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J Physiol 591.17 (2013) pp 4189–4206 4189
The Journal of Physiology
Ionic mechanisms limiting cardiac repolarization reserve
in humans compared to dogs
Norbert Jost1,2,L
´
aszl ´
oVir
´
ag2, Philippe Comtois10,11,Bal
´
azs ¨
Ord¨
og2,9,Vikt´
oria Szuts2,Gy
¨
orgy Sepr´
enyi3,
Mikl ´
os Bitay4,Zs´
ofia Kohajda1,Istv
´
an Koncz2,NorbertNagy
1,Tam
´
as Sz´
el1,J
´
anos Magyar7,M
´
aria Kov´
acs2,
L´
aszl ´
oG.Pusk
´
as6, Csaba Lengyel2,5, Erich Wettwer8,UrsulaRavens
8,P
´
eter P. N´
an´
asi7,JuliusGy.Papp
1,2,
Andr´
as Varr ´
o1,2and Stanley Nattel9,11
1Division of Cardiovascular Pharmacology, Hungarian Academy of Sciences, Szeged, Hungary
Departments of 2Pharmacology and Pharmacotherapy, 3Medical Biology, and 4Cardiac Surgery, Faculty of Medicine, University of Szeged, Szeged,
Hungary
5First Department of Internal Medicine, Faculty of Medicine, University of Szeged, Szeged, Hungary
6Laboratory for Functional Genomics, Biological Research Center, Hungarian Academy of Sciences, Szeged, Hungary
7Department of Physiology, Faculty of Medicine, University of Debrecen, Hungary
8Department of Pharmacology and Toxicology, Dresden University of Technology, Germany
Department of 9Medicine and 10Physiology, Universit´
edeMontr
´
eal, Quebec, Canada
11Research Center, Montreal Heart Institute, Montreal, Quebec, Canada
Key points
•Cardiac repolarization, through which heart-cells return to their resting state after having fired,
is a delicate process, susceptible to disruption by common drugs and clinical conditions.
•Animal models, particularly the dog, are often used to study repolarization properties and
responses to drugs, with the assumption that such findings are relevant to humans. However,
little is known about the applicability of findings in animals to man.
•Here, we studied the contribution of various ion-currents to cardiac repolarization in canine
and human ventricle.
•Humans showed much greater repolarization-impairing effects of drugs blocking the
rapid delayed-rectifier current IKr than dogs, because of lower repolarization-reserve
contributions from two other important repolarizing currents (the inward-rectifier IK1 and
slow delayed-rectifier IKs).
•Our findings clarify differences in cardiac repolarization-processes among species, highlighting
the importance of caution when extrapolating results from animal models to man.
Abstract The species-specific determinants of repolarization are poorly understood. This
study compared the contribution of various currents to cardiac repolarization in canine and
human ventricle. Conventional microelectrode, whole-cell patch-clamp, molecular biological and
mathematical modelling techniques were used. Selective IKr block (50–100 nmol l−1dofetilide)
lengthened AP duration at 90% of repolarization (APD90)>3-fold more in human than dog,
suggesting smaller repolarization reserve in humans. Selective IK1 block (10 μmol l−1BaCl2)and
IKs block (1 μmol l−1HMR-1556) increased APD90 more in canine than human right ventricular
papillary muscle. Ion current measurements in isolated cardiomyocytes showed that IK1 and IKs
densities were 3- and 4.5-fold larger in dogs than humans, respectively. IKr density and kinetics
were similar in human versus dog. ICa and Ito were respectively ∼30% larger and ∼29% smaller
in human, and Na+–Ca2+exchange current was comparable. Cardiac mRNA levels for the main
N. Jost and L. Vir´
ag contributed equally to this work. Both are to be
considered first authors. A. Varr´
o and S. Nattel share senior authorship.
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2013 The Physiological Society DOI: 10.1113/jphysiol.2013.261198
4190 N. Jost and others J Physiol 591.17
IK1 ion channel subunit Kir2.1 and the IKs accessory subunit minK were significantly lower, but
mRNA expression of ERG and KvLQT1 (IKr and IKs α-subunits) were not significantly different,
in human versus dog. Immunostaining suggested lower Kir2.1 and minK, and higher KvLQT1
protein expression in human versus canine cardiomyocytes. IK1 and IKs inhibition increased the
APD-prolonging effect of IKr block more in dog (by 56% and 49%, respectively) than human
(34 and 16%), indicating that both currents contribute to increased repolarization reserve in
the dog. A mathematical model incorporating observed human–canine ion current differences
confirmed the role of IK1 and IKs in repolarization reserve differences. Thus, humans show greater
repolarization-delaying effects of IKr block than dogs, because of lower repolarization reserve
contributions from IK1 and IKs , emphasizing species-specific determinants of repolarization and
the limitations of animal models for human disease.
(Received 26 June 2013; accepted after revision 16 July 2013; first published online 22 July 2013)
Corresponding author A. Varr ´
o: Department of Pharmacology and Pharmacotherapy, Faculty of Medicine, University
of Szeged, H-6720 Szeged, D´
om t´
er 12, PO Box 427, Hungary. Email: varro.andras@med.u-szeged.hu
Abbreviations AP, action potential; APD, action potential duration; ICaL ,L-typeCa
2+current; IK1,inwardrectifierK
+
current; IKr, rapid delayed-rectifier K+current; IKs , slow delayed-rectifier K+current; Ito, transient-outward current;
NCX,Na
+–Ca2+exchanger current.
Introduction
Many drugs can affect transmembrane K+currents
and thereby cause therapeutically useful (Honhloser
& Woosley, 1994; Brendorp et al. 2001) or harmful
(Surawicz, 1989; El-Sherif, 1992) effects. Blocking cardiac
K+channels prolongs repolarization and refractoriness,
producing Class III antiarrhythmic effects both in
ventricles and atria (Sing & Vaughan-Williams, 1970).
Excessive lengthening of repolarization may induce
life-threatening ventricular tachyarrhythmias like torsades
de pointes (Hondeghem & Snyders, 1990; El-Sherif, 1992).
Predicting the risk of such serious side effects is a major
challenge in cardiac safety pharmacology. Torsade-risk
estimation is hampered by a lack of easily usable methods
and by incomplete understanding of the repolarization
process in both experimental animals and humans.
Repolarization is controlled by two major inward
currents (Na+and Ca2+) and four major outward
K+currents (rapid and slow delayed-rectifier (IKr and
IKs), transient-outward (Ito )andinward-rectifier(IK1)
currents), as well as other less well-characterized currents,
electrogenic pumps and exchangers (Nerbonne & Kass,
2005). According to the concept of ‘repolarization reserve’
(Roden, 1998), normal repolarization is accomplished by
multiple different potassium channels providing a strong
safety reserve for repolarization. Thus, in normal cardiac
tissue the pharmacological block or impairment of a
single type of potassium channel does not necessarily
lead to marked QT interval prolongation. However, in
situations where the density of one or more types of
potassium channels is decreased by congenital disorders or
remodelling, inhibition of other potassium channels may
lead to unexpectedly augmented action potential duration
(APD) prolongation resulting in proarrhythmic reactions.
In genetic channelopathies certain potassium channels,
which normally contribute to repolarization, can attenuate
the capability of the heart to repolarize (Biliczki et al. 2002;
Jost et al. 2005).
Transmembrane ion currents flow through channel
complexes composed of α-andβ-subunit proteins
including ERG (encoded by KCNH2 ), minK (KCNE 1),
MiRP1–4 (KCNE25), KvLQT1 (KCNQ1), Kv4.3 (KCND3 ),
Kv1.4 ( KCNA4), KChIP2 (KCNIP2) and Kir2.1–2.4
(KCN J2,KCNJ12 ,KCNJ4, KCNJ14 ). These proteins are
abundantly expressed in mammalian hearts, but their
relative contributions vary considerably among species
(Varr ´
oet al. 2000; Zicha et al. 2003). Differential K+
current expression causes interspecies differences in the
response to K+channel blocking drugs, affecting pre-
dictive value for their effects in humans (Nerbonne &
Kass, 2005). Despite the very common use of the dog in
evaluating long-QT risk in man, there is little quantitative
information available about the relative responses of
human versus canine hearts to QT-prolonging inter-
ventions or regarding underlying differences in ionic
currents. Here, we compared the contribution of three
particularly important K+currents, IKr ,IK1 and IKs,to
repolarization in dog and human hearts, studied the
molecular basis of differences observed, and analysed their
importance with a mathematical model.
Methods
For methodological details, please see Supplemental
Methods.
Ethical approval and species
Patients. Hearts were obtained from organ donors
whose non-diseased hearts were explanted to obtain
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pulmonary and aortic valves for transplant surgery.
Before cardiac explantation, organ donors did not receive
medication apart from dobutamine, furosemide, and
plasma expanders. The investigations conformed to the
principles of the Declaration of Helsinki. Experimental
protocols were approved by the University of Szeged
and National Scientific and Research Ethical Review
Boards (Nos. 51-57/1997OEj and 4991-0/2010-1018EKU
(339/PI/010)). After explantation, each heart was perfused
with cardioplegic solution (for contents see Online Data
Supplement) and kept cold (4–6◦C) for 2–4 h prior to
dissection.
Animals. All experiments complied with the Guide for
the Care and Use of Laboratory Animals (NIH publication
No 85-23, revised 1985). The protocols were approved
by the Review Board of the Department of Animal
Health and Food Control of the Ministry of Agriculture
and Rural Development, Hungary (XII./01031/000/2008
and XIII./1211/2012). Adult mongrel dogs of either sex
weighing 8–16 kg were anaesthetized with pentobarbital
(30 mg kg−1I.V.). Hearts were removed through right
lateral thoracotomies and rinsed in modified Locke’s
solution containing (mmol l−1): Na+140, K+4, Ca2+
1.0, Mg2+1.0, Cl−126, HCO3−25 and glucose 11; pH
7.35–7.45, 95% O2-5% CO2,37
◦C.
Action potential measurements
Action potentials (APs) were recorded in right ventricular
trabeculae and papillary muscle preparations (<2mm
diameter), from 15 non-diseased human donor hearts
(9 male and 6 female, age =44.6 ±5.9 years) and 25 dogs,
with conventional microelectrode techniques, as described
in detail previously (Varr ´
oet al. 2000; Biliczki et al. 2002;
Jost et al. 2005).
Transmembrane current measurements
Cell isolation. Ventricular cardiomyocytes were enzy-
matically dissociated from the left ventricular mid-
myocardial free wall of 10 additional non-diseased human
donor hearts (5 male and 5 female, age =43.4 ±5.3 years)
and 21 dog hearts with previously described procedures
(Varr ´
oet al. 2000; Biliczki et al. 2002; Jost et al. 2005).
Experimental protocol. Rod-shaped, striated cardio-
myocytes were placed in a recording chamber on the
stage of inverted microscopes Olimpus, IX51 (Olympus
Ltd, Tokyo, Japan) and Nikon TMS (Nikon Ltd, Tokyo,
Japan) and allowed to adhere. The solutions, equipment
and voltage-clamp protocols (see Supplemental Methods)
were as previously detailed for K+currents (Varr ´
oet al.
2000; Biliczki et al. 2002; Jost et al. 2005) and for L-type
Ca2+current (ICaL)andNa
+–Ca2+exchanger (NCX)
current (Hobai et al. 1997; Birinyi et al. 2005).
Molecular biology
Reverse transcription (RT) quantitative polymerase chain
reaction (qPCR). Left ventricular midmyocardial free-wall
samples were obtained from eight human (7 male and
5 female, age =45.2 ±3.7 years) and eight dog hearts,
and snap-frozen in liquid N2. RNA was isolated with
the Qiagen RNase Tissue kit (Amersham). Reverse
transcription (RT) was performed with Superscript-II
RNase H-Reverse Transcriptase (Invitrogen). QPCR
was performed on a RotorGene-3000 instrument
(Corbett Research, Australia) with gene-specific primers
(Supplemental Table 1) and SybrGreen. Expression
values were normalized to β-actin. Triplicate standard
curves were run for each experiment. Data analysis
was performed with the Pfaffl method (Pfaffl, 2001),
correcting for amplification efficiency differences.
Western blot. Membrane proteins were obtained from
the same samples used for qPCR. Samples were suspended
in lysis buffer, dounced and centrifuged (2000 ×g,10min,
4◦C). The supernatant was resuspended in lysis buffer
containing 2% Triton X-100. After 1.5 h incubation
on ice, samples were ultracentrifuged (100 000 ×g,
35 min, 4◦C), supernatants collected and stored at
−70◦C. Protein concentration was measured by the
Lowry method and samples diluted in loading buffer
for SDS–polyacrylamide gel electrophoresis. Fractionated
proteins were transferred onto polyvinylidine difluoride
(PVDF) membranes, blocked in Tris buffer supplemented
with Tween-20 (TBST) and 10% non-fat milk (BioRad,
USA), then incubated overnight (4◦C) with rabbit
polyclonal primary antibodies against Kir2.1, Kir2.2,
Kir2.3, ERG, minK and KvLQT1, goat anti-Kir2.4
(Santa Cruz Biotechnology) or mouse monoclonal
anti-α-sarcomeric actin (DAKO). Bound primary anti-
bodies were detected with anti-rabbit, anti-goat or
anti-mouse secondary antibodies conjugated to horse-
radish peroxidase. Immunoreactivity was visualized
with enhanced chemoluminescence and analysed with
ImageJTM. All values were quantified relative to internal
controls on the same samples (α-actin for Kir2.x, KvLQT1
and minK, GAPDH for ERG).
Immunohistochemistry. Isolated dog (n=6) and human
(3 male, 1 female, age =48.3 ±4.7 years) left ventricular
midmyocardial free-wall ventricular cardiomyocytes on
glass coverslips were fixed with acetone. Samples were
rehydrated with calcium-free phosphate-buffered saline
(PBS) and blocked for 2 h with PBST (PBS with
0.01% Tween) containing 1% BSA at room temperature.
Incubation with the primary polyclonal rabbit anti-
body for 1.5 h at room temperature was followed by
1 h incubation with secondary antibodies (Alexafluor
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448-conjugated goat anti-rabbit IgG). Control samples
were incubated only with secondary antibody.
Fluorescence images were obtained with an Olympus
FV1000 confocal laser-scanning microscope and
standardized parameter settings. Images were quantified
in greyscale TIFF format with ImageQuantTM software.
On each image, three to five random strips were selected
and fluorescence profiles plotted. Baseline pixels were
identified and subtracted from total profile area.
Statistics. Results are expressed as means ±SEM.
Statistical significance was determined by two-tailed
Student’s ttests and ANOVA with Bonferroni-corrected
post hoc t tests as appropriate. Results were considered
significant for P<0.05.
Results
Current densities
IK1 was recorded with 300 ms 0.33 Hz test pulses from
a holding potential of −80 mV (Fig. 1A) and quantified
based on end-pulse amplitude. IK1 was significantly larger
in dog than human cardiomyocytes (Fig. 1B). Maximum
outward current density at −60 mV was almost 3-fold
greater in dog versus human (1.72 ±0.07 pA pF−1vs.
0.65 ±0.1 pA pF−1,n=21–28, Fig. 1C).
Mean IKr and IKs data are shown in Fig. 2. IKr data
are shown in panels A–C and IKs data in panels D–F.
Examples of original IKr recordings are in the top row,
and IKs recordings in the middle row. IKr tail current at
−40 mV after 1000 ms test pulses (0.05 Hz) did not differ
significantly between species (Fig. 2C). In contrast, IKs tail
current at −40 mV after 5000 ms test pulses (0.1 Hz) was
about 4.5-fold larger in dog versus human (Fig. 2F).
To estimate the magnitude of IK1 ,IKr and IKs activated
during the cardiac action potential, we compared the
amplitudes of the BaCl2-sensitive (IK1), E-4031-sensitive
(IKr) and L-735,821-sensitive (IKs) currents during ‘action
potential’ test pulses. These test pulses were obtained
by digitizing representative right ventricular human and
canine action potentials recorded with conventional
microelectrodes (Fig. 3A). Under these conditions, the
BaCl2-sensitive IK1 difference current flowing during
the AP was substantially larger in dog than in human
(Fig. 3B), while the E-4031-sensitive IKr difference
current was similar (Fig. 3C). The L-735,821-sensitive
IKs during the action potential plateau phase was very
small and not clearly different between the two species
(Fig. 3D).
The activation and deactivation kinetics of IKr and IKs
measured at the whole range of activating and deactivating
membrane potentials are shown in Fig. 4. The IKs kinetics
of human and dog are quite similar (Fig. 4Aand B). IKr
Figure 1. Inward-rectifier potassium current (IK1) in human and dog ventricular cardiomyocytes
A, original IK1 recordings in a human (top traces) and a dog (bottom traces) ventricular myocyte. Voltage protocol
shown above traces. B, mean ±SEM IK1 density–voltage relations. C, mean ±SEM IK1 density at −60 mV (left)
and −140 mV (right) membrane potentials. ∗P<0.05, ∗∗P<0.01 dog versus human. n=number of experiments.
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J Physiol 591.17 Weak IK1,IKs limit human repolarization reserve 4193
deactivation (Fig. 4C) at voltages (−70 and −60 mV)
relevant to physiological current deactivation (i.e. near
the resting potential) consisted predominantly of a rapid
phase with a time constant of 200–400 ms, not significantly
different between human and dog. At more positive
voltages, the kinetics became more clearly biexponential.
The rapid-phase time constants were similar at all voltages
for human and dog. At voltages negative to −30 mV, the
slow-phase time constant was also similar, whereas at more
positive voltages the slow-phase time constant was greater
in dog.
Species-dependent contributions of IK1,IKr and IKs to
repolarization
The contribution of IK1,IKr and IKs to repolarization was
investigated (Fig. 5) by selectively blocking these currents
with BaCl2(10 μmol l−1), dofetilide (50 nmol l−1)and
HMR-1556 (1 μmol l−1), respectively. We previously
reported that 10 μmol l−1BaCl2blocks over 70% of IK1
without affecting IKr ,IKs and Ito (Biliczki et al. 2002). In
human ventricular muscle, selective inhibition of IK1 only
marginally prolonged AP duration (APD, by 4.8 ±1.5%),
Figure 2. IKr and IKs in human and dog ventricular cardiomyocytes
Aand B, original IKr recordings from a human (A) and a dog (B) ventricular cardiomyocyte. C, mean ±SEM IKr
tail current density–voltage relations. Dand E, original IKs recordings from a human (A) and a dog (B) ventricular
cardiomyocyte. F, mean ±SEM IKs tail current density–voltage relations. n=number of experiments. ∗P<0.05,
∗∗P<0.01 and ∗∗∗ P<0.001.
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while it caused significant APD prolongation in dog
(17.9 ±2.1%, P<0.05 vs. human, n=7–11). In contrast,
selective inhibition of IKr caused markedly greater APD
prolongation in humans (56.3 ±8.4%) compared to the
dog (21.7 ±2.5%, P<0.05, n=17–20). The differential
response was due to differences in maximal effects and not
drug sensitivity per se, as shown by similar dofetilide IC50
values between species (Supplemental Fig. 1). IKs block
did not significantly alter APD in either studied species.
Contributions to repolarization reserve
We then studied the role of IK1 and IKs differences
in contributing to the larger APD increases produced
by IKr block in human versus canine cardiomyocytes.
Tissues were exposed to dofetilide in the absence or pre-
sence of 10 μmol l−1BaCl2to inhibit IK1 (Fig. 6A)or
HMR-1566 to block IKs (Fig. 6B). The change in APD
(relative to BaCl2-free control) caused by dofetilide alone
indicates the effect of the drug with repolarization reserve
intact, whereas the change caused in the presence of
BaCl2(dofetilide +BaCl2vs. BaCl2alone) indicates the
effect with IK1 suppressed, i.e. the contribution of IK1 to
repolarization reserve. In human cells, dofetilide increased
APD by 59 ±5% in the presence of BaCl2,versus 44 ±4%
in the absence of BaCl2. The relative increase from 44%
prolongation with IK1 intact to 59% prolongation with IK1
removed indicates a 34% increase in IKr blocking effect
with IK1 suppressed. For dog cells, dofetilide increased
Figure 3.
A, currents recorded with action potential voltage-clamp waveforms, obtained by recording typical normal human
or canine ventricular action potentials with a conventional microelectrode in a multicellular papillary muscle
preparation. B–D, original BaCl2(IK1, purple recordings, B), E-4031 (IKr, red recordings, C) and L-735,821 (IKs,
green recordings, D) sensitive currents obtained by digitally subtracting currents elicited by action potential test
pulses in the presence of the blocker from current in the same cell prior to the blocker in human (left panels) and
dog (middle panels) ventricular myocytes. Right panels represent corresponding mean amplitudes of drug-sensitive
IK1,IKr and IKs currents in 4–13 cells per measurement. Arrows indicate the points at which current amplitudes
were determined. Bars represent means ±SEM; corresponding nvalues are provided for each current and species.
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J Physiol 591.17 Weak IK1,IKs limit human repolarization reserve 4195
APD by 25 ±2% in the presence of BaCl2,versus 16 ±2%
in the absence of BaCl2, indicating a 56% increase in IKr
blocking effect with IK1 suppression. This result confirms
a larger contribution of IK1 to repolarization reserve in
the dog versus man. For IKs (Fig. 6B), dofetilide increased
APD by 63 ±4% in the absence of HMR-1566-induced
IKs block in humans, versus 73 ±2% in the presence of
HMR-1566, an increase of 16% attributable to the loss
of the IKs contribution. In the dog, dofetilide prolonged
APD by 29 ±5% in the absence of HMR-1566, versus
43 ±4% in its presence, indicating a 49% enhancement
attributable to loss of IKs. Thus, the larger IKs of canine
tissues also contributes to greater repolarization reserve
versus humans.
Ion channel subunit expression
To assess the potential molecular basis for the observed
differences in IK1 and IKs densities, qPCR was applied
for subunits underlying IK1,IKr and IKs . Gene expression
values for IK1-encoding subunits are shown in Fig. 7A.
Kir2.1-encoding mRNA (KCNJ2 )was>2-fold more
abundant in the dog than the total mRNA level for Kir2.1,
Figure 4. The voltage dependence of the activation and deactivation kinetics of human and canine IKr
and IKs
A, voltage dependence of activation kinetics. IKr and IKs were activated by test pulses with durations from 10 to
5000 ms, to test potentials ranging from 0 to 50 mV; then the cells were clamped back to −40 mV. The amplitudes
of tail currents as a function of the duration of the depolarization were well fitted by single exponentials. B,the
voltage dependence of IKs deactivation kinetics was determined by activating IKs with 5000 ms test pulses to
50 mV from a holding potential of −40 mV. Then the cells were clamped back for 2 s to potentials ranging from
−50 to 0 mV (pulse frequency 0.1 Hz) and the deactivation time course of the tail current was fitted by a single
exponential function. C, the voltage dependence of IKr deactivation kinetics was determined by activating IKr
with 1000 ms test pulses to 30 mV from a holding potential of −40 mV. Then the cells were clamped for 16 s
to potentials ranging from −70 to 0 mV (pulse frequency 0.05 Hz) and the deactivation time course of the tail
current was fitted by a double exponential function. The left panel shows the voltage dependence of slow and fast
time constants. An expanded version of the results for voltage dependence of the fast time constants is provided
in the right bottom panel. The right top panel shows the relative amplitudes of the fast and slow components at
different voltages in dog (black) and human (red) ventricular myocytes.
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Kir2.2, Kir2.3 and Kir2.4 combined in the human. The
KCN H2 gene encoding IKr was equivalently expressed
in canine and human ventricle (Fig. 7B). KCNQ1 gene
expression was not significantly different between human
and dog (Fig. 7C), but the KCNE1 gene encoding the
IKs β-subunit protein minK was ∼6-fold more strongly
expressedindog.ExamplesofWesternblotsforKir2.x,
ERG, KvLQT1 and minK proteins are shown in Fig. 7D–F.
Mean data are provided in Table 1. In agreement with
qPCR-findings, Kir2.1 was significantly stronger in canine
than human hearts, whereas Kir2.2 was stronger in
humans. ERG was detected as two larger molecular
mass bands (Fig. 7E) corresponding to ERG1a (∼150
and 165 kDa) and two smaller bands corresponding to
ERG1b (∼85 and 95 kDa). ERG1a was less abundant
in human samples, while ERG1b band intensities were
not significantly different from dogs. The very similar
expression of ERG1b, in agreement with physiological
data (Figs 2Cand 3), is consistent with recent evidence
for a particularly important role of ERG1b in forming
functional IKr (Sale et al. 2008) and with a recent study
of Purkinje fibre remodelling with heart failure (Maguy
et al. 2009). MinK bands were also stronger in dog
hearts, whereas KvLQT1 band intensity was greater in
human.
We also performed immunohistochemical analyses on
isolated cardiomyocytes (Fig. 8), with identical image
settings for human versus canine cells. Examples are shown
in Fig. 8A. Anti-Kir2.1 showed significantly stronger
staining for canine cells (Fig. 8B), and Kir2.3 staining
was also slightly but significantly greater for dog. In
contrast, ERG staining was comparable for the two species
(Fig. 8C). KvLQT1 staining was modestly but significantly
greater for human cells (Fig. 8D), but in keeping
with the qPCR data, mink staining was much greater
(∼5-fold) for dog cells versus human. Supplemental
Fig. 2 presents negative controls for immunostaining
measurements.
Figure 5. Effect of selective IK1 (10 μMBaCl2), IKr (50 nmol l−1dofetilide) or IKs (1 μmol l−1HMR-1566)
block on APs measured with standard microelectrode techniques in canine and human right papillary
muscles
A, recordings (at 1 Hz) before and after 40 min superfusion with BaCl2(left), dofetilide (middle) or HMR-1566
(right). Corresponding mean±SEM values for controls (C) and drug (D) effects are given under each action potential
recordings. B, mean ±SEM AP duration at 90% of repolarization (APD90 ) under each condition. n=number of
experiments, ∗∗P<0.01 and ∗∗∗ P<0.001.
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Other ionic current differences and in silico
assessment
The functional, pharmacological, and biochemical data
described above all point to reduced repolarization reserve
due to smaller IKs and IK1 expression in human hearts
as the basis for their larger APD prolonging response to
IKr inhibition. To assess the potential role of other ionic
current differences, we compared several other currents
between canine and human hearts. Ito,recordedasthe
difference between peak and end-pulse current during
300 ms depolarizing pulses from −90 mV (0.33 Hz), was
smaller in human versus dog (Fig. 9A). ICaL evoked by
400 ms test pulses from −40 mV was ∼30% larger in
human (Fig. 9B). Recovery kinetics of Ito (Supplemental
Fig. 3A)andICa (Supplemental Fig. 3B) currents were
not statistically different in myocytes from human and
dog ventricle. Ni2+(10 mmol l−1)-sensitive NCX current
was not significantly different between species (Fig. 9C
and D).
To assess the contribution of ionic current components
to repolarization reserve in human versus canine hearts, we
initially adapted the Hund–Rudy dynamic (HRd) canine
ventricular AP model (Hund & Rudy, 2004). We then
adjusted the current densities in the dog model according
to the experimentally observed differences in humans,
to obtain ‘humanized’ APs (see Supplemental Methods).
Supplemental Fig. 4 shows the resulting simulations:
APD90 at 1 Hz in the dog model was 209 ms, versus human
264 ms, close to experimentally determined values (APD90
at 1 Hz: dog 227 ms, human 270 ms). IKr block increased
APD90 by 26% in the human AP model (Supplemental Fig.
4A)versus 15.5% in the dog model (Supplemental Fig. 4B),
Figure 6. Effect of combined IKr +IK1 and IKr +IKs inhibition in human and dog ventricular muscle pre-
parations (endocardial impalements)
A, representative APs at baseline (circle), following exposure to 10 μmol l−1BaCl2(triangle), 50 nmol l−1dofetilide
(diamond), and combined 10 μmol l−1BaCl2+50 nmol l−1dofetilide (rectangle) in human (top traces) and
dog (bottom traces) ventricular muscle. Brackets show average differences between conditions indicated. B,
representative APs at baseline (circle), following exposure to 1 μmol l−1HMR-1566 (triangle), 50 nmol l−1
dofetilide (diamond), and combined 1 μmol l−1HMR-1566 +50 nmol l−1dofetilide (rectangle) in human (top
traces) and dog (bottom traces) ventricular muscle. Brackets show average differences between conditions
indicated.
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qualitatively consistent with experimental findings (56%,
22% respectively). IKr inhibition increased human APD90
by 71.2% in the presence of IK1 block, indicating a 173.8%
increase in IKr blocking effect with the IK1 contribution to
repolarization reserve suppressed (Supplemental Fig. 4A).
For the canine model (Supplemental Fig. 4B), IKr block
increased APD90 by 45.4% in the presence of IK1 block,
indicating a 193.5% increase in IKr blocking effect
when IK1 is decreased. This result is consistent with
experimental data suggesting a larger contribution of IK1
to repolarization reserve in the dog. IKr block prolonged
human APD90 by 29.4% (Supplemental Fig. 4C)inthepre-
sence of IKs inhibition, an increase of 14.6% attributable
to the loss of IKs contribution to repolarization reserve.
ForthedogAPmodel(SupplementalFig.4D), IKr
block prolonged APD by 23.8% in the presence of IKs
inhibition, indicating a 53.6% enhancement attributable
to loss of the repolarization reserve effect of IKs. Thus,
the model also confirms the importance of larger IKs to
greater repolarization reserve in dogs. Finally, we used
the model to explore the contributions of ICaL and Ito
differences. Supplemental Fig. 5 shows the APD changes
induced by IKr inhibition in canine (panel A) and human
(panel B) models. The effect of IKr inhibition in the
human model was then verified with ICaL (panel C)or
Ito (panel D) modified to canine values. APD90 increases
in the human model resulting from IKr inhibition were
minimally affected by substituting canine Ito in the human
model. Substituting canine ICaL into the human model
enhanced the IKr blocking effect on APD, whereas if
canine ICaL contributed to the larger repolarization reserve
in the dog it should reduce the APD prolonging effect.
These results indicate that ICaL and Ito differences do not
contribute to the enhanced repolarization reserve in the
dog.
To assess further the contribution of ionic current
components to repolarization reserve in human versus
canine hearts, we performed the analysis in a reverse
Figure 7. Expression of IK1-related (Kir2.x), IKr
pore-forming (ERG) and IKs-related subunits
(KvLQT1 and minK)
A–C, mean ±SEM mRNA levels of Kir2.x (A), ERG
(B) and KvLQT1/minK (C) subunits in left
ventricular human (n=6–8) and dog (n=816)
preparations. ∗P<0.05, ∗∗P<0.01 and
∗∗∗P<0.001. n=number of experiments. D–F,
representative Western blots for Kir2.x (D), ERG
(E) and KvLQT1/minK (F) in human and dog left
ventricular preparations.
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Table 1. Protein expression data for ion channel subunits in human versus dog ventricular tissues
Currents/subunits Subunit†Human†Dog
IK1 subunits Kir2.1 (n=4/4) 0.22 ±0.01 0.45 ±0.06∗
Kir2.2 (n=4/4) 0.64 ±0.03∗∗ 0.37 ±0.02
Kir2.3 (n=4/4) 0.10 ±0.01 0.09 ±0.007 (P=NS)
Kir2.4 (n=4/4) 0.01 ±0.002 0.20 ±0.009∗∗
IKr subunits ERG1a (n=5/4) 0.30 ±0.16 0.97 ±0.27∗∗
ERG1b (n=5/4) 0.71 ±0.05 0.73 ±0.07 (P=NS)
IKs subunits KvLQT1 (n=4/4) 0.15 ±0.01∗∗ 0.05 ±0.003
MinK (n=4/4) 0.31 ±0.01 0.40 ±0.05∗
Mean ±SEM data. ∗P<0.05, ∗∗P<0.01, ∗∗∗ P<0.001. ndesignates number of samples from humans/dogs.
†All values are expressed as arbitrary optical density units, quantified relative to an internal control on the same
sample (α-actin for Kir2.x, KvLQT1 and minK, GAPDH for ERG).
fashion, with the more recently published O’Hara–Rudy
dynamic (ORd) human ventricular AP model (O’Hara
et al. 2011, see Supplemental Methods). Figure 10 shows
the resulting simulations: APD90 at 1 Hz in the canine
and human models were 210 ms and 271 ms (versus
experimental APD90 at 1 Hz: dog 227 ms, human 270 ms).
IKr block increased APD90 by 42.4% in the human versus
29.4% in the dog model, consistent with experimental
findings (56%, 22% respectively). With the human ionic
model (Fig. 10A), IKr block increased APD by 58.7% in
the presence of IK1 block, versus 42.4% in the absence
of IK1 block. These results indicate a 38.3% increase in
IKr blocking effect on APD with IK1 blocked. For the dog
ionic model (Fig. 10B), IKr block increased APD by 45.8%
in the presence of IK1 block, versus 29.4% in the absence
of IK1 block, indicating a 55.7% increase in IKr blocking
effect when IK1 was decreased. This result confirms the
notion based on our experimental data, indicating a larger
contribution of IK1 to repolarization reserve in the dog
compared to man. IKr block increased APD by 42.4% in the
absence of IKs block in the human model (Fig. 10C), versus
50.3% in the presence of IKs block, an increase of 18.5%
attributable to the loss of IKs contribution torepolarization
reserve. In the dog ionic model (Fig. 10D), IKr block
prolonged APD by 29.4% in the absence of IKs block, versus
46.9% in its presence, indicating a 59.4% enhancement
attributable to loss of the repolarization reserve effect of
IKs. Thus, the model also confirms the importance of larger
IKs to greater repolarization reserve in dogs. Finally, we also
used this modelling approach to explore the contributions
of ICaL and Ito differences, and found no evidence that they
contribute to the differences in IKr blocking effects between
human and dog (Supplemental Fig. 6).
Discussion
In this study, we found that IKr inhibition causes
substantially greater APD prolongation in human
versus canine ventricular muscle, indicating reduced
repolarization reserve in man. Ionic current
measurements showed larger IK1 and IKs densities
in canine versus human hearts and APD studies with
selective blockers indicated larger repolarization reserve
in canine hearts due to stronger IK1 and IKs contributions.
Expression studies suggested that the ionic current
differences are due to species-related differences in mRNA
expression of underlying subunits.
Experimental model considerations
We compared experimental data between non-diseased
human donor hearts and canine hearts. There is a
potential difference in relative maturity/age between the
humans and dogs that provided our tissue samples, which
were essentially impossible to control, other than by
virtue of the fact that both study populations comprised
adult and not senescent individuals. Important trans-
mural and regional differences in ion channel subunit
protein expression and current densities exist within
the heart. Extrapolation of our findings to the whole
heart must therefore be cautious. We were careful to
perform all measurements in corresponding regions
of canine and human hearts to ensure comparability.
Current and mRNA/protein densities were measured
from the left ventricular midmyocardial free-wall,
but APs were recorded from right ventricular sub-
endocardial tissue. This was done both for technical
reasons (standard microelectrode recordings from left
ventricular tissue were difficult to obtain and more
likely to be contaminated by subendocardial Purkinje
fibres) and to maximize data from each human heart
by using all available tissues. We had to optimize the
information obtained from each human heart, because
functional measurements were greatly limited by the
unpredictable and infrequent availability of human
donor tissue and because of the short time window
for meaningful functional measurement after tissue
procurement. Of note, our patch-clamp/biochemical
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results in left ventricular free-wall were fully compatible
with our AP data from right ventricular tissues, indicating
that at least for these two widely separated regions the
observations are consistent.
Relationship to previous studies of repolarizing
currents and repolarization reserve
Our data suggest important expression differences in
Kir2.x channel mRNA expression between human and
Figure 8. Immunofluorescence confocal microscope image analysis for IK1-related (Kir2.x), IKr
pore-forming (ERG) and IKs-related (KvLQT1 and MinK) subunits in left ventricular cardiomyocytes
A, representative immunofluorescence images of human (left) and dog (right) cardiomyocytes. Dark-field images
of typical human and dog ventricular cardiomyocytes are shown at the bottom. B–D, mean ±SEM fluorescence
intensities for various subunits in human versus dog cardiomyocytes. Results are shown for Kir2.x (B), ERG (C)
and KvLQT1 and minK (D) subunits. n=number of experiments. ∗P<0.05 and ∗∗∗P<0.001 for dog versus
human.Constant image-settings were maintained for each construct for all cells studied.
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J Physiol 591.17 Weak IK1,IKs limit human repolarization reserve 4201
dog ventricle. Kir2.1 expression was about 3-fold greater
in the dog than human, but Kir2.2 and Kir2.4 levels
were negligible in dogs. In human hearts, we found
Kir2.3 mRNA expression comparable with that of Kir2.1,
generally considered the principal subunit underlying
IK1 (Dhamoon & Jalife, 2005). Significant Kir2.3 protein
expression in human ventricle was also detected by
Western blot (Fig. 7D). Kir2.1 currents display strong
inward rectification, whereas Kir2.3 inward rectification
is incomplete and negative slope conductance is less steep
(Dhamoon et al. 2004). In our study, the current–voltage
relation of IK1 in dog strongly resembles that previously
reported for Kir2.1 channels, but in human cells resembles
better a mixture of Kir2.1 and Kir2.3 properties (Dhamoon
et al. 2004) corresponding to mRNA data.
Protein quantification showed lesser ERG1a abundance
in human compared to dog tissue while expression
of ERG1b was not different. A higher ERG1b:ERG1a
expression ratio in humans suggests the possibility of
different channel subunit stoichiometry in human tissue
versus dog. This difference might have two functional
consequences. First, partially due to the accelerated
activation kinetics of heteromeric channels compared
to homomeric channels consisting of ERG1a only, the
relative contribution of IKr to the repolarization reserve
is expected to be higher in humans (Sale et al. 2008;
Larsen & Olesen, 2010). Secondly, ERG1a–ERG1b sub-
unit stoichiometry could also affect drug binding affinity
of dofetilide to IKr channels, as slightly higher IC50
values were obtained for ERG1a–1b heteromeric channels
Figure 9.
A,Ito current–voltage density (I–V relationship) relation obtained with the inset protocol. ∗P<0.05 and +P<0.05
for human versus dog. I–V relationships for Ito are determined and depicted as peak current (open circles and
squares) and as sustained current (closed circles and squares) as well. B,ICaL current–voltage density relation
obtained with the insetprotocol. ∗P<0.05 for human vs. dog. I–V relationships for ICa are determined and
depicted as peak current (open circles and squares) and as sustained current (closed circles and squares) as well. C,
ramp protocol was applied to measure current before and after application of Ni2+(10 mmol l−1) under conditions
to isolate NCX. Representative Ni2+-sensitive difference currents from dog and human cells are shown below. D,
mean inward (at −80 mV) and outward (at +50 mV) NCX current density values.
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4202 N. Jost and others J Physiol 591.17
as compared to ERG1a homomer channels (150 nMvs.
100 nM, respectively; Abi-Gerges et al. 2011). We have
not detected any significant difference in the kinetic
behaviour of IKr in humans versus dogs and dofetilide
affinity was not different based on concentration–response
curves (Supplemental Fig. 1). Thus, relative expression on
Western blots may not reflect accurately relative local sub-
unit expression in ion channels.
Relatively little information is available about the
molecular basis of differential repolarization patterns
among species. APD prolongation and early after-
depolarization formation upon exposure to IKr blocking
drugs varies widely, with rabbits being the most sensitive,
guinea-pigs, swine and sheep the least, and dogs inter-
mediate (H. R. Lu et al. 2001). Guinea-pigs have
particularly large, and rabbits particularly small, IKs (Z.
Lu et al. 2001). This difference results from weaker
mink expression in the rabbit, despite stronger KvLQT1
expression in rabbits (Zicha et al. 2003). Interestingly,
this expression difference resembles what we observed
for human versus dog in the present study, with dogs
having much larger minK, but smaller KvLQT1, expression
than humans, along with considerably larger IKs density.
Dumaine & Cordeiro (2007) also observed larger IK1 and
IKs, along with similar IKr , for dog compared to rabbit.
MinK, on the other hand, has also been found to modulate
hERG and Kv4.3 current densities and gating of the
channels (Pourrier et al. 2003). Therefore, other currents
in addition to IKs,suchasIKr and Ito mightbepotentially
influenced by the relatively lower minK expression level in
human ventricles we found in this study.
Possible implications
Larger APD prolongation in human tissues versus dog
in response to IKr blockade, despite similar IKr,isa
novel finding that may have important implications.
Based on the present results, despite observations that
Figure 10. Simulations of effect of combined IK+IK1 and IKr +IKs inhibition on human and dog
ventricular muscle APs by applying the O’Hara dynamic (ORd) canine ventricular AP model
A, simulated human APs at control, following IK1 block (70% reduction), IKr block (50% reduction), and combined
IK1 +IKr block. B, corresponding data for dog IK1 +IKr block. C, simulated human APs at control, following IKs
block (50% reduction), IKr block (50% reduction), and combined IKs +IKr block. D, corresponding data for dog
IKs +IKr block.
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J Physiol 591.17 Weak IK1,IKs limit human repolarization reserve 4203
the properties of individual K+channels in dog resemble
those of humans (Varr ´
oet al. 2000; Jost et al. 2005),
the reserve to repolarization-delaying drugs may differ
substantially between the two species. Consequently,
the clinical repolarization-delay potential of drugs with
IKr/HERG blocking properties could be underestimated
based on experiments in dogs, and using dogs in
safety-pharmacology studies to estimate QT-lengthening
liability could be misleading. However, there is greater
similarity of individual currents in human and dog, and
better heart rate correlation between human and dog
than human and rabbit (Lengyel et al. 2001) and the
similar relative profile of rabbit versus dog K+currents
in the Dumaine–Cordeiro study (Dumaine & Cordeiro,
2007) to the human versus dog results in the present
work raise the issue of whether the commonly used,
simpler and cheaper rabbit model might be more pre-
dictive. QT prolongation by non-cardiovascular drugs is a
major problem and considerable resources are expended to
optimize QT-liability drug screening in drug development
(Vargas, 2008). Our findings have potentially important
implications for the optimization of drug screening.
Based on our data, IK1 block or downregulation/
mutation would not necessarily lead to substantial QT
prolongation in humans, unlike in the dog, but a reduction
of repolarization reserve would be expected (Roden, 1998;
Biliczki et al. 2002; Silva & Rudy, 2005; Roden, 2006).
Therefore, an IK1 (Kir2.x) channel defect due to ion
channel mutations or drug-induced malfunction may
not significantly prolong human QT intervals, but could
produce excess QT prolongation and life-threatening
torsades de pointes in the face of additional repolarization
impairment.
The present study is, to our knowledge, the first
detailed analysis of the molecular and ionic determinants
of repolarization reserve in the human heart, and the
first to compare these determinants with those of an
animal species commonly used as a model for human
cardiac electrophysiology. Our results therefore provide
novel fundamental insights into this clinically crucial
process.
Potential limitations
IK1 flows through a variety of channel subtypes that may be
constituted by different alpha-subunits including Kir2.1,
Kir2.2, Kir2.3, Kir2.4, TASK and TWIK (Wang et al. 1998;
Lopatin & Nichols, 2001; Melnyk et al. 2002; Dhamoon
et al. 2004). The latter two-pore channels do not rectify
(Lesage & Lazdunski, 2000) and were not studied in our
experiments, although their contribution to IK1 cannot
be ruled out. Previous reports indicate important species
and regional differences in relative expression of Kir2.x
proteins (Wang et al. 1998; Melnyk et al. 2002; Dhamoon &
Jalife, 2005). The densities of IK1 and distribution of Kir2.x
proteins differ in atria versus ventricles (Melnyk et al. 2002;
Dhamoon & Jalife, 2005). In the present study, we focused
on ventricular tissue exclusively. Kir2.2 has been reported
absent in rabbit ventricle but present in human (Wang
et al. 1998) and dog (Melnyk et al. 2002) ventricles. Kir2.x
proteins not only form homomeric channels, but can
also show heteromeric co-assembly (Zobel et al. 2003),
complexifying interpretation. Heteromeric assembly of
Kir2.1 and Kir2.3 proteins produces IK1 channels with
lower conductance than homomeric Kir2.1 assembly (Yan
et al. 2005; Fang et al. 2005). Since the mRNA expression
of Kir2.1 and Kir 2.3 in human ventricle was relatively
similar, unlike the dog, heteromeric Kir2.1–2.3 channels
may be more likely in the human than in the dog
ventricle, contributing to the lower IK1 density that we
observed in humans. Indirect evidence indeed points to a
significant role for heteromeric Kir2.x channels in human
IK1 (Schram et al. 2003).
All of our human samples were stored in cardio-
plegic solution following harvesting during transportation
to our facility. In preliminary studies in which we
stored canine heart samples in cardioplegic solution
and then recorded ionic currents and APs, we did not
observe any electrophysiological effects of cardioplegic
storage. Donors received haemodynamic support with
dobutamine prior to heart explantation, a ubiquitous
practice in cardiac transplantation. We cannot exclude
possible effects of dobutamine infusion on the properties
of explanted hearts.
The effects of pharmacological blockade on canine
APs vary among different laboratories. For example,
the Antzelevitch laboratory has reported larger increases
in canine ventricular APD with K+channel blockade
(Shimizu & Antzelevitch, 1999; Tsuboi & Antzelevitch,
2006) than we observed in the present study. The
discrepancies are likely to relate to differences in
experimental conditions. For this reason, it is important
that comparative studies between species responses
are produced within a single laboratory rather than
comparing changes observed for one species in one
laboratory with those for another species in a different
laboratory.
The Na+–Ca2+exchanger current (NCX) current was
defined and measured as Ni2+-sensitive current. This
approach has limitations, because it cannot be excluded
that Ni2+blocks other ionic currents. However, for
the measurement of the NCX, we blocked other ionic
currents (including K+,Na
+and Ca2+currents, along with
Na+–K+pump current) according to the experimental
approach described by Hobai et al. (1997), which is a
relative widely used method for studying NCX current
(T ´
oth et al. 2009).
The Na+–K+pump is critically dependent on extra-
and intracellular Na+and K+concentrations, voltage, sub-
cellular space and cAMP levels, and is not well explored
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4204 N. Jost and others J Physiol 591.17
in dog and human cardiomyocytes (Fuller et al. 2013).
Since we have no experimental data regarding this current,
we cannot exclude a contribution of species difference in
the function of Na+–K+pump currents to repolarization
reserve discrepancies.
Although IKs is several-fold larger under square-wave
voltage-clamp conditions in dog than man (Fig. 2), there
was no significant difference under AP-clamp conditions
(Fig. 3). We believe that the apparent discrepancy is due to
the fact that during the normal AP, cells spend very little
time at potentials for which there is a significant difference
in IKs (positive to +20mV;Fig.2).
The enhanced density of IKs in canine versus human
heart appears to be due, at least in part, to stronger
expression of minK in the dog. However, there is a
discrepancy between the Western blot results, showing
a 33% greater expression level in the dog (Table 1),
and the immunofluorescence results (Fig. 8), showing
an approximately 5-fold greater expression in canine
cardiomyocytes. In addition, if minK overexpression were
responsible for greater IKs in the dog, kinetics should have
differed markedly between the species, which they do not.
Therefore, while differences in minK may be involved
in the species differences in IKs, other factors are likely
involved and should be addressed in future work.
Conclusions
Human ventricular cardiomyocytes have reduced
repolarization reserve compared to dog. The differential
response occurs despite similar IKr densities, due to lower
IK1 and IKs densities in human hearts. The underlying
molecular basis appears to be differential expression
of Kir2.x and minK subunits between human and
canine hearts. These results suggest that the protection
afforded by IK1 and IKs against repolarization stress
is limited in humans, making humans susceptible to
excess repolarization impairment from IKr blocking
agents. Animal models are widely used to study cardiac
pathophysiology and pharmacological responses. Our
findings highlight the importance of caution when
extrapolating results from animal models to man, even
from species as apparently similar in ionic current
mechanisms as dogs.
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Additional information
Conflict of interest
None declared.
Author contributions
Conception and design of the experiments: N.J., L.V., J.Gy.P.,
A.V., S.N.; collection, analysis and interpretation of data: N.J.,
L.V., P.C., B. ¨
O., V.Sz., Gy.S., M.B., Zs.K., I.K., N.N., T.Sz., J.M.,
M.K., L.G.P., Cs.L., A.V., S.N.; drafting the article and revising
it critically for intellectual content: N.J., L.V., P.C., B. ¨
O., V.Sz.,
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2013 The Authors. The Journal of Physiology C
2013 The Physiological Society
4206 N. Jost and others J Physiol 591.17
Gy.S., J.M., L.G.P., E.W., U.R., P.P.N., J.Gy.P., A.V., S.N. All
authors approved the final version of the manuscript.
Funding
This work was supported by grants from the Hungarian Scientific
Research Fund (CNK-77855, K-82079 and NK-104331), the
National Office for Research and Technology-Baross
Programmes (REG-DA-09-2-2009-0115-NCXINHIB), the
National Development Agency and co-financed by the European
Regional Fund (T ´
AMOP-4.2.2/B-10/1-2010-0012; T ´
AMOP-
4.2.2.A-11/1/KONV-2012-0035; T ´
AMOP-4.2.2A-11/1/KONV-
2012-0073 and T ´
AMOP-4.2.2.A-11/1/KONV-2012-0060), the
Hungarian Academy of Sciences, the Canadian Institutes for
Health Care Research (MOP 68929), German Hungarian
Research Cooperation DFG Grant (436 UNG 113/176/0-1)
HU-RO Cross-Border Cooperation Programmes (HURO/0802/
011_AF-HURO_CARDIOPOL and HURO/1001/086/2.2.1_
HURO-TWIN,) and the Hungarian Academy of Sciences.
Acknowledgements
The authors thank France Th´
eriault for secretarial help with the
manuscript.
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2013 The Authors. The Journal of Physiology C
2013 The Physiological Society
