Connexin 43 gap junction dynamics in the diabetic heart

提供空间。 糖尿病患者表现出致命性心律失常的风险增加，心肌损伤后恢复减少，以及心肌病导致心室功能障碍，与小动脉硬化和高血压无关。 导致心功能不全的机制尚不清楚，但最近的研究表明心脏缝隙连接重构参与其中。封闭小带-1（ZO-1）结合连接蛋白43（Cx43），这种相互作用对缝隙连接重塑很重要。据推测，这两种蛋白质的相互作用是致命的下游生理后果的主要贡献者，包括心室 糖尿病心脏的功能障碍和心律失常。这一假设将通过以下目标进行检验： 凌晨1确定糖尿病啮齿动物模型心室中Cx43间隙连接大小和细胞分布是否以与ZO-1 Cx43结合变化相关的方式发生改变。 1B.确定糖尿病啮齿动物模型心室中舒张功能障碍和易诱发心律失常与ZO-1 Cx43结合变化的相关程度。 2A.确定高血糖诱导的Cx43间隙连接大小、细胞分布和磷酸化的变化与ZO-1体外结合Cx43的相关程度。 2B.确定高血糖诱导的Cx43间隙连接重塑是否通过设计用于抑制ZO-1与Cx43体外相互作用的膜永久肽（ACT-1）正常化。 将使用几种方法，包括免疫共聚焦共定位分析，蛋白质印迹，FRET（荧光共振能量转移），免疫沉淀，蛋白质-蛋白质交联，和电子显微镜。此外，将使用心电图、超声心动图和荧光电压敏感染料的光学标测来评估心脏功能。此外，在目标2中， 还将在高和低葡萄糖条件下进行Cx43和ZO-1的荧光标记构建体。 在美国，80%的糖尿病患者死于心血管并发症，导致每年超过50000人死亡。该项目将更好地了解糖尿病心脏病的基本机制，并可能导致血压和血糖控制以外的新治疗方法。

SPACE PROVIDED. Diabetic patients exhibit an increased risk of fatal arrhythmias, decreased recovery after a myocardial insult, and a cardiomyopathy leading to ventricular dysfunction independent of arteriolosclerosis and hypertension. The mechanism leading to cardiac dysfunction is not known, however, recent studies have suggested involvement of cardiac gap junction remodeling. Zonula occludens-1 (ZO-1) binds Connexin 43 (Cx43), and this interaction is important for gapjunction remodeling. It is hypothesized that the interaction of these two proteins is a major contributor to the lethal downstream physiological consequences including ventricular dysfunction and arrhythmias in the diabetic heart. This hypothesis will be tested through the following aims: 1 A. Determine whether Cx43 gap junction size and cellular distribution are altered in the ventricle of rodent models of diabetes in a manner that correlates with changes in ZO-1 Cx43 binding. 1B. Determine the extent to which diastolic dysfunction and susceptibility to inducible arrhythmia correlate with changes in ZO-1 Cx43 binding in the ventricle of rodent models of diabetes. 2A. Determine the extent to which hyperglycemia induced changes in Cx43 gap junction size, cellular distribution, and phosphorylation correlate with ZO-1 binding Cx43 in vitro. 2B. Determine if hyperglycemia induced remodeling of Cx43 gap junctions are normalized by a membrane permanent peptide (ACT-1) designed to inhibit the interaction of ZO-1 with Cx43 in vitro. Several approaches will be used including immuno-confocal colocalization analysis, Western blotting, FRET (fluorescent resonance energy transfer), immunoprecipitation, protein-protein crosslinking, and electron microscopy. In addition cardiac function will be assessed using electrocardiography, echocardiography and optical mapping of fluorescent voltage sensitive dyes. Additionally in Aim 2, live cell imaging of fluorescently tagged constructs of Cx43 and ZO-1 in high and low glucose conditions will also be performed. Eighty percent of diabetics in the US die of cardiovascular complications resulting in over 50000 deaths/year. This project will provide a better understanding of the fundamental mechanism of diabetic heart disease and may lead to new treatments beyond blood pressure and sugar control.