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Cardiovascular Research 2007 74(3):406-415; doi:10.1016/j.cardiores.2007.01.020
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Copyright © 2007, European Society of Cardiology

Adrenergic control of a constitutively active acetylcholine-regulated potassium current in canine atrial cardiomyocytes

Yung-Hsin Yeha,b, Joachim R. Ehrlichc, Xiaoyan Qia, Terence E. Hébertd, Denis Chartiera and Stanley Nattela,d,*

aDepartment of Medicine and Research Center, Montreal Heart Institute and Université de Montréal, Montreal, Quebec, Canada
bFirst Cardiovascular Division, Chang Gung Memorial Hospital, Chang Gung University, Tao-Yuan, Taiwan
cDivision of Cardiology, J.W. Goethe University, Frankfurt, Germany
dDepartment of Pharmacology and Therapeutics, McGill University, Montreal, Quebec, Canada

* Corresponding author. 5000 Belanger Street, Montreal, Quebec, Canada H1T 1C8. Tel.: +1 514 376 3330x3990; fax: +1 514 376 1355. Email address: stanley.nattel{at}icm-mhi.org

Received 4 August 2006; revised 15 January 2007; accepted 31 January 2007


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 
Objectives: Canine atrial cardiomyocytes display a constitutively active, acetylcholine-regulated, time-dependent K+ current (IKH) that contributes to atrial repolarization and atrial tachycardia-induced atrial-fibrillation promotion. Adrenergic stimulation favors atrial arrhythmogenesis but its effects on IKH are poorly understood.

Methods and results: Adrenergic modulation of IKH was studied in isolated canine atrial cardiomyocytes with whole-cell patch-clamping, and action-potential consequences were assessed in multicellular preparations with fine-tipped microelectrodes. Isoproterenol increased IKH in a concentration-dependent manner (maximum 103±22% increase), an effect mimicked by forskolin and 8-bromo-cyclic AMP. Isoproterenol effects were prevented by propranolol and the selective β1-adrenoceptor blocker CGP-20712A, but not the β2-blocker ICI-118551. Isoproterenol enhancement was prevented by pipette-administered protein kinase A (PKA) inhibitor peptide or by superfusion of H89 (PKA blocker). Phenylephrine decreased IKH in a reversible, concentration-dependent way. This effect was blocked by the {alpha}-antagonist prazosin and the selective {alpha}1A-blocker niguldipine, but not the {alpha}1B-blocker chloroethylclonidine or the {alpha}1D inhibitor BMY-7378. Phenylephrine effects were prevented by the phospholipase C (PLC) inhibitor U73122 [GenBank] and the protein kinase C (PKC) inhibitor bisindolylmaleimide. The PKC-activating phorbol ester PDD (but not its inactive analogue {alpha}-PDD) mimicked phenylephrine effects. Action potential recordings in the presence and absence of the selective IKH blocker tertiapin indicated a functional role of {alpha}- and β-adrenergic actions on IKH. Adrenergic regulation of cholinergic agonist-induced K+ current paralleled that of IKH.

Conclusions: IKH is under dual regulation by the adrenergic system: β1-adrenergic stimulation enhances IKH via cAMP-dependent PKA pathways, whereas {alpha}1A-adrenergic stimulation inhibits IKH via PLC-mediated PKC activation. Modulation of constitutive acetylcholine-regulated K+ current is a novel potential mechanism for adrenergic control of atrial repolarization.

KEYWORDS Ion-channels; Second-messengers; Signal transduction


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 
We recently characterized a hyperpolarization-activated, time-dependent constitutively active acetylcholine-regulated K+ current in canine atrial cardiomyocytes, named IKH because of characteristic time-dependent activation on hyperpolarization [1]. IKH is sensitive to a highly selective Kir3 current blocker, tertiapin-Q [1]. Atrial tachycardia-induced remodeling increases IKH, which plays a significant role in remodeling-associated atrial fibrillation (AF) promotion and action potential (AP) duration (APD) abbreviation [2]. A corresponding constitutive acetylcholine-regulated, tertiapin-Q sensitive current is up-regulated in atrial cardiomyocytes from AF patients [3].

Adrenergic stimulation is an important regulator of cardiac ion-channel function [4,5] and arrhythmias [6,7]. We previously found IKH to be enhanced by β-adrenoceptor stimulation [1], but underlying receptor-subtype and signal-transduction systems were not probed, nor was functional importance studied. In addition, the {alpha}-adrenergic modulation of IKH is unexplored. The present study assessed the adrenergic regulation of IKH with respect to receptor-subtypes, signal-transduction pathways and role in mediating effects on atrial repolarization.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 Acknowledgments
 References
 
2.1 Tissue and cell preparations
All animal-care procedures were consistent 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). Adult mongrel dogs of either sex (19–38 kg) were anaesthetised with pentobarbital (30 mg/kg I.V.) and artificially ventilated with room air. Hearts were excised through a left lateral thoracotomy and immersed in oxygenated Tyrode solution. A left atrial (LA) preparation was dissected and perfused at ~ 10 mL/min via the left circumflex coronary artery, for either cardiomyocyte isolation with previously detailed methods [1,2] or AP recording.

2.2 Electrophysiology
Currents were recorded with whole-cell patch-clamp techniques at 36±0.5 °C as previously described [1,2]. Borosilicate-glass electrodes (tip resistances 1.5–3.0 M{Omega}) were used to record whole-cell currents. Junction potentials were zeroed prior to formation of gigohm seals. Compensated series-resistances and capacitive time-constants averaged 3.4±0.2 M{Omega} and 286±88 µs, respectively. Cell-capacitances averaged 94±6 pF. Original recordings are presented in terms of current amplitude, but mean data are shown as current–density (pA/pF). Currents were recorded with hyperpolarizing and depolarizing pulses from a holding potential (HP) of –40 mV. IKH was quantified [1,2] as the time-dependent current elicited under conditions designed to minimize other potential current contributions.

AP-recordings were obtained with fine-tipped standard borosilicate-glass microelectrodes in intact multicellular coronary artery-perfused LA preparations [1,2]. Floating microelectrodes (resistances 10–20 M{Omega} when filled with 3 mol/L KCl) connected to a high-input impedance amplifier were used to record APs at 2 Hz in the presence and absence of adrenergic agonists and tertiapin-Q.

2.3 Solutions
Tyrode solution contained (mmol/L): NaCl 136, KCl 5.4, MgCl2 1, CaCl2 1, NaH2PO4 0.33, HEPES 5 and dextrose 10 (pH 7.35 with NaOH). The cell-storage solution contained (mmol/L): KCl 20, KH2PO4 10, dextrose 10, mannitol 40, L-glutamic acid 70, β-OH-butyric acid 10, taurine 20, EGTA 10 and 0.1% bovine serum albumin (pH 7.3, KOH). Nifedipine (5 µmol/L) was used to suppress L-type Ca2 current in all experiments. 4-Aminopyridine (2-mmol/L) was added to suppress transient outward current (Ito). Atropine (1 µmol/L) was added to the extracellular solution to suppress muscarinic receptor-activated currents. Na+ current (INa) contamination was avoided by using a HP of –40 mV. The standard internal solution contained (mmol/L): K+-aspartate 110, KCl 20, MgCl2 1, MgATP 5, GTP (lithium salt) 0.1, HEPES 10, Na+-phosphocreatine 5 and EGTA 5.0 (pH 7.3 with KOH). For standard-microelectrode experiments (37 °C) the external solution contained (mmol/L): NaCl 120, KCl 4, KH2PO4 1.2, MgSO4 1.2, NaHCO3 25, CaCl2 1.25 and dextrose 5 (95% O2–5% CO2, pH 7.4).

Stock solutions of isoproterenol (1 mmol/L) were prepared under protection from light on the day of experiments and freshly-prepared ascorbic acid (100 µmol/L) was added to prevent oxidization. Tertiapin-Q was dissolved in 0.1% acetic acid. Unless otherwise specified, chemicals were obtained from Sigma.

2.4 Data analysis
Clampfit 6.0 (Axon) and GraphPad Prism 3.0 were used for data analysis. Group data are presented as mean±SEM. Two-way ANOVA and Bonferroni post-hoc tests were used for statistical comparisons, with an interaction variance analysis to determine the statistical significance of isoproterenol–tertiapin and phenylephrine–tertiapin interaction effects on APD. P values <0.05 were considered statistically significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 Acknowledgments
 References
 
3.1 Effects of isoproterenol on IKH
Fig. 1A and B shows typical IKH recordings from one cell at baseline and after isoproterenol (1 µmol/L). Isoproterenol increased currents both during voltage-steps and upon repolarization. Fig. 1C shows the response of the same cell to propranolol (1 µmol/L) in the continued presence of isoproterenol. Mean time-dependent IKH density–voltage relations before and after isoproterenol are shown in Fig. 1D. Isoproterenol significantly increased IKH. The effects of isoproterenol were concentration-dependent (Fig. 1E): IKH density at –120 mV was increased 26±3.8% by 0.01 µmol/L, 78±19% by 0.1 µmol/L, and 103±22% by 1 µmol/L isoproterenol. Isoproterenol effects were reversed by propranolol, e.g. IKH at –120 mV increased with isoproterenol from –1.53±0.12 to –3.05±0.59 pA/pF, and returned to –1.69±0.18 pA/pF (n=6) with the addition of propranolol. Washout reversed the effects of the drug. Ascorbic acid alone did not affect the current.


Figure 1
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  		Fig. 1 A, B, C. Representative IKH recordings (voltage protocol shown in the inset) under control condition, in the presence of isoproterenol (Iso, 1 µmol/L) and then after the addition of propranolol (Prop, 1 µmol/L) to the isoproterenol-containing superfusate. D. Mean±SEM IKH density–voltage relations from 6 cells under control conditions (baseline), and then in the presence of isoproterenol (1 µmol/L). The inset shows outward currents on an expanded scale. E. Concentration-dependence of isoproterenol effects on IKH at –120 mV shown as mean±SEM percentage of control current density. (n=8 cells/group, *P<0.05, **P<0.01 versus baseline).

 
To evaluate the β-adrenergic receptor subtype involved, we applied selective β-adrenergic receptor blockers along with 1 µmol/L isoproterenol: CGP-20712A methanesulfonate (CGP, 300 nmol/L, β1-selective) and ICI-118,551 hydrochloride (ICI, 50 nmol/L, β2-selective). The β1-receptor blocker CGP fully prevented isoproterenol-enhancement of IKH, whereas the β2-receptor blocker ICI failed to alter isoproterenol's action (Fig. 2, top).


Figure 2
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  		Fig. 2 A. Mean±SEM IKH density–voltage relations under control conditions and after the addition of 1 µmol/L isoproterenol along with the β1-adrenoceptor antagonist CGP-20712A (300 nmol/L, n=7 cells). B. Results before and after 1 µmol/L isoproterenol administered with the β2-selective antagonist ICI-118,511 (50 nmol/L, n=8 cells). C, D. Mean±SEM IKH current density–voltage relations from cells monitored continuously under control conditions and then in the presence of 3 µmol/L forskolin (C, n=6 cells) or 50 µmol/L 8-br-cAMP (D, n=8 cells), and then after washout.

 
3.2 Effects of forskolin and 8-bromo-cyclic AMP
To evaluate the role of cyclic AMP (cAMP), studies were performed with forskolin, which activates adenylyl cyclase independently of β-adrenergic receptors, and 8-bromo-cAMP, a membrane-permeable form of cAMP. Both compounds significantly enhanced IKH (Fig. 2, bottom). For example, forskolin increased IKH density at –120 mV from –4.1±0.3 to –8.1±0.7 pA/pF. At the same voltage, 8-bromo-cAMP increased IKH from –3.3±0.5 to –4.8±0.7 pA/pF. The effects of both compounds were reversible upon washout. These results indicate that the enhancement of IKH induced by β-adrenergic stimulation is due to increases in intracellular cAMP concentrations.

3.3 Role of protein kinase A
To determine the role of cAMP-dependent protein kinase A (PKA), we first used the protein kinase A inhibitor H89 (10 µmol/L). To study isoproterenol effects in the presence and absence of H89 in the same cell, we assessed two 10-minute isoproterenol (1 µmol/L) exposures in each cell, one without and the other with simultaneous superfusion of H89. Parallel experiments were performed in an identical fashion, but without H89 during the second isoproterenol exposure, to assess the reproducibility of isoproterenol's effect. Mean results are shown in Fig. 3A and B. Repeated isoproterenol exposure in the absence of H89 enhanced IKH during each exposure, but H89 fully prevented isoproterenol's action.


Figure 3
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  		Fig. 3 A, B. IKH density at –120 mV. A. Repeated exposure to isoproterenol in the absence of H89 produced consistent enhancement of IKH (n=6 cells). B. A second isoproterenol exposure failed to alter IKH in presence of H89 (n=6 cells). C. Mean±S.E.M. IKH density–voltage relations at baseline and in the presence of 100 nmol/L PDD (n=7 cells). D. Mean±S.E.M. IKH density–voltage relations at baseline and in the presence of phenylephrine (1 mmol/L, Phe) with bisindolylmaleimide-I (BIM I) pre-treatment (50 nmol/L, n=5 cells).

 
We used H89 because it allows for control exposures in the same cell. However, H89 can have complex inhibitory interactions with β-adrenoceptors [8]. We performed additional experiments with a specific intracellular PKA inhibitor peptide (PKI). IKH was recorded at –120 mV immediately after rupture, then after 20 min of dialysis with a pipette solution containing 50 nmol/L PKI. IKH was unaffected by pipette application of PKI, averaging 2.3±0.7 pA/pF immediately upon cell rupture and 2.3±0.6 pA/pF after PKI dialysis in 6 cells (at –120 mV). Isoproterenol (1 µmol/L) application failed to affect IKH in the presence of intracellular PKI: in 6 cells, IKH density averaged 2.3±0.7 pA/pF before, versus 2.6±0.6 pA/pF after, isoproterenol exposure (P=NS).

3.4 Alpha-adrenergic effects on IKH
To study {alpha}-adrenergic effects on IKH, phenylephrine was infused in the presence of 1 µmol/L propranolol to block any potential collateral β-adrenergic stimulation. Fig. 4A and B shows representative recordings before and after 1 mmol/L phenylephrine. Both time-dependent activating and tail current were reduced. Mean IKH density–voltage relations before and after phenylephrine are shown as a function of step potential in Fig. 4C. Panel D shows the concentration-dependent inhibitory effect of phenylephrine on IKH at –120 mV: IKH was decreased 23.1±3.7% by 10 µmol/L, 36.5±5.4% by 100 µmol/L, and 47.0±3.8% by 1 mmol/L phenylephrine. The inhibitory effect was reversible upon drug washout (data not shown). The effect of phenylephrine (1 mmol/L) was completely prevented upon co-administration with the {alpha}1-adrenoceptor inhibitor prazosin (2 µmol/L).


Figure 4
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  		Fig. 4 A, B. Representative IKH recordings under control condition and after phenylephrine (1 mmol/L). C. Mean±S.E.M. IKH density–voltage relations at baseline and under 1 mmol/L phenylephrine (n=12 cells). The inset shows outward currents on an expanded scale. D. Concentration-dependent effects on IKH induced by phenylephrine (Phe) (n=8 cells for each concentration, *P<0.05 and **P<0.01 versus baseline) and IKH changes caused by phenylephrine with pre-treatment of 2 µmol/L prazosin or 1 µmol/L U73122, shown as relative percentage of current density at –120 mV. (n=8 cells for prazosin group and n=6 cells for U73122 group). Mean±S.E.M. IKH density–voltage relations, E. under Phe (1 mmol/L) plus 10 mmol/L niguldipine (n=6 cells, Nig, selective {alpha}1A-selective antagonist) or F. under Phe (1 mmol/L) plus 10 µmol/L chloroethylclonidine (n=6 cells, CEC, selective {alpha}1B antagonist) or G. under Phe (1 mmol/L) plus 1 nmol/L BMY-7378 (n=6 cells, BMY, selective {alpha}1D antagonist), *P<0.05 versus baseline.

 
To evaluate the {alpha}1-adrenergic receptor subtype involved, selective {alpha}1-adrenergic receptor blockers, niguldipine (Nig, 10 nmol/L, {alpha}1A-selective), chloroethylclonidine (CEC, 10 µmol/L, {alpha}1B-selective) or BMY-7378 (BMY, 1 nmol/L, {alpha}1D-selective), were applied together with 1 mmol/L phenylephrine (in the presence of 1 µmol/L propranolol). The {alpha}1A-antagonist niguldipine fully prevented phenylephrine-induced suppression of IKH (Fig. 4E), whereas the {alpha}1B and {alpha}1D-receptor blockers CEC (Fig. 4F) and BMY (Fig. 4G) did not.

3.5 Role of phospholipase C and protein kinase C in {alpha}-adrenergic effects on IKH
Many {alpha}-adrenergic responses are mediated by phospholipase C (PLC)-induced hydrolysis of phosphatidylinositol-(4,5)-bis-phosphate (PIP2), which releases diacylglycerol leading to protein kinase C (PKC) activation. When cardiomyocytes were pre-treated with the PLC inhibitor, U73122 [GenBank] (1 µmol/L), the addition of phenylephrine (1 mmol/L) failed to suppress IKH (Fig. 4D, right). The potential role of PKC was then evaluated with the protein kinase C-activating phorbol ester, phorbol-12,13-didecanoate (PDD). PDD substantially reduced both activating and tail current IKH components. Mean activating current density–voltage relations before and after PDD are shown in Fig. 3C. Overall, 100 nmol/L PDD decreased IKH at –120 mV by 52±6%. The non-PKC-activating PDD congener 4{alpha}-PDD had no effect on IKH – e.g., at –120 mV IKH averaged 1.5±0.2 pA/pF before, versus 1.6±0.4 pA/pF after, 100 nmol/L {alpha}-PDD superfusion (n=5 cells, P=NS).

We then assessed the ability of the highly-selective PKC inhibitor bisindolylmaleimide-I (50 nmol/L), to prevent the inhibitory action of phenylephrine. Cells were pre-treated with bisindolylmaleimide for 10 min and phenylephrine (1 mmol/L) was then added along with bisindolylmaleimide. In the presence of bisindolylmaleimide, phenylephrine failed to alter IKH (Fig. 3D). IKH density (at –120 mV) averaged 2.2±0.4 pA/pF before, and 2.1±0.4 pA/pF after, bisindolylmaleimide (n=5 cells, P=NS). The PDD and bisindolylmaleimide results confirm the role of PKC.

3.6 Role of isoproterenol and phenylephrine effects on IKH in AP repolarization
To assess the functional role of adrenergic modulation of IKH, we tested the effects on left atrial action-potential (AP) duration (APD) of suppressing IKH with the highly selective blocker tertiapin-Q (TQ, 100 nmol/L). If adrenergic effects on IKH contribute to changes in repolarization, the result of IKH inhibition should be increased in the presence of β-adrenergic stimulation (which increases IKH) and decreased in the presence of {alpha}-adrenergic stimulation (which reduces IKH). Coronary-perfused multicellular preparations studied with standard fine-tipped microelectrodes were used to obtain as physiological conditions as possible. Tissue preparations were subjected to AP recordings from multiple cells under baseline conditions and then at steady state after perfusion with (1) tertiapin-Q alone (n=4 dogs), (2) isoproterenol alone (n=6 dogs), (3) isoproterenol plus tertiapin-Q (n=5 dogs), (4) phenylephrine alone (n=4 dogs) or (5) phenylephrine plus tertiapin-Q (n=4 dogs).

Fig. 5A–C shows representative left atrial APs under the various conditions studied. Tertiapin alone prolonged APD to 90% repolarization (APD90) by 22%. In the presence of isoproterenol, tertiapin increased APD90 by 72% (values in the presence of both isoproterenol and tertiapin compared to values with isoproterenol alone). In the presence of phenylephrine tertiapin increased APD90 by only 10% (values in presence of phenylephrine and tertiapin compared to values with phenylephrine alone). These results indicate that isoproterenol-enhancement of IKH results in a larger APD-prolonging effect of IKH-inhibition by tertiapin, whereas phenylephrine-induced IKH suppression attenuates tertiapin-Q's ability to prolong APD. The results of an analysis of the statistical significance of the interaction between the effects of isoproterenol or phenylephrine and tertiapin (based on raw data, not percentage changes) are shown in Table 1. There were statistically significant interactions between the effects of tertiapin-Q and isoproterenol and between tertiapin-Q and phenylephrine, confirming the relevance for effects on repolarization of the IKH-modulating actions of isoproterenol and phenylephrine.


Figure 5
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  		Fig. 5 Representative AP recordings and mean±SEM APD90 at 2 Hz are shown A. under control conditions (CTL) and then in the presence of 100 nmol/L tertiapin-Q (TQ) alone; B. under 5 µmol/L isoproterenol alone (Iso) and under isoproterenol plus tertiapin-Q (Iso+TQ); C. under 100 µmol/L phenylephrine alone (Phe) and under phenylephrine plus tertiapin-Q (Phe+TQ). For each panel, the mean % increase caused by tertiapin-Q is shown.

 

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  		Table 1 Effects of the IKH-selective blocker tertiapin-Q in the absence and presence of adrenergic agonists

 
3.7 Adrenergic effects on other inward-rectifying K+ currents
IK1 was measured with 300-ms voltage steps, as 1 mmol/L Ba2+-sensitive current in cells without IKH (Fig. 6A). IK1 was not changed by isoproterenol but was significantly reduced by phenylephrine. Neither isoproterenol nor phenylephrine significantly changed outward IK1, suggesting that IK1 contributes little to APD-changes induced by adrenergic stimulation.


Figure 6
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  		Fig. 6 A, mean±S.E.M. IK1 density–voltage relations at baseline and under 100 nmol/L isoproterenol (left) or under 100 µmol/L phenylephrine (right) (n=6 cells/observation). B, mean±S.E.M. IKACh density–voltage relations at baseline and under 100 nmol/L isoproterenol (left) or 100 µmol/L phenylephrine (right) (n=6 cells/observation). *P<0.05.

 
IKACh was induced by 500 nmol/L carbachol (Fig. 6B). Consecutive carbachol exposures were performed in each cell, first without and then with simultaneous superfusion of 100 nmol/L isoproterenol or 100 µmol/L phenylephrine (along with 1 µmol/L propranolol). The effect of carbachol washed out following carbachol exposures. IKACh was significantly increased by isoproterenol (n=5 cells, P<0.05) but was significantly decreased by phenylephrine (n=5 cells, P<0.05). Repeated carbachol exposure without adrenergic agonists reproducibly activated IKACh of the same amplitude (n=4 cells).


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
Acknowledgments
 References
 
In the present study, we demonstrated that β1- and {alpha}1A-adrenergic receptor systems modulate IKH. The current-enhancing effects of β1-adrenergic stimulation are mediated by cyclic AMP-induced activation of PKA, whereas the inhibitory effects of {alpha}1A-adrenergic stimulation proceed by PLC-mediated activation of PKC. The adrenergic modulation of IKH contributes to changes in repolarization.

4.1 Relationship to previous studies of adrenergic actions on K+-channels
Adrenergic modulation is well-known to modulate the properties of K+-channels. β-Adrenergic stimulation via a cAMP-dependent PKA mechanism enhances a variety of cardiac K+ currents including IK, in particular the slow component IKs [9–11] and IKur [12], but has been reported to inhibit IK1 [13,14]. We did not observe significant β-adrenergic effects on IK1 in the present study. {alpha}-Adrenergic stimulation inhibits several K+ currents, including Ito [15,16] and IK1 [[17,18] and present study], but enhances delayed-rectifier K+ currents in guinea pig ventricle [19]. {alpha}-Adrenergic stimulation decreases IKur in human atrial cardiomyocytes [12] but enhances the corresponding current in canine atrial myocytes [20].

In both rat atrial myocytes and Xenopus oocytes, β-adrenergic stimulation enhances acetylcholine-evoked Kir3-based currents (IKACh) via cAMP-dependent protein kinase [21]. In Xenopus oocytes injected with Kir3.1/Kir3.4 subunits, M2-cholinergic and β-adrenergic receptors, isoproterenol elicited Kir3.1/3.4 currents even in the absence of acetylcholine [21], a result analogous to our observation of β-adrenergic enhancement of IKH in the absence of muscarinic agonists. Phenylephrine accelerates the decay of IKACh via depletion of phosphatidylinositol 4,5-bisphosphate (PIP2) in mouse atrial cardiomyocytes [22]. In rabbit atrial cardiomyocytes, {alpha}-adrenergic stimulation also reduces IKACh [18]. On the other hand, {alpha}-adrenergic stimulation activates IKACh via 5'-lipoxygenase or metabolites of arachidonic acid in guinea pigs [23]. These observations indicate the potential diversity of {alpha}-adrenergic effects on Kir 3-related currents. The present study is the first to show that adrenergic stimulation modulates constitutive Kir3-related currents in mammalian atrial cardiomyocytes. We also noted β-adrenergic enhancement and {alpha}-adrenergic suppression of agonist-induced IKACh, suggesting congruent adrenergic regulation of both constitutive and agonist-induced Kir3 currents in canine atrial cardiomyocytes. Because adrenergic stimulation affects a large number of currents involved in cardiac repolarization [4,5], it is challenging to determine whether adrenergic effects on one specific current play a functional role. In this study, we addressed the functional significance of adrenergic regulation of IKH by observing the effect of blocking the current with a highly selective inhibitor, tertiapin-Q, in the absence of adrenergic stimulation and then in the presence of {alpha}- or β-adrenoceptor agonists.

4.2 Mechanisms of adrenergic modulation of IKH
Protein kinases are important components of the signal transduction system for adrenergic stimulation. β-Adrenergic stimulation enhances inward-rectifier K+ currents in glial cells [24] and acetylcholine-activated currents in rat atrial cardiomyocytes [25], with substantial evidence for a central role of PKA-phosphorylation in mediating such actions [21,24–27]. Stimulation of {alpha}-adrenergic receptors leads to various biochemical responses, including enhanced Ca2+ influx, PLC activation, and changes in intracellular cyclic nucleotide levels. The activation of PLC results in PIP2 hydrolysis and activation of PKC. PIP2 depletion has been shown to inhibit Kir3.x-related currents [28,29]. PKC is also frequently implicated as an inhibitor of Kir3-related currents [30–35], with specific serine phosphorylation sites in Kir3.1 and 3.4 being essential for PKC-dependent inhibition of GIRK channels [35]. In atrial cardiomyocytes, Kir3 channels are assembled in a signalling complex along with Gβ{gamma}, G protein-coupled receptor kinase, cyclic adenosine monophosphate-dependent protein kinase, and receptor for activated C kinase 1 (RACK1) [33]. Other recent studies have demonstrated that these signalling complexes may be formed during biosynthesis [36,37], and provide a basis for distinct signalling arrays in different cell types (for review, see Ref. [38]). It is clear that cellular context is a key feature of how signal integrators such as Kir3 channels are regulated. All of the modulatory effects on IKH were mediated by protein kinases, with no evidence for protein-kinase independent direct membrane-delimited mechanisms. Receptor-mediated inhibition of Kir3 related currents may occur independently of PKC activation [18,22,29,39]. Complicated interactions between PLC-induced PKC activation and PIP2 depletion may lead to varying consequences. In rabbit atrial myocytes, reductions in membrane PIP2 content prevent Kir3 channel–PIP2 interactions and decrease corresponding currents [40]. PKC-dependent phosphorylation of phosphatidylinositol transfer protein can reduce membrane PIP2 content [41]. In our studies, PKC inhibition prevented the inhibitory effect of phenylephrine on IKH, suggesting that in canine atrial cardiomyocytes PKC activation plays a primary role in mediating {alpha}-adrenergic inhibition of the Kir3-based current IKH.

4.3 Potential physiological and clinical relevance
Adrenergic stimulation plays a role in governing atrial APD and refractoriness. Both {alpha}- and β-adrenergic stimulation modulates various ion channels and therefore exerts potentially complex effects on APD [4,5,42]. Diverse effects of adrenergic stimulation on APD in different tissues and species have been reported [43,44]. In our study, β-adrenergic stimulation abbreviated canine atrial APD markedly, whereas {alpha}1-adrenergic stimulation tended to prolong APD slightly. Our studies of the AP response to tertiapin-Q under varying adrenergic-stimulation conditions (Fig. 5, Table) indicate functional significance for isoproterenol's effects on IKH and suggest that they contribute to β-adrenergic APD shortening. The small, statistically non-significant net effect of {alpha}1-adrenoceptor stimulation on APD suggests that the APD prolongation that would result from IKH inhibition is offset by {alpha}-adrenoceptor actions on other currents.

The autonomic nervous system is known to contribute to arrhythmogenesis [6,7]. We previously showed that IKH block by tertiapin-Q suppresses both APD-shortening and arrhythmia-promoting effects of atrial tachycardia remodeling, pointing to a significant role of IKH in atrial arrhythmogenesis [2]. Patients with AF have enhanced constitutive IKACh analogous to IKH [3]. In addition, IKACh single channel events compatible with IKH have been observed in the absence of cholinergic agonists in human atrial cardiomyocytes [45,46]. The present observations point to the potential role of constitutive acetylcholine-regulated current as a mediator of adrenergic effects on atrial APD and arrhythmias. β-Adrenoceptor blocking drugs can prevent AF [47]. β-Adrenergic modulation of constitutive acetylcholine-regulated K+ current may contribute to this action. The importance of interactions between the results of cholinergic and adrenergic stimulation on the heart is well established [48,49]. The present study provides evidence for the functional importance of a novel mechanism of cardiac adrenergic–cholinergic system interactions: adrenergic modulation of acetylcholine-regulated currents that are active in the absence of cholinergic agonists.

4.4 Potential limitations
We relied heavily on pharmacological tools for dissection of protein kinase systems, so specificity is always a potential issue. Bisindolylmaleimide is a highly selective inhibitor of PKC [50]. H89 inhibits PKA without effects on PKC, although it can exert effects on other kinases [51] and can directly inhibit β-adrenoceptors [8]. We therefore performed complementary experiments in which PKI peptide was used as a specific inhibitor of PKA [52]. We nevertheless show both sets of data, because since H89 is given by extracellular superfusion results can be obtained before and after the drug in the same cell, whereas the adrenergic response cannot be examined in the same cell before and after administration of PKI (which is dialyzed intracellularly). Tertiapin-Q was a key tool in studies of the functional significance of adrenergic regulation of IKH. Drici et al. have shown that tertiapin, at concentrations that strongly suppress IKACh, has no effect on IKr, IKs, Ito, IKl, INa, or ICaL [53]. In addition, we verified that under the conditions in this study there was no detectible "funny" current (If) before or after isoproterenol (data not shown).

In the present study, we assessed adrenergic regulation of the constitutive Kir3-based current IKH. We also noted {alpha}-adrenergic modulation of IK1 and {alpha} -and β-adrenergic modulation of agonist-induced IKACh, which could contribute to in-vivo APD-regulation. However, the role of IKH in APD-regulation that we observed with the use of tertiapin (Table, Fig. 5) is unlikely to be contaminated by effects on IK1 because IK1 is tertiapin-insensitive, nor by effects on agonist-induced IKACh because these experiments were performed in the presence of the muscarinic-receptor blocker atropine.


   Acknowledgments
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Acknowledgments
References
 
The authors thank Chantal St Cyr, Chantal Maltais and Nathalie L'Heureux for the technical assistance, and France Thériault and Luce Bégin for the secretarial help with the manuscript. Funding was provided by the Canadian Institutes of Health Research and the Quebec Heart and Stroke Foundation. T.E.H. is a Fonds de la recherche en santé du Québec senior scholar.


   Notes
 
Time for primary review 37 days


   References
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 

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