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Cardiovascular Research 2005 65(1):128-137; doi:10.1016/j.cardiores.2004.09.030
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Copyright © 2004, European Society of Cardiology

Expression of human ERG K+ channels in the mouse heart exerts anti-arrhythmic activity

Anne Royera,1, Sophie Demolombea,1, Aziza El Harchia, Khaï Le Quanga, Julien Pirona, Gilles Toumaniantza, David Mazuraisa, Chloé Bellocqa, Gilles Landea, Cécile Terrenoireb, Howard K. Motoikeb, Jean-Christophe Chevalliera, Gildas Loussouarna, Colleen E. Clancyc, Denis Escandea and Flavien Charpentiera,*

aINSERM U533, Institut du Thorax, Faculté de Médecine, 1 rue G. Veil, 44035 Nantes cedex, France
bDepartment of Pharmacology, College of Physicians and Surgeons of Columbia University, New York, NY 10032, USA
cDepartment of Physiology and Biophysics, Institute for Computational Biomedicine, Weill Medical College of Cornell University, New York, NY 10021, USA

* Corresponding author. Tel.: +33 240 41 28 44; fax: +33 240 41 29 50. Email address: flavien.charpentier{at}nantes.inserm.fr

Received 7 May 2004; revised 13 September 2004; accepted 29 September 2004


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 
Objective: The K+ channel encoded by the human ether-à-go-go-related gene (HERG) is crucial for repolarization in the human heart. In order to investigate the impact of HERG current (IKr) on the incidence of cardiac arrhythmias, we generated a transgenic mouse expressing HERG specifically in the heart.

Methods and results: ECG recordings at baseline showed no obvious difference between transgenic and wild-type (WT) mice with the exception of the T wave, which was more negative in transgenic mice than in WT mice. E4031 (20 mg/kg) prolonged the QTc interval and flattened the T wave in transgenic mice, but not in WT mice. Injection of BaCl2 (25 mg/kg) induced short runs of ventricular tachycardia in 9/10 WT mice, but not in transgenic animals. Atrial pacing reproducibly induced atrial tachyarrhythmias in 11/15 WT mice. In contrast, atrial arrhythmia was inducible in only 2/11 transgenic mice. When pretreated with dofetilide (10 mg/kg), transgenic mice were as sensitive to experimental arrhythmias as WT mice. Microelectrode studies showed that atrial action potentials have a steeper slope of duration-rate adaptation in WT than in transgenic mice. Transgenic mice were also characterized by a post-repolarization refractoriness, which could result from the substantial amount of IKr subsisting after repolarization as assessed with action potential-clamp experiments and simulations with a model of the transgenic mouse action potential.

Conclusion: HERG expression in the mouse heart can protect against experimental induction of arrhythmias. This is the first report of such a protective effect of HERG in vivo.

KEYWORDS Arrhythmia; Repolarization; K+ channel; Transgenic animal models


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 References
 
The K+ channel encoded by the human ether-à-go-go-related gene (HERG, or KCNH2) is crucial for normal action potential repolarization in the human heart. HERG encodes the pore-forming subunit of the rapidly activating cardiac delayed K+ current (IKr) channel. The classical paradigm regarding IKr and arrhythmias is that blocking IKr should be anti-arrhythmic, secondary to prolongation of repolarization and refractoriness. However, this approach has been unsuccessful in reducing mortality in patients with left ventricular dysfunction and inversely has been associated with proarrhythmia [1]. Prolongation of repolarization is a typical characteristic of the congenital long QT syndrome, a rare disease implicating many ion channels, including HERG [2,3]. In this disease, mutations in the HERG gene lead to a reduced IKr and increased incidence of lethal ventricular arrhythmias. Finally, blocking HERG channels by many non-cardiac pharmaceutical agents may produce arrhythmias and unexpected sudden cardiac death [4].

More recently, several studies suggested that increasing HERG could actually be a better approach to prevent arrhythmias [5–7]. Understanding how HERG K+ channels normally provide protection against arrhythmias is therefore of considerable interest. In the present work, we generated a transgenic mouse expressing HERG specifically in the heart. Our objective was to investigate the functional consequences of HERG expression on cardiac repolarization and on the initiation of cardiac arrhythmias. Our results show that HERG expression can protect against experimental induction of arrhythmias at both atrial and ventricular levels, an effect that could be secondary to increased repolarization reserve [8]. Together with the recent observation that gain-of-function mutations of HERG can lead to short QT syndrome and sudden cardiac death [9], our findings suggest that increasing HERG current could exert both antiarrhythmic and proarrhythmic activities.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 References
 
2.1. Generation of transgenic mice
The human ERG was subcloned into the {alpha}-MHC vector (a kind gift of Dr. Jeffrey Robbins, University of Cincinnati, USA). The transgene fragment included the mouse {alpha}MHC promoter, introns and the three non-coding exons of the mouse {alpha}MHC gene, the HERG coding sequence and the human growth hormone polyadenylation signal sequences. It was microinjected into FVB mouse oocytes. A total of 83 offspring was obtained and screened for the presence of the transgene by PCR analysis of genomic DNA. Sixteen founders were positive and were backcrossed to wild-type (WT) FVB to produce heterozygous F1 offspring. Ten founders transmitted the transgene to their lineage. Three transgenic lines were selected based on Western blots. Finally, only one line (#52) was selected based on ECG data.

All experiments were performed on adult sex- and age-matched transgenic and non-transgenic mice from the same litters (as controls). The investigation conforms 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).

2.2. Electrocardiography
Three-lead ECGs were recorded with 25-gauge subcutaneous electrodes on a computer through an analog–digital converter (IOX 1.585, EMKA Technologies, France) for monitoring and later analysis (ECG Auto 1.5.7, EMKA Technologies). ECG channels were filtered between 0.5 and 250 Hz. For phenotypic characterization, mice were anesthetized with intra-peritoneal (i.p.) injection of etomidate (15 mg/kg). ECGs were first recorded under baseline conditions, then 10 min after atropine sulfate (0.5 mg/kg, i.p.) and propranolol (1 mg/kg, i.p.) and finally 10 min after E4031 (20 mg/kg, i.p.), an inhibitor of IKr [10]. Reported measurements were averaged from five consecutive PQRST complexes in lead I. Standard criteria were used for interval measurements. On lead I, repolarization is biphasic with an initial rapid positive J wave and a slower negative T wave. The end of the T wave was defined as the point at which the slow component returned to the isoelectric line. QT intervals were corrected for heart rate using the formula, QTc=QT/(RR/100)1/2 with QT and RR measured in ms [11].

In a subgroup of mice, tedisamil (5 mg/kg, i.p.), an inhibitor of the transient outward (Ito) and delayed rectifier K+ currents [12], was injected with atropine sulfate and propranolol. ECGs were recorded 15 min later. Mice were then injected with E4031 (20 mg/kg, i.p.) and ECGs recorded 10 min after injection.

2.3. Barium-induced ventricular arrhythmias
Ten transgenic mice and 10 WT littermates were included in a blind study of barium effects on cardiac electrical activity. Under anesthesia (etomidate 15 mg/kg, i.p.), a bolus of BaCl2 (25 mg/kg) was injected into the tail vein. The effect of BaCl2 was assessed by continuous ECG monitoring and recording. In an additional subgroup of mice, dofetilide (10 mg/kg, i.p.), a blocker of the IKr potassium current [13], was injected 20 min before BaCl2 challenge.

2.4. Endocavity recording and pacing
Eleven transgenic mice and 15 WT littermates were included in a blind study of pacing-induced atrial arrhythmias. Under anesthesia with etomidate (8 mg/kg) and pentobarbital (30 mg/kg), an intubation was achieved with a cannula for continuous mechanical ventilation (respiratory rate, 140 ml/min; tidal volume, 200 µl; Minivent Type 845, Hugo Sachs Electronik, Germany). An octopolar 2 F electrode catheter with an electrode spacing of 0.5 mm (Cordis Webster®, USA) was introduced into the right atrium and ventricle through the right internal jugular vein and used for simultaneous atrial and ventricular pacing and recording. Intracardiac electrograms were filtered between 30 and 500 Hz. Surface ECG (lead I) and intracardiac electrograms were recorded to computer for monitoring and later analysis. Intracardiac pacing was performed with a Biotronik® UHS20 stimulator (Germany), modified by the manufacturer to pace at short coupling intervals. Atrial arrhythmias were induced with a protocol derived from the one described by Wakimoto et al. [14]. Briefly, mice received an i.p. injection of the muscarinic agonist carbamylcholine (50 µg/kg). Although atrial tachyarrhythmias can be induced with burst pacing in mouse without vagal stimulation [15], carbamylcholine through its activation of the acetylcholine-activated K+ current, IK,Ach, predisposes to atrial fibrillation [16]. Ten minutes later, programmed right atrial single, double and triple extrastimuli were applied at 100-ms drive cycle length. Right atrial burst pacing was also performed as 1- to 2-s train episodes at cycle lengths ranging from 10 to 30 ms separated by 5-s intervals (unless arrhythmias were induced). In an additional subgroup of mice, dofetilide (10 mg/kg, i.p.) was injected 20 min before starting atrial pacing.

2.5. Patch-clamp studies
The methods and equipment used to dissociate adult ventricular myocytes and to record whole-cell currents have been described elsewhere [17]. Patch pipettes had tip resistances of 1.8–3.0 M{Omega}. They were filled with a solution containing (in mmol/l): KCl, 20; K-aspartate, 110; HEPES, 5; EGTA, 5; MgCl2, 2; K2ATP, 5; Phosphocreatine-Na2, 5; pH 7.2 with KOH. The extracellular medium contained (in mmol/l): N-methyl-D-glucamine, 130; KCl, 5.4; MgSO4, 1.2; HEPES, 10; glucose, 10; pH adjusted to 7.4 with HCl. Nifedipine (3.10–6 mol/l) was used to block the L-type Ca2+ current. Experiments were performed at 35 °C. The IKr current was elicited by applying 500-ms depolarizations in 10-mV increments at 3-s intervals from –40 mV up to +60 mV. Holding potential was –50 mV.

Cos7 cells were transfected with pSI-HERG or pCDNA3–KCNQ1–KCNE1 plasmids together with pTR-GFP plasmid (to tag transfected cells) complexed with JetPEI (Polyplus-transfection, France), according to the protocol recommended by the manufacturer. Patch-clamp recordings were obtained 24–48 h after transfection as previously described [18]. Atrial action potentials used to clamp the cells were obtained during transmembrane microelectrodes studies.

In patch-clamp experiments, E4031 and HMR 1556 were used to block respectively IKr [10] (or HERG current) and IKs [19].

2.6. Microelectrode studies
Six transgenic and 7 WT adult mice of either sex were killed by cervical dislocation. The hearts were quickly removed and immersed in a cool modified Tyrode solution containing (in mmol/l): NaCl, 108; NaHCO3, 25; NaH2PO4, 1.8; KCl, 27; MgCl2, 1; CaCl2, 0.6; glucose, 55 and previously saturated with a 95% O2–5% CO2 gas mixture (pH 7.3). Left atria were mounted in a tissue bath chamber, the endocardial surface facing up. Preparations were superfused with an oxygenated (95% O2–5% CO2) Tyrode solution warmed to 37 ± 0.5 °C and containing (in mmol/l): NaCl, 120; NaHCO3, 27; NaH2PO4, 1.2; KCl, 5.4; MgCl2, 1.2; CaCl2, 1.8; glucose, 10 (pH 7.4). Transmembrane recordings were obtained using standard methods. The tissues were allowed to recover for at least 1 h before the experiments started. During this period, they were paced at a cycle length of 300 ms by bipolar stimulation through Teflon-coated silver wire electrodes. Stimulus pulse width was 1 ms and amplitude was twice diastolic threshold. The preparations were then paced at cycle lengths decreasing from 500 to 75 ms. Action potentials characteristics were measured at steady-state for each pacing cycle length (PCL). Effective refractory period was determined using extra-stimulus pacing at a PCL of 200 ms.

2.7. Simulation studies
The model was generated by adding the equation for HERG [20] to the previously described mouse action potential model [21]. This resulted in a set of 45 ordinary differential equations solved by a fourth-order Runge–Kutta method. Some modifications were applied on the original mouse model: to gain accuracy in the action potential calculation, the Runge–Kutta algorithm used a variable time step with a minimal time step set to 0.0001 ms and an error tolerance set to 10–4. If the error tolerance is reached, the time step is divided by two. For HERG, the macroscopic conductance was adapted to correspond to the transgenic mouse HERG current amplitude: GKr=0.12324418. [K+]out0.59 mS/µF, with [K+]out in mmol/l. The background Na+ current was modified to fit more accurately the resting potential observed in APs used for action potential-clamp studies (GNa=0.0078 mS/µF).

2.8. Western blots and immunochemistry
Protein extracts from cardiac samples were prepared in RIPA buffer as described previously [22]. After centrifugation, total cell lysates were denatured with electrophoresis sample buffer, separated on 10% SDS-PAGE and transferred to Hybond C super membrane (Amersham, France). Membranes were incubated with the rabbit anti-erg1 polyclonal antibody (kindly provided by Dr. Eckhard Ficker, Case Western Reserve University, Cleveland, USA), followed by a peroxidase conjugated goat anti-rabbit secondary antibody (Sigma, USA). The antibody complexes were detected using a commercial enhanced chemiluminescence kit (Amersham ECL plus).

For immunohistochemistry analysis, transgenic and WT mouse hearts were frozen in isopentane. The frozen preparations were then mounted on a cryostat tissue holder using Tissue-Tek® (Sakura, Japan) and cut into 8-µm thick sections. The sections were mounted onto slides, air-dried, fixed in cold acetone and blocked with bovine serum albumin. They were then incubated overnight at 4 °C with the same primary antibody used for Western blot (rabbit anti-erg1, 1:100). The sections were then incubated with biotinylated anti-rabbit secondary antibody (1:300, Santa Cruz Biotechnology). After washing in PBS, they were incubated with Alexa 488 conjugated streptavidin (1:100), washed and finally mounted using Vectashield (Vector Laboratories, Burlingame, USA). Control slides were performed by omission of the primary antibody. Slides were analyzed using a laser scanning microscope (Leica Microsystems, Germany).

2.9. Statistical analysis
All data are expressed as means ± S.E.M. Statistical analysis was performed with Fisher exact test, Student t-test and one- or two-way analysis of variance completed by a Tukey test when appropriate. A value of p<0.05 was considered significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 References
 
3.1. HERG-expressing mice
Western blotting showed that 3 different mouse lines strongly expressed HERG in the heart (Fig. 1A). Based on ECG recordings associated with E4031 challenges, line #52 was selected for further studies. Immunohistochemistry showed that erg-1 antibody strongly reacted with HERG protein (Fig. 1B). A background staining was observed in WT heart and probably represented cross-reactivity of erg-1 antibody with endogenous m-erg. The pattern of staining in heart sections suggested that HERG transgene was expressed uniformly across all regions of mouse atrial and ventricular myocardium.


Figure 1
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  		Fig. 1 Transgene expression. (A) Transgene expression detected by Western blot in wild-type (WT) and in HERG lines 52, 56 and 20 (Tg-HERG) mice. (B) Immunohistochemical analysis of HERG in frozen atrial and ventricular sections from WT and line 52 transgenic mice. The staining in WT mice was due to a cross-reactivity of the antibody with the endogenous m-erg. M-erg was not detected in Western blot because the time of exposure to the antibody was not long enough. (C) Top: Voltage-gated outward K+ currents from ventricular myocytes isolated from wild-type (WT) and transgenic (line 52) mice. From a holding potential of –50 mV, currents were elicited using 500-ms depolarizing pulses to potentials between –40 and +60 mV at 3-s intervals. Bottom: Current–voltage relationships for the tail current in wild-type (circles; six cells from three mice) and transgenic (triangles; seven cells from four animals) mice. **, ***, p<0.01 and p<0.001 vs. corresponding value in WT mice.

 
Histopathological examination of transgenic mouse hearts indicated no structural abnormalities compared with WT controls (data not shown). There was no evidence of inflammation or increased interstitial fibrosis within the heart. Body weight, heart weight and heart-to-body weight ratios were similar in both WT and transgenic mice. The cell capacitance of isolated ventricular myocytes in both groups was unchanged, averaging 169 ± 9 and 152 ± 6 pF in WT (n=10) and transgenic (n=19) myocytes, respectively.

Fig. 1C displays typical currents from WT and HERG-expressing ventricular myocytes. Myocytes from transgenic animals exhibited a tail current with maximal amplitude averaging 5.4 ± 0.8 pA/pF after a step to +50 mV. The HERG channel blocker E4031 at 10–7 mol/l reduced the amplitude of this tail current from 2.8 ± 0.2 to 1.6 ± 0.2 pA/pF (step to +10 mV; p<0.05; n=4). Myocytes from WT mice had no sizeable IKr tail under our experimental conditions. Thus patch-clamp experiments demonstrated that transgene expression led to functional channels.

3.2. Electrocardiographic studies
In order to determine the consequences of transgene expression on cardiac electrical activity, 3-lead ECGs were obtained from 8- to 10-week-old transgenic and WT littermates. As illustrated in Fig. 2A, there was no obvious difference between transgenic and WT mouse recordings with the clear exception of the T wave which was constantly more negative in transgenic mice than in WT mice (lead I). No significant differences were observed between WT (n=50) and transgenic (n=37) mice in terms of RR interval (149 ± 4 ms in WT vs. 155 ± 5 ms in transgenic mice, respectively), PR interval (29 ± 1 vs. 28 ± 1 ms) and QRS interval (15 ± 1 ms in both groups). The QTc interval was slightly, albeit not significantly (p=0.051), shorter in transgenic mice than in WT mice (51 ± 2 vs. 54 ± 1 ms).


Figure 2
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  		Fig. 2 Surface electrocardiograms in wild-type (WT) and transgenic (Tg-HERG) mice. Representative ECG recordings (lead I) obtained in WT and transgenic mice (A) under baseline conditions, (B) after i.p. injection of propranolol (1 mg/kg) and atropine sulfate (0.5 mg/kg), and (C) after i.p. injection of propranolol (1 mg/kg), atropine sulfate (0.5 mg/kg) and tedisamil (5 mg/kg), before (Control) and after i.p. injection of E 4031 (20 mg/kg). For clarity, QRS peaks have been erased. (D) Effects of E4031 (20 mg/kg) in absence or in presence of tedisamil (5 mg/kg) on the corrected QT interval (QTc; Y-axis) in WT and transgenic mice. Ctl, control. **, ***, p<0.01 and p<0.001 vs. corresponding control value. #, p<0.05 vs. corresponding value in WT mice. The number of mice was 16 WT and 10 Tg-HERG in the absence of tedisamil, 16 WT and 12 Tg-HERG in the presence of tedisamil.

 
Fig. 2B and Table 1 summarize the effects of the IKr blocker E4031 in WT and transgenic mice recorded under blockade of the autonomic nervous system. E4031 slowed the sinus rate similarly in both groups of mice. In contrast, the effects of E4031 on ventricular repolarization differed between the groups. As previously observed [23], E4031 had no effect on repolarization of WT mice, the prolongation of the QT interval resulting essentially from bradycardia (unchanged QTc). In contrast, in transgenic mice, E4031 induced a consistent prolongation of the QTc interval, suggesting that the transgene-induced IKr contributes to the late phase of ventricular repolarization (Fig. 2D). Of interest, E4031 flattened the T wave in transgenic mice making the wave shape very similar to that of WT animals (Fig. 2B).


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  		Table 1 Lead I ECG parameters in WT (n=16) and HERG-expressing mice (Tg-HERG; n=10) pretreated with propranolol (1 mg/kg) and atropine sulfate (0.5 mg/kg) before (control) and after injection of E4031 (20 mg/kg)

 
In the mouse heart, the main repolarizing currents are the transient outward current Ito, generated mainly by Kv4.2 channels and the slow K+ currents IK,slow1 and IK,slow2 generated respectively by Kv1.5 and Kv2.1 channels [24]. In order to potentiate the relative contribution of IKr to ventricular repolarization, Ito and IK,slow were partially inhibited using tedisamil. Fig. 2C and Table 2 shows the effects of E4031 on ECG characteristics in animals pretreated with tedisamil. As expected, the RR and QTc intervals were longer under tedisamil both in the transgenic and control groups (compare with Table 1). In addition, the J wave was prolonged by tedisamil similarly in WT and transgenic mice. Inversely, the QTc interval was significantly more prolonged in WT mice than in transgenic mice (see Fig. 2D) suggesting that the transgene-induced IKr partially compensated for the decrease in Ito and IK,slow currents. Confirming this hypothesis, injection of E4031 abolished the differences between WT and transgenic mice (Table 2). Fig. 2D clearly illustrates that pretreatment with tedisamil magnified the effects of E4031 on QTc in transgenic mice.


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  		Table 2 Lead I ECG parameters in WT (n=16) and HERG-expressing mice (Tg-HERG; n=12) pretreated with tedisamil (5 mg/kg), propranolol (1 mg/kg) and atropine sulfate (0.5 mg/kg) before (control) and after injection of E4031 (20 mg/kg)

 
3.3. Cardiac arrhythmias
3.3.1. Barium-induced ventricular tachycardia
Fig. 3 shows typical ECGs recorded in WT and transgenic mice immediately after challenge with BaCl2 (25 mg/kg, i.v.). In 9/10 WT mice, BaCl2 induced short runs of fast ventricular tachycardia (upper panels). These arrhythmias were preceded and followed by an increase in T wave amplitude and duration. In striking contrast, in 9/10 transgenic animals, BaCl2 altered ventricular repolarization but did not provoke ventricular tachycardia (middle panels). In the remaining transgenic mouse, injection of BaCl2 produced an accelerated idio-ventricular escape rhythm. To further confirm that protection against ventricular arrhythmia was related to the expression of HERG, transgenic mice were pretreated with dofetilide (10 mg/kg, i.p.) and then challenged with BaCl2 (rescue experiments). When pretreated with dofetilide, 8/10 transgenic mice developed short runs of ventricular tachycardia under the action of BaCl2 (see lower panels) similarly to WT mice. Pre-treating WT mice with dofetilide did not modify their response to BaCl2 (data not shown). These results strongly suggest that expression of HERG protected against the proarrhythmic effects of BaCl2.


Figure 3
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  		Fig. 3 Representative ECG tracings (lead I) showing the effects of BaCl2 injection (25 mg/kg) in two wild-type and two transgenic (Tg-HERG) mice under baseline conditions (top and middle panels) and in Tg-HERG mice pretreated with dofetilide (10 mg/kg; bottom). Note the short runs of ventricular tachycardia induced by BaCl2 in wild-type mice and Tg-HERG mice pretreated with dofetilide (circles).

 
3.3.2. Pacing-induced atrial arrhythmias
Fig. 4 shows representative surface ECGs and intracavity recordings obtained in WT and transgenic mice submitted to a burst of rapid atrial pacing (cycle length of 15 ms; 2-s duration). In 11/15 WT mice, atrial pacing reproducibly induced non-sustained atrial tachycardia and/or atrial fibrillation. The duration of atrial arrhythmias ranged from 200 ms to 146 s with an minimal cycle length of 22 ± 2 ms and a maximal cycle length (before spontaneous cessation) of 30 ± 1 ms. In contrast, atrial arrhythmia was inducible in only 2/11 transgenic mice (p<0.05 vs. WT) with durations of the salvoes ranging from 170 ms to 74 s and a cycle length ranging from 21 to 25 ms. Therefore, expression of HERG in the mouse atria prevented the initiation of atrial arrhythmias. However, when induced, atrial arrhythmias in transgenic mice were comparable to those in WT animals. Again, rescue experiments were conducted in transgenic mice. Transgenic mice pretreated with dofetilide were as sensitive to atrial arrhythmias as WT mice (6/8 mice had arrhythmias; p<0.05 vs. transgenic mice not receiving dofetilide; NS vs. WT mice), further demonstrating the role of HERG expression in the protection against cardiac arrhythmias.


Figure 4
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  		Fig. 4 Representative ECG and intracardiac recordings showing the effects of 2-s right atrial pacing at a cycle length of 15 ms following carbamylcholine injection (50 µg/kg) in wild-type and HERG-expressing (Tg-HERG) mice under baseline conditions (top and middle panels) and in a Tg-HERG mouse pretreated with dofetilide (10 mg/kg; bottom).

 
In order to determine whether the protection against arrhythmias produced by HERG expression could eventually be reproduced by another human delayed rectifier K+ current, similar protocols were conducted in transgenic mice expressing a hKCNQ1–hKCNE1 fusion protein (human KvLQT1 and its regulator mink) [25]. Fig. 5 shows representative surface ECGs and intracavity recordings obtained in WT and transgenic littermates. Both groups of mice were equally responsive to pacing. Indeed, atrial arrhythmias were reproducibly induced in 4/6 WT and 5/6 transgenic mice.


Figure 5
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  		Fig. 5 Representative ECG and intracardiac recordings showing the effects of 1.5-s right atrial pacing at a cycle length of 20 ms following carbamylcholine injection (50 µg/kg) in wild-type and hKCNQ1–hKCNE1 expressing mice.

 
3.4. Atrial action potential recording and action potential-clamp
To gain further information into the mechanisms by which HERG prevents induction of arrhythmias, transmembrane action potentials (APs) were recorded in left atrial tissue from WT and HERG-expressing mice. Action potential-clamp studies were also conducted in cells expressing HERG. Finally, simulations were performed using a mathematical model of mouse ventricular AP [21] modified to incorporate the expression of human ERG [20]. As shown in Fig. 6A, the slope of adaptation of atrial action potential duration (APD) over a range of diastolic intervals was steeper in WT animals (0.130 ± 0.008; n=5) than in transgenic mice (0.051 ± 0.007; n=6; p<0.001). We also observed that HERG expression in transgenic mice led to post-repolarization refractoriness: at a PCL of 200 ms, the ratio "effective refractory period/APD90" was prolonged from 0.9 ± 0.1 in WT left atria (n=7) to 1.3 ± 0.1 in transgenic atria (n=6; p<0.05). In cells expressing HERG, human-type action potentials induced an outward current with a characteristic waveform (Fig. 6B) [6]. Fig. 6C shows typical recordings in the same cell clamped with mouse atrial APs at physiological (100 ms) and long PCL (600 ms). At both PCL, a consistent IKr could be recorded during a mouse atrial AP. At PCL=100 ms, IKr was not entirely deactivated before the occurrence of the next AP (in Fig. 6C, the current is still outward at the end of the action potential). The time constants of deactivation (at –50 mV) of the HERG current expressed in Cos7 cells (n=5) were 54 ± 12 ms for the first one and 222 ± 32 ms for the second one. These values were similar to those obtained in isolated myocytes from transgenic mice, i.e. 44 ± 5 and 249 ± 66 ms (n=6). As a consequence, a portion of HERG channels were still open at the time of the upstroke, leading to the genesis of a transient outward current caused by a large instantaneous driving force. This was not seen at PCL=600 ms (the current at the diastole approximated zero). At the long PCL, more current was activated during the repolarization phase. This may explain the reduced slope of APD-rate adaptation observed in HERG-expressing mice. Cells transfected with hKCNQ1–hKCNE1 fusion protein did not have any measurable HMR 1556-sensitive current when paced with mouse atrial APs (data not shown) although they presented a typical current when paced with human APs (Fig. 6B). Simulation studies with the mouse model [21] predicted a comparable HERG current during the time course of a mouse action potential (Fig. 6C, bottom panels). It also predicted that at PCL=100 ms, IKr will not be entirely deactivated before the occurrence of the next AP, leading to an increasing transient outward current, as in HERG expressing cells. One difference between tissue preparations and the single cell model is the absence of action potential lengthening at long pacing cycle lengths in simulated mouse APs. As a consequence, at PCL=600 ms, the HERG current in the model was smaller than at PCL=100 ms. The mouse model also offered the opportunity to investigate further the transgene-induced current during protocols used to induce atrial arrhythmias experimentally. As shown in Fig. 6D, because of its slow deactivation kinetics, the HERG current was potentiated by short coupled extrasystoles and episodes of fast stimulation.


Figure 6
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  		Fig. 6 (A) Relationship between the diastolic interval (X-axis) and the action potential duration at 90% of full repolarization (APD90; Y-axis) in left atrial preparations from wild-type (open circles) and HERG-expressing (open triangles) mice. (B) Representative E4031- and HMR 1556-sensitive currents (thick lines; respectively HERG and KvLQT1-minK) induced by human ventricular action potential-clamp (thin lines; PCL=1000 ms) in Cos-7 cells transfected with HERG or hKCNQ1–hKCNE1. (C) Representative E4031-sensitive currents (thick lines) induced by mouse atrial action potential-clamp (thin lines) in a Cos-7 cell transfected with HERG (top panel) and action potentials (APs; thin lines) and HERG current (thick lines) simulated using the mouse ventricular myocyte model from Bondarenko et al. [21] with Markov human ERG[20] (bottom panel) at pacing cycle lengths (PCL) of 100 ms and 600 ms. The dotted lines indicates the 0 current level. Arrows indicate the peak outward currents. (D) Simulated HERG current of the mouse ventricular myocyte model induced during a burst of 100 APs with coupling intervals of 15 ms after the last AP of a series of 100 APs at PCL=100 ms. The trace within the circle is enlarged in the right panel.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
References
 
Our study shows that expression of HERG in the mouse heart can exert anti-arrhythmic effects at both atrial and ventricular levels. To our knowledge, this is the first report of such a protective effect of HERG in vivo. Under baseline conditions, the ECG of HERG-expressing mice is almost undistinguishable from WT littermates with the exception of a slight shortening of the QT interval and of a more negative T wave aspect. In the mouse as in other larger animals, the polarity of the T wave is determined predominantly by the transmural gradient that occurs as a consequence of differences in APD [26]. A negative T wave is seen when the epicardium to endocardium repolarization gradient is small; i.e. in the order of 5 ms. Because mouse endocardial APs are longer than epicardial APs, one can speculate that more time is allocated for HERG channels to activate in the endocardium than in the epicardium. Thus, overexpression of HERG should have a greater effect on the endocardial than on the epicardial repolarization thereby reducing the transmural gradient. This likely explains the E4031-sensitive negative T wave in transgenic mice. Under basal conditions, other ECG parameters were normal including duration of repolarization. The role of HERG in transgenic ventricular repolarization was unmasked under the effects of E4031 and even more dramatically when transgenic mice had a reduced Ito current under tedisamil. These data strongly suggest that HERG channels participated to cardiac repolarization in transgenic animals even if their ECGs were close to normal.

Antiarrhythmic effects of HERG expression has already been described in a model of adult rabbit ventricular myocytes maintained in primary culture [5]. Adenoviral delivery of HERG gene to cultured myocytes (which normally develop prolongation of repolarization and early afterdepolarizations after a few days in culture) resulted in significant shortening of APD, increase in refractory period and decreased incidence of early afterdepolarizations (EAD). It is conceivable that overexpressing a voltage-dependent delayed rectifier K+ channel, by increasing repolarization reserve, could compensate for a loss of repolarizing current [8]. This mechanism may very well explain the prevention of barium-induced ventricular tachycardia in our mice. Barium is a well-known blocker of the inwardly rectifying IK1 current that was shown to depolarize the myocytes and generate abnormal automaticity in atria, ventricles and Purkinje fibers of different species [27–29], a mechanism that we also observed in mouse ventricular preparations with concentrations of BaCl2 as low as 50–100 µM (unpublished observations). Although it is difficult to predict the level of IK1 inhibition in vivo, this mechanism might be responsible for cardiac arrhythmias.

Protection against pacing-induced atrial arrhythmias is more difficult to understand. Based on the "leading circle" hypothesis of Allessie et al. [30], it is hard to conceive that increasing a repolarizing current could in fact protect against reentrant mechanisms. However, recent studies suggest that APD alternans are key elements in the genesis of reentry [31,32]. More recent studies demonstrate that overexpression of HERG suppresses electrical alternans in cardiac myocytes in vitro [33]. In our study, the slope of atrial APD adaptation to the rate was lower in transgenic animals than in wild-type mice, a phenomenon thought to prevent repolarization alternans and occurrence of arrhythmias [7]. In addition, as in cultured rabbit myocytes [5], expression of HERG in mice prolonged the refractory period in atrial myocytes and led to post-repolarization refractoriness that should again prevent arrhythmia genesis. Moreover, simulation studies show that fast pacing leads to an accumulation of HERG current. Since repolarization is governed by a fine balance between inward and outward currents, this abrupt increase in repolarizing capacity may account for the lower excitability of transgenic mice cardiac myocytes during initiation of tachyarrhythmias.

Mice have become an increasingly important model for studying cardiac channelopathies and arrhythmias, largely because of the development of technologies allowing manipulation of the mouse genome [34]. Although cardiac electrophysiology in mouse clearly differs from that in human, several mouse models of channelopathies convincingly recapitulate the clinical phenotype in patients. Some mouse models have spontaneous cardiac arrhythmias, despite their small heart size and rapid heart rate, previously thought as limiting factors for triggering arrhythmias. Although species differences must be kept in mind when analyzing the results, the mouse heart, presents the advantage of allowing both molecular and integrated investigations of cardiac arrhythmias physiopathological mechanisms.

In conclusion, the present study shows that HERG expression can protect against experimental induction of arrhythmias at both atrial and ventricular levels. Inversely, it was recently shown that gain-of-function mutations of HERG can lead to short QT syndrome and sudden cardiac death [9]. Therefore it appears that increasing HERG current could have both antiarrhythmic and proarrhythmic activities, as also the case when HERG is reduced. However, evaluating further the antiarrhythmic potential of increasing IKr could be of interest in patients with left ventricular dysfunction, for which class III antiarrhythmic drugs are useless in preventing sudden death, and also in patients with the long QT syndrome.


   Acknowledgements
 
This work was supported by grants from the Ministère de la Recherche (ACI "Biologie du Développement et Physiologie Intégrative" 2001) and the Fondation de France. The authors wish to thank Béatrice Le Ray and Marie-Jo Louérat (INSERM U533) for expert technical assistance.


   Notes
 
1 Contributed equally to this work. Back

Time for primary review 17 days


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

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