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.

Abstract-The electrical activity in heart is generated in the sinoatrial node and then propagates to the atrial and ventricular tissues. The gap junction channels that couple the myocytes are responsible for this propagation process. The gap junction channels are dodecamers of transmembrane proteins of the connexin (Cx) family. Three members of this family have been demonstrated to be synthesized in the cardiomyocytes: Cx40, Cx43, and Cx45. In addition, each of them has been shown to form channels with unique and specific electrophysiological properties. Understanding the conduction phenomenon requires detailed knowledge of the spatiotemporal expression pattern of these Cxs in heart. The expression patterns of Cx40 and Cx43 have been previously described in the adult heart and during its development. Here we report the expression of Cx45 gene products in mouse heart from the stage of the first contractions (8.5 days postcoitum [dpc]) to the adult stage. The Cx45 gene transcript was demonstrated by reverse transcriptase-polymerase chain reaction experiments to be present in heart at all stages investigated. Between 8.5 and 10.5 dpc it was shown by in situ hybridization to be expressed in low amounts in all cardiac compartments (including the inflow and outflow tracts and the atrioventricular canal) and then to be downregulated from 11 to 12 dpc onward. At subsequent fetal stages, the transcript was weakly detected in the ventricles, with the most distinct expression in the outflow tract. Cx45 protein was demonstrated by immunofluorescence microscopy to be expressed in the myocytes of young embryonic hearts (8.5 to 9.5 dpc). However, beyond 10.5 dpc the protein was no longer detected with this technique in the embryonic, fetal, or neonatal working myocardium, although it could be shown by immunoblotting that the protein was still synthesized in neonatal heart. In the major part of adult heart, Cx45 was undetectable. It was, however, clearly seen in the anterior regions of the interventricular septum and in trace amounts in some small foci dispersed in the ventricular free walls. Cx45 gene is the first Cx gene so far demonstrated to be activated in heart at the stage of the first contractions. The coordination of myocytes during the slow peristaltic contractions that occur at this stage would thus appear to be controlled by the Cx45 channels. (Circ Res. 1999;84:1365-1379.)Key Words: connexin 45 Ⅲ heart Ⅲ development T he gap junctions are clusters of transmembrane channels that mediate direct communication between the cytoplasmic compartments of adjacent cells. These channels, permeable to ions and small molecules (Ͻ900 Da), including second messengers, are the structural components responsible for intercellular electrical and metabolic coupling. The proteins that form these channels are encoded by a multigene family, the connexin (Cx) family. Each Cx forms channels that have unique properties of conductance and permeability. The structure and the oligomerization of Cxs into gap junction channels, and the propertie...