Establishment of genetically modified biological pacemaker cell based on the in-silico model

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Since the characteristics and the lineage of ES-derived cardiac pacemaker cells are still unknown, we established in vitro culture system to differentiate mouse ES cells into cardiac cells via formation of embryoid bodies(EB) and to purify Nkx2.5(+) cell lineages, using green fluorescence protein(GFP) as a reporter and studied the changes in electrophysiological characteristics of the ES-derived cardiac cells with automaticity. The ES cells differentiated into cardiac cells within 6 to 8 days after formation of EB. Cardiac myocytes in embryoid bodies immunohistochemically expressed the sarcomeric tropomyosine, of which levels depends on their frequency of pulsation. The frequency of their pulsation was influenced by isoproterenol, carbachol, adenosine, lidocaine, nifedipine, Ni^<2+>,Ba^<2+>,Cs^+ and E-4031, indicating the expression of various cardiac-specific receptors (β1-, M2-, A1-receptors) as well as ion channels (Na^+ channels, T-type, L-type Ca^<2+> channels, I_<Kl> channels I_f … More channels, and I_<kr> channels) pharmacologically. Nkx2.5/GFP(+) cardiomyocytes purified from EB expressing cardiac-specific contractile proteins and receptors represented electrophysiological phenotypes corresponding to the pacemaker-cell, atrial or ventricular types cells of the heart. There were significant positive correlations between V_<max>, maximum diastolic potential and V_<th> of the ES-derived cardiac cells with automaticity, suggesting the ES-derived cardiac cells with automaticity were composed of Na^+ channel-dependent and Ca^<2+>-channel-dependent cells, although their automaticity was mediated by expression of Na^+ channels, T-type, and L-type Ca^<2+> channels, I_<kl>, channels I_f channels, and I_<kr> channels, pharmacologically. Furthermore, we found that the response of their automaticity to Ni^<2+> was significantly attenuated in a differentiation time-dependent manner with their constant sensitivities to Cs^+. These results suggested that T-type Ca^<2+> channels might play the pivotal role for the pacemaker activities in ES-derived pacemaker cells in an early stage of their differentiation, while I_f channels persistently attributing to the automaticity during entire differentiation period. In conclusion, 1)Nkx2.5(+) lineage cells with automaticity possess the potential to differentiate into various pacemaker cell types, 2)the differences of T-type Ca channel and I_f channel in ES-derived cardiac cells with automaticity might be the key for collecting the pacemaker cells. Less