Intercellular communication plays diverse roles in the embryological and postnatal development of the heart. Correspondingly, insight into developmental mechanisms governing the organization of cell-cell coupling may significantly inform the origins of electrophysiological instabilities in the diseased heart. The disorganized patterns of cell coupling common to various pathologies of the myocardium, re-capitulate patterns of gap junction (gj) distribution found in differentiating myocardium. As detailed herein, we have made significant progress in understanding patterning of gjs in the developing heart - also laying a foundation for renewal of this project. In particular, we have developed strong evidence for a novel potential function of a connexin interacting protein, ZO-1, in gj remodeling. Our specific hypothesis is that a ZO-1 basedmechanism has a role in a gj endocytosis process that may impact developmental remodeling of electrical coupling patterns between myocytes. In addition, we have derived evidence for a role for coronary arteriogenesis in determining gj distribution and connexin expression in studies of a transgenic model of sudden cardiac death (the HF1b knockout mouse). To build on these observations we aim to; 1) use membrane translocating peptides and adenoviral vectors to target ZO-1-Cx43 interactions, investigating the role of ZO-l in Cx43 gj remodeling in Langendorff perfused heart and cell culture preparations ; 2) use fluorescent ZO-1 and Cx43 fusion proteins, and stably transfected cell lines, to directly observed ZO-I-Cx43 interactions and Cx43 gj function in vivo ; 3) use adenoviral vectors and inhibitory peptides targeting ZO-l-Cx43 interactions to investigate the role of ZO-1 in Cx43 gj patterning in cardiac development and disease; and ; 4) characterize whether loss of ZO-I function and/or abnormalities in coronary arteriogenesis have a role in defective gj patterning in the working myocardium and ventricular conduction tissues of the HF lb knockout mouse heart.
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