Abstract 2957: Regulation of Cx43 GJ Aggregation by ZO-1 Potentially Modulates Differential Adhesion Between Cardiac Myocytes and Fibroblasts
Gap junctions (GJs) comprised of Cx43 have been linked to cardiac arrhythmia and infarct size. Cx43 GJs have been reported to occur between myocytes and fibroblasts, though the significance of this interaction remains unclear. Zonula occludens-1 (ZO-1) is a pan-junctional MAGUK protein that binds the Cx43 C-terminus (CT). We have previously reported that disruption of this interaction results in increases in GJ size. Based on these data, we hypothesized that inhibition of Cx43/ZO-1 interaction promotes aggregation of free membrane connexons into GJs. To test this, acute transitions in GJ intercellular communication level, GJ size and free connexon density in response to inhibition of Cx43/ZO-1 interaction were studied in HeLa cells expressing Cx43. Consistent with our hypothesis, a peptide previously shown to disrupt ZO-1 interaction with Cx43 (αCT1) prompted increases in intercellular communication and concomitant decreases in connexon hemichannel activity as assayed by confocal, live-cell imaging. Similarly, cell-surface protein biotinylation assays indicated that disruption of Cx43/ZO-1 interaction was associated with a biochemical shift of Cx43 from non-junctional plasma membrane connexon pools into GJ aggregates. It was surmised that regulation of connexon aggregation into GJs by ZO-1 could have effects on differential adhesion between cells expressing differing levels of Cx43 (e.g., myocytes and fibroblasts). Aggregates of differentially fluor-tagged cardiac fibroblasts (red) and myocytes (green) were thus prepared in the presence of αCT-1 or control conditions. Inhibition of Cx43/ZO-1 interaction promoted myocyte-myocyte interaction, but resulted in fibroblast exclusion from the aggregates. We conclude that ZO-1 dynamically regulates levels of connexon aggregation into GJs with potential downstream consequences for patterns of differential adhesion between myocytes and fibroblasts. Our data provide insight into a mechanistic link between Cx43 remodeling in the myocardial-fibrous tissues of the infarct border zone and arrhythmia.