Mechanism of spontaneous excitability in human embryonic stem cell derived cardiomyocytes

Authors

  • Jonathan Satin,

    1. Department of Physiology, University of Kentucky College of Medicine, Lexington, KY 40536-0298, USA
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  • Izhak Kehat,

    1. 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
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  • Oren Caspi,

    1. 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
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  • Irit Huber,

    1. 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
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  • Gil Arbel,

    1. 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
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  • Ilanit Itzhaki,

    1. 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
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  • Janos Magyar,

    1. Department of Physiology, University of Kentucky College of Medicine, Lexington, KY 40536-0298, USA
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  • Elizabeth A. Schroder,

    1. Department of Physiology, University of Kentucky College of Medicine, Lexington, KY 40536-0298, USA
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  • Ido Perlman,

    1. 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
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  • Lior Gepstein

    1. 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
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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@tx.technion.ac.il

Abstract

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.

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