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 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.
Satin
Jonathan
J
Department of Physiology, University of Kentucky College of Medicine, Lexington, KY 40536-0298, USA.
Kehat
Izhak
I
Caspi
Oren
O
Huber
Irit
I
Arbel
Gil
G
Itzhaki
Ilanit
I
Magyar
Janos
J
Schroder
Elizabeth A
EA
Perlman
Ido
I
Gepstein
Lior
L
D000200:Action Potentials / Q000187:drug effects / Q000502:physiology*; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D004622:Embryo, Mammalian; D006801:Humans; D007473:Ion Channels / Q000037:antagonists & inhibitors / Q000502:physiology; D032383:Myocytes, Cardiac / Q000187:drug effects / Q000502:physiology*; D009543:Nifedipine / Q000494:pharmacology; D013234:Stem Cells / Q000187:drug effects / Q000502:physiology*; D013779:Tetrodotoxin / Q000494:pharmacology
D007473:Ion Channels; D013779:Tetrodotoxin; D009543:Nifedipine