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Cardiovascular Research 2001 50(2):314-327; doi:10.1016/S0008-6363(01)00231-0
© 2001 by European Society of Cardiology
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Copyright © 2001, European Society of Cardiology

Transgenic mice overexpressing human KvLQT1 dominant-negative isoform Part I: Phenotypic characterisation

Sophie Demolombe1,a, Gilles Landea, Flavien Charpentiera, Marian A van Roonb, Maurice J.B van den Hoffb, Gilles Toumaniantza, Isabelle Baroa, Gilles Guiharda, Nathalie Le Berrec, Alain Corbierc, Jacques de Bakkerb, Tobias Opthof2,b, Arthur Wildeb, Antoon F.M Moormanb and Denis Escandea,*

aINSERM U533, Laboratoire de Physiopathologie et de Pharmacologie Cellulaires et Moléculaires G & R Laennec, Faculté de Médecine, 1 rue Gaston Veil, 44035 Nantes cedex 01, France
bExperimental and Molecular Cardiology Group, and Facility for Genetically Modified Mice, Academic Medical Center, Amsterdam, The Netherlands
cHMR, Romainville, France

* Corresponding author. Tel.: +33-2-4041-2949; fax: +33-2-4041-2950 denis.escande{at}nantes.inserm.fr

Received 7 October 2000; accepted 25 January 2001


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objectives: The KCNQ1 gene encodes the KvLQT1 potassium channel, which generates in the human heart the slow component of the cardiac delayed rectifier current, IKs. Mutations in KCNQ1 are the most frequent cause of the congenital long QT syndrome. We have previously cloned a cardiac KCNQ1 human isoform, which exerts a strong dominant-negative effect on KvLQT1 channels. We took advantage of this dominant-negative isoform to engineer an in vivo model of KvLQT1 disruption, obtained by overexpressing the dominant-negative subunit under the control of the {alpha}-myosin heavy chain promoter. Results: Three different transgenic lines demonstrated a phenotype with increasing severity. Functional suppression of KvLQT1 in transgenic mice led to a markedly prolonged QT interval associated with sinus node dysfunction. Transgenic mice also demonstrated atrio-ventricular block leading to occasional Wenckebach phenomenon. The atrio-ventricular block was associated with prolonged AH but normal HV interval in His recordings. Prolonged QT interval correlated with prolonged action potential duration and with reduced K+ current density in patch-clamp experiments. RNase protection assay revealed remodeling of K+ channel expression in transgenic mice. Conclusions: Our transgenic mouse model suggests a role for KvLQT1 channels not only in the mouse cardiac repolarisation but also in the sinus node automaticity and in the propagation of the impulse through the AV node.

KEYWORDS Antiarrhythmic agents; Congenital defects; ECG; K-channel; Long QT syndrome; Repolarisation


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
The inherited long QT (LQT) syndrome is a familial disorder characterised by prolonged cardiac repolarisation, syncope and high incidence of sudden death. Five human loci have been identified so far. Four of these encode for specific cardiac ion channel subunits [1]: (1) KCNQ1 encodes for KvLQT1 K+ channels [2]; (2) HERG generates the fast component, IKr, of the cardiac delayed rectifier current; (3) SCN5A encodes a cardiac voltage-sensitive sodium channel; (4) KCNE1 encodes for minK (or IsK), a single transmembrane domain protein, which does not form a channel by itself but modulates KvLQT1 K+ channels to generate the slow component, IKs, of the cardiac delayed outward rectifier current [3,4]. KCNQ1 mutations are the most frequent causes for the LQT syndrome in human.

In the adult mouse heart, KCNQ1 is also expressed at a high level [5] in the virtual absence of the minK regulator which expression markedly declines with ageing [5,6] 1 week after birth. As a result, adult mouse myocytes usually lack typical IKs delayed rectifier [7]. In the absence of minK, KvLQT1 K+ channels generates a voltage-dependent K+ current with very fast activation kinetics distinct from the IKs current [8]. Furthermore, other single transmembrane domain regulators such as the KCNE3 gene product could associate with KvLQT1 channels and profoundly affect its electrophysiological characteristics [9].

We have previously reported that alternative splicing of the KCNQ1 gene produces at least two isoforms: isoform 1 with a long N-terminal end [4] and isoform 2 with a very short amino-terminal end constituted by only two amino-acids [8]. Isoform 2 does not form functional channels but exerts strong dominant-negative effects on isoform 1 channels. We aimed at exploring the functional role of KvLQT1 in the mouse heart. To that end, we took advantage of the human endogenous dominant-negative isoform to functionally invalidate KvLQT1 in transgenic mice overexpressing isoform 2 under the control of the {alpha}-myosin heavy chain promoter [10,11]. Our model shares some phenotypic characteristics with the LQT syndrome including markedly prolonged QT interval and sinus node dysfunction. In addition, functional suppression of KvLQT1 channels in the mouse heart led to an unexpected atrio-ventricular block not observed in LQT patients.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
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).

2.1 Generation of transgenic mice
The human KvLQT1 isoform 2 ([8]; Fig. 1A) was subcloned into the mammalian expression vector pCI (Promega). Using polymerase chain reaction (PCR), the stop codon was removed and a tag (11-amino acid T7 epitope) was introduced in frame at the carboxyl terminus. Oligonucleotides were generated by GibcoBRL. The epitope-tagged KvLQT1 isoform 2 construct (KvLQT1-iso2-T7) was checked by sequencing, and then removed from pCI plasmid using XhoI and SmaI restriction enzymes. The generated sites were blunted and subcloned into the {alpha}-myosin heavy chain ({alpha}-MHC) vector (a kind gift from Dr Jeffrey Robbins, University of Cincinnati, USA) at the blunted SalI–HindIII sites. The {alpha}-MHC-KvLQT1-iso2-T7 plasmid was digested to isolate a 7.3-kb fragment that included the {alpha}-MHC promoter, introns and the three noncoding exons of the {alpha}-MHC gene, the KvLQT1-iso2 coding sequence, the T7 tag and the human growth hormone polyadenylation signal sequences (Fig. 1B). The 7.3-kb transgene fragment was gel-purified, further purified by cesium chloride density gradient centrifugation and three times dialysed against 5 mmol/l Tris (pH 7.4) and 0.1 mmol/l EDTA. The fragment was then diluted to a concentration of 5 ng/µl and microinjected into the male pronucleus of fertilised FVB mouse embryos. A total of 63 offsprings were obtained, and screened for the presence of the transgene by PCR analysis of genomic DNA isolated from tail clips. Twelve founders were positive and were back-crossed to wild-type (WT) FVB to produce heterozygous F1 offsprings. Nine founders transmitted the transgene to their lineage. Three transgenic lines, called H02, H05 and H08, were selected for further phenotypic characterisation. Transgenic and non-transgenic mice (as controls) from a littermate were explored between 6 and 8 weeks except if otherwise stated. Animal care was in accordance with institutional guidelines.


Figure 1
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  		Fig. 1 In vitro and in vivo KvLQT1-iso2-T7 expression. (A) Schematic diagram of KvLQT1 isoforms. (B) Schematic representation of the transgene. A T7 tag was placed in frame at the C-terminus of human KvLQT1 isoform 2 (H iso2). A human growth hormone (Hgh) poly(A) sequence guaranteed mRNA transgene stability. (C) Typical whole-cell K+ current traces in COS-7 cells injected with mouse KvLQT1 isoform 1 cDNA (M iso1; 5 µg/ml) without and with human KvLQT1-iso2-T7 cDNA (H iso2-T7; 2 µg/ml). Voltage steps, 20 mV increment, applied from –100 to +60 mV (holding potential: –80 mV; current scale: 20 pA/pF without and 5 pA/pF with H iso2-T7; time scale: 500 ms). The right panel shows averaged current tail density related to mouse isoform 1 expression. (D) Transgene expression detected by Western blot in H02, H08 and H05 lineage and in wild-type (WT) mice. Endogenous biotinylated protein (*; 90 kDa) used as internal marker.

 
2.2 Western blots
Protein extracts from cardiac samples were prepared by RIPA buffer. After centrifugation, total cell lysates were denatured with electrophoresis sample buffer (200 mmol/l DTT), separated on 10% SDS-PAGE and transferred to Hybond C super membrane (Amersham). Nonspecific bindings were blocked by incubating membrane in TBST-0.1%/5% non fat dry milk for 30 min at room temperature. Membranes were incubated with the anti-T7 tag monoclonal antibody (Novagen), followed by an alkaline phosphatase-conjugated goat anti-mouse secondary antibody (Sigma). The membranes were then washed three times for 5 min each and antibody complexes were detected by NBT/BCIP substrate (Boehringer) (Fig. 1D).

2.3 RNase protection assays
FVB adult cardiac samples were dissected out and immediately frozen in liquid nitrogen. Total RNA was isolated from each sample using the guanidinium isothiocyanate method. Assessment of the integrity of RNA samples was based on the appropriate 28S-to-18S ribosomal RNA ratios. Our method to perform RNase protection assay has previously been published [12]. The mouse KvLQT1 probe consisted of a 446-bp segment corresponding to nucleotides 2397–2843 of the 3' untranslated region subcloned into pSK(–). The minK probe corresponded to nucleotides 1–512 containing the full coding sequence. Kv4.2, Kv4.3 and Kv1.5 probes were a generous gift from Dr Wayne Giles (University of Calgary, Canada) and consisted, respectively, of 181-, 111- and 181-bp segments. All of these probes begin at the initiation Met codon of the cDNA, a region where little homology exists between K+ channels. A rat cDNA probe for GAPDH (Ambion) was used as an internal marker. Antisense RNA probes were produced using appropriate polymerase (T3 or T7, Ambion) in the presence of [{alpha}-32P]UTP (DuPont NEN).

2.4 Electrocardiograms and telemetry recordings
Surface ECGs were recorded from male and female transgenic mice and compared with age- and weight-matched littermate wild-type controls. Our method to record mouse ECG can be found in detail elsewhere [16]. All reported measurements were averaged from five consecutive PQRST complexes in lead I. The end of the T wave was defined as the point where the T wave returned to the iso-electric line. All measurements were done independently by two experienced examiners. Measurements were accepted only in case of accordance between the two examiners. The QT interval was corrected for heart rate using the formula, QTc=QT/(RR/100)1/2 established for mice with QT and RR expressed in ms [13]. The sinus node function was evaluated by assessing the intrinsic sinus period under blockade of the autonomic nervous system (with atropine sulfate 0.5 mg/kg i.p.) and propanolol (1 mg/kg i.p.) [14] and the shortest sinus period measured under isoproterenol stimulation (0.5 mg/kg i.p.).

Telemetry device implantation was performed in transgenic and control mice ranging from 23 to 31 g body weight. The telemetry system consisted of an implantable transmitter (TA10ETA-F20, Data Sciences International) with a pair of flexible leads, a telemetry receiver (RPC1, Data Sciences International) placed under the home cage of each mouse under study and an analog adapter (ART System, Data Sciences International). The data were collected on a computer using an IOX data acquisition system (EMKA Technologies). The surgery was performed as described previously [13]. All analysis started when mice recovered from surgery.

2.5 AH interval measurement
Experiments were carried out on six WT and six H02 transgenic mice (45±3.1 days) of either sex. Following cervical dislocation, the thorax was opened and heart exposed. One hundred microliters heparin (5000 IU/ml) was injected into the left ventricle, after which the heart was excised and submerged in a modified Tyrode's solution at 4°C. The apex was excised and the free wall of the right atrium and right ventricle were removed to expose the atrio-ventricular (AV) junction area and right ventricular septum. The preparation was pinned to a silicon rubber support on the bottom of a tissue bath and superfused with Tyrode's solution at 37°C. A stimulation electrode was placed posterior from the coronary sinus ostium. Extracellular recordings of the atrial septum and the bundle of His were made in the bipolar mode. Extracellular electrograms were band-path filtered (low cut-off 0.3 Hz, high cut-off 2 kHz), amplified 500 times and displayed on a two-channel oscilloscope (Tektronix 2214). Signals were stored on an eight-channel digital recorder (DTR 1801, Biologic).

2.6 Microelectrode studies
Five transgenic (H02 strain; mean age: 42.4±4.1 days) and eight wild-type mice (mean age: 43.6±4.6 days) of either sex were used. Mice were killed by cervical dislocation. The heart was removed quickly and immersed in a cool Tyrode's solution. The preparations were mounted in a tissue bath chamber, the endocardial surface facing up. Preparations were superfused with an oxygenated (95% O2, 5% CO2) Tyrode's solution containing (in mM): NaCl, 120; NaHCO3, 27; NaH2PO4, 1.2; KCl, 5.4; MgCl2, 1.2; CaCl2, 1.8; glucose, 10 (pH 7.4 and 37±0.5°C). Transmembrane recordings were obtained using standard methods.

2.7 Patch-clamp experiments
The kidney-derived COS-7 cells (American Type Culture Collection), grown on glass coverslips were microinjected with plasmids at day 1 after plating. Our protocol to microinject cultured cells using the Eppendorf ECET microinjector 5246 system, has been reported in detail elsewhere [15]. Mouse KvLQT1-isoform 1 (a kind gift from Jacques Barhanin, Sophia, France) and human KvLQT1 isoform 2 were subcloned into the mammalian expression vector pCI. Murine pCI-KvLQT1 isoform 1 (5 µg/ml) was injected alone or co-injected with 2 µg/ml human pCI-KvLQT1 isoform 2. A pCI-green fluorescence protein was used as an inert plasmid to ensure that cells were always injected with a constant 15 µg/ml plasmid concentration.

Ventricular myocytes were isolated from five wild-type, five H02, two H05 and three H08 mice. Briefly, mice of either sex were injected with heparin (5000 U/kg i.p.). After 15 min, they were anesthetised with pentobarbital sodium (50 mg/kg i.p.). The heart was quickly removed and put in ice-cold, oxygenated (100% O2) Tyrode solution (solution 1) of the following composition (in mM): NaCl, 130; NaH2PO4, 1.2; KCl, 5.4; MgSO4, 1.2; CaCl2, 1; Hepes, 10; glucose 10; 2,3-butanedione monoxime (BDM), 10; pH 7.4 with NaOH. The aorta was cannulated and flushed with the Tyrode solution. The cannula was then attached to a Langendorff apparatus and the heart was perfused for a few minutes through the aorta with solution 1 warmed to 37°C until beating was regular and the perfusate was blood-free. Solution 1 was then switched to a nominally Ca2+-free solution without BDM (solution 2) for 5 min. The heart was subsequently perfused for another 9–14 min with a 25 µM Ca2+-containing solution supplemented with 0.25 mg/ml collagenase (Sigma type I; 350 U/mg; Sigma, St Louis, MO, USA), 0.065 mg/ml protease (Sigma type XIV; 4.4 U/mg) and 20 mM taurine (solution 3). It was then rinsed with a 100 µM Ca2+-containing solution (solution 4) supplemented with 20 mM taurine and 10 mM BDM for 5 min. Ventricles were cut into 1 mm3 cubic chunks. Single myocytes were obtained by gentle trituration of the chunks with polished Pasteur pipettes. The cell suspension was filtered through a 250-µm mesh collector and placed into centrifuge tubes placed at room temperature. After permitting the cells to settle under gravity for 5 min, the cells were re-suspended in solution 4 without BDM. The Ca2+ concentration was then increased to 900 µM by 200-µM increments every 20–30 min.

Whole-cell currents were recorded at 37°C either from COS-7 cells injected with plasmids and ventricular myocytes as previously described [15]. In studies with ventricular myocytes, for K+ currents recordings, pipettes were filled with a solution containing (in mM): KCl, 74.5; K-aspartate, 70.5; Hepes, 5; EGTA, 2; MgCl2, 0.3 (free-Mg2+: 0.1); K2ATP, 5; pH 7.2 with KOH. The extracellular medium contained (in mM): N-methyl-D-glucamine, 130; KCl, 5.4; MgSO4, 1.2; CoCl2, 2; Hepes, 10; glucose, 10; pH 7.4 with HCl. For the Ca2+ current studies, patch pipettes were filled with a solution containing (in mM): aspartic acid, 75; CsCl, 50; MgATP, 5; TEA-Cl, 20; EGTA, 10; Hepes, 10; Na2-phosphocreatine, 3.6; pH 7.2 with CsOH. The external solution contained (in mM): TEA-Cl, 140; MgCl2, 2; CaCl2, 2; Hepes, 10; glucose, 10; pH 7.4 with CsOH.

2.8 Statistical analysis
Patch-clamp and microelectrode data are expressed as means±S.E.M. Statistical analysis was performed with the Student's t-test and one- or two-way analysis of variance completed by a Bonferroni t-test when appropriate. ECGs data are expressed as means±S.D. Mean data were compared using analysis of variance. Multiple comparison procedure (Fisher's PLSD test) was then performed to determine which pair(s) of means were statistically different. A value of P<0.05 was considered significant.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 The human KvLQT1-isoform 2-T7 exerts dominant-negative control of mouse KvLQT1 isoform 1
We first checked that the human isoform 2 conserves its dominant-negative properties on the mouse isoform 1. Whole-cell patch-clamp experiments performed in intranuclearly injected COS-7 cells demonstrated that the peak and tail currents related to mouse pCI-KvLQT1 isoform 1 expression were markedly reduced in amplitude by co-expression of the human pCI-KvLQT1 isoform 2 (Fig. 1C). On average, the tail current density at –40 mV was 6.50±1.71 pA/pF (n = 19) in cells expressing mouse KvLQT1 isoform 1 alone and 0.02±0.01 pA/pF (n = 7; P<0.001) in cells coexpressing mouse KvLQT1 isoform 1 plus human isoform 2. In cells co-expressing the human isoform 2, the K+ current amplitude was too small to explore voltage-dependence, activation and inactivation kinetics. The presence of the T7 tag at the 3' end of the transgene did not modify its dominant-negative properties: the tail current amplitude was 0.04±0.03 pA/pF (n = 6) at –40 mV in cells co-expressing mouse isoform 1 and T7 tagged human isoform 2.

3.2 Control screening of transgenic mice
Only the nine founders that were able to transmit the transgene to their offsprings in a Mendelian distribution were screened for transgene expression using an antibody against the T7 tag epitope. Based on results from Western blots (Fig. 1D), three transgenic lines (H02, H05, H08) were established which exhibited an increasing level of transgene expression with H02>H08>H05. On both gross and detailed histopathological examination of the heart, no differences were observed between transgenic animals and nontransgenic animals, all merged sexes. The cellular organisation of specific locations, such as sinus node and atrio-ventricular node was also similar. Body weight, heart weight, heart-to-body weight ratio from 1-month-old transgenic mice revealed no significant difference from control data. In addition, there was no histological evidence of myocyte hypertrophy in the selected transgenic lines. In accordance with this, the membrane capacitance of ventricular myocytes isolated from KvLQT1-iso2-T7 mice and wild-type animals was not statistically different: 127±10 pF (n = 32) versus 142±6 pF (n = 17), respectively. Finally, transgenic mice remained healthy for at least 1 year (see below).

3.3 Electrocardiographic phenotype of transgenic animals
In order to determine the consequences of transgene expression, six-lead surface ECGs were obtained and compared between transgenic (H02, H05 and H08) and nontransgenic littermates. Typical recordings as displayed in Fig. 2A demonstrated pronounced QT and PR interval prolongation in transgenic animals. In contrast, the QRS duration was not modified in transgenic animals (10–15 ms in both WT and transgenic mice). In H02 mice, the P wave often superposed on the terminal phase of the previous T wave because of dramatic QT and PR lengthening. In both WT and transgenic animals, the T wave had a biphasic appearance with an initial rapid component (positive in lead I) and a late slower component (negative in lead I). The rapid component was slightly prolonged in H05 mice but drastically prolonged in H08 and H02 mice (Fig. 2A).


Figure 2
Figure 2
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  		Fig. 2 Surface electrocardiograms in wild-type (WT) and transgenic mice. (A) Top and middle panel: six-lead ECG recording in WT and H08 mice (100 mm/s paper speed). Bottom panel: lead I recordings in H05 and H02 mice. The arrows indicate the end of the T wave. (B, overleaf) One year longitudinal ECGs (lead I) in the same H08 mouse. ECGs were recorded at the age of 10, 20, and 30 days (D10, D20, D30), and at 3, 6, and 12 months (M3, M6, M12). (C) Telemetry ECG recordings in non anesthetized WT and H02 mice.

 
Lead I was further selected as the most appropriate lead allowing discrimination of both T and P waves. Average ECG values are summarised in Table 1. These data demonstrate that transgenic animals had a longer P wave duration with WT<H05<H08<H02. In H05 mice, the PR interval duration was comparable to WT mice but markedly prolonged in H08 and H02 lineage. The corrected QT interval was prolonged in all lineage (WT<H05<H08<H02). No statistically significant difference in QTc was observed between transgenic males and females: in the H08 line, QTc was 93.9±16.5 ms in males (n = 42) and 95.5±24.3 ms in females (n = 42). Sinus node dysfunction was diagnosed on the basis of a longer intrinsic sinus period (intr PP) under autonomic nervous blockade and an increased minimum sinus period under isoproterenol infusion ({Sigma}+ PP). The sinus rate under isoproterenol accelerated to 685±38 bpm (n = 10) in WT, to 657± 36 bpm (n = 10) in H05, to 495±45 bpm (n = 10) in H08 and to 482±40 bpm (n = 10) in H02 mice. The intrinsic sinus period under autonomic nervous blockade, increased in TG mice with WT<H05<H08<H02 (Table 1). Therefore, the severity of the sinus node dysfunction differed depending on the mouse strains with H05<H08<H02.


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  		Table 1 Average ECG data in wild-type (WT) and transgenic (H05, H08, H02) lineagea

 
Telemetry recordings (Fig. 2C) demonstrated similar ECG anomalies in freely moving animals from an H02 littermate. In telemetry recordings, the rapid component of the T wave corrected for heart rate was 64.7±1.4 ms in TG mice (vs. 19.9±0.7 ms in WT mice; n = 7), the PP duration was 128.4±2.2 ms (vs. 107.4±1.8 ms), the PR interval was 38.8±0.8 ms (vs. 29.1±0.5 ms) and the heart rate was 471±3 beats per min (vs. 588±7 beats per min in WT mice). We never observed arrhythmias either in anesthetised animals (in the presence or absence of isoproterenol stimulation) or during telemetry recordings.

Age-related variation in the ECG phenotype was assessed between birth and 1 month and between 1 month and 1 year (Fig. 2B, Table 2). It is known that the {alpha}-MHC promoter is expressed in the atria during the embryonic life, in atria and part of ventricles after birth and in total heart around 1 week after birth [10]. All ECG parameters were progressively modified during the first 30 days of life and more particularly between day 20 and day 30 after birth (Table 2). In contrast, ECG parameters remained roughly stable between 1 month and 1 year with the exception of the P wave duration and of the intrinsic sinus period which slightly but significantly increased with ageing.


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  		Table 2 One-year follow-up of ECG parameters in five H08a

 
In 4- to 6-week-old H02 lineage, a Wenckebach phenomenon was observed in 27 out of 117 H02 mice (i.e. 23%) as illustrated in Fig. 3A, suggesting an intranodal conduction defect. In vitro experiments were conducted to explore in more details the atrio-ventricular conduction in six KvLQT1-invalidated and in six WT mice (Fig. 3B). Measures of the AV interval at the spontaneous cycle length demonstrated that the prolonged PR interval corresponded to a prolonged AH interval (49±5 ms vs. 25±3 ms in preparations from WT mice; P<0.05) with a normal HV interval (8±1 ms). In the example illustrated in Fig. 3B, the AH interval duration increased from 60 to 95 ms, leading to a non conducted A wave. These recordings revealed that functional suppression of KvLQT1 channel led to an AV block originating from an intranodal conduction defect and not from a His-bundle conduction defect.


Figure 3
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  		Fig. 3 Atrio-ventricular block in transgenic mice. (A) Typical Wenckebach phenomenon in an adult H02 mouse. A progressive lengthening of the PR interval (ms) led to a non-conducted P wave. (B) AH and HV interval measurements in vitro in the heart from an adult H02 mouse. Wenckebach periodicity is shown with progressive increase in the AH interval.

 
3.4 Prolonged action potentials in KvLQT1-iso2-T7-expressing mice
Fig. 4A shows typical action potentials recorded in right ventricular preparations from WT and H02 transgenic adult mice. Functional suppression of KvLQT1 channels induced a marked prolongation of both early and late repolarisation phases in transgenic mice. Action potential prolongation was so pronounced that, at shorter cycle lengths, the upstroke of the next action potential arose from the terminal repolarisation phase of the preceding action potential (Fig. 4A bottom). At cycle lengths >100 ms, the resting potential (RP) and the maximum upstroke velocity of phase 0 depolarisation (Vmax) were similar in both wild-type and H02 mice (Fig. 4B). In contrast, at cycle lengths <100 ms, the take-off potential was less negative in H02 than in wild-type mice, because of incomplete repolarisation. As a consequence, Vmax decreased in H02 mice at faster stimulation rates. The early phase of repolarisation (APD30) was significantly longer in H02 mice at all cycle lengths. The APD90 was also markedly increased at all cycle lengths. As shown in Fig. 4C,D, the action potentials from left atrial preparations were also markedly prolonged in transgenic animals.


Figure 4
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  		Fig. 4 Action potential recordings in transgenic mice. (A) Superimposed typical action potentials recorded in ventricular strips from a wild-type (WT) and a transgenic (H02) mouse at pacing cycle lengths (PCL) of 300 and 100 ms. Vertical bar, 50 mV; horizontal bar, 100 ms. (B) Effects of pacing cycle length (PCL; X-axis) on resting or take-off potential (RP), maximum upstroke velocity of phase 0 (Vmax) and action potential duration at 30% (APD30) and 90% (APD90) repolarisation in WT (open circles; n = 6) and H02 mice (filled triangles; n = 5). *P<0.05, **P<0.01, ***P<0.001 versus wild-type with Bonferroni t-test. (C) Superimposed typical action potentials recorded in atrial strips from a wild-type (WT) and a transgenic (H02) mouse at pacing cycle lengths (PCL) of 300 and 100 ms. Vertical bar, 50 mV; horizontal bar, 100 ms. (D) Effects of pacing cycle length (PCL; X-axis) on action potential duration at 30% (APD30) and 90% (APD90) repolarisation in WT (open circles; n = 6) and H02 mice (filled triangles; n = 5). *P< 0.05, **P< 0.01, ***P< 0.001 versus wild-type with Bonferroni t-test.

 
3.5 Reduced Ito and Isus current density in functionally invalidated mice
Patch-clamp experiments were performed on isolated ventricular myocytes from control and transgenic mice of matching age and sex. Fig. 5A shows representative K+ current density traces from wild-type, H05, H08 and H02 mice. Fig. 5B shows the corresponding mean current—voltage relationships of the peak current (Ipeak) and of the current remaining at the end of the 500-ms pulses (Isus; see legend for description of the protocol). This set of data demonstrates that the transient outward current, Ito, and the sustained current, Isus, densities were reduced in transgenic mice. The decrease in current densities (Fig. 5B) paralleled the severity of the phenotype as assessed by surface ECG with H05<H08<H02. Fig. 5C shows that the background K+ current density (IK1) was also decreased in H02 mice at voltages negative to the reversal potential. In H05 and H08 transgenic mice, the IK1 current density was not significantly decreased at any potentials (not illustrated). Finally in H02 mice, the density of L-type Ca2+ current, ICa,L, was not different from that of control mice (Fig. 5D).


Figure 5
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  		Fig. 5 Whole-cell current recordings in wild-type (WT) and transgenic ventricular myocytes. (A) Superimposed K+ current tracings recorded in myocytes from a WT, a H05, a H08 and a H02 mouse. The voltage was stepped for 300 ms from –80 mV to potentials between –60 and +60 mV in 10-mV increments. Only the currents recorded at –60, –40, –20, 0, +20, +40 and +60 mV are shown. Vertical bar, 10 pA/pF; horizontal bar, 100 ms. (B) Current—voltage relationships for the peak transient outward K+ current (Ipeak; top) and the sustained potassium current (Isus; bottom; measured at the end of the pulses). Averaged data from 15 WT (open circles), 8 H05 (filled squares), 7 H08 (filled inverted triangles), and 10 H02 (filled triangles) myocytes. (C) Top: Superimposed current tracings of the inward rectifier K+ current IK1 recorded in ventricular myocytes isolated from a WT and a H02 mouse. The voltage was stepped for 300 ms from –50 mV to various potentials between –110 and 0 mV in 10-mV increments. Only the currents recorded at –110, –90, –70, –50, –30 and –10 mV are shown. Vertical bar, 10 pA/pF; horizontal bar, 100 ms. Current—voltage relationships for IK1. Averaged data from 10 WT (open circles) and 10 H02 (filled triangles) myocytes. **P< 0.01, ***P< 0.001 versus wild-type corresponding value with Bonferroni t-test. (D) Top: superimposed L-type Ca2+ current tracings recorded in ventricular myocytes isolated from a WT and a H02 mice. The voltage was stepped for 200 ms from –60 mV to potentials between –40 to +50 mV in 10-mV increments. Only the currents recorded at –40, –20, 0, +20 and +40 mV are shown. Vertical bar, 10 pA/pF; horizontal bar, 100 ms. Bottom: current—voltage relationship for the L-type Ca2+ current. Averaged data from 6 WT (open circles) and 9 H02 (filled triangles) myocytes.

 
3.6 K+ channel expression in KvLQT1 transgenic mice
RNase protection assays were performed using total cardiac RNA (Fig. 6). The GAPDH probe was used as an internal marker for RNA quantification. Results demonstrated some degree of K+ channel remodeling in transgenic mice. Comparable findings were obtained from three independent experiments. When corrected for GAPDH expression, expressions of endogenous KvLQT1 or MinK were comparable in transgenic and WT mice. In transgenic hearts, the expression of Kv1.5 was consistently decreased, although this was seen in the H02 lineage mainly. Kv4.2 channel expression was the most remodeled with about 70% down-regulation in H02 mice compared to non-transgenic mice. In contrast, Kv4.3 expression was consistently up-regulated in all transgenic lines. Remodeling of Kv4.2 and of Kv4.3 expression was more pronounced in H02 than in H08 and H05.


Figure 6
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  		Fig. 6 RNase protection assays in KvLQT1 invalidated transgenic mice. The cardiac mRNA expression of minK, KvLQT1, Kv4.2, Kv4.3 and Kv1.5 was compared in H02, H08 and H05 transgenic and wild-type (WT) mice. GAPDH probe used as an internal marker.

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
We have established a transgenic model in which KvLQT1 has been functionally suppressed by overexpression of its dominant-negative isoform. The phenotype of transgenic animals comprises prolonged QT interval, sinus node dysfunction and intranodal conduction defect. This ECG phenotype was accompanied by prolonged action potential duration and reduced outward K+ current. In the three different transgenic lineage investigated, the severity of the ECG phenotype paralleled modifications in ionic currents as assessed by electrophysiological techniques and also paralleled the severity of K+ channel expression remodeling as assessed by RNase protection assay. In a companion paper [16], we show that our mouse model demonstrates some value in identifying in vivo pharmacological drugs that block IKr which are at risk for proarrhythmia in the clinical setting [1].

As yet, genetically engineered animal models with genes known to be involved in the LQT syndrome comprise invalidation of minK [5,17] and HERG [7]. However, suppression of the minK β-subunit or expression of a dominant-negative HERG subunit did not lead to QT prolongation at physiological rates. Other murine transgenic lines have been established which displayed a surface electrocardiogram with a long QT interval at physiological rates. These models imply K+ channels, such as Kv2 [18], Kv4.x [19,20], and Kv1.5 [21], which are not yet involved in the congenital long QT syndrome.

4.1 IKs current in adult mouse
Expression of minK increases during embryonic development to reach a maximum 7 days after birth [5,6]. In the adult mouse heart, minK expression is low and has been described as homogenous [22,23] or inversely restricted to the conductive tissue [17]. From 20 days before birth to the first week of life, the IKs current density increases to a maximum during the first week of life [24]. Seven days after birth, IKs is recorded in approximately 10% of isolated cardiomyocytes [5,17], although its pharmacological characterisation may be confounded by extremely rapid run-down. In adult mice, IKs may represent less than 25% of the cardiac delayed rectifier K+ current [7]. At the mRNA level, expression of KvLQT1 in the adult mouse heart has been demonstrated by Drici et al. [5] and also by our laboratory using either in situ hybridisation [23] or RNase protection assay (the present study). Therefore the age-related changes in IKs current better match minK expression rather than KvLQT1 expression profiles. In the adult mouse heart, it is conceivable that minK is replaced by another member of the KCNE family of K+ channel regulators such as KCNE3 which renders KvLQT1 a permanently open channel with a linear I/V relationship [9] and which expresses in the adult heart.

4.2 Remodeling of potassium channels expression
We found a remodeling of K+ channel expression in our mice. This remodeling comprises both up-regulation (i.e. Kv4.3) and down-regulation (i.e. Kv4.2 and Kv1.5) of K+ channel expression. We believe that K+ channel remodeling was responsible for a significant part of the phenotype observed in transgenic mice. Previous studies have revealed that {alpha}-subunit expression of Kv channels is modified by physiological stimuli [25,26] and in pathophysiological situations [27,28]. The specificity of the suppressing effects of isoform 2 on KvLQT1 isoform 1 channel function has previously been demonstrated (Ref. [29] and our own unpublished data). Several hypothesis may account for K+ channel remodeling in our model: (1) KvLQT1 activity is crucial during development for the correct expression of other potassium channels; (2) KvLQT1 suppression per se leads to action potential prolongation which in turn produces elevated Ca2+ influx and [Ca2+]i transients, and may modify gene expression [20,30]; (3) finally, a non-specific effect of the transgene expression cannot formally be ruled out. The ion current profile of transgenic animals overexpressing KvLQT1 dominant-negative isoform is reminiscent of that observed during cardiac hypertrophy and failure [31], including reduced Ito, reduced Isus, reduced IK1, and normal ICa,L. In line with this, it has recently been shown that cardiomyopathy could result from overexpression of a supposedly biological inert protein such as the green fluorescent protein [32]. However, the three transgenic lines that were investigated in the present study exhibit no histological sign of cardiac hypertrophy and had normal cardiac myocyte surface as measured by cell capacitance. In addition, these mice remain healthy and reproduce for more than 1 year. One additional transgenic line (H03) not retained for the present study, exhibited atrial and ventricular dilatation and a low body weight. These mice died between 3 and 4 weeks of age because of heart failure. We interpreted this phenotype as resulting from a huge transgene expression. K+ channel remodeling has previously been observed in transgenic mice expressing a dominant-negative Kv4.2 subunit under the control of the {alpha}-MHC promoter [20]. In this model however, remodeling was associated with development of cardiac hypertrophy and heart failure. Most importantly and in accordance with our own findings, K+ current remodeling has previously been demonstrated in a mouse model expressing dominant negative Kv4 subunit, lacking heart disease [19].

4.3 Atrio-ventricular block associated with KvLQT1 functional suppression
Our model comprises impaired intranodal conduction. To our knowledge, the membrane currents underlying the mouse AV node electrical properties have not been previously reported. In rabbit AV nodal cells, the transient outward current and the delayed rectifier are both present and participate in repolarisation and diastolic depolarisation [33,34]. In these cells, Mitcheson and Hancox [35] have previously shown that the Ito blocker, 4-aminopyridine, prolongs repolarisation, decreases the diastolic potential and blocks spontaneous activity. In mice, neither IKr blockers nor the Ito blocker tedisamil prolong the PR interval in WT or in TG animals [16]. Regardless of the nature of the reduced potassium current, a prolongation of nodal action potentials would lead to a slower recovery of excitability, a major determinant of action potential propagation through non-homogeneous tissues such as the AV node, therefore leading to a decreased conduction and to Wenckebach periodicity.

4.4 Sinus node dysfunction
Bradycardia was constantly observed in anesthetised transgenic mice after blockade of the autonomic nervous system and also in non constrained awake animals. This suggests that KvLQT1 participates in automaticity in mouse sinoatrial node. The membrane currents underlying diastolic depolarisation in the mouse sinoatrial node are largely unknown. In rabbit or in guinea-pig, it is well established that the delayed rectifier potassium current (IK) participates in pacemaker activity because of its progressive deactivation during diastole. In these two species, both IKr and IKs are present [36,37]. Inhibition of IKr with E-4031 produces a slowing in the spontaneous rate [38,39]. In our mouse model, similar results were obtained in vivo using various blockers of IKr [16]. The present results suggest that IKs is also involved since its functional suppression makes the sinus rate of transgenic mice even more sensitive to IKr blockers [16]. An alternative explanation could be that bradycardia observed in transgenic mice was caused by decreased expression of Kv4.2, assuming that the remodeling of K+ channels also affects the sinus node. Indeed, it is likely that transient outward currents also contribute to sinoatrial repolarisation and automaticity [40].

4.5 Limitation and relevance of the model
Our KvLQT1 mouse model shares some common features with the long QT syndrome in patients. Transgenic mice exhibit a long QT and sinus bradycardia both under baseline conditions and under β-adrenergic stimulation. Bradycardia at rest and also during exercise is a common finding in the long QT syndrome related to KCNQ1 mutations [41,42]. The mouse model also shows intranodal conduction defect, an anomaly not reported in the long QT syndrome. The difference between the mouse model and the human disease may be related to different currents controlling intranodal conduction velocity and/or to K+ channel remodeling in mice. Whether the myocardium of patients affected by the long QT syndrome undergoes K+ channel remodeling, i.e. whether the remodeling observed in our model augurs a reality in humans remains elusive. Also, the ionic currents governing repolarisation and automaticity in the mouse are likely to be different from those in the human heart. For example in the present study, IKs was not detectable in adult WT mouse myocytes although it was previously described in neonatal mouse myocytes [6]. In this line, the pharmacology of the mouse heart [16] markedly differs from that of larger animals and human. In spite of their markedly prolonged QT interval, transgenic mice never underwent arrhythmias either under isoproterenol challenge or under pharmacological stress, a major difference from the human disease. Most recently, the KCNQ1 gene has been invalidated by homologous recombination in the mouse [43]. Similarly to transgenic mice, KCNQ1 knock-out mice demonstrate prolonged QT and altered atrio-ventricular conduction.

Time for primary review 24 days.


   Acknowledgements
 
This work was supported by La Ligue contre le Cancer foundation, the G.I.P. ‘Fonds de Recherche’ Hoechst Marion Roussel, the CNRS and INSERM Institutes. Jeffrey Robbins is gratefully acknowledged for the {alpha}-MHC promoter construct. We thank Jacques Barhanin for providing the minK and KvLQT1 clones, and Wayne Giles for providing the Kv1.5, Kv4.2 and Kv4.3 clones. We are grateful to Dr Jane-Lyse Samuel (INSERM U127) for her help with histological examination of transgenic mice. We also thank Patricia Charpentier, Sylvie Leroux, Marie-Joseph Louerat, Saskia Haast, Charly Betterman and Jung-Sun Kim for their technical assistance.


   Notes
 
1 S. Demolombe held a post-doctoral position at the Academic Medical Center. Back

2 Present address: Department of Medical Physiology and Sports Medicine, University of Utrecht, The Netherlands. Back


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