Connexin40 (Cx40) is a member of the connexin family of gap junction proteins. Its mRNA, abundant in lung, is also present in mammalian heart, although in lower amount. Rabbit antipeptide antibodies directed to the COOH terminus (residues 335 to 356) of rat Cx40 were characterized to investigate the distribution of Cx40 in rat and guinea pig cardiac tissues. The affinity-purified antibodies detect specifically a major protein (Mr, 40000) in immunoblots of total extracts from rat lung and rat and guinea pig heart. In sections of guinea pig atrial tissue treated for immunofluorescence, a strong labeling associated with myocytes was seen with a distribution consistent with that of intercalated disks. The results of immunoelectron microscopy carried out with guinea pig atrial tissue showed that epitopes recognized by these antibodies were exclusively associated with gap junctions. These results, added to those of control experiments, demonstrate that antibodies 335-356 are specific for Cx40. Doublelabeling experiments carried out with lung sections using anti-factor VIII and anti-Cx40 antibodies suggest that Cx40 is expressed in blood vessel endothelial cells. In guinea pig and rat heart sections, investigated using both immunofluorescence and immunoperoxidase techniques, a signal was also found to be associated with vascular walls. In guinea pig heart, only atrial myocytes are Cx4O-positive. No labeling was detected in ventricular myocytes, including those of the His bundle and the bundle branches, which otherwise do express connexin43 (Cx43). In rat heart Cx4O -expressing myocytes are localized in the conduction system, ie, the His bundle, the bundle branches, and the Purkinje fibers. Cx43 is not detected either in the His bundle or in the proximal parts of the bundle branches, and consequently, Cx4O is the first connexin demonstrated in this region of the rat conduction system. Cx40 was not detected in the working ventricular myocytes. Doublelabeling experiments carried out with hen anti-Cx43 antibodies and rabbit anti-Cx4O antibodies demonstrated that, in tissues expressing both Cx43 and Cx4O, these two connexins were localized in the same immunoreactive sites. A few sites, however, appear to contain only one or the other of these two connexins. (Circ Res. 1994;74:839-851

Using immunohistochemical staining, the distribution of connexin40 (Cx40) and connexin43 (Cx43) was studied in rat, guinea pig, porcine, bovine and human hearts. These species display differences in the degree of morphological differentiation of the conduction system. This study was performed in the anticipation that comparison of the distributions of Cx40 and Cx43 in young and adult specimens may provide clues as to the physiological role of connexins in the heart. To a large extent, the distribution patterns of Cx40 and Cx43 are comparable between species. In neonates and adults, Cx43 was immunolocalized throughout the working myocardium, but in the conduction system Cx43 was detected only after birth. Cx40 was found to appear slightly earlier in development than Cx43 and to disappear when levels of Cx43 became more abundant. This time course was seen in working myocardium and in the ventricular conduction system. Together these data suggest that expression of Cx40 induces or facilitates expression of Cx43, while abundant expression of Cx43 in turn leads to suppression of Cx40 expression. The exceptions to this may represent blocks in this potential regulatory sequence. A second conclusion is that Cx40 and Cx43 containing gap junctions appear in the ventricular conduction system from distal to proximal and only after birth. This indicates that terminal differentiation of the conduction system occurs unexpectedly late in development.

Myocytes are electrically coupled by gap junctions, which are composed of low-resistance intercellular channels. The major cardiac gap junction protein is connexin43 (Cx43). The distribution of Cx43 has been studied by immunofluorescence to visualize the electrical coupling between atrial tissue and sinoatrial node. From modeling studies, this coupling was inferred to be gradual in order to shield the sinoatrial node from the atrial hyperpolarizing influence. The actual Cx43 labeling pattern did not show the expected gradient but instead a rather black and white staining in a striking pattern of strands of cells. We used an immunohistochemical marker (anti-alpha-smooth muscle actin [alpha SMA]) that specifically cross-reacts with guinea pig sinoatrial node cells together with Cx43 antibody to stain previously electrophysiologically mapped sinoatrial nodes. We found that in the guinea pig sinoatrial node the impulse originates in an alpha SMA-positive, virtually Cx43-negative, region (primary pacemaker region). The impulse then travels obliquely upward to the crista terminalis through a region where layers of alpha SMA-positive cells alternate with layers of Cx43-positive SMA-negative cells. The layers of Cx43-positive cells appear to become broader and thicker in the direction of the crista terminalis, whereas the layers of alpha SMA-positive cells become thinner and narrower. Lateral contacts between Cx43- and alpha SMA-positive cells were very sparse and only detected where the Cx43-positive strands ended (the region where alpha SMA-positive cells fill the whole space between endocardium and epicardium, ie, the putative primary pacemaker region). From these results, we conclude that the primary pacemaker is shielded from the hyperpolarizing influence of the atrium by a gradient in coupling brought about by tissue geometric factors rather than by a gradient of gap junction density.

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