Adrenergic regulation of the rapid component of delayed rectifier K+ currents in guinea pig cardiomyocytes

Introduction

Cardiac action potential repolarization is carried out by different potassium currents. The rapid component of the delayed rectifier potassium current (I_kr) is the most important component for phase 3 repolarization and is required to control orderly repolarization at the end of each cardiac action potential, particularly near the threshold potential of early afterdepolarizations that can trigger tachyarrhythmias (1). The human ether-a-go-go-related gene (hERG) encodes the alpha subunit of I_kr current (2-5). HERG channels are a primary target for the pharmacological management of cardiac arrhythmias using class III antiarrhythmic agents, such as dofetilide and amiodarone (6-8). Reduced hERG currents due to hERG mutations or excessive blockade of hERG channels by antiarrhythmic or non-antiarrhythmic drugs may lead to congenital or acquired long QT syndrome (9,10). The sympathetic nervous system modulates the I_kr current though both α1 and β-adrenoceptors (ARs) (11-14). Separate stimulation of α₁-ARs or β-ARs decreased I_kr current and prolonged action potential. In recent years, considerable progress has been made toward a detailed understanding of these two signaling pathways. Bian and colleagues revealed that hERG potassium channels or the I_kr current of rabbit cardiomyocytes can be regulated by the α₁-adrenoreceptor agonist phenylephrine (PE), which is consistent with our previous work in guinea pig (12,15). In an early report by Heath and Terrar, a concentration-independent increase in I_kr currents was observed at low concentrations of the β-adrenergic agonist isoprenaline (16), whereas Karle found activation of β-adrenoceptors elicits an inhibitory effect on I_kr or hERG via a cAMP/PKA-dependent pathway (17). Morever, at least three α₁ adrenoceptor subtypes (α_1A, α_1B, and α_1D) and three β adrenoceptor subtypes (β₁, β₂, and β₃) have been pharmacologically identified, it is unclear which AR subtypes primarily regulate I_kr. In addition, the interaction between the α₁ and β-adrenergic components during sympathetic regulation of the cardiac I_kr current were not examined, and their functional importance was not determined.

Methods

Cardiomyocyte preparation and whole-cell patch clamp

Guinea pigs (male, weighing 300±50 g) were purchased from Nanjing Medical University Institutional Animal Care. Single left ventricular myocytes were isolated from the heart of guinea pigs using the Langendorff apparatus as described previously (12). All experiments were performed in accordance with animal care protocols approved by the Nanjing Medical University Institutional Animal Care and Use Committee.

Cells were transferred to a recording chamber that was continuously perfused with bath solution. Whole-cell patch-clamp recordings were performed with an EPC-9 amplifier (HEKA, Germany) at 37±0.5 °C. Pipettes, filled with the pipette solution, had resistances of 3-6 MΩ. The flow rate of bath solution through the chamber was maintained at 2-3 mL/min.

Data analyses

Currents were acquired with Pulse + Pulsefit V8.53 and analyzed using SPSS 13.0 software. Statistical data are expressed as the mean ± standard error of the mean (S.E.M). Paired-sample t-tests were used to determine significant differences before and after PE or ISO intervention. One-way analyses of variance (ANOVA), with post-hoc comparisons using Newman-Keuls tests, were performed to compare differences among groups. Differences were considered significant if P<0.05.

Solutions and drug administration

For I_kr recordings, the solution was prepared as described by Wang et al. (12). The pipette solution contained 140 mM KCl, 1 mM CaCl₂, 2 mM MgCl₂, 10 mM HEPES, 11 mM EGTA, 5 mM Na₂-ATP, and 5 mM creatine phosphate (disodium salt). The pH was adjusted to 7.4 using 8 M KOH. The bath solution contained 140 mM NaCl, 3.5 mM KCl, 1.5 mM CaCl₂, 1.4 mM MgSO₄, and 10 mM HEPES. The pH was adjusted to 7.4 using 10 M NaOH. Nifedipine (10 μM) was added to the bath solution to block calcium currents and 10 μM chromanol 293B was added to ablate the slow component of delayed rectifier potassium currents (I_Ks). Na₂-ATP, EGTA, creatine phosphate, L-glutamic acid, HEPES, taurine, bovine serum albumin (BSA), nifedipine, chromanol 293B, dofetilide, PE, and ISO were purchased from Sigma (St. Louis, MO). Collagenase II was purchased from Worthington (Lakewood, NJ). All other reagents were obtained from Amresco.

For stock solutions, nifedipine and chromanol 293B were dissolved in dimethyl sulfoxide (DMSO) to a concentration of 10 mM. ISO was dissolved in distilled water to 10 mM, and PE and dofetilide were dissolved in distilled water to 1 mM. All stock solutions were stored at –20 °C, except nifedipine, which was stored at 4 °C.

Results (Figures 1-3)

Figure 1 Effect of α-adrenoceptor and subtype on I_kr. (A,B) Representative I_kr current trace before and after PE application; (C) I_kr density-voltage relations (mean ± S.E.M) from five cells under control conditions (baseline) or in the presence of PE; (D-G) I_kr density-voltage relations (mean ± S.E.M) under control conditions and after the addition of 0.1 µM PE plus the α₁ antagonist prazosin (1 µM), α_1A-selective antagonist 5-MU (1 µM), α_1B-selective antagonist CEC (10 µM), or α_1D-selective antagonist BMY (1 nM); (H) current amplitudes were measured at +40 mV and amplitudes were normalized to the values before PE perfusion in the control, prazosin, 5-MU, CEC, and BMY groups (n=5, **P<0.01).

Figure 2 Effect of β-adrenoceptor and subtype on I_kr. (A) Representative I_kr current trace before and after ISO application; (B) I_kr density-voltage relations (mean ± S.E.M) from four cells under control conditions (baseline) and in the presence of ISO; (C-E) I_kr density-voltage relations (mean ± S.E.M) under control conditions and after the addition of 10 µM ISO plus the β₁ antagonist propranolol (10 µM), the β_1A-selective antagonist CGP-20712A (10 µM), or the β_1B-selective antagonist ICI-118551 (10 µM); (F) current amplitudes were measured at +40 mV and amplitudes were normalized to the value before ISO perfusion in control, propranolol, CGP-20712A, and ICI-118551 groups (n=4, *P<0.05).

Figure 3 Interaction of α- and β-adrenoceptors on I_kr. (A) Representative current changes in the amplitude of peak I_kr tail current (test pulse at +40 mV) following exposure to PE and PE plus ISO; (B) the relative current at test pulse +40 mV in relative groups of Figure 3A; (C) representative current changes in the amplitude of peak I_kr tail current (test pulse at +40 mV) following exposure to ISO and ISO plus PE; (D) the relative current at test pulse +40 mV in relative groups of Figure 3C; (E) comparison of decreased I_Kr tail currents after application of different adrenergic agonists. Column baseline-PE represents the relative I_Kr tail current upon application of α₁-adrenergic agonist alone, column ISO-PE represents the α₁-AR activation-induced percentage of I_Kr after preactivation of β₁-AR, column baseline-ISO indicates the β₁-AR alone stimulation-induced inhibition percentage, and column PE-ISO shows the β₁-AR stimulation-induced inhibition percentage after pre-stimulation with α₁-AR (n=3; *P<0.05).

I_kr tail currents are inhibited by PE and α_1A subtype mediates this effect

When cell chambers were perfused with a bath solution containing 0.1 μM of the specific α₁-adrenereceptor agonist PE, the tail current amplitude significantly decreased (Figure 1A,B). The mean I_kr current density-voltage relations before and after PE are shown in Figure 1C. I_kr tail current density at +40 mV decreased to 0.66±0.02 upon the addition of 0.1 μM PE (first column of Figure 1H). When cells were pretreated with the non-selective α₁-adrenergic antagonist prazosin (1 μM), PE does not decrease I_kr current (Figure 1D, second column of Figure 1H). To further evaluate the α₁-adrenergic receptor (AR) subtype involved, selective α₁-AR blockers, α_1A-selective 5-methylurapidil (5-MU; 1μM), α_1B-selective chloroethylclonidine (CEC; 10 μM), or α_1D-selective BMY7378 (BMY; 1 nM) were applied together with PE (0.1 μM). When 5-MU, CEC, or BMY7378 was applied together with PE, the I_kr amplitudes decreased to 0.99±0.07, 0.62±0.03, and 0.70±0.04, respectively, indicating the α_1A-receptor antagonist 5-MU prevented PE-induced suppression of I_kr, whereas the α_1B and α_1D-receptor blockers did not (Figure 1E-H). These results suggested that stimulation of the α₁ adrenereceptor may reduce I_kr tail current, and that α_1A, rather than the α_1B or α_1D receptor subtypes, is involved in this effect.

I_kr tail currents are inhibited by isoproterenol and β₁ subtype mediates this effect

When cell chambers were perfused with a bath solution containing 10 μM of the non-specific β-adrenereceptor agonist isoproterenol (ISO), the tail current amplitude significantly decreased (Figure 2A). The mean I_kr current density-voltage relations before and after ISO are shown in Figure 2B. I_kr tail current density at +40 mV decreased to 0.62±0.03 upon the addition of ISO (first column of Figure 2F). Propranolol blocked this effect because when cells were pretreated with the non-specific β-adrenereceptor blocker propranolol (10 μM), ISO no longer decreased the I_kr tail current (Figure 2C, second column of Figure 2F). To evaluate the β-AR subtype involved, we added selective β-AR blockers along with ISO: β₁-selective CGP-20712A (10 μM) and β₂-selective ICI-118551 (10 μM). The β₁-receptor blocker CGP completely prevented the ISO-induced reduction in I_kr (the I_kr tail current density at +40 mV decreased to 0.97±0.08), whereas the β₂-receptor blocker ICI did not alter the action of ISO (the I_kr tail current density at +40 mV decreased to 0.55±0.06) (Figure 2D,E; first, third, and fourth columns of Figure 2F). These results suggested that stimulation of the β-adrenereceptor reduced I_kr tail current, and that the β₁ rather than β2 receptor subtype is involved in the regulation of I_kr.

PE prevents the inhibitory effect of ISO on I_kr

We evaluated the effects of ISO in the presence of PE (0.1 μM). ISO (10 μM) was applied after the PE-induced changes stabilized (approximately 8-10 min after PE application). Changes in current amplitude are expressed relative to the amplitude before application of any drug (PE or ISO). Figure 3A shows a typical current trace of cells without drugs, then followed by PE and the last ISO. Figure 3B shows the relative current at test pulse +40 mV in each group. When cells were acutely stimulated with PE, the I_kr tail currents did not decrease with ISO addition (10 μM ISO after 0.1 μM PE). The relative current amplitudes were 1.01±0.12, which was significantly different from the ISO effect on I_kr without PE (third and fourth columns of Figure 3E). Thus, ISO does not reduce I_kr tail currents in cardiomyocytes pretreated with PE, which significantly differed from cardiomyocytes not acutely stimulated by PE. These results suggested that the PE prevents the current reduction effects of ISO on I_kr.

ISO attenuates the inhibitory effect of PE on I_kr

We also evaluated the effects of PE in the presence of ISO. PE (10 μM) was applied after the ISO-induced changes had stabilized. A typical current trace and the relative current at +40 mV test pulse in each group are shown in Figure 3C,D. When cardiomyocytes were acutely stimulated with ISO, the I_kr tail currents only decreased to 0.80±0.02 upon PE addition. This result significantly differed from cardiomyocytes that were not acutely stimulated by ISO (0.58±0.04, P<0.05) (first and second columns of Figure 3E). These data suggested that the inhibitory effects of PE on I_kr can be attenuated by ISO.

Discussion

In the present study, we report that the β₁ and α_1A-AR systems modulate I_kr. The current reduction effects of ISO can be prevented by preactivation of α₁-adrenoceptors whereas the inhibitory effects of PE can be attenuated by preactivation of β-adrenoceptors. Signaling crosstalk between the α_1A- and β₁-adrenergic cascades might be involved in I_kr tail current regulation in guinea pig ventricular myocytes.

ARs bind and are activated by the endogenous catecholamine hormones epinephrine and norepinephrine (NE). Epinephrine is primarily produced and released into circulation from the adrenal gland. However, NE is synthesized and released by sympathetic nerve terminals in the peripheral nervous system and brain. In the heart, the two main ARs are the β-ARs, which comprise roughly 90% of total cardiac ARs, and α₁-ARs, which account for approximately 10% (18). In this study, α₁-adrenergic activation reduced I_kr in guinea pig ventricular cardiomyocytes, which are similar results to those reported by Thomas et al. (19) and Wang et al. (12). At least three α₁-AR subtypes (α_1A, α_1B, α_1D) have been described using molecular and pharmacological techniques (20,21). The ventricular α1-adrenoceptor densities did not significantly differ between guinea pig and human (22). A previous study found that different adrenoceptor subtypes have different effects on I_to (23) and I_ca-L (24). We found that α₁ adrenergic action on I_kr was via α_1A. Currently, there are no data regarding the effects of α_1B or α_1D on I_kr. Moreover, we also found that in addition to α₁, β-adrenergic activation also reduced I_kr, which is consistent with the study by Karle et al. (17). These results differ from the data of Heath and Terrar who reported that isoprenaline increased I_kr (16). This discrepancy could be related to ISO concentrations, different techniques inherent to whole-cell perforated patch clamp and switched electrode voltage clamp (SEVC), and/or different effects of β adreneoceptor subtypes on I_kr. The β₁ and β₂ adreneoceptor subtypes have different effects in regulating Ica-L (25) and cardiac function (26,27). Our study found that β-adrenoceptor action is mediated by the β₁-adrenoceptor subtype rather than the β₂-adrenoceptor subtype.

It is noteworthy that α₁-AR activation-induced inhibition of I_kr tail current in the presence of β-AR activation differs significantly from α₁-AR activation alone-induced inhibition of I_kr tail current. These data suggests that pre-activation of β-ARs suppresses the effect of α₁-AR activation on I_kr tail current, and pre-activation of α₁-ARs inhibits the effect of β-AR activation on I_kr tail current. Thus, pre-activation of one subtype adrenoceptor restrains the effect of another adrenoceptor subtype on I_kr tail current. That is, crosstalk exists in regulating I_kr current via acute adrenergic stimulation in physical circumstances. We propose that this is a protective regulatory mechanism against severe inhibition of I_kr tail current that occurs during excessive release of catecholamines in pathological circumstances.

Conclusions

I_kr in guinea pig cardiomyocyte can be regulated by both the α₁ adrenergic system through the α_1A subtype and the β adrenergic system through the β₁ subtype. The absence of an inhibitory effect on I_kr by ISO was noted in cells pretreated with PE. The PE effect on I_kr was also attenuated in cells pretreated with ISO. Our results suggest a negative feedback loop intrinsic to norepinephrine stimulation in the heart and signaling crosstalk between α_1A- and β₁-adrenergic cascades, which might be involved in I_kr regulation in guinea pig ventricular myocytes. Signaling crosstalk may have a crucial role when plasma levels of both endogenous regulators are elevated.

Limitations

Our study found that crosstalk between α_1A-ARs and β₁-ARs in I_kr regulation exists. However, the precise adrenergic effects require further study to elucidate the molecular mechanisms that underlie the functional crosstalk as well as to identify putative intermediate proteins in the signal transduction pathways involved in modulation of I_kr current via adrenergic activation.

Acknowledgements

We thank Xiang-Jian Chen, Yang-Di, and Dao-Wu Wang for their assistance, and the Research Institute of Cardiovascular Disease of the First Affiliated Hospital of Nanjing Medical University.

Funding: This study was supported by the National Scientific Foundation of China (NSFC No. 81100123).

Disclosure: The authors declare no conflict of interest.

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