Cardiac Myocyte Protein Partners in Heart Function

心肌细胞蛋白在心脏功能中的伙伴

• 批准号: 10502152
• 负责人: Joshua I Goldhaber
• 金额: $ 73.28万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-07-18 至 2026-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10502152
• 关键词: Abbreviations; Adult; Affect; Arrhythmia; Binding; Biochemical; Black race; Cardiac; Cardiac Myocytes; Cardiac conduction system; Cardiomyopathies; Cell Communication; Cell-Cell Adhesion; Cells; Classification; Complex; Confocal Microscopy; Connexins; Coupling; Data; Defect; Dilated Cardiomyopathy; Elements; Etiology; Evaluation; Functional disorder; Future; Genetic; Genetic Predisposition to Disease; Genetic Screening; Genetic Transcription; Giant Cells; Grant; Hand; Heart; Heart Atrium; Heart Diseases; Heart failure; Human; Hypertension; Intercalated disc; Intercellular Junctions; Ion Channel; Knowledge; Lateral; Lead; Link; Location; Mechanics; Membrane; Membrane Proteins; Microscopic; Modeling; Molecular; Morbidity - disease rate; Mus; Myocardium; Optics; Patients; Perinatal; Physiological; Positioning Attribute; Property; Proteins; Proteomics; Resolution; Role; Severities; Sex Differences; Sinoatrial Node; Stress; Sudden Death; Symptoms; Systemic disease; Testing; Tight Junctions; Tissues; Ventricular; Ventricular Arrhythmia; Vinculin; Work; atrioventricular node; base; cardiac muscle disease; genetic manipulation; heart function; heart preservation; heart rhythm; hemodynamics; human disease; intercellular connection; molecular phenotype; mortality; mouse model; non-genetic; novel strategies; novel therapeutics; patch clamp; postnatal; screening; sex; sudden cardiac death; transcriptomics; voltage

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.

心肌病（CMY）是一个世界性的问题，与高发病率和死亡率。CMY患者可能 出现心力衰竭（HF）、心律失常、心脏传导系统异常， 和心脏性猝死最近，越来越多的人认识到， CMY是重要的，并且细胞连接中的心肌细胞（CM）蛋白的缺陷可引起CMY。 CM的机械和电偶联发生在CM之间的细胞间连接处，称为 闰盘（ID）。ID提供了使心脏能够发挥合胞体功能的基本属性。结构 心脏传导系统（CCS）的元件，如房室（AV）和窦房（SA）结 具有与心房和心室中的工作CM不同的细胞排列和细胞间连接。 了解更多关于工作CM和CCS细胞正常功能所必需的蛋白质对于 推进我们对心脏病基础的认识，并且仍然研究不足。 该建议集中在与人的免疫球蛋白（VCL）和闭锁小带（ZO）蛋白，它们直接结合到 彼此位于工作CM的ID和侧膜中，并且具有已知的链接， 人类疾病我们的总体假设是，VCL、ZO-1和ZO-2在保存心脏中具有独特的作用， 功能、传导和节律，考虑到它们在CM和CCS中的位置。我们提出两个目标。 AIM 1将评估CM中ZO和VCL蛋白的丢失如何改变收缩功能， 工作CM和成人心脏的分子表型。我们假设，随着出生后CM ZO的丢失， 和VCL蛋白，细胞间通讯和工作心肌的完整性将受到干扰，导致 收缩功能障碍CM ZO损失预计与CM VCL损失引起的损失不同，尽管存在这些问题。 蛋白质彼此直接结合。手中独特的老鼠模型将被用来评估损失的影响 成熟CM基础及心脏应激后ZO和VCL的变化。我们将评估整个心脏， 组织和单细胞使用生理，生物化学和显微镜评价，以及一种新的 单细胞转录组学和单细胞蛋白质组学的方法，以追求如何ZO 而VCL蛋白缺失导致CMY。将考虑性别和心房与心室的差异。 AIM 2将确定ZO-1和VCL在心脏传导和节律中的功能。删除VCL CM ZO-1的缺失导致室性心律失常和猝死，而CM ZO-1的缺失导致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， 尽管它们由于与连接蛋白和膜离子通道的相互作用而直接结合。使用Patch 钳位，高分辨率共聚焦显微镜检测Ca 2+瞬变，光学电压/Ca 2+映射，和单 细胞转录和蛋白质组学研究，我们将测试如何损失ZO-1导致改变SAN和AVN功能 和兴奋-收缩偶联，以及VCL的丧失如何导致室性心律失常和猝死。