Molecular characterization of adult cardiac stem cells during differentiation towards a pacemaker phenotype

Several cardiac diseases cause disturbances in rhythm propagation, requiring a pharmacological treatment, and/or the implantation of electronic pacemakers. An alternative approach to electronic devices is the “biological” pacemaker. Possible biological pacemakers consist of cells with pacemaker-like properties, able to pace spontaneously and rhythmically. This work aims to characterize a cellular substrate derived from adult murine stem cells. Self-renewing clones of mesoangioblasts (MABS), obtained from ventricle biopsies, were analyzed for the presence of common stem cell markers. Flow cytometry experiments indicated that these cells are positive for the CD34, c-kit, Sca-1 and CD44 antigens, and for the endothelial antigen CD31. When grown in differentiating medium (low serum, 2%) about 5 days, a fraction of cells started to contract spontaneously. Electrophysiological analysis showed that these cells are able to fire action potentials spontaneously and express the pacemaker current with characteristics similar to the native If (Half-activation at -72.5±2.07 mV, n=16). Immunofluorescence was used to investigate the expression of HCN channels, the molecular components of native f-channels. HCN4 was the most expressed isoform; a signal for HCN3 could be detected in a few cells, while the HCN1 and HCN2 isoforms were not detected. As expected, only cells presenting an organized cytoskeleton showed some degree of HCN labeling. Moreover, cells with an organized sarcomeric structure expressed the connexin 43 protein, one of the major components of atrial gap junctions, in vicinity of contacts between cells. In conclusion our data show that MABS could be a potential substrate for the development of a biological pacemaker, although their properties must be further investigated.