Naunyn-Schmiedeberg's Arch Pharmacol (2014) 387:1153–1161 DOI 10.1007/s00210-014-1045-6
ORIGINAL ARTICLE
Class III antiarrhythmic drug dronedarone inhibits cardiac inwardly rectifying Kir2.1 channels through binding at residue E224 Panagiotis Xynogalos & Claudia Seyler & Daniel Scherer & Christoph Koepple & Eberhard P. Scholz & Dierk Thomas & Hugo A. Katus & Edgar Zitron
Received: 26 May 2014 / Accepted: 25 August 2014 / Published online: 4 September 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract Dronedarone is a novel class III antiarrhythmic drug that is widely used in atrial fibrillation. It has been shown in native cardiomyocytes that dronedarone inhibits cardiac inwardly rectifying current IK1 at high concentrations, which may contribute both its antifibrillatory efficacy and its potential proarrhythmic side effects. However, the underlying mechanism has not been studied in further detail to date. In the mammalian heart, heterotetrameric assembly of Kir2.x channels is the molecular basis of IK1 current. Therefore, we studied the effects of dronedarone on wild-type and mutant Kir2.x channels in the Xenopus oocyte expression system. Dronedarone inhibited Kir2.1 currents but had no effect on Kir2.2 or Kir2.3 currents. Onset of block was slow but completely reversible upon washout. Blockade of Kir2.1 channels did not exhibit strong voltage dependence or frequency dependence. In a screening with different Kir2.1 mutants lacking specific binding sites within the cytoplasmic pore region, we found that residue E224 is essential for binding of dronedarone to Kir2.1 channels. In conclusion, direct block of Kir2.1 channel subunits by dronedarone through binding at E224 may underlie its inhibitory effects on cardiac IK1 current. Keywords Class III antiarrhythmic drug . Dronedarone . Cardiac inwardly rectifying Kir2.x channels . Residue E224 . IK1 P. Xynogalos (*) : C. Seyler : D. Scherer : C. Koepple : E. P. Scholz : D. Thomas : H. A. Katus : E. Zitron Department of Cardiology, University Hospital Heidelberg, Im Neuenheimer Feld 410, 69120 Heidelberg, Germany e-mail: [email protected] D. Thomas : H. A. Katus : E. Zitron DZHK (German Centre for Cardiovascular Research), Partner Site Heidelberg/Mannheim, Heidelberg, Germany
Introduction Dronedarone is a novel class III antiarrhythmic drug that has found widespread clinical use in atrial fibrillation (Danelich et al. 2013). It was developed as an iodine-free variant of amiodarone with the aim to achieve similar electrophysiological effects but simultaneously reduce long-term side effects (Fig. 1). Pharmacologically, dronedarone has been shown to terminate atrial and ventricular fibrillation, lower heart rate and blood pressure, and exert antiadrenergic effects (Hohnloser et al. 2009). In clinical trials, dronedarone has been shown to restore sinus rhythm and to reduce rates of hospitalization and mortality in paroxysmal and persistent atrial fibrillation (Singh et al. 2007). However, in patients with permanent atrial fibrillation at risk for major vascular events, dronedarone increased rates of heart failure, stroke, and death from cardiovascular causes (Connolly et al. 2011), raising concerns about its clinical safety and potential proarrhythmic side effects. Electrophysiologically, dronedarone is a multichannel blocker with inhibitory effects on a spectrum of cardiac ion currents including INa, ICa,L, IKr, IKs, and IK1 (Gautier et al. 2003). It has been shown in guinea pig ventricular cardiomyocytes that dronedarone inhibits IK1 current dosedependently with incomplete block at 10 and 30 μM and an estimated IC50 of >30 μM (Gautier et al. 2003). This appeared comparable to previously described effects of amiodarone on IK1 current in mammalian ventricular cardiomyocytes (Gautier et al. 2003; Sato et al. 1994). Whereas the detailed molecular electrophysiology of the effects of dronedarone on IKr and IKs has already been clarified (Thomas et al. 2003; Varro et al. 2001), the molecular basis of its effects on IK1 have not been studied to date. Physiologically, the cardiac inwardly rectifying potassium current IK1 is essential to maintain the resting membrane
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Naunyn-Schmiedeberg's Arch Pharmacol (2014) 387:1153–1161
Fig. 1 Chemical structure of antiarrhythmic drugs amiodarone (a) and dronedarone (b)
potential of cardiomyocytes (Tristani-Firouzi et al. 2002). There is an increasing body of evidence that heteromeric assembly of Kir2.1, Kir2.2, and Kir2.3 potassium channels is the molecular basis of cardiac IK1 current (Preisig-Muller et al. 2002; Schram et al. 2003; Zobel et al. 2003). Kir2.1 is expressed in the whole myocardium, and IK1 current is completely abolished in Kir2.1-knockout mice (Zaritsky et al. 2000). Heteromeric assembly of Kir2.1 and Kir2.2 channel subunits probably underlies human ventricular IK1 current (Schram et al. 2003). The Kir2.3 channel subunit probably is more relevant in the atria (Melnyk et al. 2002; Wang et al. 1998). Several lines of evidence support the relevance of IK1 current as a target for the suppression of atrial and ventricular fibrillation. An increase of IK1 outward current due to a gainof-function mutation in Kir2.1 underlies Short-QT-Syndrome Type 3 (Schimpf et al. 2005b). These patients exhibit a shortened QT interval and a strong predisposition for atrial fibrillation and ventricular fibrillation and can be successfully treated with class III antiarrhythmic agents (Schimpf et al. 2005a). A causative role for another gain-of-function mutation in Kir2.1 has also been proposed in familial atrial fibrillation (Xia et al. 2005). This is in line with a model proposing that larger inward rectifier currents lead to increased stability of reentry in ventricular and atrial myocardium (Noujaim et al. 2007). Hence, overexpression of Kir2.1 channels in mouse ventricles generated stable and very fast rotors, providing direct evidence for a role of IK1 in reentry stabilization (Noujaim et al. 2007). Several studies have shown that IK1 current amplitudes are increased in chronic atrial fibrillation, and one study could even show that, in paroxysmal atrial fibrillation, this increase is confined to the left atrium (Bosch et al. 1999b; Dobrev et al. 2001; Girmatsion et al. 2009; Van Wagoner et al. 1997; Voigt et al. 2010). By contrast, IK1 current reduction caused by loss-offunction mutations in the Kir2.1 channel subunit underlies Long QT Syndrome Type 7 with a characteristic pattern of QT interval prolongation with focal ventricular arrhythmias that most probably originate from the purkinje system
(Tristani-Firouzi and Etheridge 2010). In line with this phenotype, it has been shown in a variety of experimental models that inhibition of IK1 current may induce proarrhythmia (Dhamoon and Jalife 2005). Thus, pharmacological inhibition of IK1 current and the underlying Kir2.1-2.3 channel subunits may contribute both to the antifibrillatory efficacy and to proarrhythmic side effects of dronedarone. The acute inhibition of IK1 current by dronedarone has already been studied in native cardiomyocytes (Gautier et al. 2003). However, the underlying mechanism has not been examined yet. Specifically, the effects of dronedarone on Kir2.x channels as potential molecular basis of this inhibition have not been investigated to date. Therefore, we studied the effects of dronedarone on wild-type and selected mutant Kir2.x potassium channels in the Xenopus oocyte expression system in order to elucidate further molecular mechanistic properties that may underlie the inhibition of IK1 current by dronedarone.
Methods Heterologous expression Complementary RNA was prepared from Kir2.x cDNA with the mMESSAGE mMACHINE in vitro transcription kit (Ambion) by use of T7 Polymerase (Kir2.1 and Kir2.2) and T3 Polymerase (Kir2.3). Injection of RNA into stage Vand VI defolliculated oocytes was performed using a Nanoject automatic injector (Drummond, Broomall, USA). Measurements were made 1 to 3 days after injection. The investigation conforms to 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). Electrophysiology and data analysis The two-microelectrode voltage-clamp configuration was used to record currents from Xenopus laevis oocytes. Data were low-pass filtered at 1 to 2 kHz (−3 dB, four-pole Bessel
Naunyn-Schmiedeberg's Arch Pharmacol (2014) 387:1153–1161
filter) before digitalization at 5 to 10 kHz. Recordings were performed using a commercially available amplifier (Warner OC-725A, Warner Instruments, Hamden, USA) and pCLAMP software (Axon Instruments, Foster City, USA) for data acquisition and analysis. No leak subtraction was performed during the experiments. Statistical data are presented as mean±standard error. Statistical significance was evaluated using Student’s t test or ANOVA. Differences were considered to be significant if the p value was 30 μM (Gautier et al. 2003). At a concentration of 30 μM, the inhibitory effect of dronedarone on IK1 current was approximately 25 % (Gautier et al. 2003). This appeared comparable to previously described effects of amiodarone on IK1 current in guinea pig ventricular cardiomyocytes where amiodarone also inhibited IK1 current at concentrations of 10–20 μM with a slow onset of effect (Gautier et al. 2003; Sato et al. 1994). In these studies, the effects of 10–20 μM amiodarone on IK1 current were reductions in the range of 10–15 % (Sato et al. 1994). Of note, the inhibition of IK1 current after chronic exposure to amiodarone (which better reflects the typical clinical setting where amiodarone and dronedarone are predominantly used as longterm medication) has been found to be much more pronounced. In guinea pigs treated with amiodarone for 7 days, a reduction of IK1 current by approximately 60 % was found which is much stronger than the respective acute effects (Bosch et al. 1999a). This difference by acute and chronic effects may be caused by slow diffusion of amphiphilic compounds such as amiodarone and dronedarone in multicellular tissue and protracted accumulation of the respective compounds in the myocardium or by additional inhibitory mechanisms (Gautier et al. 2003).
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different concentrations of dronedarone on Kir2.1 currents are shown in comparison to control measurements in Fig. 6: Under control conditions (i.e., either with the bath solution or with a vehicle control with ethanol) Kir2.1 currents increased by 18.0±4.1 % (n=6) and 21.8±7.3 % (n=42) in the vehicle control with ethanol, respectively, during the observation period. Dronedarone at increasing concentrations (50 μM, 100 μM and 500 μM) induced current decreases by −7.3± 7.1 %, −8.3±5.7 %, and −20.5±7.7 %, respectively, (n=6–17, p
ORIGINAL ARTICLE
Class III antiarrhythmic drug dronedarone inhibits cardiac inwardly rectifying Kir2.1 channels through binding at residue E224 Panagiotis Xynogalos & Claudia Seyler & Daniel Scherer & Christoph Koepple & Eberhard P. Scholz & Dierk Thomas & Hugo A. Katus & Edgar Zitron
Received: 26 May 2014 / Accepted: 25 August 2014 / Published online: 4 September 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract Dronedarone is a novel class III antiarrhythmic drug that is widely used in atrial fibrillation. It has been shown in native cardiomyocytes that dronedarone inhibits cardiac inwardly rectifying current IK1 at high concentrations, which may contribute both its antifibrillatory efficacy and its potential proarrhythmic side effects. However, the underlying mechanism has not been studied in further detail to date. In the mammalian heart, heterotetrameric assembly of Kir2.x channels is the molecular basis of IK1 current. Therefore, we studied the effects of dronedarone on wild-type and mutant Kir2.x channels in the Xenopus oocyte expression system. Dronedarone inhibited Kir2.1 currents but had no effect on Kir2.2 or Kir2.3 currents. Onset of block was slow but completely reversible upon washout. Blockade of Kir2.1 channels did not exhibit strong voltage dependence or frequency dependence. In a screening with different Kir2.1 mutants lacking specific binding sites within the cytoplasmic pore region, we found that residue E224 is essential for binding of dronedarone to Kir2.1 channels. In conclusion, direct block of Kir2.1 channel subunits by dronedarone through binding at E224 may underlie its inhibitory effects on cardiac IK1 current. Keywords Class III antiarrhythmic drug . Dronedarone . Cardiac inwardly rectifying Kir2.x channels . Residue E224 . IK1 P. Xynogalos (*) : C. Seyler : D. Scherer : C. Koepple : E. P. Scholz : D. Thomas : H. A. Katus : E. Zitron Department of Cardiology, University Hospital Heidelberg, Im Neuenheimer Feld 410, 69120 Heidelberg, Germany e-mail: [email protected] D. Thomas : H. A. Katus : E. Zitron DZHK (German Centre for Cardiovascular Research), Partner Site Heidelberg/Mannheim, Heidelberg, Germany
Introduction Dronedarone is a novel class III antiarrhythmic drug that has found widespread clinical use in atrial fibrillation (Danelich et al. 2013). It was developed as an iodine-free variant of amiodarone with the aim to achieve similar electrophysiological effects but simultaneously reduce long-term side effects (Fig. 1). Pharmacologically, dronedarone has been shown to terminate atrial and ventricular fibrillation, lower heart rate and blood pressure, and exert antiadrenergic effects (Hohnloser et al. 2009). In clinical trials, dronedarone has been shown to restore sinus rhythm and to reduce rates of hospitalization and mortality in paroxysmal and persistent atrial fibrillation (Singh et al. 2007). However, in patients with permanent atrial fibrillation at risk for major vascular events, dronedarone increased rates of heart failure, stroke, and death from cardiovascular causes (Connolly et al. 2011), raising concerns about its clinical safety and potential proarrhythmic side effects. Electrophysiologically, dronedarone is a multichannel blocker with inhibitory effects on a spectrum of cardiac ion currents including INa, ICa,L, IKr, IKs, and IK1 (Gautier et al. 2003). It has been shown in guinea pig ventricular cardiomyocytes that dronedarone inhibits IK1 current dosedependently with incomplete block at 10 and 30 μM and an estimated IC50 of >30 μM (Gautier et al. 2003). This appeared comparable to previously described effects of amiodarone on IK1 current in mammalian ventricular cardiomyocytes (Gautier et al. 2003; Sato et al. 1994). Whereas the detailed molecular electrophysiology of the effects of dronedarone on IKr and IKs has already been clarified (Thomas et al. 2003; Varro et al. 2001), the molecular basis of its effects on IK1 have not been studied to date. Physiologically, the cardiac inwardly rectifying potassium current IK1 is essential to maintain the resting membrane
1154
Naunyn-Schmiedeberg's Arch Pharmacol (2014) 387:1153–1161
Fig. 1 Chemical structure of antiarrhythmic drugs amiodarone (a) and dronedarone (b)
potential of cardiomyocytes (Tristani-Firouzi et al. 2002). There is an increasing body of evidence that heteromeric assembly of Kir2.1, Kir2.2, and Kir2.3 potassium channels is the molecular basis of cardiac IK1 current (Preisig-Muller et al. 2002; Schram et al. 2003; Zobel et al. 2003). Kir2.1 is expressed in the whole myocardium, and IK1 current is completely abolished in Kir2.1-knockout mice (Zaritsky et al. 2000). Heteromeric assembly of Kir2.1 and Kir2.2 channel subunits probably underlies human ventricular IK1 current (Schram et al. 2003). The Kir2.3 channel subunit probably is more relevant in the atria (Melnyk et al. 2002; Wang et al. 1998). Several lines of evidence support the relevance of IK1 current as a target for the suppression of atrial and ventricular fibrillation. An increase of IK1 outward current due to a gainof-function mutation in Kir2.1 underlies Short-QT-Syndrome Type 3 (Schimpf et al. 2005b). These patients exhibit a shortened QT interval and a strong predisposition for atrial fibrillation and ventricular fibrillation and can be successfully treated with class III antiarrhythmic agents (Schimpf et al. 2005a). A causative role for another gain-of-function mutation in Kir2.1 has also been proposed in familial atrial fibrillation (Xia et al. 2005). This is in line with a model proposing that larger inward rectifier currents lead to increased stability of reentry in ventricular and atrial myocardium (Noujaim et al. 2007). Hence, overexpression of Kir2.1 channels in mouse ventricles generated stable and very fast rotors, providing direct evidence for a role of IK1 in reentry stabilization (Noujaim et al. 2007). Several studies have shown that IK1 current amplitudes are increased in chronic atrial fibrillation, and one study could even show that, in paroxysmal atrial fibrillation, this increase is confined to the left atrium (Bosch et al. 1999b; Dobrev et al. 2001; Girmatsion et al. 2009; Van Wagoner et al. 1997; Voigt et al. 2010). By contrast, IK1 current reduction caused by loss-offunction mutations in the Kir2.1 channel subunit underlies Long QT Syndrome Type 7 with a characteristic pattern of QT interval prolongation with focal ventricular arrhythmias that most probably originate from the purkinje system
(Tristani-Firouzi and Etheridge 2010). In line with this phenotype, it has been shown in a variety of experimental models that inhibition of IK1 current may induce proarrhythmia (Dhamoon and Jalife 2005). Thus, pharmacological inhibition of IK1 current and the underlying Kir2.1-2.3 channel subunits may contribute both to the antifibrillatory efficacy and to proarrhythmic side effects of dronedarone. The acute inhibition of IK1 current by dronedarone has already been studied in native cardiomyocytes (Gautier et al. 2003). However, the underlying mechanism has not been examined yet. Specifically, the effects of dronedarone on Kir2.x channels as potential molecular basis of this inhibition have not been investigated to date. Therefore, we studied the effects of dronedarone on wild-type and selected mutant Kir2.x potassium channels in the Xenopus oocyte expression system in order to elucidate further molecular mechanistic properties that may underlie the inhibition of IK1 current by dronedarone.
Methods Heterologous expression Complementary RNA was prepared from Kir2.x cDNA with the mMESSAGE mMACHINE in vitro transcription kit (Ambion) by use of T7 Polymerase (Kir2.1 and Kir2.2) and T3 Polymerase (Kir2.3). Injection of RNA into stage Vand VI defolliculated oocytes was performed using a Nanoject automatic injector (Drummond, Broomall, USA). Measurements were made 1 to 3 days after injection. The investigation conforms to 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). Electrophysiology and data analysis The two-microelectrode voltage-clamp configuration was used to record currents from Xenopus laevis oocytes. Data were low-pass filtered at 1 to 2 kHz (−3 dB, four-pole Bessel
Naunyn-Schmiedeberg's Arch Pharmacol (2014) 387:1153–1161
filter) before digitalization at 5 to 10 kHz. Recordings were performed using a commercially available amplifier (Warner OC-725A, Warner Instruments, Hamden, USA) and pCLAMP software (Axon Instruments, Foster City, USA) for data acquisition and analysis. No leak subtraction was performed during the experiments. Statistical data are presented as mean±standard error. Statistical significance was evaluated using Student’s t test or ANOVA. Differences were considered to be significant if the p value was 30 μM (Gautier et al. 2003). At a concentration of 30 μM, the inhibitory effect of dronedarone on IK1 current was approximately 25 % (Gautier et al. 2003). This appeared comparable to previously described effects of amiodarone on IK1 current in guinea pig ventricular cardiomyocytes where amiodarone also inhibited IK1 current at concentrations of 10–20 μM with a slow onset of effect (Gautier et al. 2003; Sato et al. 1994). In these studies, the effects of 10–20 μM amiodarone on IK1 current were reductions in the range of 10–15 % (Sato et al. 1994). Of note, the inhibition of IK1 current after chronic exposure to amiodarone (which better reflects the typical clinical setting where amiodarone and dronedarone are predominantly used as longterm medication) has been found to be much more pronounced. In guinea pigs treated with amiodarone for 7 days, a reduction of IK1 current by approximately 60 % was found which is much stronger than the respective acute effects (Bosch et al. 1999a). This difference by acute and chronic effects may be caused by slow diffusion of amphiphilic compounds such as amiodarone and dronedarone in multicellular tissue and protracted accumulation of the respective compounds in the myocardium or by additional inhibitory mechanisms (Gautier et al. 2003).
1159
different concentrations of dronedarone on Kir2.1 currents are shown in comparison to control measurements in Fig. 6: Under control conditions (i.e., either with the bath solution or with a vehicle control with ethanol) Kir2.1 currents increased by 18.0±4.1 % (n=6) and 21.8±7.3 % (n=42) in the vehicle control with ethanol, respectively, during the observation period. Dronedarone at increasing concentrations (50 μM, 100 μM and 500 μM) induced current decreases by −7.3± 7.1 %, −8.3±5.7 %, and −20.5±7.7 %, respectively, (n=6–17, p
