Original articleSelective activation of adrenoceptors potentiates IKs current in pulmonary vein cardiomyocytes through the protein kinase A and C signaling pathways
Graphical abstract
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.
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