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Connexin (Cx)40 and Cx45 are the major protein subunits of gap junction channels in the conduction system of mammals. To determine the role of Cx40, we correlated cardiac activation with Connexin distribution in normal and Cx40-deficient mice hearts. Epicardial and septal activation was recorded in Langendorff-perfused adult mice hearts with a 247-point compound electrode (interelectrode distance, 0.3 mm). After electrophysiological measurements, hearts were prepared for immunohistochemistry and histology to determine Connexin distribution and fibrosis. In both wild-type and Cx40-deficient animals, epicardial activation patterns were similar. The right and left ventricular septum was invariably activated from base to apex. Histology revealed a continuity of myocytes from the common bundle to the septal myocardium. Within this continuity, colocalization was found of Cx43 and Cx45 but not of Cx40 and Cx43. Both animals showed similar His-bundle activation. In Cx40-deficient mice, the proximal bundle branches expressed Cx45 only. The absence of Cx40 in the proximal bundles correlated with right bundle-branch block. Conduction in the left bundle branch was impaired as compared with wild-type animals. Our data show that (1) in mice, a continuity exists between the common bundle and the septum, and (2) Cx40 deficiency results in right bundle-branch block and impaired left bundle-branch conduction

Cardiac malformation in connexin43 (CX43)-disrupted mice is restricted to the junction between right ventricle and outflow tract, even though CX43 is also expressed abundantly elsewhere. We analyzed cardiac morphogenesis in immunohistochemically and hybridohistochemically stained and three-dimensionally reconstructed serial sections of CX43-deficient embryos between embryonic day (ED) 10 and birth. The establishment of the D configuration in the ascending loop of CX43-deficient hearts is markedly retarded, so that the right ventricle retains a craniomedial position and is connected with the outflow tract by a more acute bend in ED10 and ED11 embryos. Because of the subsequent growth of the right ventricle, this condition usually evolves into a D loop, but when it persists, a "crisscross" configuration develops, with the atrioventricular cushions rotated 90 degrees, a horizontal muscular ventricular septum, and a parallel course of the endocardial ridges of the outflow tract. After ED12, large intertrabecular pouches develop at the ventricular side of both shelflike myocardial structures that support the endocardial ridges of the outflow tract, ie, at the location that was earlier characterized by the acute bend between the right ventricle and the outflow tract and that subsequently develops into the anterosuperior leaflet of the tricuspid valve. Retarded development of the D configuration in the ascending loop of the embryonic heart predisposes the myocardium at the junction of the right ventricle and outflow tract to excessive development of intertrabecular pouches during subsequent development

We investigated the expression pattern of gap junctional proteins (connexins, Cx) in situ and in vitro and their functional characteristics in cultured human umbilical vein endothelial cells (HUVEC) and cultured human umbilical artery endothelial cells (HUAEC). In both arteries and veins, Cx37, Cx40, and Cx43 could be detected in situ and in vitro (passages 2-4). Distribution patterns of Cx40 and Cx43 were homogeneous in situ but more heterogeneous in vitro. Cx37 is heterogeneously expressed both in situ and in vitro. Among most cells, no Cx37 staining could be detected; when present, it was found as bright spots between some clusters of cells. Cx40 was more abundant in cultured arterial endothelium than in cultured venous endothelium. Dye-coupling experiments with Lucifer yellow CH revealed extensive dye spread in HUVEC (15.2 +/- 0.4, mean +/- SE, n = 110) but was significantly restricted in HUAEC (9.8 +/- 0.3, n = 110). Electrophysiological gap junctional characteristics were determined in cultured HUVEC and HUAEC pairs by use of the dual voltage-clamp technique. In contrast to the dye-coupling experiments, mean macroscopic electrical conductance was significantly larger for HUAEC pairs (31.4 +/- 6.0 nS, n = 12) than for HUVEC pairs (16.6 +/- 2.8, n = 18). In HUVEC, we measured multiple single gap junctional channel conductances in the range of 19-75 pS. Interestingly, additional conductances of 80-200 pS were measured in HUAEC, possibly partially reflecting activity of channels formed of Cx40, which are more abundant in the cultured arterial endothelial cells

OBJECTIVES: Gap junctions have been demonstrated ultrastructurally in cardiac regions where connexin40 (Cx40) and connexin43 (Cx43) protein could not be detected immunohistochemically. We investigated therefore the distribution of their mRNAs with more sensitive techniques. METHODS: In situ hybridizations with Cx40 and Cx43 cRNA probes were performed on sections of rat hearts from 9 embryonic days (ED 9) to adults. RESULTS: From ED 13, Cx40 and Cx43 mRNA are detectable in atria and ventricles, but not in their flanking myocardium (inflow tract, atrioventricular canal and outflow tract). Even though Cx40 and Cx43 mRNA eventually become expressed in the inflow tract, they remain undetectable in the sinoatrial node, the atrioventricular canal (including atrioventricular node) and outflow tract. Expression of Cx40 is maximal in the fetal period and declines towards birth. Cx40 expression in the left and right ventricles evolves independently, its mRNA disappearing 4 days earlier from the right than from the left ventricle, and earlier from the free wall than from the trabeculations. Expression of Cx43 mRNA increases during development and changes postnatally from uniform to punctate. Prenatally, Cx43 mRNA was strongest in the subepicardial layer of the ventricular free wall. Nevertheless, we did not detect protein in this layer. CONCLUSIONS: Cardiac regions without detectable Cx40 or Cx43 mRNA either have extremely low levels of expression or express a different connexin. The temporally separate disappearance of Cx40 mRNA from the fetal ventricles implies that left and right ventricles mature independently with respect to gap-junctional communication. The division of the developing heart in compartments where Cx40 and Cx43 mRNA can and cannot be detected, implies pretranslationally regulated gene expression. The postnatally observed subcellular redistribution of Cx43 mRNA coincides with a reported increase in protein expression