰盘的解体，连接蛋白43（Cx43）的定位改变，并且通常，间质纤维化的量增加。虽然预后、LVH程度、纤维化程度和致病突变之间存在相关性，但这种危险分层方法的范围有限。特别是，在轻度至中度LVH患者中，室性心律失常的确切机制尚不明确，尤其是在儿童HCM患者中，尽管Cx43的调节可能起着关键作用。 我们最近创建了一个诱导型转基因小鼠模型，当泛素连接酶Wwp 1在全球范围内过表达时，该模型会产生HCM样表型。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早期）时突然死亡，并且这种表型是100%的渗透。与人类HCM相似，转基因动物显示心脏重量-体重比增加，中度LVH伴随肥大标志物的转录激活，肌细胞紊乱，闰盘破坏，Cx43蛋白水平显著降低，心率不规则。重要的是，我们已经能够表明，WWP 1是正常表达的囊泡在人类心肌细胞，WWP 1上调和HCM中的错误定位，和Wwp 1可以泛素化Cx43。基于这些观察，我们证实了以下中心假设：增加Wwp 1介导的Cx43泛素化导致Cx43的溶酶体降解，随后心律失常和猝死。为了验证这一假设，我们的目标是：1）确定Wwp 1过表达在心肌细胞中的作用及其促进HCM表型的能力; 2）确定Wwp 1介导的Cx43细胞内运输的机制; 3）鉴定Cx43上Wwp 1介导的泛素化位点，以确定该/这些位点泛素化的功能意义。 实现这里概述的具体目标将阐明间隙连接重塑的分子机制。然后，这些信息可以应用于HCM，这是一种常见的破坏性疾病，已知显示Cx43错误定位和WWP 1表达增加，以确定可能具有更大致命性心律失常风险的患者人群。 公共卫生相关性：肥厚型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的理解是缺乏的，尽管似乎一种名为Connexin 43的蛋白质是这个过程的关键。该应用程序定义了一个新的球员，Wwp 1，如何控制连接蛋白43的量，因此可能作为诊断，以确定那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.