Cardiac myocytes are believed to form an electrically conducting network of cells that is completely isolated from non-myocytes in the heart. Non-myocytes, like fibroblasts, form one of the largest cell populations in the heart. In particular, the healthy mammalian sino-atrial node (SAN) is very rich in fibroblasts (Shiraishi et al. 1992), which are mechanosensitive (Stockbridge et al. 1988) and, if electrically coupled to each other and/or adjacent cardiac myocytes, they would affect origin and spread of cardiac excitation (Kohl et al. 1999). The interaction of cardiac myocytes with other mechanosensitive cells (like fibroblasts), may participate in the cardiac mechanoelectric response that occurs during physiological and pathophysiological mechanical changes (Kohl et al. 1999). Preliminary findings of structural and functional studies suggest the presence of a direct electrical communication between myocytes and fibroblasts in the heart (De Maziere et al. 1992; Kohl et al. 1994).
Our aim was to solve the question of whether there is regular myocyte-fibroblast and/or fibroblast-fibroblast coupling in rabbit SAN.
We studied the distribution of the three main cardiac connexins (Cx43, Cx40 and Cx45) and myocyte-fibroblast coupling in the native SAN tissue, using a combination of immunohistochemical techniques and confocal microscopy. We used a triple labelling approach that provides, in addition to connexin localisation, identification of the coupled cell types (using anti-vimentin antibodies to mark fibroblasts and anti-myomesin antibodies for myocytes).
Our results show that there is gap junctional coupling, not only between myocyte and myocyte, but also between myocyte and fibroblast, and fibroblast and fibroblast. We found that Cx40 is mainly associated with fibroblasts and not myocytes, and that it is involved in fibroblast-fibroblast coupling in the rabbit SAN. Cx45 was expressed by both myocytes and fibroblasts of the SAN and formed junctions coupling either myocytes or fibroblasts, as well as fibroblasts and myocytes. Cx43 was found at the interface between SAN and crista terminalis, where it is longitudinally organized along the atrial myocytes arranged in bundles of atrial muscle coming into the node. Cx43 was absent in the nodal cells and fibroblasts.
We furthermore observed, in the SAN, a correlation between myocyte and fibroblast cells organisation and cell-type connexin expression. We identified two distinct fibroblast populations in the centre of the SAN: fibroblasts expressing Cx40 in fibroblast rich areas devoid of myocytes, and fibroblasts expressing Cx45 in regions of the node where both myocyte and fibroblast cells are intermingled.
In addition, dye transfer studies support the existence of functional coupling between myocytes and fibroblasts.
Our results show that the general assumption that connexins exclusively link cardiomyocytes to each other has to be modified. Here we demonstrated that, in the rabbit SAN: (i) fibroblasts and fibroblasts, as well as fibroblasts and myocytes, are structurally coupled by gap junctional channels, (ii) fibroblasts are coupled by Cx40 or Cx45, (iii) fibroblast-myocyte are coupled by Cx45 and (iv) cardiac fibroblasts can express multiple connexin isoforms in spatially distinct patterns.