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The Journal of Physiology logoLink to The Journal of Physiology
. 2007 Nov 22;586(Pt 3):701–716. doi: 10.1113/jphysiol.2007.144329

Hyperpolarization-activated cyclic nucleotide-modulated ‘HCN’ channels confer regular and faster rhythmicity to beating mouse embryonic stem cells

Yang Qu 1,2, Gina M Whitaker 3, Leif Hove-Madsen 4, Glen F Tibbits 1,2, Eric A Accili 3
PMCID: PMC2375615  PMID: 18033814

Abstract

The hyperpolarization-activated cation current (If), and the hyperpolarization-activated cyclic nucleotide-modulated ‘HCN’ subunits that underlie it, are important components of spontaneous activity in the embryonic mouse heart, but whether they contribute to this activity in mouse embryonic stem cell-derived cardiomyocytes has not been investigated. We address this issue in spontaneously beating cells derived from mouse embryonic stem cells (mESCs) over the course of development in culture. If and action potentials were recorded from single beating cells at early, intermediate and late development stages using perforated whole-cell voltage- and current-clamp techniques. Our data show that the proportion of cells expressing If, and the density of If in these cells, increased during development and correlated with action potential frequency and the rate of diastolic depolarization. The If blocker ZD7288 (0.3 μm) reduced If and the beating rate of embryoid bodies. Taken together, the activation kinetics of If and results from Western blots are consistent with the presence of the HCN2 and HCN3 isoforms. At all stages of development, isoproterenol (isoprenaline) and acetylcholine shifted the voltage dependence of If to more positive and negative voltages, respectively, and they also increased and decreased the beating rate of embryonic cell bodies, respectively. Together, the data suggest that current through HCN2 and HCN3 channels confers regular and faster rhythmicity to mESCs, which mirrors the developing embryonic mouse heart, and contributes to modulation of rhythmicity by autonomic stimulation.

The timely appearance of spontaneous and repetitive activity is a critical feature of the embryonic heart. Hyperpolarization-activated cyclic nucleotide-gated channels (HCN), which underlie the hyperpolarization-activated or ‘funny’ current Ih/If, contribute to spontaneous beating in the adult sinoatrial node (DiFrancesco et al. 1986; DiFrancesco, 1993; Accili et al. 2002; Robinson & Siegelbaum, 2003) as well as in the embryonic heart (Stieber et al. 2003). During development of the mouse heart, expression of If was detectable as early as embryonic day 8.5, approximately a day after contractions of the heart begin, and both If density and beating rate peak at embryonic day 9.5–10 (Porter & Rivkees, 2001; Stieber et al. 2003). Cardiomyocytes from embryonic mice lacking HCN4 have significantly lower levels of If and lower beating frequencies (Stieber et al. 2003). Because the morphology of their hearts was normal, it was suggested that these HCN4-lacking mice die at embryonic day 9.5–11.5 because of inadequate perfusion.

Because of the dependence of the murine embryo upon blood flow, it can be difficult to investigate the molecular basis of cardiac pacemaker activity during development in the mouse. To overcome this problem, embryonic stem (ES) cell-derived cardiomyocytes have been utilized because they recapitulate features of embryonic cardiac development (Maltsev et al. 1994; Zhang et al. 2002). Upon differentiation, mESCs exhibit cardiac specific genes and ionic currents and action potentials typical of different parts of the heart, and they beat spontaneously (Robbins et al. 1990; Maltsev et al. 1993, 1994; Klug et al. 1996; White & Claycomb, 2005). If and HCN channel RNA are expressed in a heterogeneous proportion of mESCs at both early and late stages (Maltsev et al. 1994; Abi-Gerges et al. 2000; Kolossov et al. 2005; Wang et al. 2005; White & Claycomb, 2005), and If density increases during mESC development (Abi-Gerges et al. 2000). However, the contribution of HCN channels to spontaneous beating in mESCs at each of these stages has yet to be assessed directly.

In this study, we examined the properties of If and investigated its contribution to spontaneous activity in mESCs at different stages of development using the perforated current and voltage patch clamp approaches, as well as Western blotting. We found that beating rates of mESCs increase during development as does If density. This confers more regular and faster rhythmicity, and parallels the increase in beating rate observed in the embryonic mouse heart. Taken together, the activation kinetics of If channel and results from Western blots are consistent with the presence of the HCN2 and HCN3 isoforms. Our data also suggest that the modulation of If contributes to autonomic stimulation of beating rate over the course of the development. Together with previous studies, our findings support the use of mESCs as a model for the developing mouse heart.

Methods

Culture of mESCs cells and differentiation into cardiomyocytes

Mouse embryonic stem cell line R1 (ATCC, USA) was used in the study (Nagy et al. 1993). To maintain the ES cells at an undifferentiated state, cells were cultured on 0.1% gelatin-coated culture dishes in ES-DMEM, consisting of DMEM (Invitrogen), supplemented with 15% fetal bovine serum (Wiscent), 2.0 mm l-alanyl-l-glutamine (ATCC), 0.1 mm non-essential amino acids (ATCC), 100 U ml−1 penicillin (Invitrogen), 100 μg ml−1 streptomycin (Invitrogen), 0.1 mm 2-mercaptoethanol (Sigma) and 1000 U ml−1 mouse leukaemia inhibitory factor (LIF) (Chemicon). The hanging drop method was used for differentiation of cardiomyocytes (Wobus et al. 1991; Maltsev et al. 1993). On day 1 of differentiation, ES cells were plated on a 100 mm Petri dish cover in 50–60 single drops. Each drop contained 400–800 cells in 20 μl of differentiation medium (ES-DMEM without LIF). The cover was gently inverted and put on top of the Petri dish containing PBS. The hanging drops were cultured for 2 days. On day 3, cell clusters (embryoid bodies, EBs) formed in hanging drops were flushed with the differentiation medium and grown further for 4 days. On day 7, EBs were plated out onto 0.1% gelatin-coated 24-well culture plates (Falcon) at 1–2 Ebs per well in 1 ml of medium. Spontaneous beating was observed ∼1–2 days after plating (day 7 + 1–2).

Cell isolation

Single beating cells were isolated from embryoid bodies as previously described (Maltsev et al. 1994). Beating areas of ∼10–20 EBs at each development stages were dissected at room temperature using two 23G1 needles. The whole process altogether took no more than 30 min. Tissue fragments were then incubated in low-Ca2+ medium with 1 mg ml−1 collagenase (Yakult, Japan, 500 U mg−1; (mm): NaCl 120, KCl 5.4, MgSO4−7H2O 5, CaCl2 0.03, sodium pyruvate 5, glucose 20, taurine 20, Hepes 10; pH 6.9 adjusted with NaOH) for 30 min at 37°C, while being titrated gently every 10 min. The dissociation process was continued in high-K+ solution (mm): KCl 85, K2HPO4 30, MgSO4 5, EDTA 1, Na2ATP 2, pyruvic acid 5, creatine 5, taurine 20, glucose 20, pH 7.4) for another 1 h with the gentle shaking at room temperature. Isolated cells were plated on poly d-lysine-coated glass bottom culture dishes (No.1.5, MatTek Corporation) and cultured overnight in differentiation medium. Single spontaneous beating cells and beating clusters could be observed the next day.

Electrophysiology studies

Perforated whole-cell patch-clamp technique (using amphotericin) was performed on single spontaneous beating cells using a MultiClamp 700A computer-controlled patch amplifier (Axon Instruments, Union City, CA, USA). The glass bottom culture dishes were mounted on the stage of microscope (Eclipse TE300 Nikon) and cells were viewed using a ×60 oil immersion objective. The dish was perfused by gravity at a rate of 0.2 ml min−1 with extracellular solution. The patch pipettes (1–2 MΩ) were pulled from thin-walled glass capillaries (World Precision Instruments) using a vertical puller (Narishige PP-830, Japan). Seals with a resistance of 1–2 GΩ were formed under the voltage-clamp configuration. The access resistance dropped to less than 30 MΩ within 10–20 min of seal formation. Junction potential was not corrected. Only recordings in which voltage error was less than 10% of the command voltage were accepted. Ninety per cent of the currents measured were less than 0.5 nA at −150 mV. The clamping mode was switched between current clamp and voltage clamp for the measurement of action potentials or currents. The data were digitized (Digidata, 1322A) at 20 kHz and filtered at 1 kHz acquired using Clampex (version 8.2, Axon Instruments). All experiments were done at 22°C.

The standard external solution contained (mm): NaCl 140, KCl 5.4, CaCl2 1.8, MgCl2 1, Hepes 10 and glucose 10, and adjusted to pH 7.4 with NaOH. For the measurement of If, the standard solution was modified by adding BaCl2 (2 mm) and MnCl2 (2 mm) to suppress potassium and calcium currents, as previously described (DiFrancesco et al. 1986; Accili et al. 1997). The internal pipette solution contained (mm): NaCl 10, KCl 50, KOH 80, aspartic acid 80, MgCl2 1, Hepes 10 and MgATP 3, pH 7.2 adjusted with KOH. The final concentration of amphotericin B (Sigma; solubilized in dimethyl sulfoxide) was 250 μg ml−1 in the patch pipette. ZD7288 was purchased from Tocris (Ellisville, USA).

Western blot analysis

Beating areas from mouse embryonic stem cells were isolated from embryoid bodies as previously described (Maltsev et al. 1994). Undifferentiated stem cells and Chinese hamster ovary (CHO) cells were detached from culture plates with a rubber policeman. Cells were washed with PBS and processed in RIPA lysis buffer (50 mm Tris at pH 8.0, 1% NP-40, 150 mm NaCl, 1 mm EDTA, 1 mm PMSF, 2 mm each of Na3VO4 and NaF, and 10 μg ml−1 each of aprotinin, pepstatin and leupeptin) by multiple passes through a 23G syringe, and incubated on ice for 30 min, followed by centrifugation to remove cell debris. All experiments using embryonic mouse tissue were carried out at the University of British Columbia. In accordance with guidelines from the Canadian Council for Animal Care and the University of British Columbia Committee on Animal Care, female CD1 mice (n = 3) were deeply anaesthetized using CO2 and killed by cervical dislocation. Day 18 embryos were removed (18–20 per mouse), and whole hearts were isolated and processed in RIPA lysis buffer as described above. Cell lysates were loaded into 8% SDS–polyacrilamide gels. Gels were transferred to polyvinylidene difluoride membranes, and blots were washed twice in TBS-T and then blocked with 5% non-fat dry milk in TBS-T for 1 h. Blots were incubated with primary antibody overnight at 4°C in 5% non-fat milk. After three washes with TBS-T, blots were incubated with horseradish peroxidase-conjugated secondary antibodies at a dilution of 1 : 3000 in 5% non-fat milk for 1 h at room temperature. After three washes with TBS-T, signals were obtained with ECL detection reagents (GE Healthcare). The following primary antibodies were used: Rb polyclonal HCN1, HCN2, HCN3 and HCN4 (Alomone Laboratories), goat polyclonal HCN1, HCN4 (Santa Cruz Biotechnology Inc.), Rb polyclonal HCN3 (kind gift from R. Shigemoto), mouse monoclonal HCN4 (Affinity Bioreagents), and guinea pig polyclonal HCN2 (kind gift from R. Shigemoto), as well as goat polyclonal GAPDH (SantaCruz Biotechnology Inc.). HCN antibodies were chosen because they identified appropriate bands in CHO cells transfected with the corresponding isoform. For a positive identification of isoforms, bands on each blot were required to be at or near both the predicted molecular weight as well as the weight detected from CHO cell lysates expressing individual isoforms, and recognized by at least two different antibodies. Western blotting experiments were carried out on three or more preparations of mESCs, and five times using mouse embryonic tissue.

Data analysis

If was measured as the difference between the instantaneous currents and the steady-state currents, at the beginning and end of test voltage pulses. Currents were normalized to membrane capacitance. Rates of If activation were determined using the following function (Clampfit 8.2, Axon Instruments),

graphic file with name tjp0586-0701-m1.jpg

where i = 1 or 2 (a single or double exponential fit), A is the amplitude of the fitting component(s), τ is the time constant, and C is the shift of the fitted trace from zero. A delay in If activation was noted in each current trace. As this delay is not well fitted by simple exponential functions, this portion of the trace was not utilized in our fits (Santoro et al. 2000; Altomare et al. 2001; Macri et al. 2002).

To obtain steady-state If activation curves, the relations between normalized tail currents and test voltage were fitted with the following Boltzmann equation:

graphic file with name tjp0586-0701-m2.jpg

where V is the test voltage, V1/2 is the mid-activation voltage and k is the slope factor (Origin 6.0, MicroCal Software Inc., Northampton, MA, USA). Activation thresholds were determined by fitting these relations from individual cells with the Boltzmann equation, and empirically determining the point at which these relations are continuously larger than zero.

Action potential frequency was measured as the total number of spikes within a 1 min recording. The average rates of diastolic depolarization (DDR) were determined by averaging the measurements from 10 consecutive action potentials.

Unless otherwise stated, data are presented as means ±s.e.m. Statistical significance of the results was tested using a one-way or two-way ANOVA, Tukey test for multiple comparisons or Student's t test for paired samples as indicated. A P value of < 0.05 was considered significant.

Results

Properties of If in mouse embryonic stem cells (mESCs)

The properties of If were studied in early (day 7 + 2–4), intermediate (day 7 + 5–8) and late stage (day 7 + 9–15) cells. Hyperpolarizing pulses elicited slowly activating inward currents, characteristic of If, in early, intermediate and late stage myocytes. If was present in these cells as early as day 7 + 2, the earliest time tested. If density was significantly larger in late stage cells compared to the early stage cells (Fig. 1A).

Figure 1. The density and kinetics of If change during development.

Figure 1

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A, left, representative If traces recorded in the presence of 2 mm Ba2+ and 2 mm Mn2+ from both early and late stage cells. Cells were held at −35 mV and stepped to hyperpolarizing test pulses from −50 to −150 mV for 2 s in 20 mV intervals. The voltage protocol is shown below the current traces. If was measured as the difference between the instantaneous currents and the steady-state currents, at the beginning and end of test voltage pulses. The currents were normalized by membrane capacitance to reduce variability in amplitude due to variations in cell surface area. Right, If as a function of voltage is plotted at each stage (*P < 0.05). B, upper, representative current traces from both early (left) and late stage cells (right) at −90 mV, fitted with a single exponential function; and at −150 mV, fitted with a double exponential function (see Methods). The fast and slow components for the double exponential fit are shown in black. Lower, slow time constants (τs, lower left) and fast time constants (τf, lower right) at each stage are plotted against the test voltages. τ values at −90 mV at each stage are plotted individually in the lower left panel (open symbols) Differences between early and late stage values of τs were significantly different (P = 0.0003, two-way ANOVA analysis). **Significant difference in τ value between early and late stage at −90 mV (t test, P < 0.05). C, the amplitude of slow and fast exponential components (As and Af) were determined from double exponential fits (see Methods). The relative amplitude of Af was calculated as Af/(Af+As) and plotted against voltage.

To study the activation kinetics of If, cells were stepped to test voltages for a relatively long duration (8 s) to achieve full activation and allow for better fitting (Fig. 1B). At −150 mV, −130 mV and −110 mV, If at both early and late stages was fitted better with a standard double exponential function rather than a single exponential function. At these voltages, the fast time constants determined by fitting did not differ significantly between early and late stages. However, the slow time constants were consistently and significantly larger at these voltages in the early stage cells (P < 0.0005). At −90 mV, If recorded from both early and late stage cells activated more slowly and the current traces were fitted better with a single exponential function. At this voltage, the time constant of activation in cells at early stage was significantly larger, indicative of a slower rate of If activation in these cells. These rates of activation are slower, and faster, than those obtained for If in mammalian cells transfected with HCN2 and HCN3, respectively.

We also determined the amplitudes of each exponential component at −110 mV, −130 mV and −150 mV, and the relative amount of the fast component as a fraction of the total amplitude. Previous studies have shown that the relative amount of the fast component increases at more negative potentials in oocytes expressing HCN2 but not HCN1. Here, we found that the amplitude of the slow exponential component was relatively constant at each test voltage, while the amplitude of the fast exponential component increased when the holding potentials became more hyperpolarized. Thus, the relative amount of the fast component was larger at more negative voltages, consistent with previous studies of HCN2, but not HCN1. This is plotted in Fig. 1C, which also shows that this relationship was similar between the early and late stages.

Since repetitive long pulses were not tolerated by the cells, a 2 s prepulse was applied at each test potential to measure the steady-state activation of If. As shown in Fig. 2A, the voltage dependence of If activation at the early and late stages was similar. The half-activation voltages (V1/2) and the slopes of activation (k) for the early stage cells were −90.29 ± 3.35 mV and 10.22 ± 0.94 (n = 7), respectively; for the late stage cells they were −88.02 ± 0.75 mV and 11.45 ± 0.67 (n = 4, P > 0.05), respectively. The activation thresholds, determined from Boltzmann curves from individual cells, were also similar and were −47.72 ± 1.88 mV at early stage and −46.06 ± 1.92 mV at late stage (P > 0.05).

Figure 2. The steady-state activation and reversal potentials of If do not change during development.

Figure 2

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A, left, representative current traces from a late stage cell. The voltage protocol is shown above the current traces. If tails were elicited in response to a pulse to −150 mV, following test voltages, to minimize contamination by other conductances at less negative and positive pulses. Right, steady-state If activation curves of the early and late stage cells. Tail currents were determined from the difference between the peak current and the steady-state current, normalized and plotted against test voltage. The relations were fitted with the Boltzmann equation (see Methods). B, left, representative current traces to determine the instantaneous If from both early and late stage cells. The voltage protocol is shown below the current traces. Right, instantaneous If was plotted against voltages negative to −30 mV. The voltage-independent/leakage currents were subtracted from the total instantaneous currents at each test voltage to yield instantaneous If. To determine the actual reversal potential, a straight line fit was performed for each cell and an average value was obtained.

Instantaneous If–V relations were generated as described in our previous studies of If/HCN channels (e.g. Proenza et al. 2002; Macri & Accili, 2004). Values for slope conductance (Gf) and reversal potential (Ef) were determined by fitting current amplitudes negative to −30 mV with a straight line, for each individual cell (Fig. 2B). Values for Gf were 20.71 ± 4.46 pS pF−1 at early stage and 54.42 ± 16.59 pS pF−1 at late stage (P < 0.01), confirming the increase in If density. The values for Ef were −28.17 ± 4.99 mV for the early stage cells and −22.14 ± 7.12 mV for the late stage cells (P > 0.05) (Fig. 2B). These values for Ef in physiological intracellular and extracellular solutions are consistent with the mixed permeability of If channels (DiFrancesco, 1981).

HCN2 and HCN3 isoforms are found in mESCs and in the embryonic mouse heart

In order to determine which HCN isoforms underlie If in mESCs, we carried out Western blotting using antibodies specific for the four mammalian isoforms. For a positive identification of isoforms, bands on each blot were required to be at or near both the predicted molecular weight as well as the weight detected from CHO cell lysates expressing individual isoforms. Additionally, isoforms had to be clearly recognized by at least two antibodies. Based on these criteria, HCN2 and HCN3 isoforms were identified in both the early and late stage mESCs, as well as in undifferentiated mESCs. We also found both HCN2 and HCN3 in day 18 embryonic mouse hearts (Fig. 3B and C). Lastly, we found that HCN3 isoform expression in mESCs decreased over the time course of their development in culture (Fig. 3C). We were not able to identify HCN1 or HCN4 protein in mESCs, before or after differentiation (Fig. 3A and D).

Figure 3. HCN2 and HCN3 are the predominant isoforms in differentiated and undifferentiated mESCs.

Figure 3

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A, Western blot probed with antibodies raised in rabbit (Alomone) and specific for HCN1 (upper), or raised in goat and specific for GAPDH (lower). Left, HCN1 expressed in CHO cells gives two bands at 123 and 114 kDa, as shown by the arrows. Centre, HCN1 was not detected in undifferentiated (UD), early stage EBs (ESEB) and late stage EBs (LSEB). Right, HCN1 was not detected in day 18 embryonic mouse. Arrows point to 95 and 130 kDa which encompass the area that HCN1 would be expected to migrate to if present. B, Western blot probed with antibodies raised in rabbit (Alomone) and specific for HCN2 (upper), or raised in goat and specific for GAPDH (lower). Left, HCN2 expressed in CHO cells gives two bands at 127 and 107 kDa, as shown by the arrows. Centre, HCN2 in undifferentiated (UD), early stage EBs (ESEB) and late stage EBs (LSEB). The arrow identifies the band at 106 kDa. Right, HCN2 expressed in day 18 embryonic mouse at a molecular weight of approximately 97 kDa as identified by the arrow. C, Western blot probed with antibodies raised in rabbit (Shigemoto) and specific for HCN3 (upper), or raised in goat and specific for GAPDH (lower). Left, HCN3 expressed in CHO cells shows two bands at 107 and 89 kDa, as shown by the arrows. Centre, HCN3 in undifferentiated (UD), early stage mESCs (ESEB) and late stage mESCs. The arrow identifies the band at 87 kDa. Right, HCN3 expressed in day 18 embryonic mouse at a molecular weight of approximately 86 kDa as identified by the arrow. D, Western blot probed with antibodies raised in rabbit (Alomone) and specific for HCN4 (upper), or raised in goat and specific for GAPDH (lower). Left, HCN4 expressed in CHO cells gives two bands at 174 and 149 kDa, as shown by the arrows. Centre, HCN4 was not detected in undifferentiated (UD), early stage EBs (ESEB) and late stage EBs (LSEB). Right, HCN4 was not detected in day 18 embryonic mouse. Arrows point to 130 and 170 kDa which encompass the area that HCN4 would be expected to migrate to if present. The predicted molecular weights for the unmodified proteins are: HCN1 ∼100 kDa, HCN2 ∼95 kDa, HCN3 ∼86 kDa, HCN4 ∼132 kDa. GAPDH was used as a loading control for total amount of protein. Data shown are representative of three independent experiments, from three separate preparations of mESCs.

If density correlates with action potential frequency and rate of diastolic depolarization of mESCs during development

We next examined the action potential, the ability of the cells to beat, and the contribution of If to beating frequency over the course of development. The beating cells from ES cells were classified into the same three stages used for the examination of If. At the early stage, spontaneous action potentials were irregular and intermingled with small fluctuations in membrane potential. This type of action potential feature was accompanied by an irregular beating pattern. Cells at the intermediate and late stages beat more regularly, faster and more vigorously (Fig. 4A). The frequency of action potentials (Fig. 4B, top) and DDR (Fig. 4B, middle) were both increased in the intermediate and late stage cells. The maximal diastolic potential (MDP) and the threshold of action potentials did not change significantly over this period of time (Fig. 4B, bottom). Thus, this increase in beating frequency was due primarily to the shortening of the time required to reach the threshold. The MDP recorded from spontaneously active cells at early and late stage were −43.75 ± 1.74 mV and −47.71 ± 1.82 mV, respectively, which were close to the values for If activation threshold.

Figure 4. The frequency of action potential firing and the rate of the diastolic depolarization (DDR) increase over the course of mESC development in culture.

Figure 4

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A, representative action potential recordings from single beating cells at early, intermediate and late stages. Membrane currents were held at 0 pA and the lines indicate a membrane potential of 0 mV. B, top, frequency of spontaneous action potential firing at each stage (**compared to the late stage cells, P < 0.01). Middle, the rate of diastolic depolarization (DDR) at each stage (**compared to the late stage cells, P < 0.01). Bottom, the maximal diastolic potential (MDP) and action potential threshold at each stage. The parameters are illustrated in the inset, on the left. DDR was determined by the slope from the MDP to the threshold for AP firing. The threshold was considered as the base of the fast uprising phase. The number of cells for each group is shown above or below each bar. There were no significant differences in these parameters among action potentials recorded at the different stages of development.

To determine whether the increase in beating frequency and rate of diastolic depolarization were related to increases in If, action potentials and If were recorded from the same cells by switching from current to voltage clamp. If was activated by stepping the cells to −150 mV for 2 s, from a holding potential of −35 mV. Of the cells that beat, 47% displayed If at the early stage whereas 84% of beating cells possessed If at the late stage (Fig. 5A, upper panel; P < 0.001). It is possible that this difference is due to our inability to detectIf, which could underestimate the proportion of cells expressing If to a greater extent at the early stage. However, the currents would have to be very small in order for them to go undetected, less than ∼5 pA in our system. This is much smaller than the average current amplitudes measured, which were ∼80 pA for early stage cells and ∼320 pA for late stage cells at −150 mV.

Figure 5. If density correlate with beating rate and diastolic depolarization rate in mESCs.

Figure 5

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A, upper, the percentage of beating cells that express If at each stage (P < 0.001, Chi-square contingency analysis, the P value was drawn from the table of critical values of the t distribution). The number of cells in each group is shown above the bars. These measurements were carried out in a complete set of cells, including recordings with and without Ba2+ and Mn2+. Middle, cell membrane capacitance at each stage (**compared to the late stage cells, P < 0.01). Lower, If density measured at −150 mV at each stage (*compared to the late stage cells, P < 0.05). These measurements were recorded specifically from beating cells in the absence of Ba2+ and Mn2+. Successful recordings of If and action potentials were obtained from a subset of these cells (see B and C). Only cells expressing If were included in the calculation. B, rates of diastolic depolarization (DDR) correlate with If density at −150 mV (n = 32, P < 0.01). C, action potential frequency correlates with If density at −150 mV (n = 32, P < 0.01). For B and C, points represent values from individual cells from which recordings of both If and action potentials were obtained, and were fitted by linear regression.

If density was also significantly larger in the intermediate and late stage cells (Fig. 5A, lower panel), which was consistent with the increase in DDR and action potential frequency. This increase occurred despite the concomitant increase in the cell capacitance (Fig. 5A, middle panel). The difference in If density between the early and late stage cells may be underestimated for two reasons. First, the inability to detect very small currents would preferentially overestimate If density in small cells. Second, the voltage error would preferentially underestimate If density in large cells. However, in both cases, the errors induced are limited and do not affect our conclusions regarding a difference in If density.

The faster beating rates of EBs at the late stage may be related to both an increase in the If density and the number of cells that possess If. We examined this more closely by correlating If density with DDR and action potential frequency. If density was positively correlated with DDR and action potential frequency (Fig. 5B and C). Together, the data are consistent with a contribution of If to maintain higher and more regular beating rates in mESCs.

Inhibition of If reduces beating frequency

We examined the effect of If inhibition on beating frequency with the selective inhibitor ZD7288. To reduce the possibility of non-specific actions, we utilized a relatively low concentration of ZD7288, 0.3 μm, on the single beating EBs. The action of this drug is slow but its perfusion for ∼30 min strongly and significantly slowed beating rates down to ∼20 beats min−1 in the early and intermediate stage, and to ∼35 beats min−1 in the late stage. The observed effect of ZD7288 was not due to time-dependent rundown of If as the beating rate of EBs in the absence of the drug was unchanged over the same period of time (Fig. 6A).

Figure 6. Inhibition of If reduces the beating frequency of embryoid bodies (EBs).

Figure 6

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A, the beating frequency of single EBs is significantly reduced after 30 min perfusion with 0.3 μm ZD7288 (*P < 0.05; **P < 0.01). The inset (on the right) shows control group data, measured before and after 30 min without the drug (P > 0.05). B, left, If was reduced after 30 min perfusion with 0.3 μm ZD7288. Right, the change in If at −150 mV at both stages was calibrated from the change in the control group and normalized to the currents recorded 30 min after the seal opening (P < 0.05, one sample t test).

The inhibitory effect of ZD7288 on If in mESCs was also tested. If was slowly reduced in the presence of 0.3 μm ZD7288 at voltage steps ranging from −70 mV to −150 mV. After 30 min perfusion with ZD7288 a significant decrease in If by approximately 15% was seen at −150 mV at both stages (Fig. 6B). The data in Fig. 6 suggest that the amount of block of If was similar over the range of potentials examined. The time course of the effect of ZD7288 on If is consistent with its effect on beating EBs. The similarity in the amount of inhibition of If in early and late stage cells is also consistent with its actions on beating EBs at the two stages. Taken together, these data strongly support a contribution of If to maintenance of faster beating in mESCs at all stages of development.

Autonomic regulation of If contributes to the regulation of heart rate at early and late stages of development in mESCs

Autonomic regulation has not been reported in the R1 stem cell line. To determine whether β-adrenergic and/or muscarinic modulation of If is associated with modulation of rhythmicity and/or frequency of spontaneous beating, in the R1 cell line, the actions of the β-adrenergic agonist isoproterenol (Iso) and the muscarinic agonist acetylcholine (ACh) were examined on the beating frequency of EBs. As shown in Fig. 7A, perfusion of Iso (1 μm) significantly increased beating rate in EBs, to a similar extent in both early and late stage cells (P > 0.05).

Figure 7. Isoproterenol (Iso) shifts the If activation curve to more positive potentials and increases beating rate, in both early and late stage cells.

Figure 7

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A, a bar graph of the beating rate of single EBs before and during perfusion with 1 μm Iso at each stage (*P < 0.05; **P < 0.01). B, upper, If traces elicited at −90 mV for 2 s in a late stage cell before (grey) and during (black) perfusion of Iso. Middle, bar graph of the change of If at −90 mV in response to Iso at early and late stages (*P < 0.05; **P < 0.01). Lower, If traces from a late stage cell before applying Iso (grey), during perfusion of Iso after 1 min (dark grey) and after 3 min (black). Cells were held at −90 mV for 5 s then stepped to −130 mV for 3 s. Note the increase in If amplitude at −90 mV in response to Iso but the lack of any change at −130 mV after 1 min. C, upper, action potentials recorded from a late stage cell before (continuous) and during perfusion of Iso (dashed). Middle, bar graphs of action potential frequency and DDR before and during perfusion of Iso (*P < 0.05). Below, bar graph of MDP and activation threshold before and during perfusion of Iso (P > 0.05).

To test if the observed effects of Iso could be attributed to an effect on If, two protocols were utilized. First, cells were pulsed to −90 mV, from −35 mV, and If was measured every 10 s before and during the application of Iso. Perfusion with Iso (1 μm) for 1 min increased If at −90 mV, as shown in Fig. 7B (upper traces, compare black and grey traces). The increase was approximately 18% at both early and late stages (Fig. 7B, bar graph). To test for a shift in the voltage dependence of If activation in mESCs, a two-pulse protocol was used. The cells were pulsed first to −90 mV for 5 s, and then to −130 mV for 3 s. After 1 min, Iso increased If at −90 mV but did not alter the maximum amount of current (Fig. 7B, lower traces, compare light and dark grey traces). The increase of If in the mid-activation range and the lack of any increase in the fully activated range indicates that Iso shifted the voltage dependence of If activation to more positive voltages. Rundown of If was not apparent after 1 min perfusion, but it was apparent after 3 min in Iso (compare black and dark grey traces), which was noticeable especially at −130 mV. At −90 mV, the rundown after 3 min in Iso was slight; the current was still larger than the control current. Thus, the effect of Iso on If was still notable in the face of current rundown.

To determine the effects of autonomic stimulation on the action potential, spontaneous electrical activity was recorded in the absence and presence of Iso (1 μm). Reliable recordings of action potentials from early stage cells, before and during perfusion of Iso, were difficult to obtain because they often stopped beating or the beating became very irregular during the recordings. Therefore, these experiments were carried out only at the late stage. In these cells, Iso increased both DDR and action potential frequency without the change of MDP and action potential activation threshold (Fig. 7C).

The effects of ACh were the opposite to those of Iso. Perfusion of ACh (1 μm) significantly decreased beating rate in EBs. The effects were similar between early and late stages (Fig. 8A), but smaller in the intermediate stage EBs (P < 0.01), compared to the late stage EBs. The same protocols used for Iso were utilized to determine the effects of ACh on If. Perfusion with ACh (1 μm) for 1 min decreased If at −90 mV, as shown in Fig. 8B (upper traces, compare light grey and dark grey traces). Upon washout of ACh, If returned to control values (black trace), indicating the reduction was not due to current rundown. The decrease was about 15% at both the early and later stages (Fig. 8B, bar graph). To test for a shift in the voltage dependence of If activation in mESCs, the same two-pulse protocol was used as above. After perfusion of ACh for ∼1 min, ACh decreased If at −90 mV but did not affect the current at −130 mV (Fig. 8B, lower traces, compare light and dark grey traces). The decrease in If in the mid-activation range and the lack of any decrease in the fully activated range indicates that ACh shifted the voltage dependence of If activation to more negative voltages. After 3 min in ACh, a small decrease in current was apparent at −90 mV and a larger decrease in current was apparent at −130 mV (compare black and dark grey traces). As, after 3 min in ACh, the decrease of If at −130 mV was similar to that as observed in Iso, it was also probably due to the current rundown.

Figure 8. Acetylcholine (ACh) shifts the If activation curve to more negative potentials and decreases beating rate, in early and late stage cells.

Figure 8

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A, bar graph of the beating rate of single EBs before and during perfusion with 1 μm ACh at each stage (*P < 0.05; **P < 0.01). B, upper, If traces elicited at −90 mV for 2 s in a late stage cell before (grey) and during (black) perfusion of ACh. Note the return of the current to control values after 1 min. Middle, bar graph of the change of If at −90 mV in response to ACh at early and late stages (*P < 0.05; **P < 0.01). Lower, If traces from a late stage cell before applying ACh (light grey), during perfusion of ACh after 1 min (dark grey) and after 3 min (black). Cells were held at −90 mV for 5 s then stepped to −130 mV for 3 s. Note the decrease in If amplitude at −90 mV in response to ACh but the lack of any change at −130 mV, after 1 min. C, above, action potentials recorded from a late stage cell before (continuous) and during perfusion of ACh (dashed). Note the decrease in the MDP of cells during perfusion of ACh. Middle, bar graph of action potential frequency and DDR before and during perfusion of ACh (*P < 0.05). Below, bar graph of MDP and activation threshold before and during perfusion of ACh (**P > 0.05).

Opposite to Iso, ACh decreased both DDR and action potential frequency, without a change in MDP and activation threshold (Fig. 8C). In either ACh or Iso, both DDR and action potential frequency were modified in parallel, which suggests that changes in beating frequency were due in large measure to changes in the time required to reach threshold. Furthermore, the changes in DDR are consistent with a contribution of If to the changes in beating rate induced by autonomic stimulation. Taken together, the data suggest that modulation of If contributes to alterations in beating frequency elicited by autonomic stimulation.

Discussion

HCN channels and If confer faster and more regular rhythmicity to beating mouse embryonic stem cells

In this paper, we show that HCN channels and If confer more regular and faster rhythmicity to beating mESCs. This conclusion is based on the following evidence. If density was correlated with action potential frequency and the rate of diastolic depolarization. A subset of mESCs without detectable If beat significantly more slowly and more irregularly than cells with If. The threshold for If activation and maximal diastolic potentials recorded from spontaneously active cells were similar and potentially overlap. The beating rate of single EBs and If were reduced by relatively low concentrations of the If blocker ZD7288. A developmental change of If is also associated to the increased beating rate during mESC development. If density, the proportion of cells expressing If, and the If activation rate all increased in the late stage mESCs, which may synergistically contribute to the faster rates of beating following the development.

As is the case for other spontaneously active cells, such as cardiomyocytes of the sinoatrial node, mechanisms not involving If contribute to the generation and modulation of spontaneous activity in mESCs. In mESCs, a contribution to beating and the diastolic depolarization from the T-type calcium channel has recently been suggested (Zhang et al. 2003). Other potential contributors include inward currents from the L-type calcium and sodium channels, instantaneous currents such as the Na+-sensitive background current (INa,b), and release of intracellular calcium from ryanodine-sensitive stores, all of which have been suggested to contribute to spontaneous activity in the neonatal and adult sinoatrial node (Hagiwara et al. 1992; Noble et al. 1992; Irisawa et al. 1993; Baruscotti et al. 1996; Bogdanov et al. 2001). Further studies will be required to determine which mechanisms contribute in mESCs, and to what extent each is involved under different conditions.

In our studies, we found a subset of beating mESCs that did not possess detectable If. These cells beat more slowly than those with detectable levels of If, consistent with a contribution from HCN channels to beating. In addition to the above mentioned mechanisms, a slower, rhythmic, release of intracellular calcium through IP3-sensitive stores may contribute to beating in mESCs that do not possess If (Viatchenko-Karpinski et al. 1999; Mery et al. 2005; Maltsev et al. 2006).

HCN isoforms and electrophysiological characteristics of If in mESC

In this study, Western blot analyses suggest that HCN2 and HCN3 isoforms are present in both early and late stage mESCs, as well as in the undifferentiated cells. The amount of HCN3 decreased over the course of development in culture. This profile is consistent with the electrophysiological data. The rate of If activation in mESCs is slower and faster than rates of If activation recorded from mammalian cells expressing HCN2 and HCN3, respectively. The decrease in the amount of more slowly activating HCN3 channels could also explain the increase in the rate of If activation from early to late stage. These findings are consistent with those of the embryonic mouse heart, which has been shown to possess large amounts of HCN2, and lesser amounts of HCN3 (Yasui et al. 2001; Kuwahara et al. 2003; Stieber et al. 2003; Whitaker et al. 2007). We were not able to detect HCN1 or HCN4 protein in our preparations. This may have been due to low amounts in, or their absence from, these cells, or because the antibodies we used were not able to detect them.

We also found both HCN2 and HCN3, but not HCN1 or HCN4, in undifferentiated mESCs. This is consistent with findings from another study which, using the same R1 cell line, were able to measure If and identified transcripts for HCN2 and HCN3, but not for HCN1 or HCN4, in undifferentiated mESCs (Wang et al. 2005).

The activation kinetics of If, and the position of the If activation curve, determined from mouse embryonic cardiomyocytes (Yasui et al. 2001; Stieber et al. 2003; Herrmann et al. 2007) are similar to those we determined in mESCs. The quickening of If activation rate found in our experiments may contribute to the increase in beating frequency observed over the course of development in culture. In contrast, the beating rates of human embryonic stem cells (hESCs) decrease along with the rate of If activation (Sartiani et al. 2007). Consistent with the developmental change in beating frequencies of stem cells, embryonic heart rates increase in mouse and decrease in human, but it is not known whether the rates of If activation change. In human embryonic stem cells, the decrease in rate of If activation is correlated with a decrease in the expression of HCN1 and HCN4, whereas levels of HCN2 remain constant over the course of development. Interestingly, these authors suggest that the change in isoform expression may be more consistent with a ventricular, rather than pacemaker, phenotype. More detailed experiments will be required to identify stem cells programmed to become specific cardiac cell types with certainty.

Beating rate and If are modulated by autonomic agonists

In this study, both the beating rate and If were reduced and increased by ACh and Iso, respectively, to a similar extent in early and late stage mESCs. These results suggest that the mechanisms responsible for autonomic modulation are already developed at the early stage in mESCs. Our findings in the R1 cell line are not completely consistent with the previous studies using the D3 mouse stem cell line. One study suggested muscarinic stimulation via If only occurs at the early stage, while β-adrenergic stimulation was observed only at the late stage (Abi-Gerges et al. 2000). Another study (Banach et al. 2003) found that the muscarinic response on beating EBs could be observed only at later stages of development in culture (after D7 + 5). Differences may be due to the heterogeneity of the mESC phenotype. Also, both of those studies used carbachol to examine muscarinic responsiveness, whose actions sometimes differ from those of ACh, which we used in our study.

As mouse HCN3 does not respond to cAMP, unlike mouse HCN2 (Mistrik et al. 2005), we might have expected that the extent of If modulation by ACh and Iso would have differed between the early and late cells, especially if they form two separate sets of homomeric channels. However, it is not clear as to whether HCN2 and HCN3 co-assemble to form heteromeric channels, and how these mixed channels would respond to changes in cAMP. The similarity in the effects of autonomic stimulation of If between the early and late stage cells suggests that HCN2 and HCN3 channels may form heteromeric channels in early stage mESCs that respond in a way that is not different from the response of channels made up of predominantly HCN2 subunits in late stage mESCs.

Summary and perspectives

In summary, we show that HCN channel expression is developmentally regulated, and that it confers more regular and faster rhythmicity to mESCs over the course of development in culture. Both If density and beating frequency increase over the course of development which, together with previous studies, suggest that the R1 cell line mirrors the developmental pattern observed in embryonic mouse hearts. The use of mESCs as a model for the development of the embryonic mouse heart is also supported by our data showing that HCN2 and HCN3 are found in both mESCs and the embryonic mouse heart. Further studies are needed to uncover the pattern of HCN isoform expression in the mouse embryonic heart which is presently not clear. An understanding of the mechanisms that regulate HCN expression during embryonic development, and how this expression is related to the emergence of a pacemaker cell phenotype, are important issues that also need to be addressed. The ease with which they can be manipulated genetically, and studied by electrophysiological and other approaches, make mESCs a useful and important model system for studying these issues.

Acknowledgments

The authors are grateful for the input of Dr Pamela Hoodless in the early phases of this work. These studies were generously supported by an operating grant from Canadian Institutes of Health Research (G.F.T. and E.A.A.), a Grant-in-Aid from the Heart and Stroke Foundation of British Columbia and the Yukon (E.A.A.), and a postdoctoral fellowship from the Heart and Stroke Foundation of Canada (Y.Q.). L.H.-M. is a recipient of a ‘Ramon y Cajal’ grant from the Spanish Ministry of Science and Technology and E.A.A. and G.F.T. are the recipients of Tier II and Tier I Canada Research Chairs, respectively. The gift of HCN2- and HCN3-specific antibodies from Ryuichi Shigemoto are gratefully acknowledged. We thank Dr S. X. Bamji (UBC) for the embryonic tissue. Finally, comments from anonymous reviewers were greatly appreciated.

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