Biophysical properties of slow potassium channels in human embryonic stem cell derived cardiomyocytes implicate subunit stoichiometry

Abstract

Non-technical summary

The human heart is a pump that works only when its internal electrical system coordinates both its filling and its capacity to eject blood. This critical electrical timing is coordinated by many different ion channels, and this study looks at the one known as I_Ks. Mutations in its α subunit, KCNQ1, constitute the majority of cases of the disorder long QT syndrome (LQT-1). Here we have examined properties of human cardiac cells during very early stages of development and found evidence for the manner in which the subunits of I_Ks assemble; our data suggest that this assembly may be flexible and may change during development and/or disease.

Abstract

Human embryonic stem cells (hESCs) are an important cellular model for studying ion channel function in the context of a human cardiac cell and will provide a wealth of information about both heritable arrhythmias and acquired electrophysiological disorders. However, detailed electrophysiological characterization of the important cardiac ion channels has been so far overlooked. Because mutations in the gene for the I_Ksα subunit, KCNQ1, constitute the majority of long QT syndrome (LQT-1) cases, we have carried out a detailed biophysical analysis of this channel expressed in hESCs to establish baseline I_Ks channel biophysical properties in cardiac myocytes derived from hESCs (hESC-CMs). I_Ks channels are heteromultimeric proteins consisting of four identical α-subunits (KCNQ1) assembled with auxiliary β-subunits (KCNE1). We found that the half-maximal I_Ks activation voltage in hESC-CMs and in myocytes derived from human induced pluripotent stems cells (hiPSC-CMs) falls between that of KCNQ1 channels expressed alone and with full complement of KCNE1, the major KCNE subunit expressed in hESC-CMs as shown by qPCR analysis. Overexpression of KCNE1 by transfection of hESC-CMs markedly shifted and slowed native I_Ks activation implying assembly of additional KCNE1 subunits with endogenous channels. Our results in hESC-CMs, which indicate an I_Ks subunit stoichiometry that can be altered by variable KCNE1 expression, suggest the possibility for variable I_Ks function in the developing heart, in different tissues in the heart, and in disease. This establishes a new baseline for I_Ks channel properties in myocytes derived from pluripotent stem cells and will guide future studies in patient-specific hiPSCs.

Introduction

I_Ks channels are major regulators of cardiac electrical activity (Kass & Wiegers, 1982; Terrenoire et al. 2005) and are formed by heteromultimers each containing four identical α-subunits (KCNQ1) assembled with auxiliary β-subunits (KCNE1). Co-assembly of KCNQ1 and KCNE1 confers distinct biophysical properties on the channels: a slowing of activation and deactivation kinetics and a large depolarizing shift in activation, critically determining the timing of their role during repolarization of the cardiac action potential (Barhanin et al. 1996; Sanguinetti et al. 1996; Schwake et al. 2003). Mutations in the genes for KCNQ1 and KCNE1 have been associated with long QT syndrome (LQTS) and familial atrial fibrillation (FAF) (Tsai et al. 2008; Lu & Kass, 2010). Human induced pluripotent stem cells (hiPSCs) were developed in addition to human embryonic stem cells (hESCs) in order to investigate the mechanistic basis of these inherited disorders and to develop patient-specific treatments (Yoshida & Yamanaka, 2010). A first step in these studies consists in determining the basic cellular electrophysiological properties of hESC derived cardiomyocytes (hESC-CMs).

The slowly activating E-4031 resistant I_Ks channel is a critical determinant of cellular cardiac electrical activity and mutations of either of its two principal subunits, KCNQ1 (α) or KCNE1 (β), underlie heritable arrhythmias. While mRNA for I_Ks channel subunits has been detected in hESC-CMs (Fu et al. 2010; Otsuji et al. 2010), the functional properties of endogenous I_Ks channels have not been characterized. Here we report the biophysical characterization of genotype-negative I_Ks channels in single hESC-CMs and hiPSC derived cardiomyocytes (hiPSC-CMs) using whole cell patch clamp recordings. The I_Ks channels in the hESC-CMs differ in their voltage-dependent properties from KCNQ1/KCNE1 channels in established heterologous systems including mammalian cell lines (Wang et al. 1998; Marx et al. 2002; Imredy et al. 2008; Chung et al. 2009) and are remarkably similar to those of I_Ks channels recorded in hiPSC-CMs. In order to determine whether hESC-CMs’ background affects I_Ks biophysical properties, we engineered charybdotoxin-sensitive KCNQ1 channels (CTX-KCNQ1) (Chen et al. 2003; Morin & Kobertz, 2008) providing a means to separate expressed from endogenous current. CTX-KCNQ1 was transiently expressed in hESC-CMs in the presence or absence of KCNE1. In this manner we show that the mid-point of activation of endogenous I_Ks in hESC-CMs lies between KCNQ1 alone and KCNQ1/KCNE1 heteromultimers. qPCR experiments show that KCNE1 is the major KCNE subunit expressed in hESC-CMs and transfection of KCNE1 alone in hESC-CMs modifies endogenous I_Ks channels so as to match the expressed KCNQ1/KCNE1 channels. Our results suggest that in hESC-CMs, endogenous I_Ks is produced by KCNQ1 subunits assembled with KCNE1 subunits, but the subunit stoichiometry is such that it permits assembly with additional KCNE1 subunits. These data thus suggest variable α and β subunit stoichiometry in hESC-CM I_Ks channels that may be modulated further with changes in KCNE1 expression that occur in the developing heart or in disease.

Methods

Human fibroblast reprogramming and characterization of iPSC

The subjects who contributed to this study gave informed written consent and the experiments were conducted in accordance with the Declaration of Helsinki.

Dermal fibroblasts were obtained from two healthy volunteers without mutations in the major LQT-related genes (KCNQ1, KCNH2, SCN5A, KCNE1 and KCNE2) by punch biopsy. Reprogramming was performed with the hSTEMCCA-loxP lentiviral vector, encoding the four human factors, OCT4, KLF4, SOX2 and cMYC, as previously described (Somers et al. 2010). Briefly, 1 × 10⁵ dermal fibroblasts were plated in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS) on a gelatin-coated 35 mm plastic tissue culture dish. The next day polybrene was added to the medium (5 μg ml⁻¹) and the cells were infected with hSTEMCCA-loxP lentiviruses at a multiplicity of infection (MOI) of 1. Candidate iPSC colonies were mechanically isolated and expanded on MEF feeder cells prior to characterization by immunostaining to confirm expression of markers SSEA4, TRA1/18 and TRA1/60. Normal karyotype was confirmed by G-banding analysis. Teratoma assay was used to confirm capacity to undergo differentiation into derivatives of the three primary germ layers.

hESCs and hiPSCs maintenance and directed cardiac differentiation

hESC line hES2 from ES Cell International (Singapore) was maintained on mouse embryonic fibroblasts as previously described (Kennedy et al. 2007). For both hES2 and hiPS cells, cardiac differentiation was induced by systematically staging, in vitro, the events that occur in a developing embryo, by exposing the hESCs and hiPSCs to cytokines using a previously reported procedure (Yang et al. 2008). In the present experiments, cell differentiation was, however, not followed by cell sorting to avoid the associated shear stress.

Cardiomyocyte dissociation

Beating embryoid bodies at 25–45 days post-differentiation were dissociated for 25–30 min at 37°C in the presence of collagenase II (200 units ml⁻¹, Invitrogen) and protease XIV (0.2 units ml⁻¹, Sigma-Aldrich) in a solution containing (in mmol l⁻¹): 120 NaCl, 5.4 KCl, 5 MgSO₄, 5 sodium pyruvate, 20 glucose, 20 taurine, 10 Hepes, pH 6.9. Isolated cells were then incubated at room temperature for 30 min in Kraft–Brühe (KB) solution containing (in mmol l⁻¹): 85 KCl, 30 K₂HPO₄, 5 MgSO4, 1 EGTA, 2 Na₂ATP, 5 sodium pyruvate, 5 creatine, 20 taurine, 20 glucose, pH 7.2. Cells were finally resuspended in DMEM supplemented with 10% FBS, 1 mmol l⁻¹ sodium pyruvate and 2 mmol l⁻¹ l-glutamine, and plated on 0.1% gelatin-coated 35 mm plastic Petri dishes. Isolated cardiomyocytes were identified the next day by spontaneous contraction at ∼37°C; individually marked with an object marker (Nikon); and used for recordings of endogenous I_Ks within 48 h after plating.

Heterologous expression

Recombinant I_Ks channel subunits were transiently expressed in human embryonic kidney (HEK) 293 cells and in hESC-CMs using Lipofectamine-PLUS Reagents (Lipofectamine for HEK293 cells or Lipofectamine LTX for hESC-CMs) (Invitrogen). All the genes of interest were subcloned into pcDNA3.1 vector (Invitrogen). Depending on experiments, the following DNAs were transfected: wild-type (WT) KCNQ1, KCNQ1_KCNE1 tandem multimer (Wang et al. 1998), charybdotoxin-sensitive KCNQ1 (CTX-KCNQ1; Chen et al. 2003; a kind gift from Dr William R. Kobertz University of Massachusetts Medical School, Worcester), KCNE1 (KCNQ1 and KCNE1 cDNAs were co-transfected at 1:1 ratio), KCNE4 (a kind gift from Dr Al George Jr, Department of Pharmacology, Vanderbilt University, Nashville). For transfection in hESC-CMs, cells were plated at the density of 10⁴ per 35 mm Petri dish. Five hours later 0.2 μg of DNA(s) of interest was transfected according to the manufacturer's protocol overnight in the culture medium. eGFP was co-transfected with channels subunits to identify transfected cells. About 50% transfection efficiency assessed by the eGFP expression was achieved in hESC-CMs within 24 h after transfection. For both HEK293 cells and transfected hESC-CMs, patch-clamp recordings were carried out on GFP positive cells 48 h after transfection.

Quantitative real time PCR (qPCR)

Total RNA was isolated from cell culture using the Qiagen Micro RNeasy kit (Qiagen, Valencia, CA, USA) following the manufacturer's protocols. One microgram of RNA was used to generate cDNA using the SuperScript III kit (Invitrogen). Preparations of cDNA were free of genomic contamination as verified by performing a reverse-transcription step without the SuperScript III enzyme and subsequent qPCR. qPCR was performed on the LightCycler 480 Real-Time PCR System using the SYBR Green I Master (Roche) and the following amplification protocol: 5 min at 95°C, 50 cycles (15 s at 94°C – 10 s at 60°C – 10 s at 72°C). All reactions were carried out in 96-well plates. Samples were amplified in triplicate. Standard curves were generated using sample cDNA in 1:10 dilutions by plotting the cycle numbers of crossing point (cp) versus the common log of the relative concentrations (Supplemental Fig. S2). The corresponding PCR efficiency was calculated from the slope of the standard curve with the formula of efficiency = 10^(-1/slope). Relative quantification was done using the standard curve method with PCR efficiency correction (E-method) of the LightCycler 480 Software (Roche) (Tellmann, 2006). All samples were normalized to GAPDH expression on the same plate. Primers used were: GAPDH F: 5′-TGTTGCCATCAATGACCCCTT-3′, R: 5′-CTCCACGACGTACTCAGCG-3′; KCNQ1 F: 5′-CCCTGAAGGTGCAGCAGAA-3′, R: 5′-GCCTTCCGGATGTAGATCTTCC-3′; KCNE1 F: 5′-TGTGGCAGGAGACAGTTCAG-3′, R: 5′-GCTTCTTGGAGCGGATGTAG-3′; KCNE2 F: 5′-CAGAACAGCCTGGCTTTGGA-3′, R: 5′-TCCAGCGTCTGTGTGAAATTG-3′; KCNE3 F: 5′-ACTGAGAGCCAGTGGATTTGC-3′, R: 5′-AGGTCTCCGTTCCATTGGTAGTC-3′; KCNE4 F: 5′-AACCCTCTTGGACTGGACGAT-3′, R: 5′-AGGCTCCATTTTCAGCATTGA-3′; KCNE5 F:5′-CCCCTACCCCGCACATC-3′, R: 5′-TTGGACGTGTTGGATTCAGTTC-3′. Primers for KCNQ1 and KCNE2-5 have been published previously (Lundquist et al. 2005).

Single cell electrophysiology

Cells were placed on the stage of an inverted microscope (Nikon) and currents were recorded at room temperature (23–25°C) using the whole-cell configuration of the patch-clamp technique carried out with Axopatch 200B amplifiers (Axon Instruments, Union City, CA, USA). Averaged cell capacitance was: 26.5 ± 2.7 pF (n = 28) for HEK293 cells, 45.1 ± 6.8 pF (n = 24) for hESC-CMs and 42.3 ± 6.1 pF (n = 12) for hiPSC-CMs. For voltage clamp experiments, we used a previously described internal recording solution (Terrenoire et al. 2005) and the following external solution (in mmol l⁻¹): 132 NaCl, 4.8 KCl, 2 CaCl₂, 1.2 MgCl₂, 0.05 NiSO₄, 10 Hepes, 5 glucose, 1 μmol l⁻¹ isradipine, pH 7.4. Endogenous I_Ks channels were recorded in the presence of E4031 (5 μmol l⁻¹) in external recording solution in order to block the rapidly activating delayed rectifier I_Kr. Endogenous I_Ks channels were dissected pharmacologically with chromanol 293B (30 μmol l⁻¹) whereas CTX-KCNQ1 channels were isolated with CTX (70 nmol l⁻¹). Control experiments confirmed that at this concentration CTX did not affect endogenous currents (Supplemental Fig. S1). All reagents were purchased from Sigma-Aldrich (St Louis, MO, USA), unless otherwise specified. The voltage protocol and analysis for all voltage clamp experiments were as follows: cells were depolarized from −40 mV to 50 mV (in 10 mV increments) for 2 s and then repolarized to −40 mV for 2 s (holding potential was −40 mV) at a pulse frequency of 0.1 Hz. Maximum tail current amplitudes were measured during this repolarizing pulse and plotted vs. pre-pulse voltage (i.e. test pulse voltage). Activation curves (2 s isochronal), determined from amplitudes of deactivating current tails normalized to maximum tail current plotted vs. test pulse voltage, were fitted with a Boltzman equation to determine channels mid-point of activation (V_1/2). For current clamp experiments, the internal solution was the same as in voltage clamp except that potassium aspartate was replaced by KCl. External solution was (in mmol l⁻¹): 132 NaCl, 4.8 KCl, 2 CaCl₂, 1.2 MgCl₂, 10 Hepes, 5 glucose, pH 7.4. Action potentials (APs) were triggered at 1 Hz by 3 ms suprathreshold stimulations.

Computer simulation

Computational models for KCNQ1 gating (Seebohm et al. 2003) and KCNQ1–KCNE1 gating (Imredy et al. 2008) were implemented, as previously described (Sampson et al. 2010).

Data analysis

Patch clamp data were acquired with pCLAMP 8.2 (Axon Instruments) and analysed with Origin 7.0 (OriginLab, Northampton, MA, USA) and Clampfit 8.2 (Axon Instruments). Data are shown as means ± SEM. Statistical data analyses were assessed with Student's t test for simple comparisons or one-way ANOVA followed by Tukey's test for multiple comparisons: differences at P < 0.05 were considered as statistically significant.

Results

Action potentials in cytokine-induced differentiation of hES2 cells: enrichment in ventricular-like cellular activity

We first sequenced the Long QT genes of the hES2 line and confirmed that KCNQ1 and KCNE1 were free of any known polymorphisms or disease causing mutations. Then we carried out current clamp experiments in single cells to characterize action potentials in single hES2 CMs. Action potentials (APs) were recorded in a total of 26 cells and were classified into three categories as reported by other groups (Mummery et al. 2003; Moore et al. 2008): (i) ventricular-like: most negative diastolic potentials, significant plateau phase and long action potential duration (APD); (ii) atrial-like: APs triangular waveform; diastolic potentials more positive and APD significantly shorter than ventricular like cells; and (iii) nodal-like APs: small amplitude, depolarized diastolic potential, and robust spontaneous activity (Fig. 1A, Table 1). Of the 26 cardiomyocytes we recorded, ∼90% (23 out of 26 cells) exhibited a ventricular phenotype whereas a very small number of cells were atrial-like (2 cells out of 26) and nodal-like (1 cell out of 26) (Fig. 1B). This clearly shows that cytokine-induced differentiation of hES2 cells produces a relatively homogeneous population of ventricular-like cardiomyocytes.

Figure 1. Action potentials phenotypes in cardiomyocytes derived from hES2 cells (hESC-CMs) by cytokine-induced differentiation.

A, in a total of 26 hESC-CMs, 3 different types of action potentials (APs) were recorded: nodal-like (left panel), atrial-like (middle panel, average of 2 cells) and ventricular-like (right panel, average of 23 cells). Atrial-like and ventricular-like APs were elicited by suprathreshold stimuli at 1 Hz. All experiments were carried out at room temperature. B, distribution of cardiomyocytes according to their AP phenotype: out of 26 cells, one cell was nodal-like, 2 cells were atrial-like and 23 cells were ventricular-like.

Table 1.

Action potential properties in cardiomyocytes derived from hES2 cells

	Overshoot (mv)	Amplitude (mV)	APD25	APD (ms) APD50	APD90	MDP (mV)	n (total)
Pacemaker-like	25.2	75.1	36.1	71.3	170.3	−49.9	1
Atrial-like	23.3 ± 2.5	67.9 ± 8.6	34.6 ± 11.1	50.7 ± 14.2	97.3 ± 28.5	−46.9 ± 12.5	2
Ventricular-like	47.3 ± 1.2	102.5 ± 1.7	333.1 ± 18.3	423.9 ± 22.7	513.6 ± 28.4	−62.4 ± 1.2	23

APD_25/50/90, action potential duration at 25%, 50% or 90% of maximal amplitude. MDP, maximum diastolic potential. For atrial-like and ventricular-like cells, parameters were measured on APs stimulated at 1 Hz. Data are expressed as means ± SEM.

Biophysical properties of endogenous I_Ks in hESC-CMs

We used pharmacological dissection to identify and separate two important delayed rectifier K⁺ channels that we detected under whole cell voltage clamp in these cells, I_Kr and I_Ks (Sanguinetti & Jurkiewicz, 1990), and illustrated this approach in Fig. 2. In the maintained presence of chromanol 293B (30 μmol l⁻¹), a known I_Ks blocker (Busch et al. 1996), I_Kr was identified as E4031 (5 μmol l⁻¹)-sensitive time-dependent current (Fig. 2A). Typical I_Kr rectification properties are apparent in these current traces and on the current–voltage relationship determined by current at the end of test pulses. In the maintained presence of E-4031 (5 μmol l⁻¹), I_Ks was identified as chromanol 293B (30 μmol l⁻¹)-sensitive time-dependent current, with kinetics and voltage-dependent activation consistent with I_Ks channels (Fig. 2B). We focused on I_Ks for the remainder of this study and thus included E-4031 (5 μmol l⁻¹) in all external recording solutions that follow to block I_Kr.

Figure 2. Pharmacological dissection of two delayed rectifier K⁺ channels in hESC derived myocytes.

A, left panel, in the presence of chromanol 293B (30 μmol l⁻¹) in the recording solution (control), E4031 (5 μmol l⁻¹) inhibits the rapidly activating current at 30 mV (2 s test pulse from holding potential (HP) of −40 mV) and blocks its corresponding tail current at −40 mV (arrows in left panel). Dashed line indicates zero current. Middle panel, representative E4031-sensitive current traces recorded at different test pulses (2 s test pulses to: −30, 0 and 30 mV; HP −40 mV; 0.1 Hz). Dashed line is zero current. Right panel, current–voltage relationship established by measuring the E4031-sensitive current in the end of 2 s depolarizing test pulses from −40 to 50 mV. B, left panel, in the presence of E4031 (5 μmol l⁻¹) in the recording solution (control), chromanol 293B (30 μmol l⁻¹) inhibits the slowly activating current at 50 mV (2 s test pulse from HP of −40 mV) and blocks its corresponding tail current at −40 mV (arrows in left panel). Dashed line indicates zero current. Middle panel, representative chromanol-sensitive current traces recorded at different test pulses (2 s test pulses to: −40, 0, 20 and 40 mV; HP −40 mV; 0.1 Hz). Dashed line is zero current. Right panel, current–voltage relationship established from the chromanol-sensitive maximum tail current measured during the repolarizing pulse at −40 mV that follows 2 s depolarizing test pulses from −40 to 50 mV. Data are shown as means ± SEM.

Biophysical properties of endogenous hESC-CM I_Ks channels: comparison with expression in hiPSC-CM and transiently transfected HEK 293 cells

Quantitative PCR (qRT-PCR) analysis of hESC-CMs revealed that the principal KCNE variant expressed in these cells is KCNE1. KCNE2, KCNE3 and KCNE5 transcripts were detectable only at very small levels, and KCNE1 transcripts are significantly greater than transcripts for the other potential I_Ks channel subunit, KCNE4 (Supplemental Fig. S2). Furthermore channels of only KCNQ1 and KCNE4 result in no current (Lundquist et al. 2005) and as such would not be observable in the hESC-CMs. We confirm that channels containing both KCNE1 and KCNE4 subunits have identical properties to KCNQ1/KCNE1 channels other than a decrease in amplitude (Grunnet et al. 2002) (Supplemental Fig. S3). Therefore, while KCNE4 may well be in complex with these channels, the lack of an effect on channel function allows us to focus on KCNQ1/KCNE1 channels.

I_Ks channels are tetrameric channels formed by co-assembly of four α (KCNQ1) subunits and auxiliary β subunits (KCNE1) (Barhanin et al. 1996; Sanguinetti et al. 1996) with subunit stoichiometry that has been reported to be variable by some groups (Wang et al. 1998; Nakajo et al. 2010) and fixed by others (Morin & Kobertz, 2008). Biophysical properties of expressed channels can be used to distinguish some aspects of subunit composition of the functioning channels. For example, it is well known that in the absence of KCNE1, KCNQ1 encodes functioning channels with rapid activation kinetics and activation voltages that are relatively negative (Fig. 3A and C) (Barhanin et al. 1996; Sanguinetti et al. 1996), and thus homomeric KCNQ1 channel activity can be distinguished from activity of heteromeric KCNQ1/KCNE1 channels expressed in the same cell line (HEK 293) both by the markedly slower activation kinetics and more positive activation voltages (Fig. 3A and C). When the KCNQ1/KCNE1 stoichiometry is fixed at a 1:1 ratio in a tandem multimer (Fig. 3A and C), both activation kinetics and voltage dependence are virtually identical to independently expressed KCNQ1 and KCNE1 with ratios of KCNE1/KCNQ1 cDNA of 1. In genotype-negative hESC-CMs and hiPSC-CMs, the biophysical properties of I_Ks channels are identical (Fig. 3B and D) and fall between homomeric KCNQ1 and heteromultimeric KCNQ1/KCNE1 channels with a 1:1 stoichiometry (Fig. 3C). As illustrated in Fig. 3D, the mid-point of activation (V_1/2) of endogenous I_Ks in hESC-CMs was 9.1 ± 1.7 mV (n = 11), not significantly different from the one measured in two different hiPSC lines (hiPS no. 1: V_1/2 = 5.1 ± 1.4 mV, n = 4; hiPS no. 2: V_1/2 = 10.3 ± 2.9 mV, n = 8). However, as shown in Fig. 3C, the mid-point of activation of endogenous I_Ks in hESC-CMs was between that of KCNQ1 (V_1/2 = −21.3 ± 1.5 mV, n = 4, P < 0.001) and KCNQ1+KCNE1 channels (V_1/2 = 29.1 ± 1.2 mV, n = 9, P < 0.001) as well as KCNQ1_KCNE1 tandem multimer (V_1/2 = 28.5 ± 2.1 mV, n = 7, N.S. compared to KCNQ1+KCNE1).

Figure 3. Current traces recorded during 2 s test pulse to 40 mV (HP = −40 mV) followed by repolarizing pulse to −40 mV.

A, HEK293 (HEK) cells were transfected with the following recombinant channels: KCNQ1 (left panel, average of 4 cells); KCNQ1+KCNE1 (middle panel, average of 9 cells); KCNQ1_KCNE1 tandem multimer (right panel, average of 7 cells). Dashed line indicates zero current. B, endogenous I_Ks current was recorded in cardiac myocytes derived from hES2 cells (left panel) and from hiPSCs cells (right panel). Dashed line indicates zero current. C, normalized tail currents measured at −40 mV vs. pre-pulse voltage for recombinant channels (KCNQ1, open squares, n = 4; KCNQ1+KCNE1, filled squares, n = 9; KCNQ1_KCNE1 tandem multimer, open triangles, n = 7) expressed in HEK293 cells and endogenous I_Ks channels recorded in cardiac myocytes derived from hES2 cells (filled circles, n = 11). Data are shown as means ± SEM. D, normalized tail currents measured at −40 mV vs. pre-pulse voltage for endogenous I_Ks channels recorded in cardiac cells derived from human induced pluripotent stem (iPS) cells (iPS cells from subject no. 1, open stars, n = 4; iPS cells from subject no. 2, filled stars, n = 8). Dashed line represents activation curve for endogenous I_Ks channels in hES2 cells (n = 11). Data are shown as means ± SEM.

Recombinant I_Ks channels expressed in hESC-CMs do not reconstitute endogenous I_Ks

Next, in order to test whether the hESC-CMs background affects I_Ks biophysical properties, KCNQ1 and KCNE1 were transiently transfected in these cells. To distinguish the transfected channels from the endogenous I_Ks channels, we engineered KCNQ1 channels to impart a sensitivity to CTX. Consistently with a previous report (Chen et al. 2003), we found that CTX-sensitive channels (CTX-KCNQ1) share the same biophysical properties as wild-type (WT) channels when expressed in a heterologous system (in HEK293 cells, CTX-KCNQ1: V_1/2 = −26.5 ± 4.5 mV, n = 4, and CTX-KCNQ1/KCNE1: V_1/2 = 26.8 ± 2.1 mV, n = 4; N.S. compared to corresponding WT channels in HEK293 cells shown in Fig. 3C).

CTX-sensitive channels were then transiently expressed in hESC-CMs with (1:1 ratio) or without KCNE1 and CTX (70 nmol l⁻¹) was used to isolate the recombinant channels from the endogenous I_Ks channels (Fig. 4). As shown in Fig. 4E, robust expression of CTX-sensitive channels is observed in hESC-CMs with tail currents ∼10-fold larger than endogenous I_Ks. Also, the voltage dependence of activation of CTX-sensitive channels was similar to that measured in HEK293 cells (CTX-KCNQ1: V_1/2 = −27.2 ± 4.5 mV, n = 4; CTX-KCNQ1/KCNE1: V_1/2 = 31.8 ± 1.3 mV, n = 5; N.S. compared to corresponding channels in HEK293 cells) (Fig. 4F). These results further indicate that the voltage dependence of activation of endogenous hESC-CMs I_Ks lies between KCNQ1 alone and KCNQ1/KCNE1 heteromultimers in the same cell type. This result suggests that either KCNE1 is expressed at low level in hESC-CMs and/or that other KCNE subunits assemble KCNQ1 therefore changing the properties of the whole I_Ks current.

Figure 4. Biophysical properties of recombinant CTX-sensitive I_Ks channels expressed in hESC-CMs.

A and B, CTX (70 nmol l⁻¹, arrows) inhibits CTX-KCNQ1 (A) and CTX-KCNQ1+KCNE1 (B) currents at 50 mV (2 s test pulse from holding potential of −40 mV) and blocks the corresponding tail currents at −40 mV. Dashed line indicates zero current. C and D, representative CTX-sensitive current traces recorded at different test pulses (2 s test pulses from −40 to 50 mV; HP −40 mV; 0.1 Hz). Dashed line is zero current. Average (n = 5) tail current (E) and normalized tail current (F) measured at −40 mV vs. test pulse voltage for CTX-KCNQ1 (circles) and CTX-KCNQ1+KCNE1 (squares). Dashed line indicates the level of current for endogenous I_Ks. Data are shown as means ± SEM.

Overexpression of recombinant KCNE1 in hESC-CMs shifts V_1/2 of endogenous I_Ks channels

To test whether further KCNE1 could modulate the channel, we over-expressed KCNE1 alone in hESC-CMs and compared the biophysical properties to that of endogenous I_Ks channels. I_Ks in KCNE1-transfected cells was still abolished by 30 μmol l⁻¹ chromanol 293B (Fig. 5A) and activated more slowly (at 10 mV, t_1/2 = 973 ± 32 ms, n = 4) than in non-transfected cells (t_1/2 = 859 ± 23 ms, n = 11, P < 0.05) (Fig. 5B). The mid-point of activation of I_Ks in KCNE1-transfected cells was shifted to more positive voltages (from V_1/2 = 9.1 ± 1.7 mV, n = 11, in non-transfected cells to V_1/2 = 33.6 ± 1.4 mV, n = 4, in KCNE1-transfected cells, P < 0.001) (Fig. 5D) and was not significantly different from CTX-KCNQ1 channels co-expressed with KCNE1 (Fig. 4F). Despite the shift in activation, which would suggest a smaller open probability for the channel at any given voltage, I_Ks maximum tail current density at +50 mV was significantly larger in KCNE1-transfected cells: 1.41 ± 0.36 pA pF⁻¹ (n = 4) vs. 0.71 ± 0.05 pA pF⁻¹ (n = 11, P < 0.001) (Fig. 5C). These characteristics, slowing of activation kinetics, increased tail current and depolarized shift in activation are hallmarks of KCNE1 modulation of KCNQ1, suggesting endogenous I_Ks channels in hESC-CMs can be further regulated by KCNE1.

Figure 5. Biophysical properties of endogenous I_Ks in hESC-CMs transfected with KCNE1.

A, chromanol 293B (30 μmol l⁻¹, arrows) inhibits the slowly activating current at 50 mV (2 s test pulse from holding potential of −40 mV) and blocks its corresponding tail current at −40 mV. Dashed line indicates zero current. B, representative chromanol-sensitive current traces recorded at different test pulses (2 s test pulses to: −40, 0, 30 and 50 mV; HP −40 mV; 0.1 Hz). Dashed line is zero current. Average (n = 4) I_Ks tail current (C) and normalized tail current (D) measured at −40 mV vs. test pulse voltage. Dashed line indicates activation curve for I_Ks in non-transfected hESC-CMs. Data are shown as means ± SEM.

Figure 6 summarizes data and compares measured V_1/2 values for all constructs and cells studied. The figure clearly shows that I_Ks channel activity in hESC-CMs and hiPSC-CMS can be readily distinguished from KCNQ1 homomeric channels and that the activation voltages measured in the hiPSC-CMs and in the hESC-CMs are not significantly different from each other, but in both cases the measured V_1/2 values are significantly different from V_1/2 values obtained from expression of KCNQ1_KCNE1 tandem multimer with a fixed KCNQ1/KCNE1 1:1 stoichiometry and from independent co-expression of KCNQ1 and KCNE1. Importantly hESC-CM transfection with additional KCNE1 subunits rescues V_1/2 of KCNQ1/KCNE1 channels with a 1:1 stoichiometry.

Figure 6. Bar graph summaries comparing the mid-point of activation (V_1/2) of endogenous I_Ks channels in cardiac cells derived from human stem cells (hESC-CMs and hiPSC-CMs, right panel) with those of recombinant channels expressed in HEK293 cells (left panel) and in hESC-CMs (middle panel).

V_1/2 measurements for endogenous I_Ks channels were the same for myocytes differentiated from hESCs and from hiPSCs but significantly different from KCNQ1 alone (P < 0.001) and all other combinations of KCNQ1/KCNE1 (P < 0.001). Left panel, HEK293 cells were transfected with either WT KCNQ1 (open boxes) or CTX-KCNQ1 (hatched boxes) in the absence (white boxes) or presence (grey boxes) of KCNE1. In the latter condition, ratios of KCNE1/KCNQ1 cDNA were 1. ‘KCNQ1_KCNE1’ corresponds to tandem multimer channels in which KCNQ1/KCNE1 stoichiometry is fixed at a 1:1 ratio. Middle panel, V_1/2 measurements of CTX-sensitive recombinant channels transfected in hESC-CMs. Right panel, The mid-point of activation of endogenous I_Ks channels in hESC-CMs and in two different lines of hiPSC-CMs (hiPS no. 1, hiPS no. 2) is similar and falls in between that of homomeric KCNQ1 channels and of heteromeric KCNQ1/KCNE1 channels expressed in HEK293 cells and in hESC-CMs. In hESC-CMs transfected with KCNE1 subunits (‘hES+KCNE1’, black box), V_1/2 measurement for endogenous I_Ks is more positive than in non-transfected cells (P < 0.001) but is not significantly different from that of heteromeric KCNQ1/KCNE1 channels and of tandem multimer channels (‘KCNQ1_KCNE1’) in which KCNQ1/KCNE1 stoichiometry is fixed at a 1:1 ratio.

Discussion

In this study, we report for the first time, a systematic investigation of the biophysical properties of I_Ks channels expressed in hESC-CMs and demonstrate that these cells can be used as a platform to express and study recombinant ion channels. Also, we find that the functional properties of endogenous I_Ks channels expressed in hESC-CMs and in hiPSC-CMs are similar and that they are consistent with KCNQ1/KCNE1 heteromultimers. However, the properties of the expressed channels are consistent with less than maximal KCNE1 to KCNQ1 ratios (stoichiometry). Co-expression of additional KCNE1 shows a marked effect on the function of expressed channels suggesting a pathway for I_Ks channel modulation that might occur in the developing heart or in human disease.

Whole cell patch clamp recordings demonstrate a large, slowly activating current with sensitivity to chromanol 293B, clearly demonstrating the presence of I_Ks channels in hESC-CMs. This finding is consistent with recently published reports in hESC-CMs suggesting the presence of I_Ks (Otsuji et al. 2010) and showing prolongation of APD by the I_Ks blocker chromanol 293B (Peng et al. 2010). Furthermore, we provide the first detailed characterization of the biophysical properties of I_Ks in hESC-CMs. The properties of I_Ks we report in hESC-CMs are very similar to those observed in human adult ventricular myocytes with half-maximal activation voltage of +12 mV (Li et al. 1996). Using over-expression of KCNQ1 and KCNE1, we were able to explore the properties of endogenous I_Ks in relation to recombinant channels. By engineering a CTX sensitivity into KCNQ1, over-expressed channels could be isolated from endogenous channels. Interestingly, we found the endogenous channel activity to be intermediate to KCNQ1 and KCNQ1/KCNE1 both in classic heterologous systems (HEK293) as well as in hESC-CMs. Specifically, we find that endogenous I_Ks channels in hESC-CMs activate at voltages between that of homomeric KCNQ1 channels and heteromeric KCNQ1/KCNE1 channels (Fig. 4). As the sequence of KCNQ1 and KCNE1 were genotyped as normal, genetic variation does not play a role in this discrepancy. Further qPCR shows KCNE1 as the primary KNCE family member in these cells and the presence of small amount of KCNE4 would only adjust the magnitude and not the biophysical properties of the channel. Altogether, these data strongly suggest that KCNE1 is the main KCNE subunit assembling with KCNQ1 in the I_Ks channels we detect in hESC-CMs, and that not all channels are assembled in a 1:1 stoichiometry. This was confirmed by experiments in which we used transient transfection to increase KCNE1 expression in hESC-CMs (Fig. 5). We found that endogenous I_Ks in transfected cells was larger than in non-transfected cells, that the current's onset of activation was slower and that channels mid-point of activation was remarkably shifted to the right and reached that of KCNQ1/KCNE1 heteromultimers (Fig. 6).

Differing studies in the literature have suggested either that I_Ks channels may be composed of KCNQ1/KCNE1 complexes with variable stoichiometries (Wang et al. 1998; Nakajo et al. 2010; Osteen et al. 2010) or have a fixed stoichiometry, with KCNQ1 co-assembling with KCNE1 in a 4:2 stoichiometry (Chen et al. 2003; Morin & Kobertz, 2008). If a fixed stoichiometry is assumed, the mid-point of activation of endogenous I_Ks can only be achieved by a mixed population composed of KCNQ1/KCNE1 channels in a 4:2 stoichiometry combined with KCNQ1 channels alone. To test whether a linear combination of channels with and without KCNE1 can reproduce our experimental findings, computational models were employed. Figure 7 shows the predicted results of KCNQ1 channels alone (Seebohm et al. 2003), KCNQ1+KCNE1 channels (simulating KCNE1 overexpression; Imredy et al. 2008), and a mixed population of the two (at a ratio that reproduces the observed half-maximal activation voltage in endogenous I_Ks). With these assumptions, the predicted response of the mixed population diverges substantially from the behaviour observed experimentally. First, the simulated step current is bi-exponential with a prominent initial fast component that shows no delayed sigmoidal activation. Secondly, the predicted I–V relation is discordant from experiment, with a voltage dependence that is much more graded. Lastly, due to the differences in single channel conductance, the simulations imply nearly 80% of the channels co-assemble without KCNE1. These results favour the alternative hypothesis that endogenous hESC-CM I_Ks is a co-assembled channel at stoichiometry less than 1:1. Our results are also consistent with endogenous I_Ks channels that could be further regulated by alterations in KCNE1 expression either in development toward a specific cardiac ventricular cell type or in disease (Radicke et al. 2006; Watanabe et al. 2007; Soltysinska et al. 2009).

Figure 7. Computer-generated models suggest distinction between monomeric KCNQ1, endogenous I_Ks and I_Ks with KCNE1 overexpression.

A, model-derived current traces for KCNQ1 monomeric channels, a linear combination (∼80%–20%) of KCNQ1 and KCNQ1+KCNE1 channels (simulating endogenous hESC-CMs I_Ks voltage dependence), and KCNQ1+KCNE1 channels alone. B, activation curves taken from maximal open probability for test voltages from −40 to +50 mV show that a population of channels composed of KCNQ1 homomers mixed with KCNQ1/KCNE1 heteromultimers would predict a substantially flatter activation curve.

Most of our current knowledge of the cardiac I_Ks channel complex and its pathological role has been obtained from studies using heterologous expression systems (Charpentier et al. 2010; Lu & Kass, 2010) and genetically altered mice (Marx et al. 2002) and rabbits (Brunner et al. 2008). However, a new cellular model in which the functional consequences of inherited mutations of the channel can be studied in the context of a human cardiac environment is still needed to address many important questions regarding the mechanistic basis of heritable arrhythmia and to explore potential patient-specific treatment. The results of this study demonstrate the fidelity of hESC-CMs as a platform for assaying the biophysical properties of the I_Ks channel's native and mutant gene products in a human CM. Consequently, hESC-CMs may serve as a baseline for studies of heritable arrhythmia caused by mutation in I_Ks associated genes KCNQ1 (LQT-1, FAF), KCNE1 (LQT-5), and AKAP9 (LQT-11).

Glossary

Abbreviations

CTX

charybdotoxin

CTX-KCNQ1

charybdotoxin sensitive KCNQ1 channel

FAF

familial atrial fibrillation

hESC

human embryonic stem cell

hiPSC

human induced pluripotent stem cell

hESC-CM

human embryonic stem cell derived cardiomyocyte

hiPSC-CM

human induced pluripotent stem cell derived cardiomyocyte

I_Kr

rapid potassium channel

I_Ks

slow potassium channel

LQTS

long QT syndrome

V_1/2

half-maximal activation voltage

Author contributions

Conception and design of the experiments: K.W., C.T., K.J.S., G.K., D.N.K., K.S.K.; collection, analysis and interpretation of data: K.W., C.T., K.J.S., V.I., J.D.O.; Drafting the article or revising it critically for important intellectual content: K.W., C.T., K.J.S., J.L., K.S.K. All authors approved the final version of the manuscript. Disclosures: none.

Supplementary material

Supplementary Figure S1

Supplementary Figure S2

Supplementary Figure S3

As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer-reviewed and may be re-organized for online delivery, but are not copy-edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors