Cardiac Myocyte Protein Partners in Heart Function

心肌病(CMY)是一个发病率和死亡率都很高的世界性难题。CMY患者可能 出现心力衰竭、心律失常、心脏传导系统异常、 和心源性猝死。最近，人们越来越认识到，对糖尿病病因的基因筛查 CMY很重要，细胞连接中的心肌细胞(CM)蛋白缺陷可导致CMY。 CM的机械和电气耦合发生在CM之间的细胞间连接，称为 插入磁盘(ID)。ID提供了允许心脏作为合胞体发挥作用的基本属性。结构性 心脏传导系统(CCS)的组成部分，如房室(AV)和窦房结(SA) 在心房和脑室有不同于正常的CMS的细胞排列和细胞间连接。 更多地了解CMS和CCS细胞正常工作所必需的蛋白质对于 提高了我们对心脏病基础的认识，但仍未得到充分研究。 这项建议的重点是纽蛋白(VCL)和闭锁小带(ZO)蛋白，它们直接与 彼此位于ID和工作CMS的侧膜上，并具有已知的连接 人类疾病。我们的全球假设是VCL、ZO-1和ZO-2在心脏保护中具有独特的作用 功能、传导和节奏，给出它们在CMS和CCS中的位置。我们提出了两个目标。 目标1将评估CMS中ZO和VCL蛋白的丢失如何改变收缩功能和 正常心脏和成人心脏的分子表型。我们假设，随着CM ZO的出生后丢失 而VCL蛋白、细胞间的通讯和工作心肌的完整性会受到干扰，从而导致 收缩功能障碍。尽管如此，CM ZO的损失预计将与CM VCL的损失不同 蛋白质之间直接结合。将使用手中独特的老鼠模型来评估损失的影响 ZO和VCL在成熟型CMS的基础上和心脏面临应激后。我们将对整个心脏进行评估， 组织和单个细胞使用生理、生化和显微评价，以及一种新的 单细胞转录组和单细胞蛋白质组学方法，探讨ZO的机制基础 VCL蛋白丢失是引起CMY的原因。性别和房室的差异将被考虑在内。 目的2确定ZO-1和VCL在心脏传导和节律中的作用。删除VCL CM引起室性心律失常和猝死，而CM ZO-1缺失引起CCS功能障碍。 我们假设这两种蛋白质在控制心律和传导方面具有独特的作用， 尽管它们直接结合，但由于与连接蛋白和膜离子通道的相互作用。使用面片 钳位，高分辨率共聚焦显微镜，以检测钙瞬变，光电压/钙映射，和单 细胞转录和蛋白质组学研究，我们将测试ZO-1的缺失如何导致SAN和AVN功能改变 以及VCL丢失如何导致室性心律失常和猝死。

Cardiomyopathy (CMY) is a worldwide problem associated with high morbidity and mortality. CMY patients may present with symptoms attributed to heart failure (HF), arrhythmias, cardiac conduction system abnormalities, and sudden cardiac death. Recently it has been increasingly recognized that genetic screening for etiologies of CMY is important, and that defects in cardiomyocyte (CM) proteins in cellular junctions can cause CMY. Mechanical and electrical coupling of CM occurs at the intercellular connection between CM, termed the intercalated disk (ID). The ID provides essential properties to allow the heart to function as a syncytium. Structural elements of the cardiac conduction system (CCS) such as the atrioventricular (AV) and sinoatrial (SA) nodes have cellular arrangements and intercellular connections that vary from working CMs in the atrium and ventricle. Understanding more about proteins essential for normal function of working CMs and CCS cells is critical to advance our knowledge of the basis of cardiac disease, and remains understudied. This proposal is focused on Vinculin (VCL) and Zonula Occludens (ZO) proteins, which bind directly to one another, are located in the ID and the lateral membrane of working CMs and have known links to human disease. Our global hypothesis here is that VCL, ZO-1 and ZO-2 have unique roles in preserving cardiac function, conduction, and rhythm, given their location in CMs and in the CCS. We propose two aims. AIM 1 will evaluate how loss of ZO and VCL proteins from CMs alters contractile function and the molecular phenotype of the working CM and adult heart. We hypothesize that with postnatal loss of CM ZO and VCL proteins, cell-cell communication and integrity of the working myocardium will be disturbed, resulting in contractile dysfunction. CM ZO loss is expected to be distinct from that caused by loss of CM VCL, despite these proteins directly binding one another. Unique mouse models in hand will be used to evaluate the effects of loss of ZO and VCL from mature CMs basally and after the heart is faced with stress. We will assess whole heart, tissue and single cells using physiological, biochemical, and microscopic evaluations, as well as a novel approach to single cell transcriptomics and single cell proteomics, to pursue the mechanistic basis for how ZO and VCL protein loss causes CMY. Differences by sex and in atria vs. ventricle will be considered. AIM 2 will determine the function of ZO-1 and VCL in cardiac conduction and rhythm. Deletion of VCL from CM caused ventricular arrhythmias and sudden death, while loss of CM ZO-1 produced CCS dysfunction. We hypothesize that these two proteins have unique roles in controlling cardiac rhythm and conduction, despite their direct binding, due to interactions with connexins and membrane ion channels. Using patch clamping, high-resolution confocal microscopy to detect Ca2+ transients, optical voltage/Ca2+ mapping, and single cell transcriptional and proteomic studies, we will test how loss of ZO-1 leads to altered SAN and AVN function and excitation-contraction coupling, and how loss of VCL leads to ventricular arrhythmias and sudden death.