Involvement of IsK-Associated K⁺ Channel in Heart Rate Control of Repolarization in a Murine Engineered Model of Jervell and Lange-Nielsen Syndrome

From the Institut de Pharmacologie Moléculaire et Cellulaire (M.-D.D., I.A., C.C., M.L., G.R., J.B.), CNRS-UPR 411, Valbonne, France, and the Beckman Research Institute of the City of Hope (J.R.M.), Duarte, Calif.

Correspondence to Jacques Barhanin, Institut de Pharmacologie Moléculaire et Cellulaire, CNRS-UPR 411, 660 route des Lucioles, Sophia Antipolis, 06560 Valbonne, France. E-mail barhanin{at}unice.fr

	Abstract

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Abstract—The Jervell and Lange-Nielsen (JLN) syndrome affects the human cardioauditory system, associating a profound bilateral deafness with an abnormally long QT interval on the ECG. It results from mutations in KVLQT1 and ISK genes that encode the 2 subunits forming the K⁺ channel responsible for the cardiac and inner ear slowly activating component of the delayed rectifier K⁺ current (I_Ks). A JLN mouse model that presents typical inner ear defects has been created by knocking out the isk gene (isk-/-). This study specifically reports on the cardiac phenotype counterpart, determined in the whole animal and at mRNAs and cellular levels. Surface ECG recordings of isk-/- mice showed a longer QT interval at slow heart rates, a paradoxical shorter QT interval at fast heart rates, and an overall exacerbated QT–heart rate adaptation compared with wild-type (WT) mice. A 300-ms increase in the heart rate cycle length induces a 309±21% increase in the QT duration of the WT mice versus a 500±50% in isk-/- mice (P<0.001). It is concluded that the isk gene product and/or I_Ks, when present, blunts the QT adaptation to heart rate variations and that steeper QT-RR relationships reflect a greater susceptibility to arrhythmias in patients lacking I_Ks.

Key Words: long-QT syndrome • KCNE1 • MinK • electrocardiography • sex difference

	Introduction

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Congenital LQTS and acquired LQTS are characterized by an abnormally prolonged ventricular repolarization, responsible for a polymorphic type of ventricular arrhythmia (known as torsades de pointes) that may lead to syncopes and sudden death. Two forms of congenital LQTS, RW and JLN, can be distinguished on the basis of the mode of transmission and specific symptoms. In the case of RW syndrome, the mode of transmission is dominant, with few clinical features, but cardiac.^{1 2} In the JLN syndrome, the disease is recessively transmitted and includes a profound bilateral deafness in addition to cardiac abnormalities.³ Recent information on the identity of the genes involved in both syndromes has permitted us to comprehend some of the complex gene interactions and mechanisms underlying the congenital LQTS. All the genes responsible for these syndromes identified so far are ion channel genes, including the voltage-sensitive Na⁺ channel gene SCN5A⁴ and the 3 K⁺ channel genes HERG,⁵ KVLQT1,⁶ and ISK (also called KCNE1).⁷ These latter have been shown to encode subunits of the same channel protein complex that is responsible for the slow component, I_Ks, of the cardiac delayed outward rectifier current, I_K.^{8 9} Moreover, mutations in SCN5A or HERG are associated with RW syndrome only,^{4 5} whereas KVLQT1 and ISK can be implicated in both JLN and RW syndromes, depending on the mutation they carry.^{6 7 10 11 12 13 14 15} Expression studies have shown that mutations found in RW syndrome abolish channel function but also display a dominant-negative effect by partially inactivating the normal channel subunits encoded by the WT allele in heterozygous patients.^{16 17 18} By contrast, mutations responsible for JLN syndrome have no pronounced dominant-negative effect but abolish the current in the homozygous state.^{16 17}

Transgenic and gene-targeted mice have gained great importance as models for cardiovascular congenital affections.^{19 20} In order to analyze the in vivo function of the IsK protein (also referred as minK), null mutant mice with a targeted disruption of the isk gene have been engineered. At the homozygous state, these mice present the genotypic characteristics of the ISK gene–associated form of the human cardioauditory JLN syndrome. Notably, they suffer from inner ear defects strikingly similar to those observed in JLN syndrome.^{21 22 23} As in JLN patients, the mice bear a profound bilateral deafness from birth that is shown to be due to the absence of K⁺ secretion in the endolymph.²¹ However, the cardiac phenotype is still unexplored. The goal of the present study was to determine the cardiac role of IsK in this mouse model and to evaluate its putative inference in the different cardiac parameters (ie, QT duration, QT-RR adaptation, and T-wave alternans) classically associated with LQTS. The patient's outcome is also known to be influenced by factors such as sex or bradycardia, which are explored in this model.

	Materials and Methods

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Animals
Knockout isk mice were generated by the gene-targeting methodology as previously described.²¹ The mutation has been maintained on the 129/Sv genetic background, and all the animals used in this study, isk-/- and WT, are inbred 129/Sv. Mice were maintained on sterile regular rodent chow (R03, Usine d'Alimentation Rationnelle [France]) and allowed free access to food and water in a facility (at 21±1°C with 12-hour light/dark cycles) monitored by the Institut de Pharmacologie Moléculaire et Cellulaire staff in full compliance with the French Government animal welfare policy.

Northern Blot Analysis
Total brain and heart RNAs were isolated from 3 to 6 days, 4 weeks, and 8 weeks in 129 Sv/J WT and isk-/- mice.²⁴ PolyA⁺ RNA (2 µg) was separated by electrophoresis on 1% agarose gel and transferred onto nylon membranes (Hybond N, Amersham). Blots were probed with ³²P-labeled specific cDNA fragments of the different cardiac delayed rectifier K⁺ channel subunits in Express Hyb solution (Clontech) at 60°C for 16 hours and washed stepwise to a final stringency of 0.2x SSC and 0.3% SDS at 60°C and exposed to X OMAT AR film (Kodak). Each blot was reprobed with ß-actin to control for variations in loading (not shown).

Electrocardiography
Animal Preparation
Twelve 3-week-old (9 to 11 g each) and thirty-one 12-week-old (23 to 29 g each) male and female mice were studied. For each experiment, a mouse was anesthetized with sodium pentobarbital (10 mg/kg IP for the young ones, 45 mg/kg IP for the female adults, and 55 mg/kg IP for the male adults, SANOFI-France). Surface 3-lead ECGs (bipolar leads DI, DII, and DIII) were obtained by placement of dry electrodes carefully wrapped around each of the 4 mouse limbs. The ECG channels were amplified and filtered between 0.1 and 100 Hz, and a stable signal was reliably obtained before we proceeded. Respiratory and heart rates were continuously monitored during the procedure. A warming light was used to maintain body temperature within a range of 36±1°C for prevention of hypothermia.

Measurements of QT Interval
Mice have a very fast heart rate, between 600 and 700 bpm. Bipolar electrodes were connected to an adjustable bandpass differential amplifier (ORTEC Inc). Signals were collected (bandwidth, 0.1 to 100 Hz), stored, and analyzed on a PC computer with PCLAMP software (Axon Instruments). The PR interval was measured from the beginning of the surface P wave to the beginning of the R-wave complex. The QRS was measured from the beginning of the Q wave, when it was present, or from the base of the R to the bottom of the S wave. The QT interval was calculated from the beginning of the Q wave (or from the base of the R wave if not possible) to the end of the T wave, defined as the point at which it returns to the isoelectric line.

QT-Interval Prolongation
Since the mouse heart rate is rapid, the QT intervals cannot reliably be corrected with Bazett's formula, QTc (ms)=QT/RR (s)^1/2, which is not applicable at short cycle lengths.²⁵ For each mouse, a set of 10 to 20 RR cardiac cycle length–QT-interval pairs was obtained from their ECG recordings. The QT versus RR relation was analyzed during each experiment and was best fitted with the linear regression formula QT=A(RR)+B, where QT and RR are the observed data, and A and B are the regression parameters. This formula has been shown to be optimal for describing the QT versus RR relation at steady-state conditions. Those 2 regression parameters were used to calculate the QT interval of each mouse corresponding to predetermined RR intervals of 100 and 400 ms. QT and PR intervals were calculated in isk-/- and in WT mice within a 150- to 600-bpm range of heart rates, which represents the RR-interval limits for which QT-interval measurement was actually feasible in our model.

Cardiomyocyte Culture and Electrophysiological Recordings
Primary cultures of ventricular cardiomyocytes from WT and isk-/- mice were prepared as previously described²⁶ with some modifications. Ventricles from 1- to 4-day-old mouse pups were dissected at 4°C and dissociated at room temperature for 15 minutes in 1.25 mg/mL trypsin in Joklik's MEM (M0518, Sigma) with gentle agitation. Ventricles were then digested for 10 minutes with 0.5 mg/mL collagenase (type CLSII, Worthington) under gentle agitation. This was followed by mechanical dissociation using a Pasteur pipette. Cells released in the medium were centrifuged (1000 rpm for 5 minutes), collected, and washed in Joklik's MEM. Cells obtained from 3 sequential collagenase digestions were pooled and plated in gelatin-coated Falcon culture dishes (diameter=35 mm). The culture medium was DMEM supplemented with 10% FCS, bovine insulin (10 µg/mL), bovine transferrin (10 µg/mL), 1% chick embryo extract, and 10 nmol/L dexamethasone. Cells were used after 4 days in culture.

Whole-cell transmembrane currents under voltage-clamp conditions were recorded using the patch-clamp technique.²⁷ The culture dish was placed on the stage of an inverted microscope (Axiovert 100, Zeiss). Experiments were conducted at room temperature (22°C to 25°C). Patch pipettes (2 to 6 M) were connected to the head stage of the recording apparatus (RK400, Bio-Logic). Stimulation and data acquisition and analysis were performed using PCLAMP software. The pipette solution contained (mmol/L) KCl 140, MgCl₂ 4, EGTA 1, and Na₂ATP 3. This solution was buffered at pH 7.3 with 10 mmol/L HEPES/KOH. The external solution contained (mmol/L) NaCl 30, trimethyl ammonium chloride 110, CaCl₂ 1, KCl 5, MgCl₂ 1, and glucose 2. This solution was buffered at pH 7.4 with 10 mmol/L HEPES/NaOH.

Statistical Analysis
Results are shown as mean±SEM. Continuous variables, such as slope of adaptation, QT values, and their increase from baseline, were analyzed by ANOVA (Statview 4.5 and SuperAnova 1.11, Abacus Corp) or a Mann-Whitney-Wilcoxon rank sum test, when applicable. The Bonferroni/Dunn correction was used to adjust for multiple comparisons. A value of P<0.05 was considered statistically significant.

	Results

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Northern Blot Analysis
In order to check for an eventual compensation of the isk gene knockout by a modification of the expression of other K⁺ channel subunits, the level of mRNA corresponding to the major cardiac I_Ks was analyzed at different developmental stages (Figure 1). As expected from previous work,²⁸ the level of IsK mRNA was high in neonatal hearts and decreased with age in WT hearts. At 8 weeks, the heart IsK message reached a low but still detectable level that did not change with aging (not shown). The IsK message was totally absent in null mutants for the isk gene (Figure 1). Conversely, the amount of mRNA for the other delayed rectifier subunits, including the IsK partner KvLQT1, Kv1.5 (encoding the sustained K⁺ current²⁹), and merg, the mouse counterpart of HERG, were not influenced by the absence of IsK at any age. It is particularly noteworthy that only IsK presented a strong developmental regulation. KvLQT1 expression was totally independent of that of its IsK partner, both in WT and knockout mice.

View larger version (63K): [in this window] [in a new window]   Figure 1. Northern blot analysis of the expression of major cardiac outward rectifier K+ channel subunits. Equal amounts (2 µg) of heart or brain polyA+ RNA from 1-, 4-, and 8-week-old animals were loaded in each lane. The expression of IsK and KvLQT1 was not detected in brain. Except for IsK, none of the intensities of the different bands in both tissues were influenced by the knockout.

ECG Characteristics
Forty-three animals allotted as indicated in the Table were analyzed. Three bipolar lead ECGs (DI, DII, and DIII) were recorded under anesthesia, with a good stability of the signal from all 43 mice studied. Representative traces are shown in Figure 2A and 2B. The P wave as well as the PR, QRS, and QT intervals could be reliably measured in all animals. The first recordings were performed within 10 minutes of the induction of anesthesia. At this time, considered as the initial condition, the average cycle length (RR interval) was 121±14 ms (95 to 150 ms, n=43), the P wave duration was 20±2 ms, the PR interval was 36±2 ms, and the QRS interval was 12±1 ms. The T wave was biphasic, with a rapid component and a slower one, and was more frequently positive than negative to the isoelectric line. The average QT duration on the first recording of the experiment was 75±3 ms (44 to 130 ms, n=43). During anesthesia, all animals progressively lengthened their RR intervals to an average of 247±11 ms (range, 134 to 445 ms) over a 45-minute period. PR and QT intervals also increased with a similar pattern (Figure 2A and 2B). The average QT-interval duration at the end of the experiment was of 160±9 ms (range, 63 to 303 ms). In our model, a linear relationship, QT (ms) or PR (ms)=AxRR (ms)+B, fitted best the QT-RR and the PR-RR interval relationships in 40 of 43 mice (average r²=0.94±0.06, Figure 2C and 2D), with A being the slope of the QT-RR regression and B being the QT or PR intercepts for a theoretical RR value of 0.

View larger version (23K): [in this window] [in a new window]   Figure 2. Representative lead I surface ECG recorded from a WT female mouse (A) and an isk-/- female mouse (B) by PCLAMP Software (Axon). P, Q, R, S, and T describe the P wave (atrial depolarization), the PR interval (atrioventricular conduction), QRS wave (ventricular depolarization), and the biphasic T wave (ventricular repolarization), respectively. A, The cycle length (RR) on the upper trace is 147 ms, with a PR at 37 ms and QT at 78 ms. On the lower trace, the increase in the cycle length (RR=247 ms) is accompanied by an increase in PR (48 ms) and QT (123 ms) intervals. The end of the T wave is indicated by an arrow in each trace. B, The cycle length (RR) on the upper trace is 162 ms, with a PR at 35 ms and QT at 109 ms. On the lower trace, the increase in the cycle length (RR=394 ms) is accompanied by an increase in PR (59 ms) and QT (239 ms) intervals. C and D, RR/QT (, C; , D) and RR/PR (, C; , D) relationships in WT (C) and isk-/- (D) mice. Each value of QT and PR interval was measured at different heart rates. The cycle lengths sampled during these experiments ranged from 110 to 400 ms. The linear regressions fitted best the relationship as follows: QT (ms)=0.52RR+25 (r2=0.96) and PR=0.10RR+21 (r2=0.97) for the WT mouse and QT=0.75RR-19 (r2=0.99) and PR=0.14RR+22 (r2=0.91) for the isk-/- mouse.

Gene-Related Modifications of the QT-RR Relationship
The lengthening of the QT subsequent to bradycardia was different according to the genotype of the mouse. The isk-/- mice had a greater adaptation of their QT interval to the lengthening of the RR interval than did the WT mice. The slope of the QT-RR adaptation was 0.637±0.03 in the WT mice (n=22) versus 0.795±0.03 in the isk-/- mice (n=21) (P<0.001, Table). The average QT values, determined from the regression line, were longer for WT mice (69±7 ms) compared with isk-/- mice (52±3 ms) (P<0.05) at an RR interval of 100 ms, corresponding to the normal heart mouse frequency of 600 bpm. At an RR interval of 400 ms (150 bpm), the QT value was greater in isk-/- mice (291±9 ms) than in WT mice (260±10 ms ) (P<0.05). The resulting increase of the QT interval over a 300-ms range in cycle length was 309±21% in WT mice and was significantly greater (500±50% increase) in isk-/- mice (P<0.001, Figure 3).

View larger version (28K): [in this window] [in a new window]   Figure 3. Left, Mean QT values (±SEM) determined from the regression line at RR intervals of 100 and 400 ms. Respective WT values were 69±7 and 260±10 ms (n=22) compared with respective isk-/- values of 52±3 and 291±9 ms (n=21, P<0.05 between strains in both cases). Right, Increase of the QT interval over a 300-ms range in cycle length is 309±21% in WT mice and 500±50% in isk-/- mice. *P<0.05; ***P<0.001.

Absence of Gene-Related Modifications of the PR Interval
The PR interval lengthened progressively according to the increase in the cycle length (Figure 2). The relationship was best fitted with a linear regression. No statistical difference was seen in the RR-induced adaptation of the PR interval according to the presence or absence of the isk gene (PR=0.15±0.01RR+17±3 in isk-/- mice [n=6] and PR=0.16±0.03RR+16±4 in WT mice [n=8], P=0.8). At an average measured cycle length of 249±3 ms, where the RR-QT relationships are diverging, the average PR interval was 55±1 and 54±2 ms in the isk-/- and WT mice, respectively.

Influence of Age on Gene-Related Differences in the QT-RR Relationship
The adaptation of the QT to RR intervals differed in WT and isk-/- mice. Since the level of IsK mRNA drastically decreases during development, it was important to analyze the influence of age of the animals on these parameters. The QT intervals differed with aging in WT mice (Figure 4A and 4B). Although the slopes of the QT-RR relationships were only moderately changed from young to adult stages (0.571±0.06 [n=5] versus 0.657±0.03 [n=17], P<0.15; Table), the absolute QT values were significantly shorter in young mice in the whole range of heart rates (Figure 4A). The influence of age observed in WT mice was almost abolished in the isk-/- mice, with overlapping QT-RR relationships in young and adult mice (Figure 4B; QT=0.812±0.08RR-34±12 and QT=0.786±0.035RR-25±5, respectively). This resulted in accentuated differences in the gene-dependent QT-RR adaptation in young compared with adult animals.

View larger version (39K): [in this window] [in a new window]   Figure 4. A, QT-RR adaptation relationships in young (n=5) and adult (n=17) WT mice. Young WT mice have a more blunted adaptation than their older counterparts, with an overall shorter QT. B, Overlapping QT-RR adaptation relationships in young and adult isk-/- mice , still presenting shorter QT intervals at fast heart rates and longer ones at slow heart rates. C, Slope mean±SEM of the QT-RR adaptation of young and adult male and female mice. The difference observed between adults (0.755±0.027 in females vs 0.632±0.043 in males, *P<0.05) is not present before puberty (0.703±0.098 in females vs 0.723±0.065 in males, P=0.68).

Sex Differences
The QT-RR relationship presented a higher slope in females than in males, regardless of the presence or absence of the isk gene (0.755±0.027 in females versus 0.632±0.043 in males, P<0.05; Figure 4C, left). This difference is related to the sexual maturity of the animals, since the slope of the QT-RR relationship was sex independent before puberty (0.723±0.065 in males versus 0.703±0.098 in females, P=0.88; Figure 4C, right). There was no statistically significant interaction between gene and sex (P=0.80), whereas both factors significantly influenced adaptability (gene, P<0.01; sex, P=0.02).

Isoproterenol Challenge
In order to increase their heart rate, isk-/- and WT mice were injected intraperitoneally with increasing doses of isoproterenol (20 and 200 nmol, n=5). The shortest RR intervals attained in sinus rhythm were 95±3 ms (range, 88 to 101 ms). The QT interval decreased accordingly to an average value of 54±2 ms (range, 44 to 57 ms), with no noticeable difference between groups. In both groups the T wave increased significantly in amplitude (Figure 5), and at the highest dose, a T-wave alternans phenomenon developed regardless of the gene status of the mice (2 of 3 WT mice and 1 of 2 isk-/- mice, Figure 5).

View larger version (17K): [in this window] [in a new window]   Figure 5. Lead III surface ECG, recorded from one isk-/- (upper trace) and one WT (lower trace) mouse, 5 minutes after an intraperitoneal injection of 200 nmol isoproterenol. The T waves become prominent in both cases, as isoproterenol induces a T-wave alternans phenomenon in both strains.

K⁺ Current Recordings in Cultured Cardiomyocytes
In order to apprehend which cellular events could be involved in the ECG changes observed in isk-/- mice, K⁺ currents in cultured ventricular myocytes from both WT and isk-/- mutant mice were analyzed. Under voltage-clamp conditions, I_Ks were present in both types of cells. Figure 6 (panels A and B, upper traces) shows representative K⁺ currents in response to depolarizing voltage pulses from a holding potential of -80 mV. Typical slow tail currents were elicited on repolarizations to -40 mV. I_Kr was the dominant component of I_K that was present in both WT and isk-/- mutant cells. This current was identified by its sensitivity to the specific blocker E-4031³⁰ (Figure 6, panels A and B, lower traces) and by its bell-shaped current-voltage relationship (Figure 6C). The I_Ks component of I_K could be detected after I_Kr blockade by E-4031, essentially by its remaining slow tail current and its noninactivating time-dependent current at membrane potentials positive to 0 mV (Figure 6C). However, even if the cells analyzed originated from neonates, I_Ks could only be recorded in a mere 10% of the WT cells (7 of 60). Conversely, none of the mutant cells (0 of 55) exhibited this current. The E-4031–sensitive current was not significantly different according to the gene status. Because of numerous studies that implicate a contributing role for IsK to I_Ks^{8 9} and I_Kr,^{31 32} the present study was limited to these currents.

View larger version (21K): [in this window] [in a new window]   Figure 6. IK present in cultured ventricular heart cells. Representative recordings were obtained in a WT cell (A) and in an isk-/- cell (B) in control conditions (upper traces) and after superfusion with 5 µmol/L E-4031(lower traces). IKs were superimposed in response to voltage pulses from -40 to +40 mV in 20-mV steps from a holding potential of -80 mV. Tail currents were elicited on repolarization to -40 mV. In panel C, and indicate the current-voltage (I-V) relationships of the E-4031–sensitive currents (obtained after subtraction) generated by experiments in panels A () and B (). and indicate corresponding I-V curves of the current remaining at the end of the depolarizing pulse, after E-4031. Ten percent of the WT cells expressed an E-4031–insensitive IKs-like current with slow deactivating tails as in panel A. None of the isk-/- cells displayed such a current.

	Discussion

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The cardiac phenotype of the mice has been thoroughly investigated. In contrast to what has been previously stated,^{33 34} we found that the QT duration in mice not only varies with heart rate but that it does so with a strict linearity over a wide range of heart rates. Such a linearity has been reported in humans when the heart rate tends to a steady state,³⁵ contrasting with the usual nonlinear QT-RR relationship observed in humans, rabbits, and guinea pigs under non–steady-state conditions.^{25 33} This is the case in the present study, with a steady anesthesia-induced lengthening of the RR interval and a beat-to-beat variability rarely exceeding a few milliseconds (not shown). Therefore, no correction of the QT values for RR intervals was necessary, avoiding correction bias.^{25 36}

The most important result is that when IsK is present, the QT adaptation to heart rate variations is blunted in WT mice compared with isk-/- mice. The knockout mice showed a larger lengthening of their cardiac repolarization on the decrease of the heart beat frequency. In fact, compared with WT mice, isk-/- mice have a longer QT interval in bradycardic conditions (by 31 ms at 150 bpm, P<0.05) and a shorter QT interval at fast heart rates (Figure 2). Such results raise several hypotheses. In bradycardic conditions, it is likely that I_Ks slowly develops during the time course of the action potential in WT mice.^{28 37} At slow heart rates, the action potential gets longer, allowing I_Ks to reach a higher level, thus limiting the increase of the APD.³⁸ As in patients suffering from LQTS resulting from ISK mutation,^{7 14 15 39} the absence of I_Ks in isk-/- mice may result in a longer QT interval at slow heart rates. At fast heart rates, the shorter QT intervals observed in isk-/- mice are more intriguing. The classic role attributed to I_Ks in shortening the APD (due to its open state accumulation at fast rates)⁴⁰ does not seem to hold in our model. This finding may be relevant to the following: (1) The gene invalidated in the present mouse model is isk, which encodes the regulatory subunit, and not kvlqt1, which is responsible for the pore-forming subunit of the channel complex. When these conditions are reproduced in COS cells transfected with KvLQT1 alone, a rapidly activating small-amplitude K⁺ current is obtained. The presence of such a current in the isk-/- mice could shorten the APD at fast heart rates. The fact that no KvLQT1 current was detected in either cultivated cardiomyocytes (Figure 6) or in the inner ear stria vascularis epithelium²¹ still does not invalidate such a hypothesis. The membrane resistance during the plateau phase of the action potential is rather high, and it is conceivable that a very small outward current (not detectable under our experimental conditions) could have a marked effect on the APD. Creation of mice with a knockout of the kvlqt1 gene instead of isk could help to verify this hypothesis. However, the human JLN syndrome was recently shown to result from mutations in either the ISK or the KVLQT1 gene, with no distinguishable clinical difference so far.^{14 15 39} (2) A modification of other currents involved in cardiac repolarization, such as I_Kur, the rapid sustained outward current, I_to, the transient outward current, or I_Kr, some of which having been previously linked to the isk gene,^{31 32} could occur. However, according to this hypothesis, the lack of IsK would diminish I_Kr even further or any other current that has a possible positive interaction with IsK, therefore tending to a longer QT at fast heart rates. (3) Compensation by overexpression of rapidly activating channels like I_Kr secondary to the isk gene knockout could contribute to a shortening of the QT at fast heart rates. (4) A dysregulation of the autonomic nervous system leading to an excessive QT shortening cannot be eliminated, given the beneficial effects of ß-blockers or left stellectomy in human patients with LQTS.^{41 42}

When cardiac parameters are compared at different developmental stages, it is found that young WT mice have shorter QT intervals than do the adults. This makes sense, considering that the amplitude of I_Ks is related to the amount of IsK⁴⁰ and that IsK is more heavily expressed in young hearts (Figure 1). The lack of difference between the 2 ages observed in isk-/- mice is in good agreement with this interpretation. In a way, young isk-/- hearts look like adult ones with regard to the QT-RR relationship.

A sex difference affects the outcome of both acquired and congenital LQTS, with more cardiac events in women than in men, especially after puberty.^{43 44 45} In fact, females are known to have longer QT interval values than males in several mammalian species.^{46 47} The mouse complies with this rule. An obvious sex difference has been observed in adult mice in the present study (Figure 4C). Moreover, this difference is lacking in sexually immature young mice. It was of interest to investigate the inference of the isk gene on the sex difference. Although sex difference has been attributed to differences in K⁺ currents through genomic and nongenomic effects of sex steroid hormones,^{47 48} no significant interaction between gene and sex could be supported by the present study.

Among several abnormalities in membrane ion currents accounting for the T-wave alternans phenomenon, I_Ks was a relevant candidate at fast heart rates, because of its peculiar slow deactivation.⁴⁹ The fact that T-wave alternans occurs regardless of the gene status renders the involvement of the KvLQT1/IsK current unlikely.

The present study clearly shows that the invalidation of the isk gene does cause alterations of the functional properties of the heart. In this study, I_Ks could be recorded only in cells originating from WT mice and in a small proportion of the cells analyzed. Conversely, the E- 4031–sensitive current was consistently recorded in all cells, regardless of the isk gene status. This first study was limited to I_Kr and I_Ks, since too extensive an analysis would be required to assess changes in other currents or at other developmental stages, possibly accounting for the ECG changes. However, no compensatory process resulting from the isk gene invalidation could be assessed by Northern blot analysis of heart transcripts of major K⁺ channel subunits.

Which lessons do we gain from this mouse model? Although one must remain cautious, it appears that the change in QT-RR adaptability, which has drawn much less attention than the QT duration itself, is cardinal to the disease. Torsades de pointes ventricular arrhythmias are favored by a slow heart rate in humans. The proposed underlying mechanism is the triggering of oscillations known as early afterdepolarizations that interrupt the normal repolarizing time course of the APD, especially at slow heart rates.^{49 50} The lack of I_Ks may facilitate the occurrence of early afterdepolarizations in 2 ways: (1) by delaying the repolarization phase and lengthening the action potential first, enabling inward currents to reactivate,⁵¹ and (2) by opposing weakened outward conductances on the emergence of such depolarizations. Furthermore, the onset of torsades de pointes is constantly preceded by a sudden increase in the RR interval with an abnormally prolonged QT interval.^{41 50} Therefore, it is likely that LQTS patients are prone to the occurrence of such arrhythmias through an instantaneous greater adaptability of their QT interval to their heart rate. Indeed, LQTS patients have previously been shown to have a greater adaptability of both monophasic APD and QT intervals to their heart rate, at rest and during exercise.^{35 52 53 54} The isk-/- mouse clearly is a relevant model for the JLN syndrome. The enhanced adaptability of the QT interval to the heart rate appears therefore to be a valuable criterion identifying patients at risk in an otherwise asymptomatic population of mutation carriers among relatives in RW families.

	Selected Abbreviations and Acronyms

 

APD = action potential duration IK = delayed rectifier K+ current IKr = rapidly activating component of IK IKs = slowly activating component of IK JLN = Jervell and Lange-Nielsen LQTS = long-QT syndrome RW = Romano-Ward WT = wild-type

	Acknowledgments

 
This study was supported by the Center National de la Recherche Scientifique (CNRS) and the Association Française contre les Myopathies (AFM). We gratefully thank Dr André Varenne for helpful discussions. Thanks are due to Franck Aguila, Jean-Daniel Barde, Martine Jodar, Maud Larroque, and Dahvya Doume for technical assistance.

Received February 19, 1998; accepted May 14, 1998.

	References

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