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Am J Physiol Heart Circ Physiol 294: H979-H985, 2008. First published December 21, 2007; doi:10.1152/ajpheart.00293.2007
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Go controls the hyperpolarization-activated current in embryonic stem cell-derived cardiocytes

Chian P. Ye,1,* Sheng Zhong Duan,2,* David S. Milstone,3 and Richard M. Mortensen2,4

1Division of Endocrinology, Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts; 2Department of Molecular and Integrative Physiology, University of Michigan Medical School, Ann Arbor, Michigan; 3Vascular Research Division, Department of Pathology, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts; and 4Department of Pharmacology and Department of Internal Medicine, Metabolism Endocrine and Diabetes Division, University of Michigan Medical School, Ann Arbor, Michigan

Submitted 8 March 2007 ; accepted in final form 18 December 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Hyperpolarization current (If) is an important player in controlling heart rate and is stimulated by cAMP and inhibited by members of the pertussis toxin-sensitive G-protein Gi/Go family. We have successfully derived cardiocytes from embryonic stem cells lacking Go or Gi2 and Gi3. We have established that both basal and isoproterenol-stimulated activities of If in these cardiocytes have typical nodal-atrial characteristics and are unaffected by targeted gene inactivation of the G proteins Go or Gi2 and Gi3. Under basal conditions, both Go and Gi are required for muscarinic inhibition of If activity via a mechanism that involves the generation of nitric oxide, whereas, with prior stimulation by β-agonists, only Go is required and Gi and nitric oxide production are not. Our findings establish an essential role for Go in the antiadrenergic effect of muscarinic agent on If.

G proteins; ion channels; nitric oxide; muscarinic receptors

ALTERATION IN THE CARDIAC sinoatrial node spontaneous discharge rate is a central mechanism for the control of heart rate. Heart rate is stimulated by sympathetic stimulation through β-adrenergic receptors and the activation of Gs and inhibited by parasympathetic activity through M2 muscarinic acetylcholine receptors and the activation of Gi/Go. Understanding the pathways that control heart rate will lead to the better understanding of arrhythmias of the sinoatrial node associated with aging and cardiovascular diseases. There has been considerable debate about the specific signal transduction pathways and ion channels that regulate heart rate in response to M2 muscarinic stimulation (3, 4, 10, 11, 24, 28).

Activation of the inwardly rectifying potassium current, (IK-ACh) has been hypothesized to play a major role in the negative chronotropic effects of parasympathetic stimulation (24). However, the importance of IK-ACh has been challenged because no correlation has been established between the dose response of this channel and cardiomyocyte slowing (12). In embryonic stem (ES) cell-derived cardiocytes, gene inactivation of {alpha}i2 or {alpha}i3 eliminates the muscarinic stimulation of IK-Ach but does not affect cardiomyocyte slowing (26). Inactivation of IK-Ach by G protein-activated inwardly rectifying K+ channel 4 knockout, which eliminates basal and stimulated IK-ACh activity, was shown to nearly eliminate the heart rate variability and to decrease the negative chronotropic effects of parasympathetic stimulation (27). These results show that IK-ACh plays a critical role in regulation of heart rate and heart rate variability but that other pathways and ion channels are also involved in regulation of the negative chronotropic effects.

If, the hyperpolarization-activated nonselective cation current, has been proposed to contribute to the negative chronotropic effects of parasympathetic stimulation (1, 10, 21). It has been reported that an If mutation results in reduced heart rates in zebrafish embryos (4). Like the L-type Ca2+ channel, the activity of If is modulated by G-protein-regulated second messenger systems (6, 29). A role for the adenylyl cyclase/cAMP system in transmitting β-adrenergic and muscarinic receptor input has been extensively documented (2, 25). More recently, nitric oxide (NO) was shown to stimulate basal If activity directly and to inhibit the isoproterenol (Iso)-stimulated activity of If in preparations of isolated sinoatrial nodal cells (15, 16, 31). Furthermore, the effects of acetylcholine and NO appear to involve distinct regulatory mechanisms (5, 31).

Because it has not been defined which Gi/Go protein is responsible for the negative chronotropic effects on If by parasympathetic stimulation, we hypothesize that specific G proteins (Gi2, Gi3, and/or Go) control If activity under different conditions (basal or adrenergic stimulation). We have used cardiocytes derived from wild-type (WT) and mutant mouse ES cells in which specific genes for G proteins have been inactivated (26) to study the muscarinic regulation of If.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cell lines and culture. The generation of mutant ({alpha}o- and {alpha}i2/{alpha}i3-null) mouse D3 ES cell lines has been described previously (26, 30). Briefly, constructs with a neomycin resistance gene inserted in a required exon were used to target the endogenous {alpha}o and {alpha}i3 genes in WT D3 ES cells. The {alpha}i3-null cell line was transfected with a hygromycin containing an {alpha}i2 construct to produce the cell line lacking both {alpha}i2 and {alpha}i3 ({alpha}i2/{alpha}i3-null). Both WT and mutant D3 ES cell lines were maintained and differentiated as before. After 14–18 days of differentiation, spontaneously contracting cardiocytes were dissociated by collagenase digestion or mechanically for study of If.

Electrophysiological measurements. All recordings were performed at ambient temperature (22–24°C). Action potentials were recorded with the use of a nystatin-perforated whole cell configuration in current-clamp mode (19). Pipettes with a tip diameter of 1–2 µm were filled with a solution containing (in mM) 90 potassium aspartate, 50 KCl, 4 MgCl2, 10 HEPES (pH 7.4), and 100 µg/ml nystatin. After stable basal whole cell recordings, the bath was perfused with agent-containing (Iso or carbachol) solution, and the recordings continued for 15–20 min.

Conventional whole cell recordings in the voltage-clamp mode were performed for characterization of If. The standard bath solution contained (in mM) 150 NaCl, 5.4 KCl, 0.5 MgCl2, 1.8 CoCl2, 5 HEPES (pH 7.4), and 10 glucose. Other currents (calcium, potassium, and sodium currents) were blocked by addition of 3 mM CaCl2, 2 mM BaCl2, and 30 µM tetrodotoxin, respectively. The internal solution contained (in mM) 90 potassium aspartate, 50 KCl, 5 EGTA, 3 MgATP, 0.3 NaGTP, and 5 HEPES (pH 7.3). If was activated by 7-s-long hyperpolarizing voltage steps ranging from –45 to –110 mV (in 5-mV increments) from a holding potential of –35 mV, and currents were measured at the end of the 7-s pulse. We also used the following conditions to further identify the If current in these cells: low Na+ (2.5 mM extracellular K+, 90 mM extracellular Na+) and high K+ (20 mM extracellular K+, 130 mM extracellular Na+).

The role of NO was studied by preincubating cells with NG-monomethyl-L-arginine (L-NMMA; 0.1 mM) for 30 min before the patch-clamp experiment. L-NMMA was present continuously throughout the recording period.

Calculations and statistics. The depolarization rate was calculated as dividing amplitude changes (mV) by time (ms) during the diastolic phase of each curve of each cell line under each condition. The kinetic properties of If were characterized by the relationship between the specific conductance (G) and membrane potential. At each voltage, G was obtained by dividing the current amplitude by the driving force (V – Vr), where V is the hyperpolarizing potential and Vr is the reversal potential determined through current-voltage curves of tail currents. The normalized conductance data (G/Gmax) were plotted and fitted with the Boltzmann distribution equation: G/Gmax = 1/1 + exp[(V – V50)/k]. The potential eliciting half-maximal response (V50) and the inverse slope factor (k) were obtained by a least-squares method using Graphpad Prism software. Shifts of the If activation curve in response to each agent were calculated as the difference in V50. Dose-response curves were compared by two-way ANOVA and Bonferroni posttests using Graphpad Prism software. The beating rates of cardiocytes were expressed as means ± SE, and statistical comparisons between groups were performed by Student's t-test using the same software.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
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 REFERENCES
 
Mutant ES cells lacking {alpha}o ({alpha}o-null) and both {alpha}i2/{alpha}i3 ({alpha}i2/{alpha}i3-null) were isolated and characterized previously (26, 30). Western blots confirmed gene inactivation for the individual knockout cell lines and reflected no compensatory changes in expression of other G-protein subunits (data not shown). These results are the same as reported before (26, 30). Compared with WT cells, {alpha}o-null and {alpha}i2/{alpha}i3-null cells showed no significant differences in growth characteristics or in their ability to differentiate into cardiocytes.

Spontaneous action potential. Two weeks after the induction of differentiation, the cells were put in normal Tyrode solution and observed under microscope. Only those individual spindle-shaped cells that continued to contract after 5 min were chosen for the present study. Spontaneous action potentials recorded from WT and mutant cells displayed the characteristic shape of atrial-nodal cardiocytes (Fig. 1). In the basal condition, the action potentials occurred at frequencies that were not significantly different among the cell lines. Addition of the β-adrenergic agonist Iso (20 nM) increased the rate of diastolic depolarization and increased the firing rate in all cell lines. The muscarinic agonist carbachol (50 nM) decreased the Iso-stimulated firing rate of action potentials in WT and {alpha}i2/{alpha}i3-null cells (Fig. 1, A and B) but not in {alpha}o-null cells (Fig. 1C). Addition of carbachol also brought the firing rate in Iso-stimulated WT cells to lower than basal condition (Fig. 1A). Carbachol alone decreased the firing rate in all cell lines (38 ± 2 beats/min for WT, 43 ± 5 beats/min for {alpha}i2/{alpha}i3-null, 40 ± 3 beats/min for {alpha}o-null; P > 0.05 for comparisons between different cell lines), consistent with previous data on {alpha}i2-null cells and {alpha}i3-null cells (26). Although the isolated hearts from {alpha}o-null mice respond to carbachol alone with slightly less sensitivity (14), it would be difficult to detect this small change in cell culture. Furthermore, changes in diastolic depolarization rate in all cell lines followed the same trend as the firing rate under different conditions.


Figure 1
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  		Fig. 1. Effects of carbachol [Carb (C)] and isoproterenol [Iso (I)] on spontaneous action potentials of terminally differentiated embryonic stem (ES) cell-derived cardiocytes. Nystatin-perforated whole cell recordings in current-clamp mode were performed on individual myocytes 14–18 days after induction of differentiation. A: membrane potential traces before (basal) and after superfusion of Iso (20 nM), followed by Iso plus Carb (50 nM) in wild-type (WT) cells. B: results for {alpha}i2/{alpha}i3-null cells. C: results for {alpha}o-null cells. B, basal. BPM represents the number of spikes/min. Depolarization rate represents the change of mV over time (ms) in the diastolic depolarization. *P < 0.01, Iso vs. B; **P < 0.01, Iso + Carb vs. I; ^P < 0.05, Iso + Carb vs. B.

 
Characterization of If. Examples of whole cell current traces (Fig. 2A) revealed a slowly activating inward current evoked by hyperpolarization. The voltage dependence of current activation is illustrated in the activation curve (Fig. 2B). Under basal conditions, identical whole cell currents were also obtained from {alpha}i2/{alpha}i3-null or {alpha}o-null cells (traces not shown). V50 results were –70.3 ± 0.4 mV (n = 10) for WT, –71.0 ± 0.7 mV (n = 7) for {alpha}i2/{alpha}i3-null, and 70.7 ± 0.6 mV (n = 8) for {alpha}o-null; k results were –8.5 ± 0.5 mV (n = 10) for WT, –8.8 ± 0.9 mV (n = 7) for {alpha}i2/{alpha}i3-null, and –8.3 ± 0.7 mV (n = 8) for {alpha}o-null cells. No differences were shown among the cell lines.


Figure 2
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  		Fig. 2. Characteristics of hyperpolarization current (If) in ES cell-derived cardiocytes. Conventional whole cell recordings in the voltage-clamp mode were performed. An individual cardiocyte was voltage-clamped from a holding potential of –35 mV to various hyperpolarizing voltages (–45 to –110 mV in 5-mV increments). A: representative current traces. Dotted line indicates the steady-state current at –35 mV. B: normalized specific conductance (G/Gmax) plotted vs. hyperpolarizing voltage. Half-maximal activation voltage (V50) and slope factor (k) are derived by fitting data in a Boltzmann equation (solid line). C: tail-current analysis for the If reversal potential. D: relationship between the tail current (pA/pF) and test voltage steps in different extracellular solutions. Standard condition = 5.4 mM extracellular K+ and 150 mM extracellular Na+. Low Na+ = 2.5 mM extracellular K+ and 90 mM extracellular Na+. High K+ = 20 mM extracellular K+ and 130 mM extracellular Na+.

 
A typical If in sinoatrial node cells has lower amplitude with lower concentrations of Na+ and higher amplitude with higher concentrations of K+ (1, 10, 20). To test whether If in ES cell-derived cardiocytes have these characteristics, we recorded tail currents in bath solutions containing different concentrations of Na+ and K+. The representative currents are shown in Fig. 2C. The Vr, determined from the current-voltage curves of these tail currents (Fig. 2D), demonstrated a shift in Vr from –26.2 to –37.3 mV when Na+ was lowered from 150 to 90 mM. In contrast, an increase in extracellular K+ from 5.4 to 20 mM shifted Vr to a more positive potential, confirming that the current described above is a nonselective cation current carried by both Na+ and K+. These characteristics are similar to those of If in sinoatrial node cells.

This inward current was also found to be sensitive to cesium and not altered by externally applied barium (data not shown). Overall, the electrophysiological characteristics of If expressed in the ES cell-derived cardiocytes were similar to the If from sinoatrial nodal cells (7, 9, 10). Furthermore, our data showed that these basal characteristics were not altered by inactivation of the pertussis toxin (PTX)-sensitive {alpha}-subunits. If currents were observed in ~75–80% of spontaneously contracting cardiocytes tested (a total of 108 cells from 22 preparations), and this percentage did not vary significantly between WT and mutant cells.

Inhibition of basal If requires multiple G proteins. We examined the ability of the muscarinic agonist carbachol to modulate basal If activity in WT and {alpha}-subunit-null ES cell-derived cardiocytes. The basal If traces elicited using the same voltage protocols for WT and mutant cells were quite similar. The voltage dependence of current activation is illustrated in the activation curve (Fig. 3). Externally applied carbachol (1 µM) reduced basal If amplitude at –70 mV and shifted the activation curve to a more negative V50 in WT cells; the voltage shift, {Delta}V50 (carbachol V50 – basal V50), is –6.30 ± 1.33 mV (n = 4) for WT cells (Fig. 3A). Carbachol had no significant effect on the current of either {alpha}i2/{alpha}i3-null or {alpha}o-null cardiocytes; the {Delta}V50 is –1.47 ± 0.82 mV (n = 4) for {alpha}i2/{alpha}i3-null cells and –0.85 ± 0.78 mV (n = 5) for {alpha}o-null cells (Fig. 3, B and C). A consistent pattern was observed for the mutant cell lines when the current amplitudes at –70 mV were compared. The current amplitude at –70 mV was measured because it is close to V50, at which the inhibition by carbachol has larger effects than at full-activation voltage.


Figure 3
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  		Fig. 3. Effects of Carb on If of WT (A), {alpha}i2/{alpha}i3-null (B), and {alpha}o-null (C) cells. Activation curves are shown for normalized specific conductance (G/Gmax) vs. hyperpolarizing voltage for WT, {alpha}o-null, and {alpha}i2/{alpha}i3-null cells. Curves were fit to data points by the Boltzmann distribution. Symbols and bars represent means ± SE for 4–6 individual cells. Top: representative traces. In A, Carb curve is significantly different from the basal curve (2-way ANOVA: P < 0.0001; Bonferroni posttests: **P < 0.01, #P < 0.001). Carb curve and basal curve are not statistically different by 2-way ANOVA in either B (P = 0.08) or C (P = 0.072).

 
Inhibition of β-agonist-stimulated If requires {alpha}o but not {alpha}i. β-Adrenergic agonists increase If activity in native nodal cells, and muscarinic agonists oppose the stimulation (9, 29). We examined the ability of carbachol to oppose β-adrenergic stimulation (Iso) of If in WT and {alpha}-subunit-null cardiocytes. Experiments were performed with the same protocol shown in Fig. 2A. Current amplitude at baseline (no additions), 3 min after exposure to Iso (0.5 µM), and 3 min after addition of both Iso and carbachol (1 µM) were measured at each hyperpolarizing voltage step. At –70 mV, Iso stimulated an approximately twofold increase in current amplitude; however, there was a much smaller increase at –110 mV. This voltage dependency of the adrenergic stimulation of If in these ES cell-derived cardiocytes is similar to that in ventricular myocytes (17). The activation curves (G/Gmax vs. voltage) are shown in Fig. 4. Data were analyzed with the Boltzmann equation; the effects of Iso and Iso plus carbachol on V50 and k were summarized. We found no significant differences in the effects of Iso on V50 or k between the WT and {alpha}-subunit-null cells. The positive shift induced by Iso in V50 was 9.1 ± 1.2, 9.4 ± 1.4, and 8.8 ± 1.8 mV for WT, {alpha}i2/{alpha}i3-null, and {alpha}o-null cells, respectively (n = 4 for each cell type). All of the activation curves for Iso-stimulated currents had steeper voltage dependence, indicated by the inverse slope factor k, in the range of –8.0 to –7.7 mV for all cell types. In the majority of experiments, currents reached the maximum amplitude less than 1 min after exposure to Iso.


Figure 4
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  		Fig. 4. Antiadrenergic effect of Carb in WT (A), {alpha}i2/{alpha}i3-null (B), and {alpha}o-null (C) cells with activation of If by Iso. Curves were plotted as described in MATERIALS AND METHODS and Fig. 3. Effect of Carb on Iso-stimulated If current amplitude was determined by stimulating cells with 0.1 µM Iso for at least 5 min followed by Carb at the indicated concentrations. Results are expressed as the percentage of Iso-stimulated current amplitude before the addition of Carb. Symbols and bars represent means ± SE for 4–6 individual cells. Top: representative traces. In A, Iso curve is significantly different from the basal curve (2-way ANOVA: P < 0.0001; Bonferroni posttests: *P < 0.05, #P < 0.001). Iso + Carb curve is significantly different from the basal curve (2-way ANOVA: P = 0.001; Bonferroni posttests: **P < 0.01, &P < 0.001). Iso + Carb curve is also significantly different from the Iso curve (2-way ANOVA: P < 0.0001; Bonferroni posttests: $P < 0.001). In B, Iso curve is significantly different from the basal curve (2-way ANOVA: P < 0.0001; Bonferroni posttests: #P < 0.001). Iso + Carb curve is not significantly different from the basal curve (2-way ANOVA: P = 0.11); instead, it is significantly different from the Iso curve (two-way ANOVA: P < 0.0001; Bonferroni posttests: &P < 0.01, $P < 0.001). In C, Iso curve is significantly different from the basal curve (2-way ANOVA: P < 0.0001; Bonferroni posttests: *P < 0.05, #P < 0.001). Iso + Carb curve is significantly different from the basal curve (2-way ANOVA: P < 0.0001; Bonferroni posttests: &P < 0.001), but the Iso + Carb curve is not significantly different from the Iso curve (2-way ANOVA: P = 0.19).

 
In WT cells, carbachol completely reversed the stimulatory effect of Iso on If and reduced the current below its basal level, causing a –4.1 ± 1.3 mV shift in V50 from basal (Fig. 4A). This was similar to the results seen with addition of carbachol to unstimulated cells. In cells lacking both {alpha}i2 and {alpha}i3, carbachol reversed the Iso-induced increase in If but did not decrease current activity below the basal level (Fig. 4B). In sharp contrast, carbachol had no effect on the Iso-stimulated current in cells lacking {alpha}o (Fig. 4C). A dose-response curve generated from similar experiments showed that, even at higher concentrations of carbachol (10–5 M), the Iso-stimulated current was not significantly reduced in the {alpha}o-null cardiocytes, as seen in {alpha}i2/{alpha}i3-null cells (Fig. 5). Without Iso stimulation, both {alpha}o-null and {alpha}i2/{alpha}i3-null cells showed significantly less sensitivity to a range of carbachol concentrations (Fig. 5).


Figure 5
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  		Fig. 5. Dose response of Carb effects in WT, {alpha}o-null, and {alpha}i2/{alpha}i3-null cells with and without activation of If by Iso. Currents were recorded for at least 4–5 min at each concentration. Current amplitude measured at the end of a –70-mV voltage step is expressed as the percentage of basal amplitude and plotted vs. concentration of Carb. Symbols and bars represent means ± SE for 4–6 individual cells. A: basal condition. The {alpha}o-null and {alpha}i2/{alpha}i3-null curves are both significantly different from the WT curve (2-way ANOVA: P < 0.0001; Bonferroni posttests: **P < 0.01, #P < 0.001) but not significantly different from each other (2-way ANOVA: P = 0.22). B: Iso-stimulated condition. The {alpha}o-null curve is significantly different from both WT and {alpha}i2/{alpha}i3-null cells (2-way ANOVA: P < 0.0001; Bonferroni posttests: #P < 0.001 vs. WT, $P < 0.001 vs. {alpha}i2/{alpha}i3-null). WT and {alpha}i2/{alpha}i3-null curves were not significantly different (2-way ANOVA: P = 0.38).

 
Because of previous controversial reports about the role of NO in {alpha}o-mediated pathways, we investigated the effects of a NO synthase inhibitor, L-NMMA. Pretreatment of WT cells with L-NMMA (0.1 mM) ameliorated the inhibitory effect of carbachol on basal If. The activation curves showed that the NO synthase inhibitor did not alter the basal characteristics of If (V50 or k) but that it did prevent carbachol from inducing a leftward shift in the activation curve (Fig. 6A). We also examined the effect of L-NMMA on the anti-β-adrenergic effect of carbachol in WT cells (Fig. 6B). Again, the NO synthase inhibitor had no effect on basal or Iso-stimulated current characteristics (current amplitude, V50, and k). Pretreatment of the WT cells with L-NMMA did not prevent the anti-β-adrenergic effect of carbachol. However, the NO synthase inhibitor prevented carbachol from further reducing If activity below the basal level.


Figure 6
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  		Fig. 6. Effect of the nitric oxide synthase inhibitor NG-monomethyl-L-arginine (L-NMMA) on If modulation. A: WT cells were incubated with 0.1 mM L-NMMA for 30 min before the experiment, and L-NMMA was present continuously throughout the recording period. After basal activity was recorded for 4–5 min, cells were exposed to 1 µM Carb. The effect of L-NMMA on inhibition of basal If activity by Carb is illustrated in the activation curves. Each point is the mean ± SE for 4 cells. No statistically significant differences were detected (2-way ANOVA: P = 0.084). B: effect of L-NMMA on Iso (0.1 µM) stimulation and the inhibition of Iso-stimulated If activity by Carb. By 2-way ANOVA, P < 0.0001 for Iso vs. basal and Iso vs. Iso + Carb. Bonferroni posttests: *P < 0.05 vs. basal, #P < 0.001 vs. basal, **P < 0.05 vs. Iso + Carb, &P < 0.01 vs. Iso + Carb, $P < 0.001 vs. Iso + Carb. There was no statistically significant difference between Iso + Carb group and basal group (2-way ANOVA: P = 0.091).

 

   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
To our knowledge, this is the first report to define the G-protein-signaling requirements for muscarinic-receptor modulation of If using cardiocytes lacking specific PTX-sensitive G proteins. ES cell-derived cardiocytes have been demonstrated to recapitulate many characteristics of sinoatrial node cells (1, 10, 20). More importantly, they are much easier to be genetically modified than adult pacemaker cells, which are very difficult to isolate.

We had previously established that Gi2 and Gi3 (but not Go) are required for activation of IK-ACh (26). Muscarinic inhibition of the Iso-activated L-type calcium channel (IL-Ca) requires Go-dependent regulation of NO-dependent second messenger systems (30). For IL-Ca, expression of both Gi2 and Gi3 is also required for the normal rapid inhibition of Iso-stimulated channel activity. We have now established that Go is also essential for the antiadrenergic effect of carbachol on If and that the signaling mechanism does not appear to involve NO. Finally, muscarinic inhibition of basal If activity appears to require NO generation and all three PTX-sensitive G proteins: Go, Gi2, and Gi3. The points at which these pathways converge have not yet been established. In some cell systems, NO may be a convergence point. Regulation of cAMP is less likely to be a convergence point, since it has been reported that inactivation of {alpha}i2 but not {alpha}o affects cAMP (18, 23).

For If and the L-type calcium channel, there has been considerable debate and disagreement about the role of NO in inhibition by muscarinic receptor activation. Interestingly, we find that the same G-protein subunit ({alpha}o) is required for inhibition of If whether there is prior β-adrenergic stimulation or not. However, Gi and NO synthase activity are only required when inhibiting If below the basal state and not when inhibiting If stimulated by β-adrenergic agonists. The involvement of a second messenger system in the basal state is consistent with the involvement of cAMP in the muscarinic inhibition of If in sinoatrial node myocytes observed by Accili et al. (3). It is not clear whether a second messenger system is involved in the presence of β-agonists. Thus pathways may differ based on the repertoire of other receptors. Therefore, the exact experimental conditions may influence the importance of NO in the pathway.

In these terminally differentiated ES cell-derived cardiocytes, the action potentials were characterized by a diastolic depolarization phase with the membrane potential slowly increasing from –65 to –40 mV, a voltage range well covered by the activation curves of If recorded in this study. Characteristics of the If expressed by these nodal-atrial-like cardiocytes are similar to those of If defined in adult sinoatrial node cells or cardiac ventricular myocytes, in aspects such as the trace of the tail currents and the amplitude change by concentrations of Na+ and K+ (1, 10, 20, 32). Muscarinic agonists decrease If activity via mechanisms involving their inhibitory effect on cellular cAMP levels (7, 13, 22, 25). The activation by Iso and inhibition by carbachol have larger effects at V50 than at full-activation voltage, confirming that β-adrenergic and muscarinic agonists modulate current kinetics (2). It has been reported that acetylcholine inhibits If in Purkinje fibers only after β-stimulation, whereas it also inhibits basal If in the sinoatrial node (8). The present results showed that the modulations of If by adrenergic and muscarinic agonists in the ES cell-derived cardiocytes are the same as those seen in the native If in sinoatrial nodal cells.

We also established that the principal electrophysiological characteristics of If were not altered in the {alpha}-subunit-null cell lines and that the stimulatory effect of the adrenergic agonist Iso was unaffected by inactivation of these PTX-sensitive G proteins. We did note a marked alteration in muscarinic modulation of basal If activity in cell lines lacking {alpha}o or {alpha}i2/{alpha}i3. In both mutants, the ability of carbachol to reduce If activity below basal levels was blunted. This effect was similar to that observed with the NO synthase inhibitor L-NMMA, suggesting that one or more of these PTX-sensitive G proteins may inhibit basal If activity via NO-dependent mechanisms.

Absence of {alpha}o completely prevented the anti-β-adrenergic effect of carbachol on If, whereas absence of both {alpha}i2 and {alpha}i3 did not significantly alter the inhibition by carbachol of Iso-stimulated currents. These data demonstrate that pathways regulated by Go signaling are critical for the anti-β-adrenergic effect of carbachol on If and that Gi2/Gi3 signaling is not required for this response. Furthermore, the inability of L-NMMA to alter the antiadrenergic effect of carbachol on If suggests that NO is not the principal second messenger involved.

These results establish a critical role for {alpha}o-containing heterotrimers in the muscarinic regulation of negative chronotropy and If. The specific G proteins and second messenger systems required for regulation of heart rate, contractility, and cardiac ion channels (If, IL-Ca, and IK-ACh) are different and can vary depending on the cell system analyzed.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Institutes of Health Grants K01 AG-00891-01 (C. P. Ye), PO1 HL-36028 (D. S. Milstone), R01 HD-04089 (D. S. Milstone), and HL-790902 (R. M. Mortensen).


   FOOTNOTES
 

Address for reprint requests and other correspondence: R. M. Mortensen, Depts. of Molecular and Integrative Physiology, 7744 MSII, 1150 W. Med. Ctr. Dr., Ann Arbor, MI 48109-0622 (e-mail: rmort{at}umich.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

* C. P. Ye and S. Z. Duan contributed equally to this work. Back


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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