The Role of SNAP29, a Novel Desmoplakin Interactor, in Cardiac Pathophysiology

描述（由申请人提供）：致心律失常性心肌病（AC）是一种遗传性心脏病，其特征为心室功能障碍、心肌纤维脂肪替代和室性心律失常，导致心源性猝死。最近的人类临床数据显示，只有40%的AC患者在桥粒组分（即桥粒斑蛋白（DSP）、桥粒芯蛋白、桥粒胶蛋白、斑珠蛋白、斑嗜蛋白）中存在突变，桥粒是一种在相邻细胞之间建立机械连接的细胞间结构。其余的致病突变在很大程度上是未知的，这表明新的桥粒相互作用蛋白的鉴定和表征是至关重要的。为了解决这一挑战，我们开始确定新的桥粒相关蛋白进行酵母双杂交筛选DSP作为诱饵在成人心脏cDNA文库。在这个屏幕上，我们确定了突触体相关蛋白29（SNAP 29），囊泡运输蛋白与介导的膜融合的作用，虽然它在心脏中的功能还没有建立。在人类中，SNAP 29突变与桥粒疾病、CEDNIK综合征相关，其包括神经学表现以及与桥粒不稳定相关的皮肤缺陷。我们的初步数据表明，SNAP 29与DSP共定位在细胞的远端，其中桥粒位于成年小鼠和人类心脏，这表明这种相互作用发生在完整的心脏。在表现出AC特征的DSP心脏特异性敲除（DSP-cKO）小鼠的心脏中，SNAP 29在连接部分中减少，表明SNAP 29和DSP之间在心脏中的功能性相互作用。相反，使用来自我们最近产生的SNAP 29整体敲除（SNAP 29-gKO）小鼠的心脏，其表现出断奶前致死性、心脏重量与体重比增加和皮肤缺陷，在连接部分中DSP减少，为SNAP 29、DSP和桥粒之间的功能关系提供了惊人的证据。SNAP 29在心脏功能中的重要性进一步得到了我们在斑马鱼中敲除SNAP 29的实验的支持，该实验导致心脏缺陷和心律失常。我们假设SNAP 29是心脏桥粒的一种新成分，SNAP 29的缺失将导致心脏桥粒超微结构和稳定性的缺陷，从而导致心脏功能受损和心律失常。为了验证这一假设，我们的目标如下：i）使用突变体序列的强制酵母-2-杂交和心肌细胞过表达来表征SNAP 29和DSP之间的分子相互作用以及理解AC中这些相互作用的相关性，以及ii）通过评估整体SNAP 29缺失小鼠和心肌细胞的心脏表型来表征SNAP 29在发育和成年心脏中的作用。通过确定a）使用荧光免疫染色和蛋白质印迹确定桥粒和囊泡运输蛋白的水平和定位，b）使用透射电子显微镜确定桥粒超微结构，c）使用组织学技术、超声心动图、MRI和表面心电图确定特定SNAP 29缺失小鼠。B）使用透射电子显微镜确定桥粒超微结构。

DESCRIPTION (provided by applicant): Arrhythmogenic cardiomyopathy (AC) is a genetic based heart disease characterized by ventricular dysfunction, fibrofatty replacement of the myocardium and ventricular arrhythmias leading to sudden cardiac death. Recent human clinical data has revealed that only 40% of AC patients harbor mutations in components of the desmosome (i.e. desmoplakin (DSP), desmoglein, desmocollin, plakoglobin, plakophilin), an intercellular structure that establishes a mechanical link between adjacent cells. The remaining causative mutations are largely unknown, suggesting that identification and characterization of novel desmosomal interacting proteins is of utmost importance. To address this challenge, we set out to identify novel desmosome associated proteins by performing a yeast-2-hybrid screen with DSP as bait in an adult human heart cDNA library. In this screen, we identified synaptosomal associated protein 29 (SNAP29), a vesicular trafficking protein with roles in mediating membrane fusion, though its function in the heart has not been established. In humans, SNAP29 mutations are associated with the desmosomal disease, CEDNIK syndrome, which includes neurological manifestations as well as skin defects associated with desmosomal instability. Our preliminary data show that SNAP29 colocalizes with DSP at the distal ends of cells where desmosomes are located in adult mouse and human hearts, suggesting that this interaction occurs in the intact heart. In hearts of DSP cardiac specific knockout (DSP-cKO) mice which exhibit features of AC, SNAP29 is reduced in the junctional fraction indicating a functional interaction between SNAP29 and DSP in the heart. Conversely, using hearts from our recently generated SNAP29 global knockout (SNAP29-gKO) mice which exhibit preweaning lethality, increased heart weight to body weight ratios, and skin defects, there is a reduction in DSP in the junctional fraction providing striking evidence for a functional relationship between SNAP29, DSP, and the desmosome. The importance of SNAP29 in heart function is further supported by our experiments knocking down SNAP29 in zebrafish which result in cardiac defects and arrhythmias. We hypothesize that SNAP29 is a novel component of the cardiac desmosome and that loss of SNAP29 will lead to defects in cardiac desmosome ultrastructure and stability leading to impaired heart function and arrhythmias. To test this hypothesis, our aims are as follows: i) to characterize the molecular interactions between SNAP29 and DSP as well as understand the relevance of these interactions in AC using forced yeast-2-hybrid and cardiomyocyte overexpression of mutant sequences and ii) to characterize the role of SNAP29 in the developing and adult heart by assessing the cardiac phenotype of global SNAP29 null mice and cardiac-specific SNAP29 null mice by determining a) levels and localization of desmosomal and vesicular trafficking proteins using fluorescent immunostaining and western blot, b) desmosome ultrastructure using transmission electron microscopy, c) cardiac morphology and function using histological techniques, echocardiography, MRI, and surface electrocardiography.