Download Rapid Communications - Circulation Research

Rapid Communications
Involvement of IsK-Associated K1 Channel in Heart Rate
Control of Repolarization in a Murine Engineered Model of
Jervell and Lange-Nielsen Syndrome
Milou-Daniel Drici, Isabelle Arrighi, Christophe Chouabe, Jeffrey R. Mann, Michel Lazdunski,
Georges Romey, Jacques Barhanin
Downloaded from http://circres.ahajournals.org/ by guest on June 11, 2017
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 K1 channel responsible for the cardiac and inner ear slowly activating component
of the delayed rectifier K1 current (IKs). A JLN mouse model that presents typical inner ear defects has been created by
knocking out the isk gene (isk2/2). This study specifically reports on the cardiac phenotype counterpart, determined
in the whole animal and at mRNAs and cellular levels. Surface ECG recordings of isk2/2 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
309621% increase in the QT duration of the WT mice versus a 500650% in isk2/2 mice (P,0.001). It is concluded
that the isk gene product and/or IKs, 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 IKs. (Circ Res. 1998;83:95-102.)
Key Words: long-QT syndrome n KCNE1 n MinK n electrocardiography n sex difference
C
function but also display a dominant-negative effect by
partially inactivating the normal channel subunits encoded by
the WT allele in heterozygous patients.16 –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.21–23 As in JLN patients, the mice bear a
profound bilateral deafness from birth that is shown to be
due to the absence of K1 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.
ongenital 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 Na1 channel gene SCN5A4 and the 3 K1 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, IKs, of the
cardiac delayed outward rectifier current, IK.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 –15 Expression studies have shown
that mutations found in RW syndrome abolish channel
Received February 19, 1998; accepted May 14, 1998.
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 [email protected]
© 1998 American Heart Association, Inc.
95
96
QT–Heart Rate Adaptation in isk Knockout Mice
Selected Abbreviations and Acronyms
APD 5 action potential duration
IK 5 delayed rectifier K1 current
IKr 5 rapidly activating component of IK
IKs 5 slowly activating component of IK
JLN 5 Jervell and Lange-Nielsen
LQTS 5 long-QT syndrome
RW 5 Romano-Ward
WT 5 wild-type
Materials and Methods
Animals
Downloaded from http://circres.ahajournals.org/ by guest on June 11, 2017
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, isk2/2 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
2161°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 isk2/2 mice.24 PolyA1 RNA (2
mg) 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 K1 channel subunits in Express
Hyb solution (Clontech) at 60°C for 16 hours and washed stepwise
to a final stringency of 0.23 SSC and 0.3% SDS at 60°C and
exposed to X OMAT AR film (Kodak). Each blot was reprobed with
b-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 3661°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)5QT/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 QT5A(RR)1B, 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 isk2/2 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
isk2/2 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
Figure 1. Northern blot analysis of the
expression of major cardiac outward rectifier K1 channel subunits. Equal
amounts (2 mg) of heart or brain polyA1
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.
Drici et al
July 13, 1998
97
Comparison of the QT-RR Adaptation Slopes in WT and
Knockout Mice
Slope of the QT-RR Adaptation
isk2/2
WT
Mice
n
Mean6SE
n
Mean6SE
P Value
Total
22
0.63760.03
21
0.79560.03
0.0006
Young
5
0.57160.06
7
0.81260.08
0.0431
Adult
17
0.65760.03
14
0.78660.04
0.0072
Male
8
0.58860.03
7
0.76260.05
0.0095
14
0.67360.03
14
0.81160.05
0.0244
Female
Downloaded from http://circres.ahajournals.org/ by guest on June 11, 2017
from 3 sequential collagenase digestions were pooled and plated in
gelatin-coated Falcon culture dishes (diameter535 mm). The culture
medium was DMEM supplemented with 10% FCS, bovine insulin
(10 mg/mL), bovine transferrin (10 mg/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 MV) 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 mean6SEM. 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
Northern Blot Analysis
In order to check for an eventual compensation of the isk gene
knockout by a modification of the expression of other K1
channel subunits, the level of mRNA corresponding to the
major cardiac IKs 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 K1
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.
Figure 2. Representative lead I surface ECG recorded from a
WT female mouse (A) and an isk2/2 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 (RR5247 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 (RR5394
ms) is accompanied by an increase in PR (59 ms) and QT (239
ms) intervals. C and D, RR/QT (F, C; E, D) and RR/PR (f, C; M,
D) relationships in WT (C) and isk2/2 (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)50.52RR125 (r 250.96) and PR50.10RR121
(r 250.97) for the WT mouse and QT50.75RR219 (r 250.99) and
PR50.14RR122 (r 250.91) for the isk2/2 mouse.
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
121614 ms (95 to 150 ms, n543), the P wave duration was
2062 ms, the PR interval was 3662 ms, and the QRS interval
was 1261 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 7563
ms (44 to 130 ms, n543). During anesthesia, all animals
98
QT–Heart Rate Adaptation in isk Knockout Mice
Figure 3. Left, Mean QT values (6SEM) determined from the
regression line at RR intervals of 100 and 400 ms. Respective
WT values were 6967 and 260610 ms (n522) compared with
respective isk2/2 values of 5263 and 29169 ms (n521,
P,0.05 between strains in both cases). Right, Increase of the
QT interval over a 300-ms range in cycle length is 309621% in
WT mice and 500650% in isk2/2 mice. *P,0.05; ***P,0.001.
Downloaded from http://circres.ahajournals.org/ by guest on June 11, 2017
progressively lengthened their RR intervals to an average of
247611 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 16069 ms (range, 63 to 303
ms). In our model, a linear relationship, QT (ms) or PR
(ms)5A3RR (ms)1B, fitted best the QT-RR and the PR-RR
interval relationships in 40 of 43 mice (average
r250.9460.06, 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.
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 isk2/2
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 0.63760.03 in the WT
mice (n522) versus 0.79560.03 in the isk2/2 mice (n521)
(P,0.001, Table). The average QT values, determined from
the regression line, were longer for WT mice (6967 ms)
compared with isk2/2 mice (5263 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 isk2/2 mice (29169 ms)
than in WT mice (260610 ms ) (P,0.05). The resulting
increase of the QT interval over a 300-ms range in cycle
length was 309621% in WT mice and was significantly
greater (500650% increase) in isk2/2 mice (P,0.001,
Figure 3).
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
(PR50.1560.01RR11763 in isk2/2 mice [n56] and
PR50.1660.03RR11664 in WT mice [n58], P50.8). At
Figure 4. A, QT-RR adaptation relationships in young (n55) and
adult (n517) 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 isk2/2 mice , still presenting shorter QT intervals at
fast heart rates and longer ones at slow heart rates. C, Slope
mean6SEM of the QT-RR adaptation of young and adult male
and female mice. The difference observed between adults
(0.75560.027 in females vs 0.63260.043 in males, *P,0.05) is
not present before puberty (0.70360.098 in females vs
0.72360.065 in males, P50.68).
an average measured cycle length of 24963 ms, where the
RR-QT relationships are diverging, the average PR interval
was 5561 and 5462 ms in the isk2/2 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
isk2/2 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 (0.57160.06 [n55] versus
0.65760.03 [n517], 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 isk2/2 mice, with overlapping QT-RR relationships in young and adult mice (Figure 4B;
QT50.81260.08RR234612 and QT50.78660.035RR22565,
respectively). This resulted in accentuated differences in the
gene-dependent QT-RR adaptation in young compared with
adult animals.
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 (0.75560.027 in females versus 0.63260.043 in males,
Drici et al
Figure 5. Lead III surface ECG, recorded from one isk2/2
(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.
Downloaded from http://circres.ahajournals.org/ by guest on June 11, 2017
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
(0.72360.065 in males versus 0.70360.098 in females,
P50.88; Figure 4C, right). There was no statistically significant interaction between gene and sex (P50.80), whereas
both factors significantly influenced adaptability (gene,
P,0.01; sex, P50.02).
Isoproterenol Challenge
In order to increase their heart rate, isk2/2 and WT mice
were injected intraperitoneally with increasing doses of isoproterenol (20 and 200 nmol, n55). The shortest RR intervals
attained in sinus rhythm were 9563 ms (range, 88 to 101 ms).
The QT interval decreased accordingly to an average value of
5462 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 isk2/2
mice, Figure 5).
K1 Current Recordings in
Cultured Cardiomyocytes
In order to apprehend which cellular events could be involved
in the ECG changes observed in isk2/2 mice, K1 currents in
cultured ventricular myocytes from both WT and isk2/2
mutant mice were analyzed. Under voltage-clamp conditions,
IKs were present in both types of cells. Figure 6 (panels A and
B, upper traces) shows representative K1 currents in response
to depolarizing voltage pulses from a holding potential of
280 mV. Typical slow tail currents were elicited on repolarizations to 240 mV. IKr was the dominant component of IK
that was present in both WT and isk2/2 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 IKs
component of IK could be detected after IKr blockade by
E-4031, essentially by its remaining slow tail current and its
noninactivating time-dependent current at membrane poten-
July 13, 1998
99
Figure 6. IK present in cultured ventricular heart cells. Representative recordings were obtained in a WT cell (A) and in an
isk2/2 cell (B) in control conditions (upper traces) and after
superfusion with 5 mmol/L E-4031(lower traces). IKs were superimposed in response to voltage pulses from 240 to 140 mV in
20-mV steps from a holding potential of 280 mV. Tail currents
were elicited on repolarization to 240 mV. In panel C, f and M
indicate the current-voltage (I-V) relationships of the E-4031–
sensitive currents (obtained after subtraction) generated by
experiments in panels A (f) and B (M). F and E 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 isk2/2 cells displayed
such a current.
tials positive to 0 mV (Figure 6C). However, even if the cells
analyzed originated from neonates, IKs 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 IKs8,9 and IKr,31,32 the
present study was limited to these currents.
Discussion
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 isk2/2 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, isk2/2 mice have a longer QT interval in
bradycardic conditions (by 31 ms at 150 bpm, P,0.05) and a
100
QT–Heart Rate Adaptation in isk Knockout Mice
Downloaded from http://circres.ahajournals.org/ by guest on June 11, 2017
shorter QT interval at fast heart rates (Figure 2). Such results
raise several hypotheses. In bradycardic conditions, it is
likely that IKs 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 IKs 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 IKs in isk2/2 mice may result in a longer QT
interval at slow heart rates. At fast heart rates, the shorter QT
intervals observed in isk2/2 mice are more intriguing. The
classic role attributed to IKs 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 smallamplitude K1 current is obtained. The presence of such a
current in the isk2/2 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 IKur, the rapid sustained outward current, Ito, the
transient outward current, or IKr, 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
IKr 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 IKr 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 b-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 IKs 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 isk2/2 mice is in good agreement with this
interpretation. In a way, young isk2/2 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– 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
K1 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, IKs 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, IKs 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 IKr and IKs, 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 K1 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 IKs 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.35,52–54 The isk2/2 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.
Acknowledgments
This study was supported by the Center National de la Recherche
Scientifique (CNRS) and the Association Française contre les
Drici et al
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.
References
Downloaded from http://circres.ahajournals.org/ by guest on June 11, 2017
1. Romano C. Congenital cardiac arrhythmia [letter]. Lancet. 1965;1:
658 – 659.
2. Ward OC. A new familial cardiac syndrome in children. J Irish Med
Assoc. 1964;54:103–106.
3. Jervell A, Lange-Nielsen F. Congenital deaf-mutism, functional heart
disease with prolongation of Q-T interval and sudden death. Am Heart J.
1957;54:59 – 68.
4. Wang Q, Shen JX, Splawski I, Atkinson D, Li ZZ, Robinson JL, Moss AJ,
Towbin JA, Keating MT. SCN5A mutations associated with an inherited
cardiac arrhythmia, long QT syndrome. Cell. 1995;80:805– 811.
5. Curran ME, Splawski I, Timothy KW, Vincent GM, Green ED, Keating
MT. A molecular basis for cardiac arrhythmia: HERG mutations cause
long QT syndrome. Cell. 1995;80:795– 803.
6. Wang Q, Curran ME, Splawski I, Burn TC, Millholland JM, Vanraay TJ,
Shen J, Timothy KW, Vincent GM, Dejager T, Schwartz PJ, Towbin JA,
Moss AJ, Atkinson D L, Landes GM, Connors TD, Keating MT. Positional cloning of a novel potassium channel gene: KVLQT1 mutations
cause cardiac arrhythmias. Nat Genet. 1996;12:17–23.
7. Splawski I, Tristani-Firouzi M, Lehmannn MH, Sanguinetti MC, Keating
MT. Mutations in hminK gene cause long-QT syndrome and suppress IKs
function. Nat Genet. 1997;17:338 –340.
8. Barhanin J, Lesage F, Guillemare E, Fink M, Lazdunski M, Romey G.
K(v)LQT1 and IsK (minK) proteins associate to form the I-Ks cardiac
potassium current. Nature. 1996;384:78 – 80.
9. Sanguinetti MC, Curran ME, Zou A, Shen J, Spector PS, Atkinson DL,
Keating MT. Coassembly of K(v)LQT1 and MinK (IsK) proteins to form
cardiac I-Ks potassium channel. Nature. 1996;384:80 – 83.
10. Neyroud N, Tesson F, Denjoy I, Leibovici M, Donger C, Barhanin J,
Faure S, Gary F, Coumel P, Petit C, Schwartz K, Guicheney P. A novel
mutation in the potassium channel gene KVLQT1 causes the Jervell and
Lange-Nielsen cardioauditory syndrome. Nat Genet. 1997;15:186 –189.
11. Donger C, Denjoy I, Berthet M, Neyroud N, Cruaud C, Bennaceur M,
Chivoret G, Schwartz K, Coumel P, Guicheney P. KVLQT1 C-terminal
missense mutation causes a forme fruste long-QT syndrome. Circulation.
1997;96:2778 –2781.
12. Neyroud N, Denjoy I, Donger C, Gary F, Villain E, Leenhardt A, Benali
K, Schwartz K, Coumel P, Guicheney P. A heterozygous mutation in the
pore of the potassium channel gene KvLQT1 causes an apparently normal
phenotype in long QT syndrome. Eur J Hum Genet. 1998;6:129 –133.
13. Splawski I, Timothy KW, Vincent GM, Atkinson DL, Keating MT.
Molecular basis of the long-QT syndrome associated with deafness.
N Engl J Med. 1997;336:1562–1567.
14. Schulze-Bahr E, Wang Q, Wedekind H, Haverkamp W, Chen Q, Sun Y.
KCNE1 mutations cause Jervell and Lange-Nielsen syndrome. Nat Genet.
1997;17:267–268.
15. Duggal P, Vesely MR, Wattanasirichaigoon D, Villafane J, Kaushik V,
Beggs AH. Mutation of the gene for IsK associated with both Jervell and
Lange-Nielsen and Romano-Ward forms of long-QT syndrome. Circulation. 1998;97:142–146.
16. Chouabe C, Neyroud N, Guicheney P, Lazdunski M, Romey G, Barhanin
J. Properties of KvLQT1 K1 channel mutations in Romano-Ward and
Jervell and Lange-Nielsen inherited cardiac arrhythmias. EMBO J. 1997;
16:5472–5479.
17. Wollnik B, Schroeder BC, Kubisch C, Esperer HD, Wieacker P, Jentsch
TJ. Pathophysiological mechanisms of dominant and recessive KVLQT1
K1 channel mutations found in inherited cardiac arrhythmias. Hum Mol
Genet. 1997;6:1943–1949.
18. Shalaby FY, Levesque PC, Yang W-P, Little WA, Conder ML,
Jenkins-West T, Blanar MA. Dominant-negative KvLQT1 mutations
underlie the LQT1 form of long QT syndrome. Circulation. 1997;96:
1733–1736.
19. Paigen K. A miracle enough: the power of mice. Nat Med. 1995;1:
215–220.
20. Lin MC, Rockman HA, Chien KR. Heart and lung disease in engineered
mice. Nat Med. 1995;1:749 –751.
21. Vetter DE, Mann JR, Wangemann P, Liu JZ, McLaughlin KJ, Lesage F,
Marcus D C, Lazdunski M, Heinemann SF, Barhanin J. Inner ear defects
induced by null mutation of the IsK gene. Neuron. 1996;17:1251–1264.
July 13, 1998
101
22. Friedmann I, Fraser GR, Froggatt P. Pathology of the ear in the
cardioauditory syndrome of Jervell and Lange-Nielsen (recessive
deafness with electrocardiographic abnormalities). J Laryngol Otol.
1966;80:451– 470.
23. Friedmann I, Fraser GR, Froggatt P. Pathology of the ear in the cardioauditory syndrome of Jervell and Lange-Nielsen: report of a third case
with an appendix on possible linkage with the Rh blood group locus. J
Laryngol Otol. 1968;82:883– 896.
24. Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid
guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem.
1987;162:156 –159.
25. Funck-Brentano C, Jaillon P. Rate-corrected QT interval: techniques and
limitations. Am J Cardiol. 1993;72: 17B–22B.
26. Steinhelper ME, Lanson N Jr, Dresdner KP, Delcarpio JB, Wit AL,
Claycomb WC, Field LJ. Proliferation in vivo and in culture of differentiated adult atrial cardiomyocytes from transgenic mice. Am J Physiol.
1990;259:H1826 –H1834.
27. Hamill OP, Marty A, Neher E, Sakmann B, Sigworth FJ. Improved
patch-clamp techniques for high resolution current recording from cells
and cell-free membranes patches. Pflugers Arch. 1981;391:85–100.
28. Honoré E, Attali B, Romey G, Heurteaux C, Ricard P, Lesage F,
Lazdunski M, Barhanin J. Cloning, expression, pharmacology and regulation of a delayed rectifier K1 channel in mouse heart. EMBO J. 1991;
10:2805–2811.
29. Deal KK, England SK, Tamkun MM. Molecular physiology of cardiac
potassium channels. Physiol Rev. 1996;76:49 – 67.
30. Spector PS, Curran ME, Keating MT, Sanguinetti MC. Class III antiarrhythmic drugs block HERG, a human cardiac delayed rectifier K1
channel: open-channel block by methanesulfonanilides. Circ Res. 1996;
78:499 –503.
31. McDonald TV, Yu ZH, Ming Z, Palma E, Meyers MB, Wang KW,
Goldstein SA N, Fishman GI. A minK-HERG complex regulates the
cardiac potassium current I-Kr. Nature. 1997;388:289 –292.
32. Yang T, Kupershmidt S, Roden DM. Anti-minK antisense decreases the
amplitude of the rapidly activating cardiac delayed rectifier K1 current.
Circ Res. 1995;77:1246 –1253.
33. Hayes E, Pugsley MK, Penz WP, Adaikan G, Walker MJ. Relationship
between QaT and RR intervals in rats, guinea pigs, rabbits, and primates.
J Pharmacol Toxicol Methods. 1994;32:201–207.
34. Manoach M, Fein A, Hecht Z, Varon D. A cellular basis for the prolonged
QT interval in mammals. Ann N Y Acad Sci. 1992;644:84 –92.
35. Neyroud N, Maison-Blanche P, Denjoy I, Chevret S, Donger C, Dausse
E, Fayn J, Badilini F, Menhabi N, Schwartz K, Guicheney P, Coumel P.
Diagnostic performance of QT interval variables from 24-h electrocardiography in the long QT syndrome. Eur Heart J. 1998;19:158 –165.
36. Franz MR. Time for yet another QT correction algorithm?: Bazett and
beyond [editorial comment]. J Am Coll Cardiol. 1994;23:1554 –1556.
37. Sanguinetti MC, Jurkiewicz NK. Two components of cardiac delayed
rectifier K1 current: differential sensitivity to block by class-III antiarrhythmic agents. J Gen Physiol. 1990;96:195–215.
38. Hauswirth O, Noble D, Tsien RW. Separation of the pace-maker and
plateau components of delayed rectification in cardiac Purkinje fibres.
J Physiol (Lond). 1972;225:211–235.
39. Tyson J, Tranebjærg L, Bellman S, Wren C, Taylor J, Bathen J, Aslaksen
B, Sørland SJ, Lund O, Malcolm S, Pembrey M, Bhattacharya S, BitnerGlindzicz M. IsK and KvLQT1: mutation in either of the two subunits of
the slow component of the delayed rectifier potassium channel can cause
Jervell and Lange-Nielsen syndrome. Hum Mol Genet. 1997;6:2179 –
2185.
40. Romey G, Attali B, Chouabe C, Abitbol I, Guillemare E, Barhanin J,
Lazdunski M. Molecular mechanism and functional significance of the
MinK control of the KvLQT1 channel activity. J Biol Chem. 1997;272:
16713–16716.
41. Napolitano C, Priori SG, Schwartz PJ. Torsade de pointes: mechanisms
and management. Drugs. 1994;47:51– 65.
42. Schwartz PJ, Locati EH, Moss AJ, Crampton RS, Trazzi R, Ruberti U. Left
cardiac sympathetic denervation in the therapy of congenital long QT syndrome: a worldwide report [comments]. Circulation. 1991;84:503–511.
43. Hashiba K. Sex differences in phenotypic manifestation and gene transmission in the Romano-Ward syndrome. Ann N Y Acad Sci. 1992;644:
142–156.
44. Makkar RR, Fromm BS, Steinman RT, Meissner MD, Lehmann MH.
Female gender as a risk factor for torsades de pointes associated with
cardiovascular drugs. JAMA. 1993;270:2590 –2597.
102
QT–Heart Rate Adaptation in isk Knockout Mice
45. Lehmann MH, Timothy KW, Frankovich D, Fromm BS, Keating M,
Locati EH, Taggart RT, Towbin JA, Moss AJ, Schwartz PJ, Vincent GM.
Age-gender influence on the rate-corrected QT interval and the QT-heart
rate relation in families with genotypically characterized long QT
syndrome. J Am Coll Cardiol. 1997;29:93–99.
46. Bazett H. An analysis of the time relationship of electrocardiograms.
Heart. 1920;7:353–370.
47. Drici MD, Burklow TR, Haridasse V, Glazer RI, Woosley RL. Sex
hormones prolong the QT interval and downregulate potassium channel
expression in the rabbit heart. Circulation. 1996;94:1471–1474.
48. Busch AE, Busch GL, Ford E, Suessbrich H, Lang HJ, Greger R,
Kunzelmann K, Attali B, Stuhmer W. The role of the I-sK protein in the
specific pharmacological properties of the I-Ks channel complex. Br J
Pharmacol. 1997;122:187–189.
49. Roden DM, Lazzara R, Rosen M, Schwartz PJ, Towbin J, Vincent GM.
Multiple mechanisms in the long-QT syndrome: current knowledge, gaps,
50.
51.
52.
53.
54.
and future directions: the SADS Foundation Task Force on LQTS. Circulation. 1996;94:1996 –2012.
el-Sherif N, Bekheit SS, Henkin R. Quinidine-induced long QTU interval
and torsade de pointes: role of bradycardia-dependent early afterdepolarizations. J Am Coll Cardiol. 1989;14:252–257.
Zeng J, Rudy Y. Early afterdepolarizations in cardiac myocytes:
mechanism and rate dependence. Biophys J. 1995;68:949 –964.
Hirao H, Shimizu W, Kurita T, Suyama K, Aihara N, Kamakura S,
Shimomura K. Frequency-dependent electrophysiologic properties of
ventricular repolarization in patients with congenital long QT syndrome.
J Am Coll Cardiol. 1996;28:1269 –1277.
Krahn AD, Klein GJ, Yee R. Hysteresis of the RT interval with exercise: a
new marker for the long- QT syndrome? Circulation. 1997;96:1551–1556.
Vincent GM, Jaiswal D, Timothy KW. Effects of exercise on heart rate,
QT, QTc and QT/QS2 in the Romano-Ward inherited long QT syndrome.
Am J Cardiol. 1991;68:498 –503.
Downloaded from http://circres.ahajournals.org/ by guest on June 11, 2017
Downloaded from http://circres.ahajournals.org/ by guest on June 11, 2017
Involvement of IsK-Associated K+ Channel in Heart Rate Control of Repolarization in a
Murine Engineered Model of Jervell and Lange-Nielsen Syndrome
Milou-Daniel Drici, Isabelle Arrighi, Christophe Chouabe, Jeffrey R. Mann, Michel Lazdunski,
Georges Romey and Jacques Barhanin
Circ Res. 1998;83:95-102
doi: 10.1161/01.RES.83.1.95
Circulation Research is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
Copyright © 1998 American Heart Association, Inc. All rights reserved.
Print ISSN: 0009-7330. Online ISSN: 1524-4571
The online version of this article, along with updated information and services, is located on the
World Wide Web at:
http://circres.ahajournals.org/content/83/1/95
Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published
in Circulation Research can be obtained via RightsLink, a service of the Copyright Clearance Center, not the
Editorial Office. Once the online version of the published article for which permission is being requested is
located, click Request Permissions in the middle column of the Web page under Services. Further information
about this process is available in the Permissions and Rights Question and Answer document.
Reprints: Information about reprints can be found online at:
http://www.lww.com/reprints
Subscriptions: Information about subscribing to Circulation Research is online at:
http://circres.ahajournals.org//subscriptions/