Expression of connexin-26 in mammalian cardiomyocytes

Background. Connexins (Cxs) form membrane channels which play essential role in the propagation of electrical activity throughout the heart. Their dysfunction has been linked to congenital malformation of the heart and to a wide variety of cardiac pathologies. Over twenty isoforms of Cxs have been recognized in mammalian and they are classified according to their molecular weight. To date, seven isoforms, namely Cx30.2, Cx37, Cx40, Cx43, Cx45, Cx46 and Cx57, have been found expressed in cardiac myocites. Here, preferentially located at the intercalated disks, Cxs form gap junctions allowing the intercellular exchange of 1-1.8 kDa molecules as electrical signals, small metabolites and second messengers. However, in other tissues, has been demonstrated that Cxs may also organize in connexons free at the plasma membrane, coupling intracellular and extracellular compartments or may be involved in the modulation of cell proliferation and tumor progression, interacting with intracellular protein as oncogene products, protein kinases and cytoskeleton elements. Cx26 is present in a number of tissues, and its mutations are frequently associated with deafness and skin diseases. Altered expression of Cx26 at level of colonic smooth muscle cells may predispose to the formation of diverticular lesions. The reduced expression of Cx26 may contribute to low sensitivity of hepatocellular carcinoma to chemotherapeutic agent oxaliplatin. Purpose. The aim of this study was to demonstrate the presence of Cx26 expression also in human, pig, rat and mouse cardiomyocites. Methods. Paraffin embedded sections of human auricle, pig ventricle, mouse whole heart and H9c2 cardiac rat cell line were used. Several methods were employed to test the expression of Cx26. In particular, different immunocytochemical techniques were performed using two different primary antibodies, one of which raised against a peptide near the C-terminus and the other against a segment of cytoplasmic loop of Cx26. The isotype specificity of the antibodies was verified both by western blot and by immunoperoxidase on control tissues. Real-Time PCR was used to reveal the expression of Cx26-mRNA in H9c2 cells. Results. The presence of Cx26 has been revealed in all mammalian cardiac tissues analyzed by both the two primary antibodies used. In particular, immunoperoxidase positivity was revealed in both working and specific cardiomyocytes of ventricle and atrium. Interestingly, this Cx has been found localized scattered throughout the cytoplasm of the cells but it was not revealed at level of intercalar disks where the other cardiac Cxs are mainly located. Indeed, immunofluorescence double staining using both Cx26 and Cx43 specific antibodies, showed different localization of these Cxs, in the cytoplasm and in the intercalar disks respectively. Cx26 protein expression was also demonstrated in the cytoplasm of H9c2 rat cardiac myoblasts by immunocytochemistry confirming the data obtained on Cx26-mRNA expression by RT-PCR in this cells. Conclusion. In our knowledge, this is the first study demonstrating the presence of Cx26 in mammalian cardiomyocites. Cx26 cell localization could evoke a function for this protein in signal transduction rather than in the formation of gap junctions. Other studies are in progress to test the role of this connexin in the heart and its possible involvement in cardiac disease.