The synchronized contraction of myocytes in cardiac muscle requires the structural and functional integrity of the gap junctions present between these cells. Gap junctions are clusters of intercellular channels formed by transmembrane proteins of the connexin (Cx) family. Products of several Cx genes have been identified in the mammalian heart (eg, Cx45, Cx43, Cx40, and Cx37), and their expression was shown to be regulated during the development of the myocardium. Cx43, Cx40, and Cx45 are components of myocyte gap junctions, and it has also been demonstrated that Cx40 was expressed in the endothelial cells of the blood vessels. The aim of the present work was to investigate the expression and regulation of Cx40, Cx43, and Cx37 during the early stages of mouse heart maturation, between 8.5 days post coitum (dpc), when the first rhythmic contractions appear, and 14.5 dpc, when the four-chambered heart is almost completed. At 8.5 dpc, only the reverse-transcriptase polymerase chain reaction technique has allowed identification of Cx43, Cx40, and Cx37 gene transcripts in mouse heart, suggesting a very low activity level of these genes. From 9.5 dpc, all three transcripts became detectable in whole-mount in situ-hybridized embryos, and the most obvious result was the labeling of the vascular system with Cx40 and Cx37 anti-sense riboprobes. Cx40 and Cx37 gene products (transcript and/or protein) were demonstrated to be expressed in the vascular endothelial cells at all stages examined. By contrast, only Cx37 gene products were found in the endothelial cells of the endocardium. In heart, Cx37 was expressed exclusively in these cells, which rules out any direct involvement of this Cx in the propagation of electrical activity between myocytes and the synchronization of contractions. Between 9.5 and 11.5 dpc, Cx40 gene activation in myocytes was demonstrated to proceed according to a caudorostral gradient involving first the primitive atrium and the common ventricular chamber (9.5 dpc) and then the right ventricle (11.5 dpc). During this period of heart morphogenesis, there is clearly a temporary and asymmetrical regionalization of the Cx40 gene expression that is superimposed on the functional regionalization. In addition, comparison of Cx40 and Cx43 distribution at the above developmental stages has shown that these Cxs have overlapping (left ventricle) or complementary (atrial tissue and right ventricle) expression patterns.

In adult mouse heart, CX40 is expressed in the atria and the proximal part of the ventricular conduction system (the His bundle and the upper parts of the bundle branches). This cardiac tissue is specialized in the conduction of the electrical impulse. CX40 is the only mouse connexin known to be expressed in these parts of the adult conductive tissue and is thus considered as a marker of the conduction system. In the present report, we investigated CX40 expression and distribution during mouse heart development. We first demonstrate that CX40 mRNA is regulated throughout development, as are other heart connexin transcripts, i.e., CX37, CX43, and (2x45, with a decreasing abundance as development proceeds. We also show that the CX40 transcript and protein are similarly regulated, CX40 being expressed as two different phosphorylated and un-phosphorylated forms of 41 and 40 kDa, respectively. Surprisingly, distribution studies demonstrated that CX40 is widely expressed in 11 days post-coitum (dpc) embryonic heart, where it is detected in both the atria and ventricle primordia. As development proceeds, the CX40 distribution pattern in the atria is maintained, whereas a more dynamic pattern is observed in the ventricles. From 14 dpc onwards, as the adult ventricular conduction system differentiates, CX40 decreases in the trabecular network and it is preferentially distributed in the ventricular conduction system. CX40 is thus the marker of the early differentiating conduction system. It is hypothesized that the conduction system is present in unorganized "embryonic" form at 11 dpc and trans-differentiates by 14 dpc into the adult conduction system. 0 1995 Wiley-Liss, Inc.

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