Original article
Selective activation of adrenoceptors potentiates IKs current in pulmonary vein cardiomyocytes through the protein kinase A and C signaling pathways

https://doi.org/10.1016/j.yjmcc.2021.08.004Get rights and content

Highlights

  • The IKs channel was expressed on the membrane of pulmonary vein cardiomyocytes (PVC).

  • β- and α1-adrenoceptor stimulation enhanced IKs in PVC via the PKA and PKC pathway.

  • The IKs was more potentiated by β2-adrenoceptor stimulation than β1-adrenoceptor.

  • The β-adrenoceptor enhanced firing rate was markedly reduced by IKs inhibition.

Abstract

Delayed rectifier K+ current (IKs) is a key contributor to repolarization of action potentials. This study investigated the mechanisms underlying the adrenoceptor-induced potentiation of IKs in pulmonary vein cardiomyocytes (PVC). PVC were isolated from guinea pig pulmonary vein. The action potentials and IKs current were recorded using perforated and conventional whole-cell patch-clamp techniques. The expression of IKs was examined using immunocytochemistry and Western blotting. KCNQ1, a IKs pore-forming protein was detected as a signal band approximately 100 kDa in size, and its immunofluorescence signal was found to be mainly localized on the cell membrane. The IKs current in PVC was markedly enhanced by both β1- and β2-adrenoceptor stimulation with a negative voltage shift in the current activation, although the potentiation was more effectively induced by β2-adrenoceptor stimulation than β1-adrenoceptor stimulation. Both β-adrenoceptor-mediated increases in IKs were attenuated by treatment with the adenylyl cyclase (AC) inhibitor or protein kinase A (PKA) inhibitor. Furthermore, the IKs current was increased by α1-adrenoceptor agonist but attenuated by the protein kinase C (PKC) inhibitor. PVC exhibited action potentials in normal Tyrode solution which was slightly reduced by HMR-1556 a selective IKs blocker. However, HMR-1556 markedly reduced the β-adrenoceptor-potentiated firing rate. The stimulatory effects of β- and α1-adrenoceptor on IKs in PVC are mediated via the PKA and PKC signal pathways. HMR-1556 effectively reduced the firing rate under β-adrenoceptor activation, suggesting that the functional role of IKs might increase during sympathetic excitation under in vivo conditions.

Introduction

Atrial fibrillation (AF) is the most common of all sustained cardiac arrhythmias in humans, manifesting more frequently with age and being typically caused by stroke, although the mechanisms underlying the initiation of AF are still not fully understood [1]. Early studies reported that the walls of pulmonary veins (PVs) have a myocardial muscle layer connected to left atrial cardiac myocytes, which generate spontaneous automaticity (action potentials) [2]. This automaticity can be enhanced and propagated into the left atrium by treatment with digitalis toxicity [3]. Of note, paroxysmal AF in humans is initiated by ectopic beats originating from the PVs [4]. Many subsequent clinical and experimental studies confirmed that PVs are important sources of ectopic beats for the initiation of paroxysmal and chronic AF [5,6]. Furthermore, a reduction in the PVs focus through ablation is effective for treating acute and sustained AF, suggesting dynamic interaction between the left atrial (LA) and PVs ectopic activity [7,8].

The basic electrophysiological and pharmacological properties of PVs and pulmonary vein cardiomyocytes (PVC) have been reported in various experimental species, including dogs [9], rabbits [10], rats [11,12] and guinea pigs [2,13,14]. The electrical properties of PVs were reported to differ markedly from the left atrium, and their ectopic automaticity might also be induced via different mechanisms from the sinus node [5,6]. Previous studies have reported that PVC favor the occurrence of reentry due to shorter action potentials, apparently due to a lower voltage-dependent L-type Ca2+ current [15,16]. In addition, PVC have a lower negative resting membrane potential than the left atrium due to the lower density of the inwardly rectifying K+ current (IK1) in canine [16] and guinea pig models [13]. A recent study reported that the electrical activity of rat PVC is suppressed by treatment with inhibitors of sarcoplasmic reticulum and membrane Na+/Ca2+ exchanger, and similar results were confirmed in other experimental species [17]. These results suggest that the increases in intracellular Ca2+ induced by the sarcoplasmic reticulum and the enhanced activity of the Na+/Ca2+ exchanger might influence the ectopic automaticity of PVs.

The slow component of the delayed rectifier K+ channel (IKs) has been shown to exist in the cardiac myocytes of various mammalian species, including humans [18]. The IKs channel is formed by two subunits: KCNQ1 (pore-forming α-subunit) and KCNE1 (β-subunit) [19,20]. In human and guinea pigs, IKs is activated slowly following the upstroke of the action potential and has an important role in providing an outward current to initiate the phase 3 repolarization of atrial and ventricular action potentials [21,22]. Furthermore, a previous report showed that, in sinoatrial (SA) node cells, the deactivation process of the delayed rectifier K+ current at the repolarizing phase plays a crucial role in providing diastolic depolarization [23]. We previously reported that suppression of the IKs current by its selective blocker delays the repolarization process and markedly reduces the pacemaker activity in SA node cells of guinea pigs [24,25] suggesting that IKs can contribute to the repolarizing process in SA node cells, which is important for determining the pacemaker activity. Of note, in the presence of β-adrenergic stimulation condition the dominance contribution involved in the cardiac action potential shifted from the delayed rectifier K+ channel (IKr) to IKs [26], so IKs may regulate cardiac excitement and contraction by various extracellular signaling molecules. Indeed, we and other groups have shown that β-adrenergic stimulation preferentially enhances IKs in guinea pig ventricular myocytes [27,28], SA node cells [29] and human atrial myocytes [30]. We suspect that, during sympathetic or exercised stimulation, both Ca2+ and the IKs current are enhanced, so potentiated IKs may maintain a proper balance between the inward and outward currents, thereby regulating cardiac pacemaker activity and contraction.

Given these previous findings, we hypothesized that adrenergic signal activation might regulate the function of IKs in PVC, and IKs might modify the property of PVC automaticity, especially under adrenoceptor-stimulated conditions. We therefore investigated 1) whether or not IKs is functionally present in guinea pig PVC and 2) whether or not its activity and contribution to the automaticity are modulated during sympathetic excitation. Since the adrenergic signal pathway regulates the heart function through different receptor subtypes [31,32], the present study investigated the electrophysiological properties of IKs and compared the effects of β1-, β2- and α1-adrenergic receptor stimulation on IKs in PVC.

Section snippets

Preparations

All animal care and experimental procedures were conducted in accordance 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) and were approved by the institution's Animal Care and Use Committee of Shiga University of Medical Science (approval number, 2018-7-3). Female Hartley guinea pigs (4–6 weeks old, 250–400 g) were used in the present experiments. The studies involving animals were reported in

The expression, localization and current density of IKs in PVC and LA

The protein expression of KCNQ1 (pore-forming α-subunit of IKs channel) of PVC and LA was examined as shown in Fig. 1A, the positive single band of KCNQ1 of roughly 100 kDa was detected by Western blotting, as previously reported in mouse cardiomyocytes [39] and human iPS Cell-Derived cardiomyocytes [40]. The positive single band of KCNE1 (pore-forming β-subunit of IKs channel) was detected approximately 17 kDa in size (Supplementary material ), supported by the previous results obtained in rat

Discussion

The present study examined the electrophysiological properties of IKs and its modulation by stimulating different kinds of adrenergic receptors in guinea pig PVC. Our findings show that (1) the functional IKs channel (KCNQ1) was expressed and located on the cell membrane; (2) IKs amplitude was markedly enhanced with a negative shift in the voltage dependence of channel activation by both β1- and β2-adrenoceptor stimulation mediated via the AC-cAMP-PKA signaling pathway and also potentiated by α1

Sources of funding

This study was supported by a Grant-in-Aid for Scientific Research (No. 17K08536 to H.M.) from the Japan Society for the Promotion of Science (Tokyo, Japan).

Author contributions

W.-G.D., X.M. and H.M. designed the experiments. X.M., W.-G.D. and M.O.-K. conducted the experiments. X.M., W.-G.D., F.T. and A.K. participated in the data interpretation. X.M., W.-G.D. and H.M. wrote the manuscript. X.M. W.-G.D., F.T., A.K., M.O.-K. and H.M. approved the final manuscript.

Declaration of Competing Interest

The authors declare no conflicts of interest.

References (70)

  • D.M. Bers et al.

    Na/K-ATPase--an integral player in the adrenergic fight-or-flight response

    Trends Cardiovasc. Med.

    (2009)
  • M.E. Mangoni et al.

    Voltage-dependent calcium channels and cardiac pacemaker activity: from ionic currents to genes

    Prog. Biophys. Mol. Biol.

    (2006)
  • S. Kato et al.

    Pharmacological blockade of IKs destabilizes spiral-wave reentry under β-adrenergic stimulation in favor of its early termination

    J. Pharmacol. Sci.

    (2012)
  • S. Nattel et al.

    Electrophysiological and molecular mechanisms of paroxysmal atrial fibrillation

    Nat. Rev. Cardiol.

    (10, 2016)
  • D.W. Cheung

    Electrical activity of the pulmonary vein and its interaction with the right atrium in the guinea-pig

    J. Physiol.

    (1981)
  • D.W. Cheung

    Pulmonary vein as an ectopic focus in digitalis-induced arrhythmia

    Nature

    (1981)
  • M. Haïssaguerre et al.

    Spontaneous initiation of atrial fibrillation by ectopic beats originating in the pulmonary veins

    N. Engl. J. Med.

    (10, 1998)
  • S. Nattel

    Basic electrophysiology of the pulmonary veins and their role in atrial fibrillation: precipitators, perpetuators, and perplexers

    J. Cardiovasc. Electrophysiol.

    (12, 2003)
  • I. Namekata et al.

    Electrophysiological and pharmacological properties of the pulmonary vein myocardium

    Biol. Pharm. Bull.

    (2013)
  • P. Jaïs et al.

    A focal source of atrial fibrillation treated by discrete radiofrequency ablation

    Circulation

    (1997)
  • H. Calkins

    Has the time come to abandon the concept that “pulmonary vein isolation is the cornerstone of atrial fibrillation ablation”?

    Circ. Arrhythm. Electrophysiol.

    (2013)
  • P. Melnyk et al.

    Comparison of ion channel distribution and expression in cardiomyocytes of canine pulmonary veins versus left atrium

    Cardiovasc. Res.

    (2005)
  • V. Maupoil et al.

    Ectopic activity in the rat pulmonary vein can arise from simultaneous activation of alpha1- and beta1-adrenoceptors

    Br. J. Pharmacol.

    (2007)
  • M. Hocini et al.

    Electrical conduction in canine pulmonary veins: electrophysiological and anatomic correlation

    Circulation

    (20, 2002)
  • J.R. Ehrlich et al.

    Cellular electrophysiology of canine pulmonary vein cardiomyocytes: action potential and ionic current properties

    J. Physiol.

    (2003)
  • M.C. Sanguinetti et al.

    Two components of cardiac delayed rectifier K+ current. Differential sensitivity to block by class III antiarrhythmic agents

    J. Gener. Physiol.

    (1990)
  • M.C. Sanguinetti et al.

    Coassembly of K(V)LQT1 and minK (IsK) proteins to form cardiac I(Ks) potassium channel

    Nature

    (1996)
  • J. Barhanin et al.

    K(V)LQT1 and lsK (minK) proteins associate to form the I(Ks) cardiac potassium current

    Nature

    (1996)
  • R.F. Bosch et al.

    Effects of the chromanol HMR 1556 on potassium currents in atrial myocytes

    Naunyn Schmiedeberg’s Arch. Pharmacol.

    (2003)
  • R.F. Bosch et al.

    Effects of the chromanol 293B, a selective blocker of the slow, component of the delayed rectifier K+ current, on repolarization in human and guinea pig ventricular myocytes

    Cardiovasc. Res.

    (1998)
  • H. Irisawa et al.

    Cardiac pacemaking in the sinoatrial node

    Physiol. Rev.

    (1993)
  • Y. Xie et al.

    Ca2+/calmodulin potentiates I Ks in sinoatrial node cells by activating Ca2+/calmodulin-dependent protein kinase II

    Arch. Eur. J. Physiol.

    (2015)
  • H. Matsuura et al.

    Rapidly and slowly activating components of delayed rectifier K(+) current in guinea-pig sino-atrial node pacemaker cells

    J. Physiol.

    (2002)
  • T. Banyasz et al.

    Beta-adrenergic stimulation reverses the I Kr-I Ks dominant pattern during cardiac action potential

    Arch. Eur. J. Physiol.

    (11, 2014)
  • M. Si et al.

    Involvement of protein kinase A and C in norepinephrine- and angiotensin II-induced modulation of cardiac IKs

    Pharmacology

    (2013)
  • View full text