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 permeant 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.
