Characterization of If and HCN distribution in Embryonic Stem Cells (ESC)-derived pacemaker-like myocytes

Recent progress in stem cell research makes it possible to use stem cells in cardiac therapy. The aim of this study was to characterize the pacemaker current (If) and HCN channels distribution in murine embryonic stem cells (ESC) following differentiation towards a pacemaker-like phenotype. Methods: Mouse ES cells (D3 line, ATCC) were induced to differentiate as follows: ~500 cells in 20 µl of differentiation medium were plated on the lid of Petri dishes and cultured as hanging drops for 2 days. The embryoid bodies (EBs) formed were then cultured in suspension for 5 days; 7 day-old EBs were plated on gelatin-coated tissue culture dishes. Spontaneously contracting foci could be observed from day 7+2; beating areas from EBs at day 7+6 to 7+12 were mechanically isolated and enzimatically dissociated for electrophysiological and immunofluorescence investigation. Results: Current-clamp recordings from single cells, obtained from EBs, exhibited spontaneous action potentials (APs) characterized by the presence of the slow diastolic depolarization phase typical of native sinoatrial myocytes. Voltage-clamp recordings showed that a fraction of ES cells-derived cardiomyocytes express a Cs+-sensitive If current with fast activation kinetics while other displayed an If with a significantly slower activation kinetics. The half-activation voltage (V1/2) of the fast activating channels was significantly different than the V1/2 of the slow activating channels (V1/2 fast: -71.2 ± 1.6 n =6; V1/2 slow: -79.1 ± 2.28 n = 4). To further investigate the differences in current kinetics we analyzed the HCN isoform distribution with a video-confocal microscope. We could detect clear signals for both the HCN1 and HCN4, while the HCN2 and HCN3 isoforms were never detected. Conclusion: Our data show that ESC-derived “pacemaker” cells express two If currents with different kinetics; although it is well known that several factors contribute to determine f-current kinetics, the observed differences could be, at least in part, due to the prevalent expression of either HCN1 or HCN4 channels, the most widely expressed isoforms in the sinus node.