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1 Department of Physiology, University of Kentucky College of Medicine, Lexington, KY 40536-0298, USA
2 Department of Physiology and Biophysics, The Bruce Rappaport Faculty of Medicine and the Rappaport Family Institute for Research in the Medical Sciences. Technion-Israel Institute of Technology, Haifa, Israel
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/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.
(Received 18 May 2004;
accepted after revision 2 July 2004;
first published online 8 July 2004)
Corresponding author L. Gepstein: Cardiovascular Research Laboratory, The Bruce Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, 2 Efron Street, P.O.B. 9649, 31096 Haifa, Israel. Email: mdlior{at}tx.technion.ac.il
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