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

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

Sen Wang, Xiao-Yan Min, Si-Si Pang, Jin Qian, Di Xu, Yan Guo

Department of Geriatric Cardiology, First Affiliated Hospital of Nanjing Medical University, Nanjing, 210029, China

Correspondence to: Yan Guo, PhD. Department of Geriatric Cardiology, First Affiliated Hospital of Nanjing Medical University, Nanjing, 210029, China. Email: lnxxgk@163.com.

Background: Guinea pig ventricular cardiomyocytes display the rapid component of the delayed rectifier potassium current (Ikr) that contributes to ventricular repolarization and promotes stress-induced arrhythmias. Adrenergic stimulation favors ventricular arrhythmogenesis but its effects on Ikr are poorly understood.

Methods: Adrenergic modulation of Ikr was studied in isolated guinea pig ventricular cardiomyocytes using whole-cell patch clamping.

Results: We found that the Ikr amplitude was reduced to 0.66±0.02 and 0.62±0.03 in response to 0.1 µM phenylephrine (PE), an α1AR agonist, and 10 µM isoproterenol (ISO), a βAR agonist, respectively. The effect of PE can be blocked by the selective α1A-adrenoceptor antagonist 5-methylurapidil, but not by the α1B-adrenoceptor antagonist chloroethylclonidine or α1D-adrenoceptor antagonist BMY7378. Additionally, the effect of ISO can be blocked by the β1-selective AR antagonist CGP-20712A, but not by the β2-selective AR antagonist ICI-118551. Although PE and ISO was continuously added to cells, ISO did not decrease the current to a greater extent when cells were first given PE. In addition, PE’s effect on Ikr was suppressed by β1AR stimulation.

Conclusions: Ikr can by regulated by both the α1 and β ARs system, and that in addition to direct regulation by each receptor system, crosstalk may exist between the two systems.

Keywords: Adrenergic receptors (ARs); potassium current; crosstalk; cardiomyocytes


Submitted Aug 12, 2014. Accepted for publication Nov 13, 2014.

doi: 10.3978/j.issn.2072-1439.2014.12.27


Introduction

Cardiac action potential repolarization is carried out by different potassium currents. The rapid component of the delayed rectifier potassium current (Ikr) 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 Ikr 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 Ikr current though both α1 and β-adrenoceptors (ARs) (11-14). Separate stimulation of α1-ARs or β-ARs decreased Ikr 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 Ikr 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 Ikr 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 Ikr or hERG via a cAMP/PKA-dependent pathway (17). Morever, at least three α1 adrenoceptor subtypes (α1A, α1B, and α1D) and three β adrenoceptor subtypes (β1, β2, and β3) have been pharmacologically identified, it is unclear which AR subtypes primarily regulate Ikr. In addition, the interaction between the α1 and β-adrenergic components during sympathetic regulation of the cardiac Ikr 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 Ikr 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 (IKs). 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 Ikr. (A,B) Representative Ikr current trace before and after PE application; (C) Ikr density-voltage relations (mean ± S.E.M) from five cells under control conditions (baseline) or in the presence of PE; (D-G) Ikr 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), α1A-selective antagonist 5-MU (1 µM), α1B-selective antagonist CEC (10 µM), or α1D-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 Ikr. (A) Representative Ikr current trace before and after ISO application; (B) Ikr density-voltage relations (mean ± S.E.M) from four cells under control conditions (baseline) and in the presence of ISO; (C-E) Ikr 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 β1A-selective antagonist CGP-20712A (10 µM), or the β1B-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 Ikr. (A) Representative current changes in the amplitude of peak Ikr 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 Ikr 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 IKr tail currents after application of different adrenergic agonists. Column baseline-PE represents the relative IKr tail current upon application of α1-adrenergic agonist alone, column ISO-PE represents the α1-AR activation-induced percentage of IKr 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).

Ikr tail currents are inhibited by PE and α1A 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 Ikr current density-voltage relations before and after PE are shown in Figure 1C. Ikr 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 Ikr current (Figure 1D, second column of Figure 1H). To further evaluate the α1-adrenergic receptor (AR) subtype involved, selective α1-AR blockers, α1A-selective 5-methylurapidil (5-MU; 1μM), α1B-selective chloroethylclonidine (CEC; 10 μM), or α1D-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 Ikr amplitudes decreased to 0.99±0.07, 0.62±0.03, and 0.70±0.04, respectively, indicating the α1A-receptor antagonist 5-MU prevented PE-induced suppression of Ikr, whereas the α1B and α1D-receptor blockers did not (Figure 1E-H). These results suggested that stimulation of the α1 adrenereceptor may reduce Ikr tail current, and that α1A, rather than the α1B or α1D receptor subtypes, is involved in this effect.

Ikr 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 Ikr current density-voltage relations before and after ISO are shown in Figure 2B. Ikr 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 Ikr 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 Ikr (the Ikr 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 Ikr 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 Ikr tail current, and that the β1 rather than β2 receptor subtype is involved in the regulation of Ikr.

PE prevents the inhibitory effect of ISO on Ikr

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 Ikr 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 Ikr without PE (third and fourth columns of Figure 3E). Thus, ISO does not reduce Ikr 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 Ikr.

ISO attenuates the inhibitory effect of PE on Ikr

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 Ikr 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 Ikr can be attenuated by ISO.


Discussion

In the present study, we report that the β1 and α1A-AR systems modulate Ikr. 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 α1A- and β1-adrenergic cascades might be involved in Ikr 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 Ikr 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 (α1A, α1B, α1D) 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 Ito (23) and Ica-L (24). We found that α1 adrenergic action on Ikr was via α1A. Currently, there are no data regarding the effects of α1B or α1D on Ikr. Moreover, we also found that in addition to α1, β-adrenergic activation also reduced Ikr, 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 Ikr (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 Ikr. 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 Ikr tail current in the presence of β-AR activation differs significantly from α1-AR activation alone-induced inhibition of Ikr tail current. These data suggests that pre-activation of β-ARs suppresses the effect of α1-AR activation on Ikr tail current, and pre-activation of α1-ARs inhibits the effect of β-AR activation on Ikr tail current. Thus, pre-activation of one subtype adrenoceptor restrains the effect of another adrenoceptor subtype on Ikr tail current. That is, crosstalk exists in regulating Ikr current via acute adrenergic stimulation in physical circumstances. We propose that this is a protective regulatory mechanism against severe inhibition of Ikr tail current that occurs during excessive release of catecholamines in pathological circumstances.


Conclusions

Ikr in guinea pig cardiomyocyte can be regulated by both the α1 adrenergic system through the α1A subtype and the β adrenergic system through the β1 subtype. The absence of an inhibitory effect on Ikr by ISO was noted in cells pretreated with PE. The PE effect on Ikr 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 α1A- and β1-adrenergic cascades, which might be involved in Ikr 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 α1A-ARs and β1-ARs in Ikr 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 Ikr 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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Cite this article as: Wang S, Min XY, Pang SS, Qian J, Xu D, Guo Y. Adrenergic regulation of the rapid component of delayed rectifier K+ currents in guinea pig cardiomyocytes. J Thorac Dis 2014;6(12):1778-1784. doi: 10.3978/j.issn.2072-1439.2014.12.27