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Cardiovascular Research 2002 53(2):355-362; doi:10.1016/S0008-6363(01)00509-0
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Copyright © 2001, European Society of Cardiology

Rapid component IKr of the guinea-pig cardiac delayed rectifier K+ current is inhibited by β1-adrenoreceptor activation, via cAMP/protein kinase A-dependent pathways

Christoph A Karle, Edgar Zitron, Wei Zhang, Sven Kathöfer, Wolfgang Schoels and Johann Kiehn*

Department of Cardiology, University of Heidelberg Medical School, Bergheimerstrasse 58, D-69115 Heidelberg, Germany

* Corresponding author. Tel.: +49-6221-568-682; fax: +49-6221-565-515 johann_kiehn{at}ukl.uni-heidelberg.de

Received 13 June 2001; accepted 28 September 2001


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: The antiarrhythmic potential of betablockers contributes to their beneficial effects in the treatment of cardiac diseases, although the molecular basis of their class II antiarrhythmic action has not been clarified yet. Methods: To investigate a putative functional link between β-adrenoreceptors and the fast component of cardiac delayed rectifier K+ channels (IKr), whole-cell patch-clamp experiments were performed with isolated guinea-pig ventricular myocytes. Tail currents of IKr were measured at –40 mV after short (200 ms) test pulses to +40 mV. Results: After application of the unspecific β-receptor agonist isoproterenol (10 µM) for 12 min, the IKr tail current was decreased by 72%, with an IC50 of 1.4 µM. The specific β1-blocker CGP207120A (10 µM) significantly attenuated the isoproterenol effect (net 24% decrease). The specific β1-agonist xamoterol (10 µM), could mimic the isoproterenol effect (58% decrease). Modulators of β2- or β3-adrenoreceptors were far less effective. When isoproterenol or xamoterol were combined with KT5720 (2.5 µM), a specific inhibitor of protein kinase A (PKA), their effects were drastically reduced, indicating that PKA presumably mediates the β1-adrenergic inhibition of IKr. Tail current reductions by cAMP, forskolin, PKA catalytic subunit and a combination of PKA holoenzyme and cAMP support an involvement of PKA in the regulation of IKr. Conclusions: The functional link between IKr and the β1-adrenergic receptor involving PKA may play an important role in arrhythmogenesis and contribute to the antiarrhythmic action of clinically used β1-blockers.

KEYWORDS Adrenergic (ant)agonists; Antiarrhythmic agents; K-channel; Protein kinases


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Cardiac activity is influenced by regulatory areas in the medulla oblongata and the pons via autonomic fibres of the sympathetic and parasympathetic nervous system. Noradrenaline and adrenaline are the predominant mediators of sympathetic action. They exert their influence by binding to {alpha}- and β-adrenergic receptors located in the membrane of cardiomyocytes.

Physiological cardiac activity in systole and diastole, but also pathophysiological conditions like stress, autonomic compensation of heart failure, shock and phaeochromocytoma are linked to sympathetic activity. Thereby, stress may trigger fatal ventricular fibrillation [1], especially in patients with coronary heart disease [2]. In patients with congenital long QT syndrome, stress commonly triggers ‘Torsade de pointes’ ventricular arrhythmias in a so far unknown mechanism [3–5]. As betablockade represents one of the most common and effective cardiovascular therapy strategies, the target receptors, subdivided into isoforms β1-β4 [6], have been characterized [7–11]. However, details about the molecular mechanisms of antiarrhythmic action, i.e. the functional coupling to cardiac ion currents have not yet been clarified. One very important repolarizing potassium current in heart is the rapid component of the delayed rectifier potassium current IKr [12]. Recently it has been demonstrated, that the HERG potassium channel, which underlies IKr in cardiomyocytes, is regulated by protein kinase A (PKA) in Xenopus oocytes and in a CHO cell line [13–15]. The aim of this study was to investigate whether IKr in cardiomyocytes could be regulated by PKA and which type of β-adrenergic receptor initiates a functional coupling to IKr.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1 Electrophysiology and data analysis
Single guinea pig cardiomyocytes were isolated with the Langendorff apparatus as described previously [16]. Cells were transferred into a recording chamber which was continuously perfused with the bath solution. Pipettes had resistances of 3–4 M{Omega} and whole-cell patch-clamp currents were measured with an EPC-7 amplifier (List, Darmstadt), recorded on hard-disk and analysed with pCLAMP 6 software (Axon Instruments, Foster City, CA). To calculate the half-maximal inhibitory concentration (IC50) for isoproterenol, the data were fitted with a concentration response curve according to the following equation: Y=(Imin–Imax)/{[1+([isoproterenol]/IC50)k]+Imin}, where Imin and Imax are the normalised minimal and maximal currents, k is the slope factor and IC50 is the concentration of isoproterenol, producing half-maximal inhibition of IKr, respectively. Statistical data are presented as mean±S.E.M. Statistical significance was evaluated using the unpaired Student's t-test. Differences were considered significant when a P value <0.05 was reached.

The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996).

2.2 Solutions and drugs
The bath solution contained (in mM) NaCl 140, KCl 3.5, CaCl2 1.5, MgSO4 1.4, HEPES 10, pH adjusted to 7.4 with about 10 NaOH. The pipette solution contained KCl 140, CaCl2 0.5, MgCl2 1.5, Na2-ATP 5, EGTA 1, HEPES 10, pH adjusted to 7.4 with about 8 KOH. The calculated free Ca2+ concentration in the pipette was 1.6x10–7 M [17]. The EK was –98 mV. Adenosine triphosphate-sensitive K+ channels (KATP) were inhibited by adding 5 mM ATP to the pipette solution. The slow component of the delayed rectifier potassium current (IKs) was blocked by adding 10 µM chromanol (Hoechst, Frankfurt, Germany), a specific blocker of IKs [18], to the bath solution. Calcium currents were reduced with 10 µM nifedipine in the bath solution. Dofetilide, a specific blocker of IKr or HERG [19], was provided by Pfizer (Central Research, Sandwich, Kent, UK). KT5720 [20], forskolin and 8-bromo-adenosine-3',5'-cyclic monophosphate (a membrane-permeable analogue of cAMP) were purchased from Calbiochem (La Jolla, CA, USA). Isoproterenol, Na2-ATP, PKA holoenzyme and the catalytic subunit of PKA were purchased from Sigma GmbH (Deisenhofen, Germany). BRL37344, CGP20712A, ICI118551 and S-(–)-propranolol were purchased from RBI (Natick, MA, USA). Procaterol–HCl and xamoterol–hemifumarate were purchased from Tocris (Ballwin, WI, USA).

For stock solutions, BRL37344, CGP20712A, ICI118551, procaterol, S-(–)-propranolol, and xamoterol were dissolved in distilled water to 10–2 M; cAMP was dissolved in water to 10–1 M. Chromanol 293B and forskolin were dissolved in DMSO to 10–2 M. KT5720 was dissolved in DMSO to 10–3 M. PKA holoenzyme and the catalytic subunit of PKA were dissolved in distilled water to stocks of 1000 U/ml. For the experiments stock solutions were further diluted with the bath solution to the desired concentration of 2.5 µM (KT5720), 100 µM (cAMP) and 10 µM (other substances). PKA holoenzyme and catalytic subunit were diluted with the pipette solution to a concentration of 50 U/ml. The final concentration of maximal 0.5% DMSO in the bath had no effect on the currents measured.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
To investigate the regulation of IKr, experiments with the following test pulse protocol were performed: From a holding potential of –40 mV, test pulses to various voltages from –40 to +40 mV (step width 20 mV, step duration 200 ms each), were applied before going back to –40 mV, to measure tail currents. Substance effects were investigated on tail currents after the test pulse to 40 mV. Measurements were repeated every 2 min, starting at time 0 and ending after 12 min.

3.1 IKr shows small run-down
During an observation time of 12 min, a small gradual decrease of tail current amplitudes occurred, i.e. after 12 min tail currents had decreased by 5.9±3.8% (n=7; Fig. 1A,B). When 1 µM dofetilide, a specific blocker of IKr was applied to the bath, the IKr current was completely blocked, indicating that no other currents contribute to the tail current under the given experimental conditions (Fig. 1C,D).


Figure 1
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  		Fig. 1 The rapid component of the delayed rectifier potassium current (IKr) is reduced by isoproterenol in isolated guinea-pig ventricular cardiomyocytes. (A) Shows whole cell patch-clamp measurements in one cell over a control period of 12 min. During the test pulse calcium inward currents could be measured, which were very variable in amplitude; during the return pulse to constant –40 mV IKr tail currents could be measured, which were stable within 12 min. (B) Shows the peak tail current amplitude of IKr in the control measurement at the beginning and after 12 min. (C,D) Show complete block of the IKr current by the specific IKr blocker dofetilide (1 µM), demonstrating, that selectively IKr tail currents were measured in the return pulse, without contamination by other currents under the given experimental conditions. (E,F) Demonstrate the reduction of the IKr tail current with the β-adrenergic agonist isoproterenol. The IKr tail current amplitude was reduced by ~49% in this experiment. Protocol: holding potential –40 mV, test pulses from –40 to +40 mV in 20-mV increments (duration 200 ms), return pulse constant –40 mV (600 ms) to measure IKr tail currents.

 
3.2 IKr tail currents are inhibited by isoproterenol
When the cell chamber was perfused with a solution containing 10 µM of the unspecific beta-adrenoreceptor agonist isoproterenol [21], the tail current amplitude was decreased by 71.5±5.6% (n=6; significantly different from the run-down, t=9.993, P=3.609x10–7 at the 0.05 level; Fig. 1E,F). The time course of the isoproterenol effects is displayed in Fig. 2A. The current reduction occurred fast, reaching saturation after approximately 12 min. The IC50 for the inhibition of the IKr tail current amplitude by isoproterenol was 1.4±0.8 µM (Fig. 2B). To verify that the pharmacological action of 10 µM isoproterenol was β-adrenoreceptor-mediated, the unspecific beta-blocker propranolol (10 µM) [22], was added to isoproterenol (10 µM). This resulted in a current reduction of only 24.7±8.8% (n=4), which was significantly different compared to isoproterenol alone (t=–4.721, P=0.002 at the 0.05 level; Fig. 2C). These results indicate that IKr is regulated by β-adrenoreceptors in guinea pig cardiomyocytes.


Figure 2
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  		Fig. 2 (A) Time-dependence of the current reduction by 10 µM isoproterenol. (B) The concentration response curve for the isoproterenol effect on the IKr tail current. The IC50 was calculated to be 1.36 µM. (C) The significant inhibition of the isoproterenol (10 µM) effect by the unspecific β-blocker propranolol (10 µM). Protocols were identical to those shown in Fig. 1. * These values were significantly different.

 
3.3 β1-Adrenoreceptor subtype mediates the effect on IKr
To further elucidate which β-adrenoreceptor subtype mediates the effect we used combinations of selective agonists and antagonists. Combining the specific β1-adrenoreceptor antagonist CGP20712A (10 µM) [23] with isoproterenol (10 µM) resulted in a decrease of tail current amplitude by only 24.5±20.7% (n=4). The current amplitude reduction was significantly attenuated compared to isoproterenol (10 µM) alone (t=–2.643, P=0.030 at the 0.05 level; Fig. 3A). Combining the specific β2-adrenoreceptor antagonist ICI118551 (10 µM) [21] with isoproterenol (10 µM) resulted in a decrease of tail current amplitude by 74.1±2.6% (n=6), which was not significantly different from isoproterenol alone (t=0.384, P=0.710 at the 0.05 level; Fig. 3A). The latter two experimental conditions indicate, that the β1-adrenoreceptor and not the β2-adrenoreceptor mediates the effect on IKr. This hypothesis was further tested by applying the selective β1-adrenoreceptor agonist xamoterol [24]. Xamoterol (10 µM) alone resulted in a significant tail current reduction of 58.2±9.3% (t=–6.031, P=8.539x10–5 at the 0.05 level for n=5), comparable to the effect of isoproterenol alone (Fig. 3B). In contrast, the selective β2-adrenoreceptor agonist procaterol (10 µM) [25] and the selective β3-adrenoreceptor agonist BRL37344 (10 µM) [22] decreased IKr tail currents only by 20.6±6.2% (n=4) and 22.7±3.2% (n=3, Fig. 3B). These results support a predominant involvement of the β1-adrenoreceptor subtype rather than other beta-receptor subtypes in the regulation of IKr.


Figure 3
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  		Fig. 3 (A) Attenuation of the isoproterenol effect by the specific β1-adrenoreceptor antagonist CGP20712A (10 µM). The specific β2-adrenoreceptor antagonist ICI118551 (10 µM) showed no significant attenuation. (B) The inhibitory effects of isoproterenol and the specific β1-agonist xamoterol were similar. In contrast, neither the β2-agonist procaterol nor the specific β3-agonist BRL37344 could significantly mimic the effects of isoproterenol. Protocols were identical to those shown in Fig. 1. * These values were significantly different.

 
3.4 IKr is modulated by β1-adrenoreceptors via a protein kinase A-dependent pathway
The coapplication of KT5720 (2.5 µM), a specific inhibitor of PKA, attenuated the isoproterenol (10 µM) effect.

Under these experimental conditions the tail current reduction was 47.0±10.7% (n=4; reaching almost statistically significant difference compared with isoproterenol alone with t=–2.250 and P=0.065 at the 0.05 level; Fig. 4A). The effect of specific β1-adrenoreceptor agonist xamoterol (10 µM) was also attenuated by KT5720 (2.5 µM) (35.9±9.8% [n=3] tail current reduction; insignificant from xamoterol alone with t=–1.562 and P=0.169 at the 0.05 level; Fig. 4B).


Figure 4
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  		Fig. 4 Coapplication of 2.5 µM KT5720, a specific inhibitor of PKA, attenuated the effect of isoproterenol (A) and xamoterol (B). Protocols were identical to those shown in Fig. 1.

 
The extracellular application of the second messenger cAMP (100 µM), which is membrane permeable to a certain amount, decreased IKr tails after 10 min significantly by 70.2±20.2% (t=–7.037, P=6.074x10–5 at the 0.05 level for n=5), comparable to the isoproterenol effect. Forskolin (10 µM), an activator of the adenylyl cyclase, was even more effective and led to a 82.2±8.2% (n=3) reduction of the current, significantly different from the control (t=–9.645, P=4.829x10–6 at the 0.05 level; Fig. 5A).


Figure 5
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  		Fig. 5 Protein kinase A mediates the effect of isoproterenol on IKr. (A) The extracellular application of cAMP (100 µM), which reaches its point of action intracellularly, leads to a reduction in tail current amplitude, similar to the isoproterenol effect. Forskolin (10 µM), an activator of the adenylyl cyclase, could exceed the effect of isoproterenol. (B) Effect of intracellular (in the pipette solution) protein kinase A holoenzyme (50 U/ml) alone or in combination with extracellular cAMP (100 µM). A combination of PKA holoenzyme and cAMP only slightly exceeded the isolated cAMP effect. (C) Intracellular application of the PKA catalytic subunit (50 U/ml) resulted in a current decrease from the beginning of the measurement. Protocols were identical to those shown in Fig. 1. * These values were significantly different. # These values were also significantly different.

 
PKA holoenzyme (50 U/ml), applied to the pipette solution from the beginning of the experiment, only produced a current decrease of 10.9±3.4% (n=5). This was not significantly different from control. However, co-application of PKA holoenzyme (50 U/ml) in the pipette solution together with extracellular cAMP (100 µM) resulted in a significant current reduction of 75.8±9.8% (t=–8.277, P=1.685x10–5 at the 0.05 level for n=3), exceeding the individual effects of either substance (Fig. 5B). The catalytic subunit of PKA is able to phosphorylate proteins without prior activation by cAMP. Applying the catalytic subunit of protein kinase A (50 U/ml) to the pipette solution resulted in a current decline of 85.0±4.2% (highly significant from the control with t=–13.450 and P=9.908 at the 0.05 level, n=4; Fig. 5C). These data indicate, that PKA directly reduces IKr via PKA-dependent phosphorylation.


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
This study demonstrates the regulation of IKr in guinea-pig cardiomyocytes by the β1-adrenergic receptor, via a PKA-dependent pathway, revealing important aspects into the pathogenesis and therapy of cardiac arrhythmias. IKr is reduced by PKA activation.

In an early report by Sanguinetti et al. [27], the IKr current seemed not to be measurably affected by isoproterenol. In contrast, Heath and Terrar [28] described a concentration-independent increase of IKr tail currents by low concentrations of isoprenaline with the amphotericin B perforated-patch whole-cell technique. They further reported, that with the conventional whole cell ruptured-patch technique, isoprenaline in low concentrations was ineffective. In our study we used different experimental conditions and higher concentrations of isoproterenol in the whole-cell patch-clamp technique, where IKr was isolated by shortening the depolarizing steps to 200 ms and by blocking IKs with a high concentration of the specific inhibitor chromanol 293B. One possible explanation between the different result of our experiments and the results from Heath and Terrar may be the recently described dual regulation of this current by cAMP and PKA phosphorylation [13]. Depending on the regulatory protein, HERG current may be decreased by cAMP-dependent activation of PKA (HERG alone or with the protein MiRP1) [26], or in contrast, a putative direct binding of cAMP to the channel causes opposite effects (HERG with the protein minK). Therefore, the final consequence of the regulation of IKr by cAMP and PKA may be very complicated, depending on the expression of accessory subunits, the density of β-adrenoreceptors, the compartmentalization of PKA and AKAPs and finally on the concentration and specificity of agonists. Our results do not exclude any direct binding of cAMP to the channel; although, the overall effect of a current decrease suggest a predominant activation of PKA by cAMP.

Decreasing IKr by PKA in this study is consistent with recent data about decrease of HERG current (underlying IKr in humans [29]) by pharmacological PKA activation [13,14] or destruction of four putative PKA phosphorylations sites by site-directed mutagenesis [13,15].

The length of the cardiac action potential is largely determined by IKr and the slow component of the delayed rectifier potassium current IKs increased by isoproterenol [30,31]. To investigate the effects of isoproterenol on IKr and finally the cardiac action potential length, IKs has to be blocked before. In a study by Shimizu and Antzelevitch [32] with dog midmyocardial cells, isoproterenol (0.1 µM) produced an additional increase of the action potential duration (APD90) after blocking IKs with the specific blocker chromanol, consistent with data presented in this study. Similar results came from a study in guinea-pig papillary muscles, where isoproterenol (in presence of chromanol 293B) prolonged the cardiac action potential [33]. In dog cardiomyocytes, low concentrations of isoproterenol (0.001 and 0.01 µM), shortened the cardiac action potential, in contrast to a relative prolongation with high (1 and 10 µM) concentrations of isoproterenol [34]. The prolongation of the cardiac action potential was missing, when CGP20712A, a specific blocker of β1-adrenoreceptors, was added [34], consistent with the β1-dependent inhibition of IKr shown in this study.

Highly concentrated catecholamines are found under pathophysiological conditions and possibly at the nerve endings in heart and vessels, giving our findings with 0.1–100 µM isoproterenol a clinical relevance. In resistance arteries, norepinephrine concentrations as high as 1–10 mM have been estimated in the synaptic cleft [35]. High dosages of isoproterenol (plasma concentration of 13.9 ng/ml, i.e. 0.06 µM) are used frequently in the pediatric intensive care unit to treat patients with severe reactive airway disease [36]. In patients with haemorrhagic shock, norepinephrine concentrations of 20 nM have been measured [37]. In patients with pheochromocytoma, norepinephrine concentrations of 0.10 µM and epinephrine concentrations of 0.12 µM have been measured [38]. Pheochromocytoma is often associated with QT prolongation [39] and ventricular arrhythmias [40] like ‘torsades de pointes’ [41], successfully treated with the selective β1-adrenoreceptor blocker atenolol [42]. Another clinical application of high-dose catecholamines is the dobutamine–atropine stress echocardiography (maximal dobutamine dose of 40 µg/kg/min) associated with ventricular tachycardia [43] and ventricular fibrillation [44].

Comparing in vitro effects of the synthetic β-agonist isoproterenol with effects of naturally occurring catecholamines, their relative potency to increase cAMP has to be considered; i.e. EC50 concentrations for norepinephrine were 10-fold higher than for epinephrine and 50-fold higher than for isoproterenol [45]. From this comparison it appears that only very high concentrations of catecholamines may affect IKr through β1-receptors. But these high concentrations may be reached locally at the synaptic ends of nerve fibres in the heart. In resistant arteries, norepinephrine concentrations of 1–10 mM in the synaptic cleft have been estimated [35], presumably giving our finding a physiological relevance.

Time for primary review 20 days.


   Acknowledgements
 
This study was supported by a grant of the ‘Deutsche Forschungsgemeinschaft’ (Ki 663/1-1) to Dr Kiehn and by a grant of the University of Heidelberg to Dr Karle and Dr Kiehn. We thank K. Güth and S. Lück for excellent technical support.


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

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