High purity human-induced pluripotent stem cell-derived cardiomyocytes: electrophysiological properties of action potentials and ionic currents - PubMed (original) (raw)
High purity human-induced pluripotent stem cell-derived cardiomyocytes: electrophysiological properties of action potentials and ionic currents
Junyi Ma et al. Am J Physiol Heart Circ Physiol. 2011 Nov.
Abstract
Human-induced pluripotent stem cells (hiPSCs) can differentiate into functional cardiomyocytes; however, the electrophysiological properties of hiPSC-derived cardiomyocytes have yet to be fully characterized. We performed detailed electrophysiological characterization of highly pure hiPSC-derived cardiomyocytes. Action potentials (APs) were recorded from spontaneously beating cardiomyocytes using a perforated patch method and had atrial-, nodal-, and ventricular-like properties. Ventricular-like APs were more common and had maximum diastolic potentials close to those of human cardiac myocytes, AP durations were within the range of the normal human electrocardiographic QT interval, and APs showed expected sensitivity to multiple drugs (tetrodotoxin, nifedipine, and E4031). Early afterdepolarizations (EADs) were induced with E4031 and were bradycardia dependent, and EAD peak voltage varied inversely with the EAD take-off potential. Gating properties of seven ionic currents were studied including sodium (I(Na)), L-type calcium (I(Ca)), hyperpolarization-activated pacemaker (I(f)), transient outward potassium (I(to)), inward rectifier potassium (I(K1)), and the rapidly and slowly activating components of delayed rectifier potassium (I(Kr) and I(Ks), respectively) current. The high purity and large cell numbers also enabled automated patch-clamp analysis. We conclude that these hiPSC-derived cardiomyocytes have ionic currents and channel gating properties underlying their APs and EADs that are quantitatively similar to those reported for human cardiac myocytes. These hiPSC-derived cardiomyocytes have the added advantage that they can be used in high-throughput assays, and they have the potential to impact multiple areas of cardiovascular research and therapeutic applications.
Figures
Fig. 1.
Purity and structural organization of cardiomyocytes. A: flow cytometry analysis of cardiomyocytes stained for cardiac troponin-T (cTNT; closed histogram) or isotype control (open histogram). Cell count indicates the number of cells at each fluorescence intensity. Bar represents the troponin-T-positive gate with 99.3% of cells in this example stained for cTNT. B: immunofluorescent analysis of a cardiomyocyte monolayer stained for sarcomeric α-actinin (red) and with anti-nucleic acid stain (blue). Scale bar = 50 μm. Sarcomeric structure can be identified at higher magnification (inset, scale bar = 20 μm).
Fig. 2.
Action potentials (APs) were recorded from human-induced pluripotent stem cells (hiPSC)-derived cardiomyocytes at 35–37°C using the perforated patch-clamp method. See text for details. Interval, beat rate interval; MDP, maximum diastolic potential; Peak, peak voltage; Amp, amplitude; dV/d_t_max, maximal rate of depolarization, APD, AP duration at different levels of repolarization (APD measured at 10% increments of Amp).
Fig. 3.
Effects of ion channel blocking drugs on AP morphology. Data were recorded at 35–37°C with the perforated patch-clamp method. Cardiomyocytes in A and B were stimulated continuously at 1 Hz. A: effects of control extracellular solution after 3-, 6-, 9-, 12-, and 15-min intervals (left) and in different cardiomyocytes exposed to increasing concentrations of tetrodotoxin (TTX; middle) or E4031 (right). B: effects of control extracellular solution after 3 min followed by extracellular solution containing 0.1% DMSO at 6-, 9-, 12-, and 15-min intervals and in different cardiomyocytes exposed to increasing concentrations of nifedipine (middle) and 3R4S-chromanol 293B (right). C: APs were stimulated at 0.5 or 1 Hz in increasing concentrations of E4031 (10, 30, and 100 nmol/l). Pacing at 0.5 Hz in 100 nmol/l E4031 resulted in an early afterdepolarization (EAD; arrow). D: plot of EAD peak voltage vs EAD take-off potential with linear fit to the data. Inset: 2 representative EADs with different take-off potentials and peak voltages.
Fig. 4.
Characteristics of sodium (_I_Na) and L-type and calcium (_I_Ca) currents. A: representative _I_Na traces were elicited by the protocol shown in the inset (left). Peak _I_Na density current density (I-V) plot is shown at middle. Steady-state inactivation and activation relations for INa are shown at right. B: representative _I_Ca traces were elicited by the protocol shown in inset (left). Peak _I_Ca density I-V plot is shown at middle. Steady-state inactivation and activation plots are shown at _right. V_1/2, half-activation voltage; k, slope factor.
Fig. 5.
Characteristics of rapidly and slowly activating components of delayed rectifier potassium (_I_Kr and _I_Ks) currents at 35–37°C. A: representative current traces elicited by the protocol shown in inset. Left, middle, and right traces: control, E4031 (500 nmol/l), and E4031-subtraction, respectively. B: representative current traces elicited by the protocol shown in the inset. Left, middle, and right traces: control, 3R4S-chromanol 293B (10 μmol/l), and 3R4S-chromanol 293B-subtraction, respectively. C: I-V plots of _I_Kr at the end of the depolarizing step (left), peak tail _I_Kr normalized to maximal current following repolarization to −40 mV (middle), and I-V plot of _I_Ks at the end of depolarizing step (right).
Fig. 6.
Characteristics of hyperpolarization-activated pacemaker (_I_f), transient outward potassium (_I_to), and inward rectifier potassium (_I_K1) currents at 35–37°C. A: representative recording of _I_f (left) and the activation relation for peak _I_f (_I_f/_I_f max, right) with the protocol shown in _inset. I_f max was the current measured at −120 mV. B: I-V relation of peak _I_f in the absence and presence of 5 mmol/l Cs+ (left) and in the absence and presence of 100 μmol/l ZD7288 (right). C: averaged current traces of _I_K1 elicited by the voltage-ramp protocol shown in inset recorded before and after 500 μmol/l Ba2+ blockade and the Ba2+-sensitive current normalized to cell capacitance. D: representative I _t_o traces elicited by protocol shown in inset (left) and the I-V plot for peak _I_to normalized to cell capacitance (right).
Fig. 7.
Planar-automated patch clamp. A: representative traces for _I_Na are shown above the I-V plot for peak _I_Na. Inset: concentration-response relation for block by TTX. B: representative traces for _I_Ca are shown above the I-V plot for peak _I_Ca. Inset: concentration-response relation for block by nifedipine. C: representative traces for _I_Kr (arrow) and the voltage protocol are shown during E4031 wash-in (top). Plot of peak tail _I_Kr (middle) shows the rapid development of block with E4031 (arrow). Activation relation for _I_Kr is shown at bottom.
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