Disruption of connexin43 and ZO-1 interaction alters the size and distribution of gap junctions.

The gap junction (GJ) is an aggregate of intercellular channels that links the cytoplasm of neighboring cells and allows small molecules such as ions to pass between these cells. Within the mammalian heart, the distribution of connexin43 (Cx43)-containing gap junctions is essential to facilitating the coordinated spread of electrical activation throughout the cardiac muscle cells (cardiomyocytes) and allowing the heart to contract properly. Disruptions in GJ distribution patterns in the adult heart are thought to be linked directly to cardiac disease and arrhythmia. Recently, the protein zonula-occludens-1 (ZO-1) has been suggested to have an important role in the organization of GJs between cardiomyocytes in development and disease. However, the underlying mechanism by which ZO-1 affects GJ organization remains undetermined. In this study, the interaction between Cx43 (the major cardiac GJ protein) and ZO-1 in rat neonatal cardiomyocytes and HeLa cells was inhibited by a peptide designed to disrupt the binding of Cx43 and ZO-1. Images of the Cx43 GJs and ZO-1 in treated and control cultures were collected by confocal microscopy and quantitatively analyzed. The resulting data indicate a significant increase in Cx43 aggregate size and a shift in Cx43 distribution to the cell membrane accompanying a decrease in Cx43-ZO-1 binding mediated by the peptide in both myocytes and HeLa cells. These results suggest that ZO-1 may be important in regulating the extent of Cx43 GJ aggregates and provide insight into the presently unknown role of ZO-1 interaction with gap junction sub-unit proteins. At present, we are validating the degree to which the inhibitory peptide causes decoupling of direct protein-protein interaction between Cx43 and ZO-1 at molecular level using fluorescence resonance energy transfer (FRET).