Relating Electrical Conductance, Connexin 43 Immunostaining, and Cell Shape in Micropatterned Cardiac Cell Pairs

Abstract

Gap junctions are responsible for cell-cell electrical coupling and maintaining normal cardiac conduction patterns. Connexin 43 (Cx43) is the dominant gap junction protein in ventricular myocytes. Although the relationship between Cx43, conduction, and tissue structure have been extensively studied in engineered monolayers of cardiac tissue, there have been few studies comparing conductance, Cx43, and cell shape on the cell-cell level. We have used micropatterning to control the dimensions of myocyte pairs and study electrophysiological properties at very high resolution. We hypothesized that conductance and Cx43 immunostaining would be directly correlated. We also sought to relate our electrophysiological measurements to cell shape. Using a dual voltage clamp system, we measured the conductance of micropatterned ventricular myocyte pairs, and subsequently fixed and immunostained the same cells for Cx43. Thus, we compared conductance and Cx43 immunostaining serially in the same cell pair. The volume of Cx43 immunostaining was determined using confocal microscopy and quantitative software programs. Using brightfield images, we measured cell dimensions and 2-dimensional cell-cell contact. We studied three types of rectangular cell pairs with varying length to width aspect ratios (3.33:1, 5:1, 6.67:1). The average length of the cell-cell junction (R2=0.99, n=23), average conductance (R2=0.92, n=22), and average Cx43 immunostaining (R2=0.85, n=22) increased linearly relative to the aspect ratio. We found a linear relationship between Cx43 immunostaining and conductance (R2=0.70, n=22). A weaker linear relationship was found between conductance and 2-dimensional length of the cell-cell junction (R2=0.51, n=23). Our results suggest that cell pairs nearly maximize their contact area, which contributes to increases in both Cx43 and conductance. Cell pairs with higher length-width aspect ratios have more cell-cell contact and therefore higher Cx43 density and conductance. However, Cx43 density is the most important determinant of conductance.