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1153
Increased Right Ventricular Repolarization Gradients Promote
Arrhythmogenesis in a Murine Model of Brugada Syndrome
CLAIRE A. MARTIN, M.R.C.P.,∗YANMIN ZHANG, Ph.D.,∗,†ANDREW A. GRACE, F.R.C.P.,‡
and CHRISTOPHER L.-H. HUANG, Ph.D.∗
From the ∗Physiological Laboratory, University of Cambridge, Downing Site, Cambridge, United Kingdom; †Department of Paediatrics,
First Affiliated Hospital, Xi’an Jiaotong University, Xi’an, Peoples Republic of China; and ‡Department of Biochemistry, University of
Cambridge, Downing Site, Cambridge, United Kingdom
Repolarization Gradients in Brugada Syndrome. Introduction: Brugada syndrome (BrS) is
associated with loss of Na+channel function and increased risks of a ventricular tachycardia exacerbated
by flecainide but reduced by quinidine. Previous studies in nongenetic models have implicated both altered
conduction times and repolarization gradients in this arrhythmogenicity. We compared activation latencies
and spatial differences in action potential recovery between different ventricular regions in a murine
Scn5a+/−BrS model, and investigated the effect of flecainide and quinidine upon these.
Methods and Results: Langendorff-perfused wild-type and Scn5a+/−hearts were subjected to regular
pacing and a combination of programmed electrical stimulation techniques. Monophasic action potentials
were recorded from the right (RV) and left ventricular (LV) epicardium and endocardium before and
following flecainide (10 μM) or quinidine (5 μM) treatment, and activation latencies measured. Transmural
repolarization gradients were then calculated from the difference between neighboring endocardial and
epicardial action potential durations (APDs). Scn5a+/−hearts showed decreased RV epicardial APDs,
accentuating RV, but not LV, transmural gradients. This correlated with increased arrhythmic tendencies
compared with wild-type. Flecainide increased RV transmural gradients, while quinidine decreased them,
in line with their respective pro- and antiarrhythmic effects. In contrast, Scna5+/−hearts showed slowed
conduction times in both RV and LV, exacerbated not only by flecainide but also by quinidine, in contrast
to their differing effects on arrhythmogenesis.
Conclusion: We use a murine genetic model of BrS to systematically analyze LV and RV action potential
kinetics for the first time. This establishes a key role for accentuated transmural gradients, specifically in
the RV, in its arrhythmogenicity. (J Cardiovasc Electrophysiol, Vol. 21, pp. 1153-1159, October 2010)
arrhythmia, ion channels, sudden death, action potentials, conduction velocity, Brugada syndrome, sodium
channels, ventricular tachycardia, antiarrhythmic drugs, repolarization
Introduction
Brugada syndrome (BrS) is associated with a loss of Na+
channel function and increased risks of polymorphic ven-
tricular tachycardia (VT), potentially leading to sudden car-
diac death. The arrhythmogenicity in BrS is thought to be
a result of electrophysiological alterations localized to the
Re-use of this article is permitted in accordance with the Terms and Con-
ditions set out at http://wileyonlinelibrary.com/onlineopen# OnlineOpen
Ter m s
Funding was provided by the British Heart Foundation, the Medical Re-
search Council, the Wellcome Trust and the Biotechnology and Biological
Research Council, UK. C. Martin was supported by a Medical Research
Council Clinical Research Fellowship and a Sackler Studentship of the
University of Cambridge School of Clinical Medicine. Y. Zhang was par-
tially supported by the Chinese Nature Science Foundation (project numbers
30371571 and 30672209).
No disclosures.
Address for correspondence: Dr. Claire Martin, M.R.C.P., Physiological
Laboratory, University of Cambridge, Downing Site, Cambridge CB2 3EG,
United Kingdom. Fax: +441223333840; E-mail: clairemartin@gmail.com
Manuscript received 4 January 2010; Revised manuscript received 18 Febru-
ary 2010; Accepted for publication 22 February 2010.
doi: 10.1111/j.1540-8167.2010.01767.x
right ventricle, reflected in ST elevation in the right precor-
dial leads, right bundle branch block and changes specific to
right ventricle (RV) epicardial action potential (AP) wave-
forms.1Previous studies have implicated either slowed RV
AP conduction2,3or alterations in AP repolarization1,4in the
arrhythmogenesis associated with BrS. Thus, on one hand, re-
duced Na+currents in BrS would be expected to compromise
AP conduction. On the other, their reduction in relationship
to Ito currents in BrS would shorten the RV epicardial APD
and consequently permit reentrant waves.5
Experimental models for BrS have involved a number
of pharmacological manipulations in canine systems. These
have involved use of agents producing K+channel opening
by pinacidil, Na+channel block by flecainide, Ca2+channel
block by verapamil, increased [Ca2+]o, metabolic inhibition
or simulated ischemia. Such studies have implicated repolar-
ization gradients caused by regional heterogeneities in elec-
trical activity. However, there is uncertainty as to whether
such pharmacological systems provide accurate or complete
replication of the physiological changes underlying BrS. Fur-
thermore, the pharmacological manipulations themselves in-
herently lack a specificity of action. Experimental systems
using genetic modifications to replicate BrS may provide a
more specific model to clarify physiological abnormalities
associated with the disease condition.
Thus far, only the Scn5a gene, which encodes the car-
diac Nav1.5 α-subunit, has been extensively studied in
1154 Journal of Cardiovascular Electrophysiology Vol. 21, No. 10, October 2010
connection with BrS. Our previous studies using a het-
erozygotic Scn5a+/−mouse have recapitulated features of
the human clinical condition in demonstrating an enhanced
arrhythmogenesis that is exacerbated by flecainide and re-
lieved by quinidine. This model has previously shown a 50%
reduction in the transmembrane Na+current and evidence
for slowed ventricular and atrioventricular conduction.6As
yet, no evidence has been found for accentuated repolariza-
tion gradients in this model although to date no study of the
RV has been made.7
In the present experiments, we seek for the first time in a
genetic whole-heart BrS model to discriminate the contribu-
tions of alterations in conduction and repolarization gradients
in the arrhythmogenesis, and to localize the pathophysiolog-
ical mechanism in the RV. We were particularly interested in
the effects upon conduction velocity and repolarization gra-
dients of 2 clinically used drugs: flecainide, which is known
to unmask ventricular arrhythmogenesis in otherwise asymp-
tomatic BrS patients,8and quinidine, which has shown pro-
tective actions in symptomatic BrS.9We correlate alterations
in RV gradients in the BrS model that are specifically ex-
acerbated by flecainide, with the observed arrhythmogenic
properties and with clinical findings implicating the RV in
the arrhythmogenic mechanism. This may contribute to fu-
ture work investigating possible pharmacological treatments
for a disease for which the current mainstay of treatment is
implantable cardioverter defibrillator (ICD) implantation.5
Methods
Langendorff Perfusion
Mice aged 4–8 months were obtained from breeding
pairs of heterozygote Scn5a+/−and wild-type (WT) in-
bred 129/sv mice initially supplied by Harlan (UK). Ex-
periments used a Langendorff-perfused preparation adapted
for the murine heart, as described previously.10 Following
the start of perfusion, viable hearts suitable for subsequent
experimentation regained a pink coloration and spontaneous
rhythmic contraction with warming. Where used, 10 μM
flecainide and 5 μM quinidine (Sigma-Aldrich, Poole, UK)
dissolved in buffer solution were perfused for 15 minutes
prior to and throughout data acquisition. Concentrations were
within the same range as known clinical therapeutic levels
(flecainide: 0.2–0.9 mg L−1; quinidine: 2.0–5.0 mg L−1).11
All procedures conformed to the UK Animals (Scientific
Procedures) Act 1986.
Monophasic Action Potential Recording
Monophasic action potentials (MAPs) were recorded us-
ing an established contact-electrode technique detailed pre-
viously.12 Epicardial MAPs were recorded from the basal
surface of the ventricular epicardium, both on the left and on
the right using a miniaturized MAP electrode tip (Linton In-
struments, Harvard Apparatus, UK). Endocardial MAPs used
electrodes constructed from galvanically chlorided, Teflon-
coated 0.25 mm diameter silver wire introduced into the
ventricular cavity through a small access window created in
the ventricular wall. Pilot experiments showed that both sets
of electrodes provided equivalent MAP traces. MAP signals
were amplified and band-pass filtered between 0.1 Hz and
300 Hz (Neurolog AC amplifiers and filters Models NL104
and NL125/6, respectively: Digitimer, Welwyn Garden City,
Herts, UK), then digitized using a 1401plus interface (Cam-
bridge Electronic Design, Cambridge, UK). Paired platinum
stimulating electrodes paced the heart high on the interven-
tricular septum.
The stimulating electrode and the epicardial LV and RV
recording electrodes were clamped at a constant position
through all experiments. This was at a distance of approxi-
mately 10 mm between stimulating and each recording elec-
trode, although it was slightly larger for the LV than for
the RV. This distance allowed the hearts to be placed into
the rig, with the stimulating electrode coming into contact
with the septum and the recording electrodes coming into
contact with the left and right ventricles. While the direct
absolute distance between electrodes would not necessarily
reflect the path through which the electrical signal would be
conducted in the spherical whole heart, the fact that the dis-
tance was maintained between experiments allowed consis-
tent measurements of activation latencies. As the clamp was
secure throughout experiments, this distance was constant to
∼0.5 mm, i.e., 5% of the distance between stimulating and
recording electrodes.
For measurement of APDs, hearts were paced at 8 Hz for
5 minutes and recordings were made at LV and RV, epicardial
and endocardial sites. For activation latencies, only epicardial
sites were used, as the point of endocardial recording could
not be accurately determined. For calculations of arrhythmia
incidence, as well as regular 8 Hz pacing, the hearts also
underwent an S1S2 protocol, imposing S2 extrastimuli fol-
lowing pacing S1 stimulus trains at S1–S2 intervals, reduced
by 1 ms between successive drive trains until the preparation
became refractory, and a dynamic pacing protocol progres-
sively increasing pacing frequency every 100 beats. Thus for
each heart, 12 recordings in total were made, with 3 proto-
cols and 4 cardiac regions employed. MAP recordings were
made both before and during treatment with either flecainide
(10 μM) or quinidine (5 μM).
Data Analysis
MAP waveforms were analyzed using Spike2 software
(Cambridge Electronic Design). The point of maximum pos-
itive deflection was considered the point of 0% repolariza-
tion; that of full return to baseline of 100% repolarization.
The intervening waveform was described in terms of APDx
measurements at x =90% (APD90 ), 70% (APD70 ) and 50%
(APD50 ) repolarization. The effect of heterogeneous repo-
larization on the difference between different cardiac regions
was expressed empirically as the difference (APDx) be-
tween neighboring APD values: LV transmural =LV en-
docardial APD—LV epicardial APD, RV transmural =RV
endocardial APD—RV epicardial APD, epicardial transven-
tricular =LV epicardial APD—RV epicardial APD, endo-
cardial transventricular =LV endocardial APD—RV endo-
cardial APD.
Activation latencies for MAPs recorded from the RV and
LV epicardium were measured from the stimulus time to
peak amplitude of the MAP to give an indication of con-
duction velocities. With the positions of the stimulating and
recording electrodes constant between experiments, compar-
isons could be made between WT and Scn5a+/−hearts, and
before and after drug. However, as the distance from stimu-
lating electrode on the septum was slightly greater to the LV
recording electrode than the RV recording electrode, no direct
Martin et al. Repolarization Gradients in Brugada Syndrome 1155
Figure 1. Representative RV epicardial MAP records from Scn5a+/−
hearts during regular 8 Hz pacing. The vertical markers below each trace
indicate stimulus timings. The y-axis is the MAP voltage normalized to peak
MAP deflection. Hearts were either resistant to arrhythmia (A) or showed
VEs (B), nsVT (lasting less than 1 second) (C), or VT (lasting more than 1
second) (D).
comparison could be made between LV and RV. For the calcu-
lation of arrhythmic incidence, each run was labeled as either
nonarrhythmic or showing one of 3 possible arrhythmias: VT
(exceeding 1 second in duration), nonsustained VT (nsVT,
lasting less than 1 second), or having one or more ventricular
ectopic (VE).
Statistical Procedures
Sixteen WT and 16 Scn5a+/−mice were used in our
experiments, as this was a number sufficient to enable cat-
egorical statistical analysis for incidence of arrhythmogene-
sis. Although we also attempted quantitative measurements
in all 16, we only included measurements from hearts whose
MAPs strictly attained the accepted criteria of rapid upstroke
phases, consistent amplitudes, smooth contoured repolariza-
tion phases and stable baselines13 in all 4 cardiac regions. We
consequently used 12 hearts of each genotype for the calcu-
lation of activation latencies and repolarization gradients. In
all cases, half of each group was exposed to flecainide and 6
to quinidine.
All results were expressed as mean ±S.E.M. values. The
significance of differences in arrhythmic incidences used
TABLE 1
The Incidence of Arrhythmogenesis in WT and Scn5a+/−Hearts Characterized as Showing One of Three Possible Arrhythmias: VT (Exceeding 1 second
in Duration), nsVT (Lasting Less Than 1 second), and One or More VEs
WT Scn5a+/−
No Drug Flecainide 10 μM Quinidine 5 μM No Drug Flecainide 10 μM Quinidine 5 μM
VT 3/192 6/96 3/96 21/192 16/96 1/96
nsVT 5/192 2/96 4/96 8/192 12/96 1/96
VEs 2/192 0/96 1/96 3/192 9/96 1/96
Total 10/192 (5.2) 8/96 (8.3) 8/96 (8.3) 32/192 (16.7)∗37/96 (38.5)∗†3/96 (3.1)∗†
Hearts showing more than one of these arrhythmias were marked as showing the more potentially clinically significant arrhythmic event, VEs being the least
and VT being the most significant. Recordings were made from hearts either in the absence or presence of either flecainide (10 μM) or quinidine (5 μM).
Results are expressed both as numbers of test runs showing arrhythmia out of the total, and as percentages in parentheses. Sixteen WT and 16 Scn5a+/−
hearts were used before drug, and 8 of each were exposed to each drug treatment. Each heart was tested in 4 cardiac regions and with 3 different protocols,
leading to 192 runs before drug for each genotype, and 96 each for each drug. The results of Chi-squared tests are shown, sorted by genotype (corrected P <
0.05 significance denoted by∗) and drug condition (denoted by†).
Fisher exact and Chi-squared tests. Differences in activation
latencies, APDs and APDs between different regions were
analyzed using Student’s t-tests; these were paired where re-
sults could be compared from the same heart. Previously,
ANOVA with post-hoc Tukey’s honestly significant different
tests have been used to analyze multiple paired data sets;
however, this assumes that all data sets are compared with
each other and thus increases the likelihood of Type II er-
rors. We accordingly adopted a procedure using a modified
Bonferroni correction factor14 using the following procedure:
t-tests were grouped into independent data sets and the sig-
nificance values rank-ordered from smallest to largest. The
significance of the test with P-value at alpha/(number of tests)
was evaluated and, if statistically significant, the test result
from the test with the next smallest significance value was
selected and evaluated at alpha/(number of tests—1), and so
on. Alpha was set at 0.05.
Results
Arrhythmias Were Unmasked by Flecainide in Scn5a+/−
Mice, but Reduced by Quinidine
WT and Scn5a+/−hearts could either be resistant to
arrhythmia or could show VEs, nsVT, or polymorphic VT
during recording. Figure 1 illustrates these possible outcomes
by showing RV epicardial MAP records from Scn5a+/−
hearts during regular 8 Hz pacing. Similar traces could be
obtained from WT, and from other cardiac regions in both
Scn5a+/−and WT.
WT hearts showed low incidences of arrhythmia, even
when subjected to PES protocols or with flecainide or
quinidine treatment. In contrast, Scn5a+/−hearts showed
markedly greater arrhythmic tendencies. In both cases, Fisher
exact tests excluded significant differences in arrhythmia in-
cidence between cardiac region or stimulus protocol (regular
pacing, S1S2 or dynamic pacing protocols: see Methods).
These results were therefore combined for Chi-squared test-
ing sorted by genotype and drug condition (Table 1). WT
hearts showed only small, insignificant increases in their low
incidence of arrhythmogenesis with either drug treatment.
However, flecainide and quinidine exerted contrasting effects
in the Scn5a+/−hearts through all cardiac regions studied
and protocols employed. Scn5a+/−hearts showed markedly
increased arrhythmogenicity with flecainide, while quinidine
reduced arrhythmic incidence in Scn5a+/−hearts to levels
1156 Journal of Cardiovascular Electrophysiology Vol. 21, No. 10, October 2010
Figure 2. (A) MAP trace from a typical Scn5a+/−heart, showing measurement of activation latency from stimulus to peak amplitude of the MAP. (B)
Activation latencies in the RV and LV epicardium from WT and Scn5a+/−hearts, before and after the addition of flecainide or quinidine. Open symbols
denote LV; filled symbols denote RV. Twelve WT and 12 Scn5a+/−hearts were used, with 6 of each exposed to each drug. t-tests comparing values before
and after drug have significant values (with P <0.05) denoted by ∗.t-tests comparing WT and Scn5a+/−have significant values denoted by †.The same
significant results occurred both in RV and LV, therefore only one set of symbols is shown.
statistically indistinguishable from those normally observed
in WT.
Scn5a+/−Hearts Show Slowed Conduction, Which is
Exacerbated by Both Flecainide and Quinidine
In comparison with WT hearts, Scn5a+/−hearts showed
longer activation latencies, corresponding to slowed conduc-
tion (Fig. 2). However, this effect was not localized to the RV
epicardium, and was also true to the same extent in the LV.
Both flecainide and quinidine increased activation latencies
to similar extents (t-test: P =0.09) in both the LV and RV of
both WT and Scn5a+/−hearts.
Scn5a+/−and WT Hearts Show Regional Differences in
AP Waveforms
We then undertook a systematic comparison of epicar-
dial and endocardial MAP waveforms in both RV and LV.
Figure 3A–D shows typical MAP waveforms in a typical
Scn5a+/−heart, overlaid to demonstrate the contrasts in LV
and RV transmural (Fig. 3A,B) and epicardial and endocar-
dial transventricular gradients (Fig. 3C,D). Figure 3E shows
typical RV epicardial and endocardial MAPs from a WT heart
for comparison, while Figure 3F overlays WT and Scn5a+/−
RV epicardial MAPs. Figure 4A shows the 4 areas of a typi-
cal Scn5a+/−heart, with APD70 values for each area shown
in normal type. Arrows between the regions show the direc-
tion of each gradient, with its magnitude in italic type. Fig-
ure 4B shows APD70s for WT and Scn5a+/−hearts recorded
from the 4 examined ventricular regions and (C) the result-
ing gradients between them expressed as APD70s across
neighboring regions. Thus,
•Both untreated WT and Scn5a+/−hearts showed signif-
icant repolarization gradients varying between different
cardiac regions. These gradients were particularly strong
at earlier (i.e., 70% repolarization) phases of the AP recov-
ery and arose from the following findings of the individual
MAP durations. Endocardial MAP durations were very
similar in the LV and RV. In contrast, epicardial MAP du-
rations in the RV were shorter than those in the LV of
both WT and Scn5a+/−hearts. Finally, both the RV and
LV endocardial MAPs were longer than their correspond-
ing epicardial MAPs, giving statistically significant differ-
ences in the RV in both WT and Scn5a+/−hearts. These
differences resulted in RV transmural gradients that were
significantly greater than the corresponding LV gradients,
particularly in the Scn5a+/−hearts.
•Scn5a+/−hearts showed generally shorter APDs than
WT. Although this was true of all ventricular regions, this
only reached statistical significance in the RV epicardium.
This resulted in significantly greater RV transmural gradi-
ents, as reflected in the APD50 ,APD70 , and APD90
values in Scn5a+/−hearts compared with WT.
Effects of Flecainide and Quinidine on AP Waveforms in
Murine Scn5a+/−and WT Hearts
The findings above implicate the RV in the arrhythmo-
genicity shown by Scn5a+/−through accentuated RV trans-
mural repolarization gradients, brought about by shortened
RV epicardial APDs. This hypothesis could be tested by
assessing the extent to which the actions of flecainide and
quinidine upon these gradients in Scn5a+/−compared with
WT paralleled their respective pro- and antiarrhythmic ac-
tions in Scn5a+/−.
The resulting experiments split the WT and Scn5a+/−
hearts into age and sex matched groups for studies before and
following treatment with 10 μM flecainide or 5 μM quinidine
(n =6 hearts in each group). Figure 5 shows MAP wave-
forms from RVs of the same WT and Scn5a+/−hearts as in
Figure 3, now following introduction of (A, B) flecainide
or (C, D) quinidine, overlaying epicardial and endocardial
MAPs to illustrate how both drugs affect the RV transmu-
ral repolarization gradients. Then shown are (E) APD70s
from the RV epicardium, and (F) the resultant RV trans-
mural APD70 s for WT and Scn5a+/−hearts before and
after drug treatment.
These demonstrated the following:
•In WT hearts, flecainide shortened the APD values in all 4
ventricular regions investigated, but doing so significantly
only for RV epicardium. This resulted in increases in the
RV transmural gradients, but which only reached signifi-
cance for the APD90 .
•In WT hearts, quinidine increased the APD values in all
the ventricular regions studied, but did so most noticeably
Martin et al. Repolarization Gradients in Brugada Syndrome 1157
Figure 3. Typical MAP waveforms under 8 Hz pacing in (A)–(D) Scn5a+/−
hearts, with traces overlaid to demonstrate the different resultant gradients:
(A) LV epicardial and LV endocardial, (B) RV epicardial and RV endo-
cardial, (C) LV and RV epicardial, (D) LV and RV endocardial. (E) RV
epicardial and endocardial MAP waveforms in a typical WT heart and (F)
RV epicardial MAPs compared between typical WT and Scn5a+/−hearts.
The vertical arrow in each trace marks the pacing stimulus, which appears
as an artifact on the trace.
in the RV epicardium, reaching significance here for all 3
repolarizations. The RV transmural gradient was reduced,
but again only reaching significance for the APD90.
•In Scn5a+/−hearts, flecainide shortened the APD values
in all the ventricular regions studied. Again, this effect
was most noticeable in the RV epicardium. This resulted
in marked increases in the RV transmural gradient whether
measured as APD50 ,APD70 ,orAPD90 . There were
no significant alterations in either the transventricular or
the LV transmural gradient.
•In contrast, quinidine lengthened the APDs in all ventric-
ular regions in Scn5a+/−hearts, again reaching signifi-
cance specifically in the RV epicardium. These changes
markedly decreased the RV transmural gradients to levels
similar to that seen in WT before pharmacological treat-
ment, but did not significantly reduce either the transven-
tricular or LV transmural gradients.
The pharmacological maneuvers thus exerted similar pat-
terns of effects upon Scn5a+/−as WT hearts, particularly
involving the RV epicardium, with quinidine reducing, and
flecainide increasing, the RV transmural gradients. However,
the latter maneuvers acted upon a situation of already accen-
Figure 4. (A) Four chambers of a typical Scn5a+/−heart are displayed
diagrammatically: left epicardium, right epicaridum, left endocardium, and
right endocardium, with their respective APD70 values in normal font. Ar-
rows between the 4 areas depict the gradients between them, with APD70
values in italic font. Data are expressed in ms as mean +/−SEM. Thus the
gradients are calculated: LV transmural =LV endocardial APD—LV epi-
cardial APD; RV transmural =RV endocardial APD—RV epicardial APD;
epicardial transventricular =LV epicardial APD—RV epicardial APD; en-
docardial transventricular =LV endocardial APD—RV endocardial APD.
(B) APD70 values obtained from the 4 ventricular regions and (C) gradients
for WT and for Scn5a+/−hearts at the APD70 level. Twelve WT and 12
Scn5a+/−hearts were used. t-tests comparing APD70 values between car-
diac regions within both WT and Scn5a+/−hearts have significant values
(with P <0.05) denoted by †.t-tests for both APD70 and APD70 values
comparing WT and Scn5a+/−have significant values denoted by ∗.
tuated right transmural gradients in the Scn5a+/−, thereby
giving very strongly positive RV transmural gradients.
Discussion
The BrS has been associated with a heterogeneous group
of genotypes; nevertheless, ∼15% of BrS patients do show
a loss-of-function Na+channel mutation. This prompted the
recent development and use of a Scn5a+/−murine genetic
model in studies of the BrS condition. Having first con-
firmed arrhythmogenicity in the Scn5a+/−hearts, we sys-
tematically investigated arrhythmic incidence in all 4 car-
diac regions with 3 different stimulation protocols for the
first time. We demonstrated that this model reproduces the
clinical BrS condition by displaying ventricular arrhythmias
that were exacerbated by flecainide, in line with its clinical
effects in unmasking ventricular arrhythmogenesis in other-
wise asymptomatic BrS patients,8and reduced by quinidine,
in line with its protective action on symptomatic BrS.9Equal
arrhythmia incidence in all 4 cardiac regions is consistent
with arrhythmic activity arising in any one cardiac region
1158 Journal of Cardiovascular Electrophysiology Vol. 21, No. 10, October 2010
Figure 5. (A)–(D) MAP waveforms from RVs of (A), (C) WT and (B), (D)
Scn5a+/−hearts subject to 8 Hz pacing following introduction of (A),
(B) flecainide and (C), (D) quinidine. (E) APD70 measurements in the RV
epicardium and (F) RV transmural gradients for WT and Scn5a+/−hearts
before and following addition of either flecainide or quinidine. Six hearts
were used in each group. t-tests comparing values before and after drug
have significant values denoted by ∗.
ultimately spreading throughout both ventricles, therefore
involving the entire heart. That stimulation protocol did not
significantly affect arrhythmogenic incidence is consistent
with clinical observations that the inducibility of VT during
PES fails to predict sudden cardiac death in BrS patients.15
The differences in arrhythmic properties could be asso-
ciated with both delayed epicardial activation latencies and
increased right transventricular repolarization gradients in
Scn5a+/−hearts. Such findings complement previous re-
ports of Scn5a+/−myocytes having Na+current amplitudes
reduced to ∼50% of the levels shown by WT, as well as
slowed AP conduction.6Our study supports earlier work
measuring regional APDs in canine16 and rat17 WT sys-
tems, as well as molecular and cellular studies of regional
ion channel location.18-21 The present results also extend
findings of altered repolarization gradients in canine wedge
preparations that mimic BrS by pharmacological rather than
genetic means.22 However, our study is the first using a ge-
netic model for BrS and in which all 4 cardiac regions have
been systematically compared.
Comparison of such altered conduction and repolariza-
tion properties through the epicardia and endocardia of the
right and left ventricles demonstrated that while both slowed
conduction and accentuated repolarization gradients could
potentially contribute to the arrhythmogenic mechanism in
BrS, it was only the repolarization abnormalities that could
be localized to the RV epicardium. Thus, while conduction
slowing may provide a background arrhythmic substrate, the
RV origin of the arrhythmias in clinical BrS suggests that
the heightened repolarization gradients in this region may
be the specific trigger. These findings also shed light on the
pathophysiology of ST elevation seen in BrS, for which 2
mechanisms have been proposed: either it is due to the dif-
ference in AP morphology between the RV epicardium and
endocardium,23 or it is secondary to conduction slowing.24
The localization of the enhanced repolarization gradients to
the RV may suggest that the physiological mechanism un-
derlying ST elevation is also more closely related to a repo-
larization disorder than a delay in depolarization.
The increase in both latencies and transmural gradients
produced by flecainide correlates with its proarrhythmic ef-
fects in both the model and clinical situations. The former
finding parallels the clinical findings implicating slowed con-
duction in the RV outflow tract in Brugada patients2that is
exacerbated with class 1C drug challenge.3The latter result
supports findings in canine models, where high flecainide
concentrations result in loss of the AP dome and marked
AP abbreviation,25 and clinical studies demonstrating that
pilsicainide administration in BrS patients triggers T wave
alternans, which is known to reflect enhanced spatial and
temporal dispersion of repolarization.26 However, whereas
the action of flecainide on activation latencies occurred in
both the RV and the LV, its action on transmural repolar-
ization gradients was again significant specifically for the
RV.
The increase in conduction delay produced by quinidine
was not unexpected given its effect in blocking Na+chan-
nels, but is in contrast to its effect in decreasing arrhythmic
incidence in murine Scn5a+/−and human BrS hearts. On
the other hand, its action in reducing transmural repolar-
ization gradients correlated with its antiarrhythmic effects.
Quinidine has correspondingly been shown to reverse the
associated electrocardiographic abnormalities and prevent
phase 2 reentry and polymorphic VT in experimental ca-
nine BrS models,22 and normalize the ST segment in clinical
circumstances.27,28
The main limitations of the study are associated with the
differences between mouse and human physiology. The small
size of the mouse heart means that strong electrotonic forces
may act to minimize the effect of any spatial heterogeneities
that are created and reduce their potential to create reentrant
substrate. The AP morphology differs between humans and
mice, as mice use less L-type Ca channel current,which
means that the mouse AP does not have a plateau phase
and has a shorter APD.29 This means that the spike and
dome morphology present in larger mammals is not present
in our mouse model. However, despite these difficulties, we
were able to demonstrate a clear propensity to arrhythmia in
Martin et al. Repolarization Gradients in Brugada Syndrome 1159
our BrS mouse model and correlate this with enhanced RV
transmural gradients.
Conclusion
In summary, this study has for the first time in a geneti-
cally modified BrS model assessed activation latency, APDs
and derived AP repolarization gradients from 4 regions of
the heart under a range of stimulation protocols and a range
of pharmacological conditions. The experiments correlate
the increased arrhythmogenicity in Scn5a+/−hearts with
both increased activation latency and decreased RV epicar-
dial APD and increased RV APDs. However, while con-
duction delay was not localized specifically to the RV and
was increased by both flecainide and quinidine, the respective
pro- and antiarrhythmic effects of flecainide and quinidine
could be directly correlated with their actions in increasing
and decreasing RV APDs. Thus, these findings, while not
excluding a contribution of conduction delay, support most
closely an arrhythmogenic trigger based on reentry from ab-
normal repolarization gradients between RV epicardial and
endocardial sites.
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