Mechanism of spontaneous excitability in human embryonic stem cell derived cardiomyocytes.

摘要

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 microm) dramatically slowed conduction velocity from 5.1 to 3.2 cm s(-1) while 100 microm TTX caused complete cessation of spontaneous electrical activity in all EBs studied. In contrast, the Ca2+channel blockers nifedipine or diltiazem (1 microm) 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 microm TTX induced longer AP interpulse intervals and 10 microm 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/dtmax) of 118 +/- 14 V s(-1) in control conditions; in contrast, partial block of Na+ current significantly reduced stimulated dV/dtmax (38 +/- 15 V s(-1)). RT-PCR revealed NaV1.5, CaV1.2, 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 NaV1.5; thus, the NaV1.5 Na+ channel is important for initiating spontaneous excitability in hES-derived heart cells.