Inhibition of cardiac two-pore-domain K+ (K2P) channels by the antiarrhythmic drug vernakalant – Comparison with flecainide

doi:10.1016/j.ejphar.2013.12.030

European Journal of Pharmacology

Volume 724, 5 February 2014, Pages 51-57

https://doi.org/10.1016/j.ejphar.2013.12.030 Get rights and content

Abstract

The mixed ion channel blocker, vernakalant (RSD1235), is effective in rapid conversion of atrial fibrillation (AF) to sinus rhythm (SR). Suppression of cardiac two-pore-domain potassium (K_2P) channels causes action potential prolongation and has recently been proposed as a novel antiarrhythmic strategy. The objective of this study was to investigate acute effects of vernakalant on human K_2P2.1 (TREK-1) and K_2P3.1 (TASK-1) channels to provide a more complete picture of its antiarrhythmic mechanism of action. The class IC antiarrhythmic drug flecainide was studied as a comparator agent. Two-electrode voltage clamp and whole-cell patch clamp electrophysiology was used to record K_2P currents from Xenopus oocytes and Chinese hamster ovary (CHO) cells. Vernakalant inhibited cardiac K_2P2.1 channels expressed in Xenopus oocytes and in CHO cells. The IC₅₀ value obtained from mammalian cells (13.3 µM) was close to the range of vernakalant levels reported in patients (2–8 µM), indicating potential clinical significance of K_2P2.1 blockade. Open rectification characteristics and current–voltage relationships of K_2P2.1 currents were not affected by vernakalant. Vernakalant did not significantly reduce K_2P3.1 currents. Finally, the class I antiarrhythmic drug flecainide had no effect on K_2P2.1 or K_2P3.1 channels. In conclusion, the recently developed antiarrhythmic drug vernakalant targets human K_2P2.1 K⁺ background channels. This previously unrecognized inhibitory property adds to the multichannel blocking profile of vernakalant and extends the mechanistic basis for its anti-fibrillatory effect.

Introduction

Effective pharmacological management of atrial fibrillation (AF) still remains an unmet medical need. The antiarrhythmic drug vernakalant (RSD1235) effectively converts AF to sinus rhythm (SR) (Atrial Arrhythmia Conversion Trial Investigators, 2008, Atrial Arrhythmia Conversion Trial Investigators, 2009, Dobrev et al., 2010, Atrial Arrhythmia Conversion Trial (ACT-III) Investigators, 2010, Stiell et al., 2010, AVRO Investigators, 2011). At the molecular level, the drug targets multiple ion currents in cardiac myocytes (CM) or ion channels expressed in mammalian cells (MC), including Nav1.5 (9 µM at 20 Hz; 43 µM at 1 Hz; MC), I_K,ACh (10 µM; CM), Kv1.5 (13 µM; MC), I_to (15 µM; CM), Kv4.2 (38 µM; MC), Kv4.3 (30 µM; MC), Kv11.1 (21 µM; MC), and I_Ca,L (42 µM; CM) (Fedida et al., 2005, Orth et al., 2006, Fedida, 2007, Dobrev et al., 2010, Wettwer et al., 2013). I_K1 currents recorded from cardiac myocytes were not significantly affected by vernakalant at high concentrations (Fedida et al., 2005). A comparison between human right atrial tissue and isolated myocytes from SR versus AF patients revealed that the antiarrhythmic efficacy in AF is mainly due to rate-dependent inhibition of sodium currents, with additional contribution of I_K,ACh and early I_to inhibition (Wettwer et al., 2013). Data obtained from human cardiac myocytes further suggest inhibitory effects of vernakalant on background currents in addition to blockade of sodium and outward potassium currents (Wettwer et al., 2013).

Two-pore-domain potassium (K_2P) channels are implicated in the background K⁺ conductance (Marban, 2002) and facilitate action potential repolarization in excitable cells (Goldstein et al., 2001). Regulation of K_2P background currents serves as a dynamic mechanism for control of cellular excitability (Thomas et al., 2008, Staudacher et al., 2011a, Staudacher et al., 2011b, Gierten et al., 2012, Rahm et al., 2012, Rahm et al., 2013, Seyler et al., 2012). In the heart, genetic inactivation or inhibition of K_2P3.1 (TASK1, tandem of P domains in a weak inward rectifying K⁺ channel (TWIK)-related acid sensitive K⁺ channel 1) currents by class III antiarrhythmic drugs results in action potential prolongation (Decher et al., 2011, Petric et al., 2012). Human K_2P3.1 channels are targeted by the antiarrhythmic drugs amiodarone, carvedilol, dronedarone, mexiletine, and propafenone (Gierten et al., 2010, Staudacher et al., 2011b, Schmidt et al., 2012, Schmidt et al., 2013a). In addition, stretch-activated K_2P2.1 (TREK1, TWIK-related K⁺ channel) currents are sensitive to dronedarone, mexiletine, and propafenone (Schmidt et al., 2012, Schmidt et al., 2013a).

Effects of vernakalant on cardiac K_2P channels have not been addressed to date. The present study investigated electrophysiological effects of vernakalant on K_2P2.1 and K_2P3.1 channels to provide a more complete assessment of its antiarrhythmic mechanism of action. A class IC antiarrhythmic drug with established efficacy in converting AF to SR, flecainide, was studied for mechanistic comparison. Flecainide modulates atrial and ventricular electrophysiology and was previously shown to inhibit cardiac I_Na (1.3 µM at 1 Hz; CM), I_to (3.7–10 µM; CM), and Kv11.1 channels (1.1 µM; MC), at low micromolar concentrations (Slawsky and Castle, 1994, Wang et al., 1995, Penniman et al., 2010, Du et al., 2011).

Section snippets

Molecular biology

Complementary DNAs encoding human K_2P2.1 (GenBank accession number EF165334) and hK_2P3.1 (NM_002246) were kindly provided by Steve Goldstein (Brandeis University, Waltham, MA, USA). In vitro transcription was performed as published (Thomas et al., 2008). Complementary RNAs were transcribed after vector linearization using T7 RNA polymerase and the mMessage mMachine kit (Ambion, Austin, TX, USA). Transcripts were quantified by spectrophotometry and cRNA integrity was assessed by agarose gel

Effects of vernakalant on human K_2P2.1 and K_2P3.1 channels expressed in Xenopus oocytes

Vernakalant sensitivity of human K_2P2.1 and K_2P3.1 channels was assessed in Xenopus laevis oocytes. From a holding potential of −80 mV, currents were activated by hyperpolarizing and depolarizing pulses (500 ms) to voltages between −140 and +60 mV in 20 mV increments (Fig. 1A). Current amplitudes were measured at the end of the +20 mV-pulse. This protocol was used in all oocyte experiments in this study. Substantial K⁺ currents were recorded from oocytes expressing either K_2P2.1 (Fig. 1A) or K_2P3.1

Vernakalant inhibits human cardiac K_2P2.1 K⁺ currents

Human cardiac K_2P2.1 (TREK-1) background K⁺ channels were blocked by vernakalant in a concentration-dependent fashion, whereas the channels were insensitive to the class IC antiarrhythmic drug flecainide. K_2P2.1 channels expressed in Xenopus oocytes exhibited an apparently lower sensitivity to vernakalant compared to CHO cells. Differences in drug affinity between oocytes and mammalian cells are commonly observed and attributed to specific properties of oocytes (e.g. the vitelline membrane and

Conclusion

K_2P2.1 current inhibition by vernakalant represents a previously unrecognized mode of action that extends the multichannel blocking profile of the drug. Targeting of human cardiac K_2P2.1 channels by vernakalant suggests a mechanistic role of the channels in AF. The therapeutic significance of the K_2P ion channel family in AF requires further evaluation.

Acknowledgments

We thank Jennifer Gütermann, Bianca Stadler and Kai Sona for excellent technical assistance. This study was supported by a Grant from MSD Sharp and Dohme GmbH. Additional support was provided by Grants from the Joachim Siebeneicher Foundation (to DT), the DZHK (Deutsches Zentrum für Herz-Kreislauf-Forschung – German Centre for Cardiovascular Research) and the BMBF (German Ministry of Education and Research) (to HAK and DT).

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Atrial fibrillation (AF) is a common cardiac arrhythmia that is associated with increased mortality. Heart failure, hypertension, valvular disease, and obstructive sleep apnea are risk factors for incident AF. A common characteristic of these diseases is that they increase atrial wall stretch. Multiple experimental studies confirm a proarrhythmic effect of atrial stretch. Conversely, a reduction in stretch is antiarrhythmic. A therapeutic target for AF, therefore, lies in local reduction of atrial stretch. This review focuses on atrial stretch and its clinical associations in patients with AF and its downstream effects on electrophysiology. We discuss the possible application of targeted atrial stretch reduction in AF prevention. We conclude that a reduction in local atrial stretch should be considered an essential element in rhythm control.
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Similarly, antiarrhythmic drugs have been reported to block TREK-1 currents. Here, Vaughan Williams class III antiarrhythmic drugs dronedarone and vernakalant, class I compounds lidocaine, mexiletine and propafenone, carvedilol, a beta-blocker that possesses additional class III properties, and the late sodium current inhibitor ranolazine have been shown to inhibit TREK-1 channels in heterologous expression systems (Schmidt et al., 2012, 2013; Seyler et al., 2014; Kisselbach et al., 2014; Ratte et al., 2019). The IC50 values of TREK-1, measured in different heterologous expression systems, are summarized in Table 2.
TWIK-related K⁺ channel (TREK-1) two-pore-domain potassium (K_2P) channels mediate background potassium currents and regulate cellular excitability in many different types of cells. Their functional activity is controlled by a broad variety of different physiological stimuli, such as temperature, extracellular or intracellular pH, lipids and mechanical stress.
By linking cellular excitability to mechanical stress, TREK-1 currents might be important to mediate parts of the mechanoelectrical feedback described in the heart. Furthermore, TREK-1 currents might contribute to the dysregulation of excitability in the heart in pathophysiological situations, such as those caused by abnormal stretch or ischaemia-associated cell swelling, thereby contributing to arrhythmogenesis.
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Cardiac K<inf>2P</inf>13.1 (THIK-1) two-pore-domain K<sup>+</sup> channels: Pharmacological regulation and remodeling in atrial fibrillation
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Two-pore domain K+ (K2P) channels have recently been proposed as novel antiarrhythmic drug targets (Hancox et al., 2016; Ravens, 2010; Schmidt et al., 2014b; Wiedmann et al., 2016). K2P channels facilitate action potential repolarization, and dynamic regulation of K2P currents modulates cellular excitability (Donner et al., 2011; Enyedi and Czirják, 2010; Gierten et al., 2010; Goldstein et al., 2001, 2005; Kisselbach et al., 2012, 2014; Marban, 2002; Rahm et al., 2012, 2014; Sandoz et al., 2011, 2012; Schmidt et al., 2012, 2013, 2014a, 2017a; Seyler et al., 2012, 2014a, 2014b; Staudacher et al., 2011a, 2011b). In the heart, inhibition by class III antiarrhythmic drugs or genetic inactivation of K2P currents results in action potential prolongation (Chai et al., 2017; Donner et al., 2011; Schmidt et al., 2015).
Cardiac two-pore-domain potassium (K_2P) channels have been proposed as novel antiarrhythmic targets. K_2P13.1 (THIK-1) channels are expressed in the human heart, and atrial K_2P13.1 levels are reduced in patients with atrial fibrillation (AF) or heart failure. The first objective of this study was to investigate acute effects of antiarrhythmic drugs on human K_2P13.1 currents. Second, we assessed atrial K_2P13.1 remodeling in AF pigs to validate the porcine model for future translational evaluation of K_2P13.1-based antiarrhythmic concepts. K_2P13.1 protein expression was studied in domestic pigs during AF induced by atrial burst pacing. AF was associated with 66% reduction of K_2P13.1 levels in the right atrium at 21-day follow-up. Voltage clamp electrophysiology was employed to elucidate human K_2P13.1 channel pharmacology in Xenopus oocytes. Propafenone (−26%; 100 μM), mexiletine (−75%; 1.5 mM), propranolol (−38%; 200 μM), and lidocaine (−59%; 100 μM) significantly inhibited K_2P13.1 currents. By contrast, K_2P13.1 channels were not markedly affected by quinidine, carvedilol, metoprolol, amiodarone and verapamil. Concentration-dependent K_2P13.1 blockade by mexiletine occurred rapidly with membrane depolarization and was frequency-independent. Mexiletine reduced K_2P13.1 open rectification properties and shifted current-voltage relationships towards more negative potentials. In conclusion, atrial expression and AF-associated downregulation of K_2P13.1 channels in a porcine model resemble human findings and support a general role for K_2P13.1 in AF pathophysiology. K_2P13.1 current sensitivity to antiarrhythmic drugs provides a starting point for further development of an emerging antiarrhythmic paradigm.
Identification and functional characterization of zebrafish K<inf>2P</inf>17.1 (TASK-4, TALK-2) two-pore-domain K<sup>+</sup> channels
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Human K_2P17.1 (TASK-4, TALK-2) two-pore-domain potassium (K_2P) channels have recently been implicated in heart rhythm disorders including atrial fibrillation and conduction disease. The functional in vivo significance of K_2P17.1 currents in cardiac electrophysiology remains incompletely understood. Danio rerio (zebrafish) may be utilized to elucidate the role of cardiac K_2P channels in vivo. The aim of this work was to identify and characterize the zebrafish ortholog of K_2P17.1 in comparison to its human counterpart.
The zkcnk17 coding sequence was amplified from zebrafish cDNA. Zebrafish kcnk17 mRNA expression was assessed by polymerase chain reaction. Human and zebrafish K_2P17.1 currents were analyzed using two-electrode voltage clamp electrophysiology and the Xenopus oocyte expression system. Kcnk17 mRNA was detected in zebrafish brain. Human and zebrafish K_2P17.1 proteins exhibited 33.4% identity. Zebrafish K_2P17.1 channels conducted K⁺ selective currents with open rectification properties. Both human and zebrafish K_2P17.1 were inhibited by barium. In contrast to human K_2P17.1, zK_2P17.1 currents were not sensitive to extracellular alkalization, likely due to the lack of a lysine residue involved in pH sensing of hK_2P17.1.
In conclusion, zebrafish and human K_2P17.1 channels display similar structural and regulatory properties. Zebrafish may serve as an in vivo model to study neuronal K_2P17.1 function but does not appear appropriate for cardiac electrophysiology studies. Differences in pH sensitivity of zK_2P17.1 currents need to be considered when zebrafish data are extrapolated to human physiology.
Stretch-activated two-pore-domain (K<inf>2P</inf>) potassium channels in the heart: Focus on atrial fibrillation and heart failure
2017, Progress in Biophysics and Molecular Biology
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The channels are sensitive to antiarrhythmic drugs that are used in clinical cardiology. Human K2P2.1 current is inhibited by the antiarrhythmic drugs dronedarone, vernakalant, mexiletine, propafenone, and carvedilol (Schmidt et al., 2012, 2013; Kisselbach et al., 2014; Seyler et al., 2014). Downregulation in a porcine model of AF and HF may further indicate a clinical relevance of cardiac K2P2.1 channels (Schmidt et al., 2014c; Lugenbiel et al., 2017).
Two-pore-domain potassium (K_2P) channels modulate cellular excitability. The significance of stretch-activated cardiac K_2P channels (K_2P2.1, TREK-1, KCNK2; K_2P4.1, TRAAK, KCNK4; K_2P10.1, TREK-2, KCNK10) in heart disease has not been elucidated in detail. The aim of this work was to assess expression and remodeling of mechanosensitive K_2P channels in atrial fibrillation (AF) and heart failure (HF) patients in comparison to murine models.
Cardiac K_2P channel levels were quantified in atrial (A) and ventricular (V) tissue obtained from patients undergoing open heart surgery. In addition, control mice and mouse models of AF (cAMP-response element modulator (CREM)-IbΔC-X transgenic animals) or HF (cardiac dysfunction induced by transverse aortic constriction, TAC) were employed. Human and murine KCNK2 displayed highest mRNA abundance among mechanosensitive members of the K_2P channel family (V > A). Disease-associated K_2P2.1 remodeling was studied in detail. In patients with impaired left ventricular function, atrial KCNK2 (K_2P2.1) mRNA and protein expression was significantly reduced. In AF subjects, downregulation of atrial and ventricular KCNK2 (K_2P2.1) mRNA and protein levels was observed. AF-associated suppression of atrial Kcnk2 (K_2P2.1) mRNA and protein was recapitulated in CREM-transgenic mice. Ventricular Kcnk2 expression was not significantly altered in mouse models of disease.
In conclusion, mechanosensitive K_2P2.1 and K_2P10.1 K⁺ channels are expressed throughout the heart. HF- and AF-associated downregulation of KCNK2 (K_2P2.1) mRNA and protein levels suggest a mechanistic contribution to cardiac arrhythmogenesis.
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¹: Present address: Department of Cardiology, Angiology, Nephrology and Intensive Care, Klinikum Wolfsburg, Sauerbruchstrasse 7, D-38440 Wolfsburg, Germany.

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Cardiovascular pharmacologyInhibition of cardiac two-pore-domain K+ (K2P) channels by the antiarrhythmic drug vernakalant – Comparison with flecainide

Abstract

Introduction

Section snippets

Molecular biology

Effects of vernakalant on human K2P2.1 and K2P3.1 channels expressed in Xenopus oocytes

Vernakalant inhibits human cardiac K2P2.1 K+ currents

Conclusion

Acknowledgments

J. Am. Coll. Cardiol.

Lancet

J. Am. Coll. Cardiol.

BBA – Biomembr.

J. Pharmacol. Toxicol. Methods

Am. J. Cardiol.

J. Am. Coll. Cardiol.

Eur. J. Pharmacol.

Heart Rhythm

Am. Heart J.

Neuron

Rate-dependent effects of vernakalant in the isolated non-remodeled canine left atria are primarily due to block of the sodium channel: comparison with ranolazine and dl-sotalol

Circ. Arrhythm. Electrophysiol.

Knock-out of the potassium channel TASK-1 leads to a prolonged QT interval and a disturbed QRS complex

Cell. Physiol. Biochem.

Vernakalant

Nat. Rev. Drug. Discov.

The effect of vernakalant (RSD1235), an investigational antiarrhythmic agent, on atrial electrophysiology in humans

J. Cardiovasc. Pharmacol.

Pharmacological inhibition of the hERG potassium channel is modulated by extracellular but not intracellular acidosis

J. Cardiovasc. Electrophysiol.

Molecular background of leak K+ currents: two-pore-domain potassium channels

Physiol. Rev.

Vernakalant (RSD1235): a novel, atrial-selective antifibrillatory agent

Expert Opin. Investig. Drugs

The mechanism of atrial antiarrhythmic action of RSD1235

J. Cardiovasc. Electrophysiol.

Regulation of two-pore-domain (K2P) potassium leak channels by the tyrosine kinase inhibitor genistein

Br. J. Pharmacol.

The human cardiac K2P3.1 (TASK-1) potassium leak channel is a molecular target for the class III antiarrhythmic drug amiodarone

Naunyn-Schmiedebergs Arch. Pharmacol.

Potassium leak channels and the KCNK family of two-P-domain subunits

Nat. Rev. Neurosci.

Cardiovascular pharmacology
Inhibition of cardiac two-pore-domain K⁺ (K_2P) channels by the antiarrhythmic drug vernakalant – Comparison with flecainide

Effects of vernakalant on human K_2P2.1 and K_2P3.1 channels expressed in Xenopus oocytes

Vernakalant inhibits human cardiac K_2P2.1 K⁺ currents

Regulation of two-pore-domain (K_2P) potassium leak channels by the tyrosine kinase inhibitor genistein

The human cardiac K_2P3.1 (TASK-1) potassium leak channel is a molecular target for the class III antiarrhythmic drug amiodarone