Connexin expression in cultured neonatal rat myocytes reflects the pattern of the intact ventricle

Objective: Primary cultures of neonatal rat ventricular myocytes have become a widely used model to examine a variety of functional, physiological and biochemical cardiac properties. In the adult rat, connexin43 (Cx43) is the major gap junction protein present in the working myocardium. In situ hybridization studies on developing rats, however, showed that Cx40 mRNA displays a dynamic and heterogeneous pattern of expression in the ventricular myocardium around birth. The present studies were performed to examine the expression pattern of the Cx40 protein in neonatal rat heart, and to examine the connexins present in cultures of ventricular myocytes obtained from those hearts. Methods: Cryosections were made of hearts of 1-day-old Wistar rats. Cultures of ventricular myocytes obtained from these hearts by enzymatic dissociation were seeded at various densities (to obtain >75, ∼50%, and 75% confluency) Cx43 and Cx40 immunoreactivity could be detected. In contrast to Cx43 immunolabeling which showed a homogeneous distribution pattern, Cx40 staining was heterogeneous, i.e. in some clusters of cells abundant labeling was present whereas in others no Cx40 staining could be detected. The pattern of Cx43 immunoreactivity was not altered by the culture density. In contrast, in isolated ventricular myocytes cultured at low density (<25% confluency) the relative number of cell—cell interfaces that were Cx40-immunopositive decreased as compared to high density cultures (35 vs. 70%). Western blots did not reveal significant differences in the level of Cx40 and Cx43 expression at different culture densities. Conclusions: These results show that cultured ventricular myocytes retained typical features of the native neonatal rat ventricular myocardium with regard to their composition of gap junctions. This implicates that these cultures may serve as a good model for studying short-term and long-term regulation of cardiac gap junction channel expression and functio

Selective inhibition of gap junction channel activity by synthetic peptides

The aim of this study was to inhibit specifically one type of gap junction channel in cells expressing multiple connexins (Cx) using synthetic oligopeptides.A7r5 cells (an aortic smooth muscle cell line expressing Cx40 and Cx43) were incubated overnight with synthetic oligopeptides (P180-195) corresponding to a segment of the second extracellular loop of Cx43. This segment is different in sequence from the corresponding location in Cx40.P180-195 (500 μM) decreased cell-to-cell coupling as assessed by dye coupling and dual whole-cell voltage clamp. The decrease in permeability and junctional conductance was caused by selective inhibition of Cx43 gap junction channels. In contrast, overnight incubation of A7r5 cells with oligopeptides corresponding to a segment of the intracellular cytoplasmic tail of Cx43 was without effect.These results indicate that oligopeptides P180-195 may interact with the extracellular domain of the Cx43 protein, thereby possibly mimicking connexin-connexin binding. This apparently inhibits Cx43 channel activity without disturbing the activity of Cx40 channels.Experiments with oligopeptides corresponding to the equivalent part of the second extracellular loop of Cx40 (P177-192) pointed towards a selective inhibition of Cx40 channel activity.Competition assays using synthetic oligopeptides may help to resolve the regulatory properties of gap junction channels in primary cells expressing multiple Cx