Abstract 738: Properties of Embryonic Stem Cell Derived Cardiomyocytes with Altered Levels of T-type Ca Channel Expression
Low voltage-activated, T-type Ca2+ channels are prevalent in embryonic heart, but their expression is transient and disappears in the mature myocardium. Using embryonic stem cell-derived cardiomyocytes (ESdC) as a culture model, it is clear that T-type channels are not necessary for contraction; however, the function of these channels in developing heart is unknown. Since LVA Ca2+ channels have been implicated in cell proliferation and pacemaker activity, we are investigating the effects of varying levels of expression of Cav3.1 (α1G), the main T-type Ca2+ channel found in mouse ESdC. We constructed genetically modified mouse R1-ES cells to either over express or knock out Cav3.1, in order to study its role in cardiac differentiation and the onset of spontaneous contractility. Electrical activity of the genetically modified ESdCs was measured using both patch clamp, and multielectrode arrays (MEAs) for extracellular recording of field potentials (FP) over time in culture. Interspike interval (ISI), FP amplitude, FP duration and conduction velocity were compared over time between the over expressing and knock out lines as parameters of cardiomyocyte maturation, since both ISI and FP duration have been shown to decrease with cardiomyogenesis. Also, calcium imaging was used to study possible contribution of T-type channels on the kinetic properties of calcium transients. Preliminary characterization of the modified ES cell lines showed that in the undifferentiated state, the over expressing line proliferates 3 times faster than the knockout line. Regarding electrical activity, differences between the two lines were observed after day 7+7, when some of the Cav3.1 over expressing ESdC showed arrhythmic activity. Calcium imaging experiments have shown that the duration of the transient (expressed as half-width of the transient) was significantly smaller at day 7+8 in Cav3.1 over expressing vs. knock out lines. The rise time of the calcium transient, which can be influenced by the fast kinetics and low voltage dependence of LVA channels, was increased in the Cav3.1 knock out line starting with day 7+7. Overall, these results suggest a contribution of LVA calcium channels to stem cell proliferation and the kinetic properties of ESdC excitability.