The IgCAM CAR regulates gap junction mediated coupling on embryonic cardiomyocytes and affects their beating frequency

Abstract

The IgCAM coxsackie-adenovirus receptor (CAR) is essential for embryonic heart development and for electrical conduction in the mature heart. However, it is not well-understood how CAR exerts these effects at the cellular level. To address this question, we analysed the spontaneous beating of cultured embryonic hearts and cardiomyocytes from wildtype and CAR knockout (KO) embryos. Surprisingly, in the absence of CAR, cultured cardiomyocytes showed an increased frequency of beating and calcium cycling. Increased beating of heart organ cultures was also induced by application of reagents that bind to the extracellular region of CAR, such as the adenovirus fiber knob. However, the calcium cycling machinery, including calcium extrusion via SERCA2 and NCX, was not disrupted in CAR KO cells. In contrast, CAR KO cardiomyocytes displayed an increase in the size, but decrease in total number, of membrane-localized Cx43 clusters. This was accompanied by improved cell-cell coupling between CAR KO cells, as demonstrated by increased intercellular dye diffusion. Our data indicate that CAR may modulate the localization and oligomerization of Cx43 at the plasma membrane, which could in turn influence electrical propagation between cardiomyocytes via gap junctions. Simple summaryCAR is implicated in higher-order gap junction structure by regulating clustering of connexins in embryonic cardiomyocytes. In the absence of CAR, cardiomyocytes in culture showed an increased frequency of beating and calcium cycling. Perturbation experiments suggest that CAR could be a potential new candidate in cardiac diseases.