Mechanism of spontaneous excitability in human embryonic stem cell derived cardiomyocytes

Human embryonic stem cell-derived cardiomyocytes (hES-CMs) are thought to recapitulate the embryonic development of heart cells. Given the exciting potential of hES-CMs as replacement tissue in diseased hearts, we investigated the pharmacological sensitivity and ionic current of mid-stage hES-CMs (20-35 days post plating). A high-resolution microelectrode array was used to assess conduction in multicellular preparations of hES-CMs in spontaneously contracting embryoid bodies (EBs). TTX (10 μM) dramatically slowed conduction velocity from 5.1 to 3.2 cm s^-1 while 100 μM TTX caused complete cessation of spontaneous electrical activity in all EBs studied. In contrast, the Ca²⁺ channel blockers nifedipine or diltiazem (1 μM) had a negligible effect on conduction. These results suggested a prominent Na⁺ channel current, and therefore we patch-clamped isolated cells to record Na⁺ current and action potentials (APs). We found for isolated hES-CMs a prominent Na⁺ current (244 ± 42 pA pF^-1 at 0 mV; n = 19), and a hyperpolarization-activated current (HCN), but no inward rectifier K⁺ current. In cell clusters, 3 μM TTX induced longer AP interpulse intervals and 10 μM TTX caused cessation of spontaneous APs. In contrast nifedipine (Ca²⁺ channel block) and 2 mM Cs⁺ (HCN complete block) induced shorter AP interpulse intervals. In single cells, APs stimulated by current pulses had a maximum upstroke velocity (dV/dt_max) of 118 ± 14 V s^-1 in control conditions; in contrast, partial block of Na⁺ current significantly reduced stimulated dV/dt_max (38 ± 15 V s^-1). RT-PCR revealed Na_V1.5, Ca_V1.2, and HCN-2 expression but we could not detect Kir2.1. We conclude that hES-CMs at mid-range development express prominent Na⁺ current. The absence of background K⁺ current creates conditions for spontaneous activity that is sensitive to TTX in the same range of partial block of Na_V1.5; thus, the Na_V1.5 Na⁺ channel is important for initiating spontaneous excitability in hES-derived heart cells.