Gap Junctional Patterning in Arrhythmic Heart

描述（由申请人提供）：细胞之间的直接胞浆通讯是由质膜上称为间隙连接（GJ）的细胞间通道聚集介导的。连接蛋白43 (Cx43) GJs的重塑与心律失常有关。本研究前期完成的工作1确定了Cx43与肌动蛋白结合蛋白Zonula Occludens (ZO)-1相互作用在GJ重塑中的生物学功能。在最近的工作中，我们已经确定了一种已知抑制Cx43/ZO-1相互作用的肽，1CT1，降低左心室损伤后诱发性心律失常的频率。体外1CT1实验的初步数据表明，从膜上的自由连接子通道到细胞间通道聚集体中的配对连接子的转变是GJ重塑的基础。我们假设ZO-1调节了膜上自由连接子向gj聚集的速率，即“连接子开关”。这项更新的目的是验证这一假设及其对GJ细胞间通讯、膜兴奋性的影响，以及心肌梗死后Cx43在肌细胞和成纤维细胞之间的差异粘附中的新作用——所有这些过程都可能影响对再入性心律失常的易感性。拟议的实验将定量确定Cx43/ZO-1相互作用在“连接子开关”中的作用，方法包括活细胞成像、荧光融合蛋白、功能缺失突变体、全细胞膜片钳、单通道电生理和生化分析。在体内心脏损伤的生理框架内，Cx43/ZO-1相互作用的影响将通过超声心动图、心电图遥测、电激活光学成像和心律失常诱导方案进行评估。所产生的数据将扩大我们对Cx43功能基本机制的理解，并可能转化为预防和治疗心律失常的新疗法。

DESCRIPTION (provided by applicant): Direct cytosolic communication between cells is mediated by an aggregate of intercellular channels in the plasma membrane called the gap junction (GJ). Remodeling of connexin43 (Cx43) GJs has been linked to cardiac arrhythmias. Work accomplished during the previous period of this RO1 identified a biological function of interaction between Cx43 and the actin-binding protein Zonula Occludens (ZO)-1 in GJ remodeling. In recent work, we have determined that a peptide known to inhibit Cx43/ZO-1 interaction, 1CT1, reduces the frequency of inducible arrhythmias following injury to the left ventricle. Preliminary data from work performed with 1CT1 in vitro, indicates that the transition from free connexon channels in the membrane to paired connexons in intercellular channel aggregates underlies GJ remodeling. We hypothesize that ZO-1 regulates the rate at which free connexons in the membrane accrete to GJs-the "connexon switch". The aims in this renewal will test this hypothesis and its implications for GJ intercellular communication, membrane excitability, and a novel role for Cx43 in differential adhesion between myocytes and fibroblasts following myocardial infarction - all processes likely to impact susceptibility to re-entrant arrhythmia. The proposed experiments will quantitatively determine the role of Cx43/ZO-1 interaction in the "connexon switch" using methods including live cell imaging, fluorescent fusion proteins, loss-of-function mutants, whole cell patch clamp, single channel electrophysiology and biochemical assays. Implications of Cx43/ZO-1 interaction within the physiological framework of cardiac injury in vivo will be assessed by echocardiography, EKG telemetry, optical mapping of electrical activation and arrhythmia induction protocols. The data generated will broaden our understanding of fundamental mechanisms of Cx43 function and may translate to new therapies for the prevention and treatment of cardiac arrhythmia. PUBLIC HEALTH RELEVANCE: Injury and scarring of the heart following myocardial infarction (heart attack) is one the most frequent causes of sudden death in the USA. This project will deliver new insights on the function of a protein essential to a stable heartbeat called connexin43. The project aims to provide novel approaches to the prevention and treatment of heart attacks.