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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
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Abstract |
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(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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Introduction |
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The cardiac Na+ channel (termed NaV1.5) is expressed in relatively high density on surface membrane of mature heart cells in atria and ventricle. Despite the high NaV1.5 expression these cell types are not normally automatic because a high density of inward rectifier K+ channels (Kir) clamps the membrane potential to a value near the K+ reversal potential. At such hyperpolarized potentials the NaV1.5 channel's open probability approaches 0. In quiescent, mature heart cells, the initiating depolarization originates from neighbouring cells via gap junctions. Depolarization of membrane potential (Vm) activates NaV1.5 rapidly, driving the rapid AP upstroke, and within a few milliseconds of sustained depolarization NaV1.5 inactivates. Thus, NaV1.5 serves the role of generating a pathway for a rapid influx of depolarizing current. The maximum diastolic potential (MDP) is a key control point for NaV1.5, if MDP is relatively depolarized then NaV1.5 will be largely inactivated and unable to contribute to the AP upstroke. For this reason, there is a correlation between Na+ channel current density (not simply channel density), and the maximum upstroke velocity of the cellular AP (dV/dtmax). Moreover, in the developing heart there is a an increase in dV/dtmax from < 20 to 100–150 V s–1 that is concomitant with the onset of TTX sensitivity (McDonald et al. 1973), an increase in Na+ current density (Fujii et al. 1988), and a negative shift in the MDP (McDonald et al. 1973; DeHaan, 1980; Sperelakis, 1984).
Given the exciting potential use of hES-CMs as replacement tissue in diseased heart (Gepstein, 2002; Kehat & Gepstein, 2003), and as an in vitro model for the study of early human cardiac development it is important to characterize their functional properties. We assessed the electrical properties of these cells in spontaneously beating embryoid bodies (EBs) using a multielectrode array mapping technique and detailed patch-clamp recordings and pharmacologically dissected the critical pathways in these structures. Our results provide the first description of the ionic currents in hES-CMs and show that the basis for spontaneous electrical activity in these cells is the absence of Kir conductance, a phenomenon that provides the substrate for a relatively large voltage-gated Na+ current to drive activity.
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Methods |
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Human undifferentiated ES cells of the clone H9.2 (Amit et al. 2000) were grown on mouse mitotically inactivated (mitomycin C) embryonic fibroblast feeder layer as previously described (Amit et al. 2000; Kehat et al. 2001). To induce differentiation, hES cells were cultured in suspension for 7–10 days, where they aggregated to form EBs. At 22–35 days, beating areas were either plated on gelatin, or fibronectin-coated microelectrode array plates or dispersed into isolated cells for the patch-clamp studies.
Multielectrode array (MEA) mapping technique
Extracellular recordings from the EBs were performed with a MEA data acquisition system as previously described (Feld et al. 2002; Kehat et al. 2002). During the recording sessions, the MEA was constantly perfused with a gas mixture consisting of 5% CO2 and 95% air, at a temperature of 37.0 ± 0.1°C.
Local activation time (LAT) at each electrode was determined as the timing of the maximal negative intrinsic deflection (dV/dtmin) of the unipolar recording. The measured LATs at all electrodes were then used for generation of colour-coded activation maps. Local conduction velocity vector fields were determined from these maps, as previously described (Kehat et al. 2002).
Following baseline recording, 20 µl of stock solution of the test drug were added to the 2 ml of culture medium. The pharmacological agents included tetrodotoxin (TTX; Alomone Laboratories, Jerusalem, Israel) at final concentrations of 10 or 100 µmol l–1, and diltiazem hydrochloride (1 or 10 µmol l–1, Sigma, St Louis, MO, USA). Extracellular recordings were performed for 30 s, at baseline and 5 min following drug application. The effects of these drugs on conduction were evaluated by examining the culture's global velocity (measured as the distance between earliest and latest activation divided by the total MEA activation time), the mean magnitude of the local velocity vector, and the maximal absolute value of the first time derivative of the extracellular signal.
Electrophysiological recordings
For single cell AP and ionic current, the whole-cell configuration of the patch-clamp technique was used. After dissociation cells were replated for 1–4 days on gelatin, or fibronectin-coated glass cover slips. The patch pipette solution consisted of (mM): 120 KCl, 1 MgCl2, 3 Mg-ATP, 10 Hepes, 10 EGTA (pH 7.3). The bath recording solution consisted of (mM): 140 NaCl, 5.4 KCl, 1.8 CaCl2, 1 MgCl2, 10 Hepes, 10 glucose (pH 7.4). Upon seal formation and following patch break, analog capacitance compensation was used. APs were recorded from the current-clamp mode. Voltage recordings were filtered at 10 kHz. For voltage-clamp experiments only isolated cells were selected. Series resistance compensation was used up to 80%, and was monitored immediately prior to the initiation of each individual protocol. Currents were filtered at 10 kHz and sampled at 20 or 50 kHz. Axopatch 200B, Digidata1322, and pClamp8 (Axon, Burlingame, CA, USA) was used for data amplification, acquisition, and analysis. Only spontaneously beating cells were chosen for study as a positive identifier of heart muscle. For consideration of voltage control, roughly spherical cells were chosen (see Fig. 2 below), and the mean cell capacitance (Cm) of 21.6 ± 1.3 pF (n = 68) is small relative to Cm of typical mature heart cells. For current–voltage curves (activation curves) data were discarded for recordings showing indications of inadequate voltage that included an apparent threshold voltage for Na+ current activation, or activation that was described by a Boltzmann distribution with a slope factor steeper than 4. Although this is a rather steep voltage dependence, other protocols indicated adequate voltage control. For steady-state inactivation and recovery from inactivation protocols the currents elicited by common test potentials had indistinguishable kinetics independent of current amplitude. For HCN recordings current was filtered at 2 kHz and a split clock was used to sample at 10 kHz and 1 kHz to prolong the duration of the test step. All recordings were performed at 26–29°C. Individual voltage protocols are indicated as insets in the figures. Equations used for fitting data are presented in the figure legends. Curve fitting was performed with either Clampfit8 or non-linear curve fitting routines in Origin7.1.
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Real-time PCR was performed in 96-well optical plates in triplicate. Samples were prepared using Brilliant Sybr Green QPCR Master Mix (Stratagene). Primers used are shown in Table 1. Fetal human heart cDNA (0.1 ng Stratagene) or 0.1 ng experimental cDNA was used as a template in the real-time PCR reactions. Experimental cDNA was obtained from RT of total RNA isolated from beating EBs trimmed from non-beating areas to enrich the preparation in cardiac myocytes. EBs plated for 25–30 days were used for total RNA isolation. PCR samples were cycled for 50 cycles using an ABI 7700 Sequence Detector (Applied Biosystems). Default 7700 cycle conditions were as follows: 15 min at 95°C followed by 50 cycles of 15 s at 95°C, 30 s at 62°C and 30 s at 72°C. A standard curve was generated from dilutions of the human fetal heart cDNA by plotting the natural log of the threshold cycle (CT) against the natural log of the number of molecules. The CT was defined as the cycle at which a statistically significant increase in the magnitude of the signal generated by the PCR reaction was first detected. CT was calculated under default settings for the real-time sequence detection software (Applied Biosystems). To maximize accuracy, dilutions were made over the range of copy numbers that included the amount of target cDNA expected in the experimental cDNA samples. Specific calcium and sodium channel component cDNA molecules present in the experimental cDNA were calculated from the standard curve (Bustin, 2002; Whelan et al. 2003). To rule out contaminating genomic DNA a ‘no RT’ control was performed for each sample. Dissociation plots showed no evidence of primer-dimer and non-specific PCR products.
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Results |
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We have previously shown that spontaneously contracting areas could be generated within EBs during the in vitro differentiation of the H9.2 human ES cell line. We have also demonstrated using a multielectrode array mapping technique and detailed immunostaining studies that these beating areas are comprised of an isotropic cardiomyocyte cell network acting as a functional syncytium with stable spontaneous pacemaking activity and synchronous AP propagation (Kehat et al. 2002).
The spontaneous activity and the relatively rapid conduction in these studies suggested the presence of a high-density inward current via voltage-activated channels. Therefore, we first tested the effects of Na+ channel and Ca2+ channel blockers on spontaneous activity and conduction. A representative MEA recording of electrical activity from a beating cluster is shown in Fig. 1A and B. Note the relative fast conduction with a total activation time of 11.5 ms in this example. Similar conduction profiles were manifested in all EBs tested in control bath solution.
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Syncytial APs inhibited by Na+ channel, but not Ca2+ channel blockade
Our next step was to evaluate the action potential properties of the hES-CMs and the response to Na+ channel and Ca2+ channel blockade in clusters of spontaneously beating hES-derived cells. Figure 2A depicts a typical cluster of hES-CMs. This photograph shows the pattern of growth of a spontaneously contractile syncytium of cells. The partially dispersed cells that remain in clusters tend to grow vertically and at the periphery of these clusters individual, roughly spherical cells are accessible to patch electrodes. In all clusters tested, the cells within a cluster were beating in synchrony. Figure 3 shows representative spontaneous, rhythmic APs from a cluster of spontaneously contractile tissue. In clusters of cells under control conditions the rhythm was regular (Fig. 3A). The AP morphology (Fig. 3B) consisted of a rapid upstroke followed by a plateau phase. Upon repolarization, there was a slow, spontaneous depolarization. At 3 µM, TTX converted cluster APs from a single diastolic depolarization rate (DDR) to a biphasic DDR (Fig. 3C). As a consequence of an additional slow DDR the interpulse interval (beat rate) of cluster was slowed (Fig. 3D). Also, the maximal upstroke velocity (dV/dtmax) was slowed by Na+ channel block by 3 µM TTX. In contrast, we could not detect any significant effects on action potential durations (APD) by 3 µM TTX (Fig. 3F). At 10 µM, TTX completely inhibited spontaneous activity (Fig. 3G). This effect was readily reversed upon wash-out (Fig. 3G). We conclude that Na+ channel current contributes to both the AP upstroke and the spontaneous diastolic ‘pacemaker’ depolarization.
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Cardiac pace-making tissue such as mature nodal cells or embryonic myocardium has relatively high input resistance as a consequence of the diminished expression of background K+ currents, principally those of the inward rectifier family Kir2. To measure background current we voltage-clamped isolated, single hES-CMs. Figure 2B depicts typical single cells in isolation. The cell shape was roughly spherical and small (cell capacitance ranged from 7 to 40 pF), making them amenable to improved spatial voltage control. All cells selected for study were spontaneously contractile. Voltage steps from a holding potential (Vhold) of –40 mV to potentials negative to –70 mV elicited slowly developing inward current (Fig. 5). The Kir channel is sensitive to external Ba2+ with a half-block concentration on the order of 20 µM (Hille, 2001), but in hES cells 0.5 mM Ba2+ had no effect on ionic current (Fig. 5B and D). Also, there was inward current at the potassium reversal potential (EK 
–84 mV; Fig. 5C), which indicated that the current observed was not via K+-selective Kir channels. Taken together there is no evidence for detectable Kir in hES-CMs differentiated for less than 42 days.
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2.3 s. At the end of the hyperpolarization a large fast inward current is elicited upon return to –40 mV, but only for hyperpolarizing ‘conditioning’ potentials negative to –60 mV. The inset in Fig. 6A shows the return step to –40 mV on an expanded scale. The inset in Fig. 6A is in essence a 2.3 s ‘steady-state’ Na+ channel inactivation protocol with Na+ current activated by a depolarization to –40 mV. Note that the Na+ current persists in the presence of Cs+ (‘tail’ current in Fig. 6B). Although these recordings were made with a relatively slow sample frequency we can still see that the fast inward current is sensitive to the preceding hyperpolarized potential. Thus, the more sustained and more negative the MDP the more HCN will be activated, and the more Na+ channels will be available (the inverse of inactivated) to open. At 2 mM, Cs+ did not inhibit spontaneous activity in cell clusters (data not shown). This insensitivity of spontaneous AP activity to Cs+ argues that HCN may not necessarily contribute to spontaneous depolarization, but the sensitivity to TTX argues that Na+ current is essential.
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At steady state, it is difficult to detect ionic current at voltages corresponding to the range between MDP and the AP threshold. The absence of background K+ currents creates a high input resistance membrane; as a consequence tiny currents can have dramatic effects on membrane potential. This is illustrated in Fig. 7A. Vm was recorded from a single cell with an increasing amount of holding current. With only an additional 6 pA of current, from –35 to 41 pA, a noisy Vm recording was converted to a rhythmically beating pattern. For the largest applied holding current we observed increased diastolic intervals; however, during such prolonged intervals we observed subthreshold depolarizations (e.g. asterisk in Fig. 7A, bottom centre panel). Increasing holding current induced the following trends (Fig. 7B): MDP became more negative, the diastolic interval lengthened and DDR became slower. The slowing of DDR with hyperpolarization is counter-intuitive, given our observation of HCN expression. If HCN were contributing more current more quickly with hyperpolarization we would expect a faster DDR. We can explain the antithesis by considering the notion that Na+ current dominates diastolic depolarization. It is well established that dV/dtmax is an index of Na+ current (Cohen et al. 1984). Therefore, we plotted the MDP versus dV/dtmax (Fig. 7C). This scatter plot shows data from both cell clusters (n = 14) and single spontaneously active cells (n = 3). First, dV/dtmax increases with hyperpolarization. The continuous curve is the Boltzmann distribution with parameters for hES-CM Na+ current steady-state availability (see below), with the amplitude normalized to the fastest measured spontaneous maximum upstroke velocity. Previous published studies of hES APs (from large clusters of cells) showed relatively slow upstroke velocities of < 10 V s–1 which corresponds to an MDP of –53 mV in our data. For more negative MDPs, dV/dtmax tended to increase, with the highest spontaneous upstroke velocity being about 118 V s–1.
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The absence of Kir in hES cells necessitated the use of a holding current to maintain a Vm in the range of that measured from hES clusters because even gigaohm seals allowed sufficient leak current to artificially depolarize cells. As illustrated in Fig. 7A, single cells with hyperpolarized MDPs had prolonged diastolic intervals. By adding a constant holding current we adjusted the MDP to levels ranging from –62 to –70 mV. The long diastolic intervals allowed us to probe the characteristics underlying AP initiation. We stimulated single cells with 1 ms current pulses in 10 pA increments. In stark contrast to mature cardiac preparations, current stimuli of as small as 10–40 pA were sufficient to elicit action potentials (Fig. 8). Figure 8B shows part of the trace in Fig. 8A on an expanded time base to illustrate the rapid dV/dtmax elicited by approximately 120% of stimulus. The rapid dV/dtmax is consistent with mature APs. For supra-threshold stimuli, the dV/dtmax averaged 118 ± 14 V s–1. This value is similar to mature atrial or ventricular myocytes, suggesting a large Na+ conductance. Therefore we tested the effect of TTX on AP initiation. At 10 µM, TTX blocked single cell AP automaticity, and partially blocked INa (see below). In cells exposed to TTX, the dV/dtmax of stimulated APs was reduced to 38 ± 15 V s–1, and three times more current amplitude was required to elicit an AP. The rapid dV/dtmax combined with the sensitivity of dV/dtmax to TTX argues for the presence of a relatively high Na+ channel current density. We therefore measured Na+ current amplitude under voltage clamp. To correlate ion current results with AP properties we measured current in the same cells and under the same ionic conditions as we measured APs. Figure 8C shows that there was a positive correlation between Na+ current amplitude and dV/dtmax.
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Figure 9A shows ionic current elicited by a steady-state inactivation protocol (Fig. 9A, inset). A fast inward current peaked within 1.5 ms of depolarization to 0 mV and inactivated with a fast time constant of 0.5 ms (smooth line superimposed over current trace in Fig. 9A and B). A slower time constant was needed to fit < 5% of the total amplitude. These are well-established characteristics of mammalian cardiac Na+ channel current. TTX blocked INa and shifted the inactivation curve 10 mV in the hyperpolarizing direction (Fig. 9C). Thus, as for NaV1.5 current and native INa, TTX has apparent higher affinity for inactivated channels in hES-CMs. The mean peak inward, maximally available current density for a test potential (Vtest) of 0 mV was –244 ± 42 pA pF–1 (n = 19 cells). The peak current density of maximally available channels measured from the steady-state inactivation protocol ranged from 69 to 877 pA pF–1 (Fig. 9D). This approximate 10-fold range of current densities is not peculiar to a particular embryoid body, given that from the same EB dissociation we recorded cells in the 800 pA pF–1 range and cells in the 100 pA pF–1 range. The voltage midpoint of steady-state inactivation of Na+ current is –72.6 ± 0.7 mV (n = 19). Figure 9C shows pooled, normalized steady-state inactivation curves in control and for TTX. At 100 nM, TTX had no significant effect on current density amplitude; higher doses caused current block and hyperpolarization of the steady-state inactivation midpoint. The half-block concentration of TTX for fully available Na+ current was 6.6 µM (Fig. 9E). The measured IC50 is strongly suggestive of NaV1.5 expression.
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fast) fitted > 90% of the amplitude. The fast voltage dependence is shown in Fig. 10C. The voltage dependence of fast can be described by a single exponential decay with a limiting, voltage-independent rate of 0.44 ms. Thus the activation kinetics and voltage dependencies are consistent with functional NaV1.5 channels. The only exception is that both steady-state inactivation and activation midpoints are shifted in a positive direction on the voltage axis relative to mature preparations (Fig. 10E). Moreover, there is overlap of the steady-state inactivation and activation curves (Fig. 10F). The potential range of this overlap coincides with that of the latter phase of the spontaneous diastolic depolarization, and this latter phase of diastolic depolarization is sensitive to TTX block (Fig. 3). In Fig. 7 we document a window of diastolic potentials that are optimal for spontaneous activity. Taken together, these data suggest that INa openings within this negative potential range are critical for pacemaking activity.
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6 ms before fractional recovery can begin to be detectable. The recovery time course is then well described by a bi-exponential function with time constants of 57 and 708 ms.
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Discussion |
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25 days of differentiation hES-CMs exhibit a hybrid developmental excitable phenotype. In common with early embryonic heart myocytes, hES-CMs have no detectable Kir current, but express a prominent INa approaching levels comparable to those measured from mature cardiac preparations. The basis for automaticity
The ionic currents of hES-CMs in this study are consistent with a hybrid, or intermediate stage of embryonic development. In animal models during ontogeny there is a progressive increase of INa (Nathan & DeHaan, 1978) and inward rectifier currents in atrial and ventricular myocytes (Davies et al. 1996). Ca2+ channel currents appear early in development and are largely responsible for the relatively slow dV/dtmax of the action potential. The paucity of inward rectifier channels creates a high input resistance surface membrane. As a consequence only a few open channels allowing either Na+ or Ca2+ flux may be sufficient to bring the developing heart cell to AP threshold. A similar pattern is also reproduced in mouse ES cells (Maltsev et al. 1994). Data on human fetal development is scarce; however, this same trend apparently also exists (Tuganowski & Tendera, 1973; Janse et al. 1976). In our study, the low current stimulus threshold for AP initiation, and the rapid upstroke velocity coupled with the pharmacological evidence for Na+, but not Ca2+ channel sensitivity argues for a prominent role of NaV1.5 channels in the spontaneous excitability of hES-CMs. The high Na+ current density is indicative of more mature heart cells whereas the high input resistance is more typical of immature cells. The low background conductance facilitates AP production with only a small number of open NaV1.5 channels. Based on the present study we would predict that hES-CMs of up to 30 days differentiation would be good pacemakers, but without Kir expression pose a pro-arrhythmic risk.
In the early developing mouse myocardium, in contrast to hES-CMs, it is thought that pacemaker cells among the embryonic cardiomyocytes start with a ‘primitive’ pacemaker AP that is probably generated by only two ionic currents, ICa,L and IK,to (Hescheler et al. 1997), or even just by intracellular Ca2+ oscillations(Viatchenko-Karpinski et al. 1999). Subsequent to this primitive pacemaker stage HCN knockout in mice suggests an essential role for HCN-4 in maintaining heart development (Stieber et al. 2003). Similarly, intracellular Ca2+ may be essential for pacemaker dominance in mature pacemaker cells (Lakatta et al. 2003). Our data do not exclude a contribution of HCN or ICa,L to the upstroke, but we do show evidence that HCN or ICa,L activity is not a requirement for spontaneous AP upstroke initiation. We clearly show that AP autorhythmicity is insensitive to Cs+ or nifedipine, but is sensitive to TTX block of INa. Therefore, we conclude that the human ES-CM AP initiation is fundamentally different from that of the well-studied mouse.
NaV1.5 underlies hES-CM INa
In mature cardiac preparations pacemaker cells are spontaneously active due to a mixture of ion channel expression that includes ICa,L, If (HCN), and the absence of significant Kir expression. Recently, studies have shown that a TTX-sensitive Na+ channel (1–10 nM TTX) also contributes to spontaneous AP initiation (Maier et al. 2003). In hES-CMs we show, however, that INa is unaffected by 100 nM TTX. The TTX half-block concentration is 6.6 µM, which is very close to the widely cited range of 1–6 µM for half-block of native and cloned human Na+ current (Fozzard & Hanck, 1996). In fact, in adult human myocytes 10 µM TTX was required for half-block of INa (Schneider et al. 1994). Some of the discrepancies in reported half-block concentrations of TTX can arise from the protocol dependence of the degree of block. TTX block of Na+ channels is sensitive to both external Na+ concentration and channel state dependence (Fozzard & Hanck, 1996). It is likely that the Na+ dependence arises from competition between the guanidinium group of the toxin and the partially dehydrated permeating Na+ ion (reviewed by Fozzard & Hanck, 1996). We worked with relatively high external Na+ which may partially explain the relatively weak TTX sensitivity of hES-CM INa. The state dependence of TTX block may help to explain the tendency for TTX to have a greater effect on spontaneous excitability than would be anticipated from the measured half-block concentration of maximally available INa (Fig. 9). It should be noted that early reports of dissociation equilibrium constants for TTX block of native cardiac INa showed an almost 5-fold increase in apparent TTX affinity for inactivated versus closed state channels (Carmeliet, 1987). This finding was later confirmed in cloned, heterologously expressed cardiac Na+ channels (Satin et al. 1992). One consequence of this higher affinity TTX block is that TTX will have an apparent higher affinity for intact hES-CMs by virtue of the fact that depolarized holding potentials promote substantial channel inactivation. Given that MDP was never more negative than –65 mV, this implies that in intact hES-CMs the majority of Na+ channels are steady-state inactivated. Hence, lower TTX concentrations than that required for the fully available half-block level are required to inhibit spontaneous AP initiation.
The voltage dependence and kinetics of hES-CM INa are also consistent with the function of the human NaV1.5 channel, with some minor, but notable exceptions. hES-CM INa, native human INa, and heterologously expressed cloned human NaV1.5 current all display an approximate 43 mV difference between the midpoints for macroscopic activation and steady-state inactivation. It is notable, however, that hES-CM activation and inactivation midpoints are uniformly shifted 15 mV in the depolarized direction relative to their mature counterparts. It is unlikely that this shift is due to a unique polymorphism in the hES line in this report. Consider, for example, that a careful study of the functional characteristics of polymorphisms of NaV1.5 variants showed that different polymorphisms confer different expression levels but similar voltage dependencies (Makielski et al. 2003). Moreover, early studies in developing mammalian myocardium noted that INa is present in the earliest beating heart, increases in amplitude with development, and has unchanged kinetics, pharmacology or voltage dependencies with the exception of a progressive hyperpolarizing shift of steady-state inactivation and activation as a function of development (Shigenobu & Sperelakis, 1971; Sperelakis & Shigenobu, 1972; Lompre et al. 1979; Fujii et al. 1988; Sada et al. 1995). Thus the early embryonic shift of INa is possibly due to an uncharacterized splice variation or accessory subunit expression in hES cells, and this would form an interesting basis for future investigations. Finally, although a uniform translation of voltage dependence can be explained by a charge-screening mechanism that would be unique to the embryonic cellular membrane we think that this possibility is unlikely because the Cs+-sensitive HCN current exhibited a similar voltage dependence to that measured from mature human Purkinje fibre and ventricular myocytes (Han et al. 2002). Thus there is not a common shift towards depolarized potentials of all voltage-activated ionic conductances in hES-CMs. Regardless of the mechanism, the shift in voltage dependency towards depolarized potentials allows Na+ channels to operate in the relatively, in comparison to mature working cardiac myocytes, depolarized range of the embryonic MDP.
Kinetic parameters of hES-CM INa compared to human INa are also similar. Our measures of a fast inactivation rate of 0.4 ms at 28°C for test steps positive to 0 mV is close to values ranging from 0.2 to 0.4 ms obtained from heterologously expressed human NaV1.5 channels at 22°C at 0.35 ms and at the relatively elevated temperature of 32°C (Nagatomo et al. 1998; Dumaine et al. 1999). A consequence of the shift of the hES-CM INa gating is that kinetic parameters are also shifted. A key parameter to consider for the maintenance of spontaneous pacing is the rate of recovery from inactivation. However, for the purpose of comparison with the literature the values obtained need to be normalized to the inactivation midpoint; in our case this is slightly less than 10 mV hyperpolarized to the inactivation midpoint. For example, in a careful study of canine ventricle in which the recovery potential was varied, the recovery from inactivation was described by two time constants with rates of 116 and 454 ms (Pu & Boyden, 1997). These recovery rates are sufficient to allow > 80% of channels to recover during a 1 s interpulse interval. This slow recovery from inactivation is consistent with creating a limit to the rapidity of spontaneous ES cell beating.
Comparison to murine ES-derived heart cells
Because the mouse is an important species for designing human disease models it is important to compare the ionic currents between murine and human ES-derived cardiac myocytes. In terminally differentiated mouse ES-CMs the INa current density was 200 pA pF–1 in one study, but only 70 pA pF–1 in a more recent report (Fijnvandraat et al. 2003). We now show that human ES-CMs express a range of current densities between over 800 and 60 pA pF–1 with an average of 242 pA pF–1. The large disparity between mouse and human gestation periods must be considered. In the mouse 22 days of mES cell plating time is taken as terminal differentiation; a comparable duration in humans would be 270 days. In this study, we measured INa from hES cells between 18 and 35 days after plating of embryoid bodies. During this window of time we noted more disparity of current density within cells from a single embryoid body dissociation than between cells of different ages. If we simply scale human time to mouse time based on in utero gestation periods, our study would represent a cell differentiation period of about 1.3 mouse-days and would coincide approximately with mouse-days 2–3 of ES differentiation time (starting from the plating of embryoid bodies). Prior to 5 mouse-days of differentiation time murine ES-CMs expressed no detectable INa (Maltsev et al. 1994; Fijnvandraat et al. 2003). We highlight these differences simply to make the point that the hES INa density is extremely high for such a relatively early developmental time. Similarly, HCN current (If) density in human ES-CMs at a stage equivalent to only 2–3 mouse-days of differentiation were comparable to the 1–10 pA pF–1 levels measured from terminally differentiated mouse ES-CMs (> 12 mouse-days; (Maltsev et al. 1994). The human development of Kir (IK1) may also parallel that of mouse. In the mouse, Kir expression does not appear until the terminal differentiation stage (16 mouse-days). As in the mouse, we cannot detect Kir expression in human ES-CMs at a stage equivalent to 3–4 mouse-days of differentiation. To extend the parallel in humans, we anticipate having to investigate human ES-CM cultures over 200 days after plating in order to observe Kir expression. Such a time frame entails obvious technical and practical limitations. On the other hand, the relatively long human gestation period presents the opportunity to carefully evaluate the physiological mechanisms of human heart cell early development in future studies. Although we cannot access early human hearts for study for obvious ethical reasons, in the mouse ES-CMs parallel native development (Doevendans et al. 2000), which reinforces our proposal that hES-CMs differentiating in vitro provide a good model for early human development.
Comparison to earlier human ES studies
There are no reports of ionic currents in hES-CMs, but there are two published reports of hES-CM APs from cell clusters. All of our AP recordings have a definitive plateau and they are similar to the embryonic ventricular-like action potentials reported by He et al. (2003), even though their experiments were carried out at 37°C. We report a dV/dtmax (118 V s–1) from isolated cells that is markedly faster than the dV/dtmax in cell clusters, 8 V s–1 (He et al. 2003; Mummery et al. 2003). There are several important factors to consider in explaining this discrepancy. First, our voltage-clamp recordings of a prominent INa coupled with TTX sensitivity of spontaneous excitability and conduction furnishes proof that the Na+ channel (NaV1.5) is the dominant ion charge carrier driving the upstroke of the AP. Therefore, the MDP is a critical determinant of Na+ channel availability, which in turn is a critical determinant of maximum upstroke velocity (Fozzard et al. 1987; Sheets et al. 1988). Second, AP propagation and morphology are dependent on both active and passive properties. In modelling and experimental studies, dV/dtmax increases with cell uncoupling (Rudy & Quan, 1987; Shaw & Rudy, 1997). EBs show uniform TTX sensitive conduction velocity (see Fig. 1 and Kehat et al. 2002) that is sensitive to conduction block by gap junction uncoupling reagents (I. Kehat & L. Gepstein, unpublished observations). Thus cell–cell coupling is relatively strong. These conditions are therefore expected to reduce dV/dtmax relative to isolated cells which are small and coupled to no other active or passive electrical components. In both single cells and well-coupled clusters, slight deviations in MDP will have large effects on Na+ channel inactivation. Finally, we voltage-clamped isolated cells based on spontaneous beating and morphology and thus introduced a potential selection bias.
Summary
In conclusion, this study is the first to measure and characterize ionic currents and single cell AP properties from isolated human ES cells. TTX inhibits the automatic excitability of clusters of cardiac myocytes in EBs, and that of isolated cells. The high input resistance coupled with a high Na+ channel density creates conditions for spontaneous excitability.
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References |
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) tended to be shorter than APD in the presence of TTX (
), but the differences were not significant. n = 6 for all panels. G, APs from a representative cluster in control, 10 µM TTX and following washout. 10 µM TTX induced quiescence and was accompanied by a slow steady depolarization to 
) and slope factors (k): control (
), –74 mV and 5.8; 3 µM TTX (
), –79 mV and 5.8; 30 µM TTX (
), –81 mV and 5.7. D, distribution of current density of cells from peak current obtained from the steady-state inactivation protocol. Bin width is 100 pA pF–1. E, TTX dose–response relationship fitted with a single site blocking curve of the form: fractional block = 1/(1 + [TTX]/Kd) with Kd = 6.8 µM.
). Smooth curve is a modified Boltzmann distribution where, G(V) = Gmax(Vtest – Erev))/{1 + exp((V
