Connexin45 contributes to global cardiovascular development by establishing myocardial impulse propagation.

Among gap junction-encoding genes, the loss of connexin (Cx) 45 most profoundly obstructs embryogenesis through an endocardial cushion defect and conduction block. However, the interdependence of these defects is not known, and the details of conduction block have not been elucidated. Here, we examined mouse embryos with a region-specific deletion of Cx45 in the myocardium (CA-Cre; Cx45(flox/flox)) or endothelium (Tie2-Cre; Cx45(flox/flox)). Although the deletion of Cx45 in the myocardium was heterogeneous, the CA-Cre; Cx45(flox/flox) embryos were lethal at the same stage as the constitutive Cx45-deficient (Cx45(-/-)) embryos. We determined the onset and patterns of their conduction block through point-tracking in video recordings of embryonic heart contractions. An incomplete conduction block at the atrioventricular canal appeared at embryonic day (E) 8.5 and was predominant around the lethal E9.5 stage in both the Cx45(-/-) and CA-Cre; Cx45(flox/flox) embryos. Although the Cx45(-/-) hearts showed a consistently severe reduction in atrioventricular conduction velocity, the CA-Cre; Cx45(flox/flox) hearts had delay times within the normal range and showed frequent retrograde conduction. As previously reported, the Cx45(-/-) endocardial cushion was consistently defective, and nuclear factor of activated T-cells cytoplasmic (NFATc)1 within the endocardium showed inactive cytoplasmic distribution. In CA-Cre; Cx45(flox/flox), however, the endocardial cushion was partially formed, with active NFATc1 within the endocardium. There was no developmental abnormality in the Tie2-Cre; Cx45(flox/flox) embryos. These results indicate that myocardial dysfunction is responsible for most of the reported defects in Cx45(-/-), which are alleviated by sporadic Cx45 expression in the CA-Cre; Cx45(flox/flox) myocardium.

Conduction abnormality in gap junction protein connexin45-deficient embryonic stem cell-derived cardiac myocytes.

In early-stage heart, the cardiac impulse does not propagate through the specialized conduction system but spreads from myocyte to myocyte. We hypothesized that the gap junction protein connexin45 (Cx45) regulates early-stage contractions, because it is the only gap junction protein described in early hearts. Cx45-deficient (Cx45(-/-)) mice die of heart failure, concomitantly displaying other complex defects in the cardiovascular system. In order to determine the specific cardiac muscular function of Cx45, we created Cx45(-/-) embryonic stem (ES) cells to be differentiated into cardiac muscle in vitro. Unlike the coordinated contractions of wild-type cells, differentiated Cx45(-/-) cardiac myocytes showed high and irregular pulsation rates. Alterations of the electrophysiological properties of the Cx45(-/-) cardiac myocytes were indicated both by extracellular recording on planar multielectrode array probes and by intracellular Ca(2+) recording of the fluorescent Ca(2+) indicator fura-2. The in vitro system minimizes an influence of hemodynamic factors that complicate the phenotypes of Cx45(-/-) mice. Our results indicate that Cx45 is an essential connexin for coordinated conduction through early cardiac myocytes. The Supplementary Material referred to in this article can be found at the Anatomical Record website (http://www.interscience.wiley.com/jpages/0003-276X/suppmat).

Mice lacking connexin45 conditionally in cardiac myocytes display embryonic lethality similar to that of germline knockout mice without endocardial cushion defect.

The gap junction protein connexin45-deficient (Cx45-KO) mice die shortly after the hearts begin to beat. In addition to the heart defect, they also show defective vascular development which may be closely related with the cardiac phenotype. Therefore, we created mice whose floxed-Cx45 locus could be removed conditionally. We utilized cardiac alpha-actin-Cre transgenic mice to investigate the specific cardiac muscular function of Cx45 in vivo. The resultant conditional mutants were lethal, showing conduction block similar to that of the Cx45-KO mice. Unlike Cx45-KO, development of the endocardial cushion was not disrupted in the conditional mutants. X-gal staining was detected in the embryonic cardiac myocytes as a hallmark of Cre-loxP mediated floxed-Cx45 deletion. These results reconfirm the requirement of Cx45 for developing cardiac myocytes. These also indicate that establishing the first contractions is a crucial task for the early hearts.