RESTRICTED DISTRIBUTION OF CONNEXIN40, A GAP JUNCTIONAL PROTEIN, IN MAMMALIAN HEART

Connexin40 (Cx40) is a member of the connexin family of gap junction p roteins. Its mRNA, abundant in lung, is also present in mammalian hear t, although in lower amount. Rabbit antipeptide antibodies directed to the COOH terminus (residues 335 to 356) of rat Cx40 were characterize d to investigate the distribution of Cx40 in rat and guinea pig cardia c tissues. The affinity-purified antibodies detect specifically a majo r protein (M(r), 40 000) in immunoblots of total extracts from rat lun g and rat and guinea pig heart. In sections of guinea pig atrial tissu e treated for immunofluorescence, a strong labeling associated with my ocytes was seen with a distribution consistent with that of intercalat ed disks. The results of immunoelectron microscopy carried out with gu inea pig atrial tissue showed that epitopes recognized by these antibo dies were exclusively associated with gap junctions. These results, ad ded to those of control experiments, demonstrate that antibodies 335-3 56 are specific for Cx40. Double-labeling 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 p ig and rat heart sections, investigated using both immunofluorescence and immunoperoxidase techniques, a signal was also found to be associa ted with vascular walls. In guinea pig heart, only atrial myocytes are Cx40-positive. No labeling was detected in ventricular myocytes, incl uding those of the His bundle and the bundle branches, which otherwise do express connexin43 (Cx43). In rat heart Cx40-expressing myocytes a re 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 conseq uently, Cx40 is the first connexin demonstrated in this region of the rat conduction system. Cx40 was not detected-in the working ventricula r myocytes. Double-labeling experiments carried out with hen anti-Cx43 antibodies and rabbit anti-Cx40 antibodies demonstrated that, in tiss ues expressing both Cx43 and Cx40, these two connexins were localized in the same immunoreactive sites. A few sites, however, appear to cont ain only one or the other of these two connexins.