The molecular mechanisms underlying arrhythmogenic right ventricular dysplasia/cardiomyopathy

 描述(申请人提供)：这是我们的资助金R01 HL095780-01的竞争性续期申请书。我们最初获得资助是为了研究已知的中枢桥粒成分桥粒蛋白在与遗传性心脏病相关的临床和细胞特征中的作用机制，该疾病是一种导致年轻人猝死的遗传性右室心肌病(ARVC)，通过产生和表征新的桥粒蛋白缺乏模型系统。对于这一应用，我们通过利用我们新的ARVC遗传小鼠和人类心脏模型，在酵母双杂交筛选中发现了突触体相关蛋白29(SNAP29)作为一种新的桥粒蛋白相关蛋白存在于成人心脏中，我们发现该蛋白与ARVC相关。尽管SNAP29在心脏中的作用尚不清楚，但我们发现SNAP29共定位于成年小鼠和人类心脏以及人类诱导的多能干细胞来源的心肌细胞(HiPSC)中的心肌桥粒细胞-细胞连接。此外，在我们的ARVC和ARVC HiPSC来源的小鼠模型心脏中，SNAP29在桥粒连接处的定位和/或水平丢失，这些细胞表现出显著的桥粒缺陷和致心律失常行为。新的SNAP29缺陷小鼠模型的建立(全局的和心肌细胞特有的)显示出显著的心脏缺陷，包括(I)心肌细胞连接处的自噬缺陷(细胞连接处的自噬/溶酶体标记和结构的积累)，该缺陷特异性地影响桥粒蛋白水平和(Ii)心脏形态缺陷。来自我们的ARVC小鼠模型的数据为这一机制提供了验证，因为我们揭示了他们的心脏在心肌细胞连接处的自噬控制方面表现出类似的缺陷。我们假设SNAP29通过自噬机制控制桥粒翻转，从而调节心肌中桥粒蛋白的水平和功能，其缺失将触发桥粒蛋白水平和功能的丧失，最终导致ARVC。我们的目标是：(I)通过鉴定SNAP29功能丧失的小鼠模型，确定SNAP29在心脏中的功能作用；(Ii)通过表达人类ARVC突变并以HiPSCs为工具，确定SNAP29-DSP相互作用在人ARVC和心肌细胞中的相关性；以及(Iii)通过分析SNAP29功能丧失模型和体外ARVC模型中SNAP29的过度表达，确定自噬中控制桥粒水平/周转的SNAP29依赖事件。

 DESCRIPTION (provided by applicant): This is an application for a competitive renewal of our grant R01 HL095780-01. We were originally funded to investigate mechanisms underlying the role of the known central desmosomal component, desmoplakin, in the clinical and cellular features associated with the genetic-based heart disease, arrhythmogenic right ventricular cardiomyopathy (ARVC) that causes sudden death in the young, by generating and characterizing novel desmoplakin deficient model systems. For this application, we uncovered synaptosomal associated protein 29 (SNAP29) as a novel desmoplakin associated protein in the adult human heart in a yeast-two hybrid screen that we show has relevance to ARVC, by leveraging our novel genetic mouse and human cardiac models of ARVC. Although the role of SNAP29 is unknown in the heart, we show that SNAP29 co-localizes to cardiac muscle desmosomal cell-cell junctions in the adult mouse and human heart as well as human induced pluripotent stem cell derived cardiac cells (hiPSC). Furthermore, SNAP29 localization and/or levels at desmosomal junctions are lost in hearts from our mouse model of ARVC and ARVC hiPSC-derived cardiac cells that exhibit striking desmosomal defects and arrhythmogenic behavior. Generation of novel SNAP29 deficient mouse models (global and cardiomyocyte-specific) revealed striking cardiac defects including (i) autophagic defects at the cardiac muscle cell junction (accumulation of autophagic/lysosomal markers and structures at the cell junction) that specifically impacted desmosomal protein levels and (ii) cardiac morphology defects. Data from our mouse model of ARVC provides validation to this mechanism as we reveal that their hearts exhibit similar defects in autophagic control at the cardiac muscle cell junction. We hypothesize that SNAP29 regulates desmosomal protein levels and function in cardiac muscle by controlling desmosomal turnover via autophagy-mediated mechanisms and its loss will trigger loss of desmosomal protein levels as well as function and ultimately cause ARVC. We aim to determine: (i) the functional role of SNAP29 in the heart by characterizing SNAP29 loss of function mouse models, (ii) the relevance of SNAP29-DSP interaction in human ARVC and cardiomyocytes by expressing human ARVC mutations and using hiPSCs as a tool, and (iii) the SNAP29-dependent events in autophagy that control desmosomal levels/turnover, by analyzing defects in cardiac autophagy (induction and flux) and relevant desmosome targets using SNAP29 loss of function models and overexpression of SNAP29 in an in vitro ARVC model.