![nvestigation of the action potential (AP) phenotype in single cell simulations. ( A ) Boundaries between the spike and dome and premature repolarization phenotypes in the ([K þ ] o , g to ) parameter space for the WT, HZ and C1850S genotypes. Combinations of [K þ ] o and g to below the corresponding curves produce](https://www.researchgate.net/profile/Juerg-Schlaepfer/publication/5599024/figure/fig3/AS:277853557018634@1443256858429/nvestigation-of-the-action-potential-AP-phenotype-in-single-cell-simulations-A_Q320.jpg)
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Analyses of a novel SCN5A mutation (C1850S):
conduction vs. repolarization disorder hypotheses
in the Brugada syndrome
Se
´
verine Petitprez
1
†
, Thomas Jespersen
1
†
, Etienne Pruvot
2
, Dagmar I. Keller
3
, Cora Corbaz
2
,
Ju
¨
rg Schla
¨
pfer
2
, Hugues Abriel
1,2
*
‡
, and Jan P. Kucera
4
*
‡
1
Department of Pharmacology and Toxicology, University of Lausanne, 27, Bugnon, 1005 Lausanne, Vaud, Switzerland;
2
Service of Cardiology, CHUV, Lausanne, Switzerland;
3
Department of Cardiology, University Hospital, Basel, Switzerland;
and
4
Department of Physiology, University of Bern, Bu
¨
hlplatz 5, 3012 Bern, Switzerland
Received 28 March 2007; revised 28 January 2008; accepted 31 January 2008; online publish-ahead-of-print 5 February 2008
Time for primary review: 29 days
Aims Brugada syndrome (BrS) is characterized by arrhythmias leading to sudden cardiac death. BrS is
caused, in part, by mutations in the SCN5A gene, which encodes the sodium channel alpha-subunit
Na
v
1.5. Here, we aimed to characterize the biophysical properties and consequences of a novel BrS
SCN5A mutation.
Methods and results SCN5A was screened for mutations in a male patient with type-1 BrS pattern ECG.
Wild-type (WT) and mutant Na
v
1.5 channels were expressed in HEK293 cells. Sodium currents (I
Na
) were
analysed using the whole-cell patch-clamp technique at 378C. The electrophysiological effects of the
mutation were simulated using the Luo-Rudy model, into which the transient outward current (I
to
)
was incorporated. A new mutation (C1850S) was identified in the Na
v
1.5 C-terminal domain. In
HEK293 cells, mutant I
Na
density was decreased by 62% at 220 mV. Inactivation of mutant I
Na
was accel-
erated in a voltage-dependent manner and the steady-state inactivation curve was shifted by 11.6 mV
towards negative potentials. No change was observed regarding activation characteristics. Altogether,
these biophysical alterations decreased the availability of I
Na
. In the simulations, the I
to
density necess-
ary to precipitate repolarization differed minimally between the two genotypes. In contrast, the
mutation greatly affected conduction across a structural heterogeneity and precipitated conduction
block.
Conclusion Our data confirm that mutations of the C-terminal domain of Na
v
1.5 alter the inactivation of
the channel and support the notion that conduction alterations may play a significant role in the patho-
genesis of BrS.
KEYWORDS
Brugada syndrome;
Sodium channel;
Genetics;
Electrophysiology;
Computational analysis
1. Introduction
Brugada syndrome (BrS) is a disorder characterized by ST
segment elevation in the right precordial leads V1
–
V3 on
the surface ECG, with atypical right bundle branch block
pattern, and an increased risk for sudden cardiac death
(SCD).
1
The BrS is a primary electrical cardiac disorder,
which is inherited as an autosomal dominant trait. Many
mutations in SCN5A, encoding the a-subunit of the voltage-
gated sodium channel Na
v
1.5, have been identified,
2
though
only 10 to 30% of clinically affected individuals carry a
mutation in this gene.
3
Among the 90 mutations reported
thus far,
2
only a few of them have been characterized at the
molecular and biophysical level using cellular expression
systems.
1
Most of SCN5A mutations lead to a ‘loss-
of-function’ by reducing the sodium current (I
Na
) available
during the phases 0 (upstroke) and 1 (early repolarization)
of the cardiac action potential (AP).
1
Despite more than 10
years of research in this field, the molecular and cellular
mechanisms leading to the BrS are not yet completely
understood.
1
Interestingly, in some patients, BrS is con-
cealed on the surface ECG and can be unmasked using
sodium channel blockers
4
or during fever episodes.
5
Here, we screened SCN5A in a patient with typical clinical
manifestations of BrS. A novel mutation, C1850S, located
in the C-terminal domain of Na
v
1.5 was identified.
*
Corresponding author. Tel: þ41 21 692 5364; fax: þ41 21 692 5355 (H.A);
Tel: þ41 31 631 87 59; fax: þ41 31 631 4611 (J.P.K.).
E-mail addresses: hugues.abri el@unil.ch (H.A.); kucera@pyl.unibe.ch
(J.P.K.)
†
These authors contributed equally to this study.
‡
These authors share the last authorship of this article.
Published on behalf of the European Society of Cardiology. All rights reserved. & The Author 2008.
For permissions please email: journals.permissions@oxfordjournals.org.
Cardiovascular Research (2008) 78, 494
–
504
doi:10.1093/cvr/cvn023
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When mutant channels were expressed in HEK293 cells, the
peak I
Na
density was decreased by 62% (at 220 mV) and fast
I
Na
inactivation was accelerated.
Computer simulations were conducted using the Luo-Rudy
(LRd) model to examine the interaction of I
Na
and the tran-
sient outward current (I
to
), proposed to determine early
repolarization in the right epicardium.
6
The I
to
density
necessary to precipitate repolarization was similar in the
presence of wild-type (WT) and mutant I
Na
; however, our
computational analyses suggest that conduction in structu-
rally discontinuous tissue is prone to block and much more
sensitive to I
to
in the presence of the mutation at the het-
erozygous state. Moreover, these manifestations are modu-
lated by the extracellular concentration of K
þ
. Our data
confirm that mutations of the C-terminal domain of Na
v
1.5
alter the inactivation properties of the channel and
support the notion that conduction disturbances may be
involved in the pathogenesis of BrS.
2. Methods
2.1 Molecular screening
Genomic DNA was extracted from peripheral lymphocytes, and all
coding exons of SCN5A were amplified by polymerase chain reaction,
using primers designed in intronic flanking sequences according to
the gene sequences.
7
Denaturating high performance liquid chrom-
atography (DHPLC) was performed on DNA-amplification products on
at least one temperature condition. Abnormal DHPLC profiles were
analysed by sequence reaction in both strands of the exon, using
Big Dye termination mix, and analysed by cycle sequencing on an
automated laser fluorescent DNA sequencer (ABI prism 3100,
Applied Biosystems). The novel mutation C1850S was absent in
200 normal alleles. The investigation conformed with the principles
outlined in the Declaration of Helsinki. The index patient gave
written informed consent to participate in the study.
2.2 Electrophysiology
SCN5A mutation was engineered into WT cDNA (clone hH1a received
from Dr R. Kass) cloned in pcDNA3.1 (Invitrogen), using the Quick-
Change Kit (Stratagene) and verified by sequencing. For electro-
physiology studies, HEK293 cells were transiently transfected with
0.6 mg of WT or mutant Na
v
1.5 construct cDNAs or 0.3 mg of each.
All transfections included 2.0 mg pIRES-hb1-CD8 cDNA encoding
hb1 subunit and CD8 antigen as a reporter gene. Cells were trans-
fected using calcium phosphate or Lipofectamine
w
and I
Na
were
measured after 48 h. Anti-CD8 beads (Dynal) were used to identify
transfected cells. Patch-clamp recordings were conducted using
an internal solution containing (mmol/L) CsCl 60; CsAspartate 70;
EGTA 11; MgCl
2
1; CaCl
2
1; HEPES 10; and Na
2
-ATP 5, pH 7.2 with
CsOH; external solution NaCl 130; CaCl
2
2; MgCl
2
1.2; CsCl 5;
HEPES 10; and glucose 5, pH 7.4 with CsOH. Using these solutions,
5 min after rupturing the membrane, we observed no significant
alteration of the availability curve and the peak current. Peak cur-
rents were measured during an inactivation protocol and I
Na
den-
sities (pA/pF) were obtained by dividing the peak I
Na
by the cell
capacitance obtained from the pClamp function (pClamp suite,
v.8, Axon Instruments, CA, USA). For the activation and steady-state
inactivation curves, data from individual cells were fitted with
Boltzmann relationship, y(V
m
) = 1/{1 þ exp [(V
m
2V
1/2
)/K]}, in
which y is the normalized current or conductance, V
m
is the mem-
brane potential, V
1/2
is the voltage at which half of the channels
are activated or inactivated, and K is the slope factor. Recovery
from inactivation curves were fitted individually with a mono-
exponential relationship. I
Na
were measured using a VE-2 amplifier
(Alembic Instruments, Montre
´
al, QC) allowing a full compensation
of the series resistance, and were analysed using the pClamp soft-
ware suite. No correction of liquid junction potentials was per-
formed. All measurements were carried out at 37 + 18C using a
control system (Cell MicroControls) heating the perfusion solution.
2.3 Biochemistry
Western blotting conditions have been described previously.
8
The anti-Na
v
1.5 affinity purified antibody recognizes residues 493
–
511 of rat Na
v
1.5 (ASC-005, Alomone). These residues are
identical in the human sequence. The anti-actin antibody was
from Sigma.
2.4 Statistics
Data are presented as means + SEM. The two-tailed Student t-test
or the ANOVA test with the Bonferroni correction were used to
compare means; P , 0.05 was considered significant.
2.5 Mathematical modelling of I
Na
, the action
potential, and conduction
The LRd model of the ventricular epicardial cell
9
was used to recon-
struct I
Na
under voltage clamp conditions as well as the AP of the
epicardial myocyte. The model incorporated the updates published
by Faber and Rudy
10
as well as the ATP-sensitive K
þ
current as
described by Shaw and Rudy.
11
The transient outward K
þ
current
I
to
was introduced into the model according to the formulation of
Dumaine et al.
12
To account for the important heterogeneity of I
to
expression between the left and right ventricles and across the myo-
cardial wall, simulations were run over a broad range of I
to
maximal
conductance (g
to
, from 0 to 4 mS/mF).
In the LRd model, I
Na
is formulated as I
Na
= g
Na
.
m
3
hj(V
m
2E
Na
),
where g
Na
is the maximal conductance of I
Na
(16 mS/mF), m, h,
and j are the activation, fast inactivation, and slow inactivation
gating variables, respectively, and E
Na
is the Na
þ
Nernst potential.
The effects of the C1850S mutation (CS) were simulated by
modifying
b
h
, (the closing rate of the fast inactivation gate h)ina
voltage-dependent manner consistent with our experimental
findings to account for (i) the decrease of peak I
Na
by 62% at V
m
= 220 mV; (ii) the shift of the V
1/2
of steady-state inactivation
by 11.6 mV towards more negative potentials; and (iii) the voltage-
dependent decrease of the time constant of fast I
Na
inactivation for
V
m
240 mV.
The WT and CS formulations of
b
h
are as follows:
WT (Original formulation of the LRd model):
ForV
m
, 40mV :
b
h;WT
¼ 3:56 expð0:079V
m
Þþ3:1 10
5
expð0:35V
m
Þ:
For V
m
40 mV :
b
h;WT
¼ 1=ð0:13 ð1 þ expððV
m
þ 10:66Þ=ð11:1ÞÞÞÞ:
CS:
For V
m
, 40 mV :
b
h;CS
¼ 2:8606 ðexpð0:0672 ðV
m
þ 40ÞÞÞ:
For V
m
40 mV :
b
h;CS
¼
b
h;WT
ð1:5 þ 4:1 expððV
m
þ 40Þ=16:8ÞÞ:
The opening rate constant
a
h
of gate h as well as the rates of the
m and j gates were not modified. To simulate heterozygote (HZ)
cells (or I
Na
in cells transfected with equal amounts of WT
and mutant cDNA), g
Na
was split into two equal components of
8 mS/mF each corresponding to the two SCN5A alleles (I
Na
=
I
Na,WT
þI
Na,CS
).
Further details concerning the modelling approach are provided in
the Supplementary material online.
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3. Results
3.1 Index case
The index case was a 55-year old Caucasian male seen at our
cardiology clinic (Lausanne, Switzerland) for investigation of
a typical type-1 BrS ECG pattern recorded during cystitis
with high fever. However, the ECG remained abnormal
after fever (Figure 1A). The patient never had syncope or
palpitations but his mother died suddenly at age 35. The
echocardiography was normal. An electrophysiological
study was conducted using two quadripolar leads. Premature
ventricular stimulation was performed at cycle length of 600
and 400 ms followed by one and then two premature beats.
Ventricular fibrillation was reproducibly inducible at a drive
train of 400 ms followed by two premature beats at a coup-
ling interval of 240 and 200 ms, respectively. Sinus rhythm
was restored by an external biphasic shock of 120 J. No
ajmaline test was performed. An internal cardioverter-
defibrillator was implanted.
3.2 Identification and character ization of the
SCN5A mutation
A heterozygous G to C substitution at position 5549 of SCN5A
resulted in a C1850S mutation in the C-terminal tail of
Na
v
1.5 (Figure 1B). In HEK293 cells transiently transfected
with WT and mutant cDNAs, Western blot analyses showed
that the level of protein expression of C1850S mutant chan-
nels was comparable with that of WT (Figure 1C). In con-
trast, using the patch-clamp technique in the whole-cell
configuration, at 378C, C1850S channels generated I
Na
with
biophysical properties that were clearly different compared
with WT currents. First, the peak I
Na
density mediated by
C1850S channels was reduced by 62 + 5% at 220 mV
(Figure 1D). When cells were co-transfected with equal
amounts of WT and mutant cDNAs to mimic the heterozygous
status of the patient, I
Na
density was intermediate (Figure
1D). Furthermore, as illustrated in Figure 2A, C1850S cur-
rents were almost completely inactivated 2 ms after the
voltage step (arrows), while WT currents were still substan-
tial. This indicates that C1850S I
Na
inactivated faster com-
pared with WT I
Na
. Figure 2B shows simulated current
traces generated by the original I
Na
formulation of the LRd
model (WT) and by the formulation in which the rate of
fast inactivation
b
h
was modified. Acceleration of fast inac-
tivation without modifying other gating kinetics and without
modifying I
Na
conductance resulted in a reduction of peak I
Na
by 63% (at 220 mV) and almost complete inactivation after
2 ms, similar to the experimental findings.
In HEK293 cells, the V
1/2
of voltage-dependence of
steady-state inactivation of C1850S was shifted towards
more negative values by 11.6 mV (Table 1 and Figure 3A).
No significant alteration in the activation curve V
1/2
was
observed. When HEK293 cells were transfected with equal
amount of WT and mutant cDNA, the shift of the
steady-state inactivation curve was intermediate (Figure
3A and Table 1). In the I
Na
model, the shift of the
steady-state inactivation curve towards more negative
potentials without alterations in recovery from fast inacti-
vation (discussed later) was simulated by increasing the
fast inactivation rate
b
h
in a voltage-dependent manner
while leaving
a
h
unchanged (see Methods).
No change in the kinetics of recovery from fast inacti-
vation was seen (Figure 4A). In the mathematical model of
I
Na
(Figure 4B), modification of the rate
b
h
did not alter
recovery from inactivation.
As illustrated in Figure 2A, fast inactivation is accelerated
for the currents mediated by mutant channels compared
with WT (arrows in Figure 2A). Individual inactivating
current traces were fitted using a mono-exponential func-
tion and the resulting time constants (
t
) were plotted as a
function of the different voltages (Figure 4C). At voltages
more negative than 5 mV, the mutant
t
values were signifi-
cantly shorter compared with WT. In Figure 4D, the same
analysis is presented for the model WT and mutant I
Na
.
The voltage-dependent behaviour of the
t
values in the
model is comparable with the behaviour observed for the
experiments. In transfected HEK293 cells, we also tested
the entry into the intermediate inactivated state,
13
but no
difference was observed (see Supplementary material
online, Figure S1).
3.3 Action potential simulations
It has been postulated that the clinical manifestations of BrS
are related to the propensity of right epicardial tissue to
premature repolarization, which can explain ST-segment
elevation in the right precordial leads and phase 2
re-excitation by deeper layers of myocardium.
6
This suscep-
tibility of the right epicardial AP to lose its dome is
explained by the predominance of outward currents over
inward currents during phase 1 (notch). During this phase,
the balance of transmembrane currents is principally deter-
mined by the inactivating I
Na
, the activating L-type Ca
2þ
current, and the rapidly activating transient outward K
þ
current, I
to
. Thus, under conditions of high levels of I
to
as
encountered in the right epicardium, the loss of the depolar-
izing contribution of I
Na
caused by its accelerated
Table 1 Biophysical properties of I
Na
recorded in HEK293 cells transfected with wild-type (WT), C1850S, or
1
/
2
WTand
1
/
2
C1850S cDNA. For
the activation and steady-state inactivation curves (Figure 3A), data points from individual cells were fitted with Boltzmann relationship,
and the V
1/2
and K values of the different conditions are presented; *P , 0.05, ***P , 0.001 vs. WT, Bonferroni correction after ANOVA test
Steady state V
1/2
inactivation (mV)
Slope factor: K
inactivation (mV)
V
1/2
activation
(mV)
Slope factor: K
activation (mV)
Time to half recovery from
fast inactivation (ms)
WT 278.6 + 0.8 (n = 9) 6.4 + 0.4 (n =9) 236.9 + 1.4 (n = 7) 7.0 + 0.4 (n = 7) 3.4 + 0.3 (n = 13)
C1850S 290.2 + 0.5*** (n = 7) 5.9 + 0.4 (n =7) 235.5 + 1.3 (n = 5) 7.4 + 0.4 (n = 5) 3.8 + 0.4 (n = 15)
1
/
2
WT þ
1
/
2
C1850S
283.8 + 1.8* (n = 9) 6.9 + 0.3 (n =9) 233.6 + 1.3 (n = 8) 6.4 + 0.6 (n = 8) 3.5 + 0.6 (n =8)
S. Petitprez et al.496
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inactivation can promote early repolarization.
14
In
addition, it was reported that both hyper- and
hypokalemia could precipitate the clinical manifestations
of BrS.
15,16
This background motivated us to investigate the conse-
quences of the C1850S mutation on the epicardial AP as a
function of the level of I
to
expression and of extracellular
potassium concentration ([K
þ
]
o
). Single cell AP simulations
were conducted using the LRd model. For every [K
þ
]
o
tested (2.5 to 12.0 mmol/L in steps of 0.5 mmol/L), we
determined the critical value of g
to
above which the AP
loses its dome-shaped plateau and repeated this approach
for the WT, HZ, and homozygote C1850S (CS) genotypes.
As shown in Figure 5A, the critical g
to
was, as expected,
the largest for the WT, the smallest for the CS and inter-
mediate for the HZ over the entire range of [K
þ
]
o
tested.
Interestingly, there was a biphasic dependence of critical
g
to
on [K
þ
]
o
for all three genotypes with a maximum near
[K
þ
]
o
= 6 mmol/L. Figure 5B illustrates this loss of the AP
dome with increasing levels of I
to
at a normal [K
þ
]
o
of
4 mmol/L. Increasing g
to
led first to a loss of the dome in
the CS (b), then in the HZ (c) and finally in the WT (d).
Thus, accelerated inactivation of I
Na
may lead to premature
epicardial repolarization under conditions of elevated I
to
expression, which, in vivo, can be encountered in the right
ventricular epicardium.
17
However, the critical g
to
values
Figure 1 Electrocardiogram of the index case, mutations in Na
v
1.5 C-terminus, and expression of wild-type (WT) and C1850S Na
v
1.5 channels. (A) ECG of the
index case at baseline without fever. ( B) Membrane topology of Na
v
1.5. Location of the novel C1850S mutation (black circle) and five BrS mutations located in the
C-terminus (white circles). (C ) Western blot of HEK293 cell lysates expressing WT, C1850S, and 50/50% WT/C1850S channels. Controls are non-transfected cells.
(D) I
Na
density at 378C of HEK293 cells transiently transfected with WT, 50/50% WT/C1850S and C1850S cDNA; n = 14, 10, 14 cells, respectively; ***P , 0.001. n.s.
not significant vs. 50/50% WT/C1850S.
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differed only by a few percent between WT and CS, and the
difference between WT and HZ conditions was ,1% for all
values of [K
þ
]
o
.
It is well known that the clinical manifestations of BrS are
rate-dependent.
18
Moreover, AP characteristics during
pacing at a physiological rate differ from those of an isolated
AP elicited in a resting cell. Therefore, to evaluate the
behaviour of the model cell paced at a physiological rate,
we conducted simulations in which trains of stimuli were
applied during 30 s following the initial 1-min period
without pacing. Figure 5C compares APs in the simulated
WT, HZ and C1850S mutant cells during 30 s of pacing at a
rate of 1 Hz, for [K
þ
]
o
= 4 mmol/L and with g
to
= 1.315 mS/
mF. This value of g
to
was inferior to the thresholds at
which premature repolarization occurred for the isolated
AP (see Figure 5A). While no premature repolarization
occurred during the entire train in the WT cell, premature
repolarization occurred for the second last AP of the train
in the HZ cell (asterisk). In the C1850S cell, premature repo-
larization occurred earlier in the train. When g
to
was sub-
stantially increased (.1.5 mS/mF, not shown), the APs
exhibited an irregular sequence of spike and dome and pre-
mature repolarization morphologies for all three genotypes
and the average rate at which premature repolarization
occurred was not manifestly different.
We then extended the investigation of the critical g
to
level
leading to premature repolarization to the situation of the
paced cell. The critical level was defined as the boundary
between the complete absence of premature repolarization
during the 30-s train and the presence of at least one AP
exhibiting premature repolarization. These simulations
were conducted at pacing rates of 1 and 2 Hz (Figure 5D
and E, respectively). For all three genotypes, the boundaries
exhibited a similar shape as in the single AP simulations but
were shifted towards smaller values of g
to
. This shift was
more prominent at 2 Hz than at 1 Hz. Nevertheless, for
both pacing rates and for all values of [K
þ
]
o
, the critical
g
to
values differed only by a few percent (,1% between
WT and HZ), similar to the single AP simulations.
3.4 The C1850S mutation causes conduction block
in branching tissue
It has recently been proposed that right ventricular fibrosis
may contribute to the genesis of conduction disorders
Figure 2 Wild-type (WT) and C1850S currents recorded in HEK293 cells and
their simulations. (A) Current traces recorded at 378C from cells expressing
WT or C1850S channels in response to a series of 10-ms pulses (inset).
Arrows at 2 ms after onset of the voltage-step indicate the faster inactivation
of C1850S I
Na
compared with WT. The fast transient capacitive current is
blanked for sake of clarity. (B) Reconstruction of WT and C1850S currents
using the original I
Na
of the Luo-Rudy model (WT) and the I
Na
model in
which
b
h
was modified (see ‘Methods’).
Figure 3 Voltage-dependence of activation and inactivation. (A) Activation
(triangles) and inactivation (squares) curves at 378C. Activation properties
were determined from I/V relationships (obtained after applying the step pro-
tocol in inset) by normalizing peak I
Na
to driving force and maximal I
Na
, and
plotting normalized conductance vs. V
m
. Voltage-dependence of steady-state
inactivation was obtained by plotting the normalized peak current (25-ms
test pulse to 220 mV after a 500-ms conditioning pulse, see inserted proto-
col) vs. V
m
. Boltzmann curves were fitted to both activation and steady-state
inactivation data. Averaged values and the number of cells used are pre-
sented in Table 1.(B) Steady-state activation and inactivation curves of the
model I
Na
. These curves were generated using exactly the same voltage-
clamp protocols and analyses as in the experiments.
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leading to the characteristic BrS ECG.
19,20
Fibrous cardiac
tissue is characterized by sparsely connected strands and
sheets of myocardium intermingled with unexcitable tissue
(Figure 6A), and such structures are known to form the sub-
strate of slow conduction or conduction block.
21
Therefore, we investigated the consequences of the
C1850S mutation on conduction characteristics along a
121-cell strand releasing two branches from its centre
(Figure 6A). These simulations were conducted for a single
AP. In the simulation presented in Figure 6B, the branches
were 20 myocytes long. This branch length was several
times the space constant of the strand, which amounted to
533 mm (approximately five cells) at [K
þ
]
o
= 4 mmol/L (the
space constant was not influenced by g
to
or by the I
Na
geno-
type). This simulation corresponds to scarce lateral coupling
between fibres. The branching strand was characterized by a
mismatch between the current generated by the strand
before the branch point and the increased load represented
by the distal segment of the strand and the two branches.
22
Figure 6B shows AP upstrokes, WT and mutant I
Na
currents
as well as I
to
in the vicinity of the branch point for both the
WT and the HZ (g
to
= 0.5 mS/mF, [ K
þ
]
o
= 4 mmol/L). In the
WT, the current-to-load mismatch resulted in slow upstrokes
and a local conduction delay (0.92 ms). In the HZ, the
reduced total I
Na
shifted the current-to-load balance in
favour of the load, which resulted in conduction block.
In Figure 6C, the dependence of the conduction delay and
the occurrence of block were investigated as a function of
g
to
in strands releasing branches of different lengths (20,
10, 7, 5, and 2 myocytes).
In the absence of I
to
(g
to
= 0), conduction was successful
for both genotypes but, irrespective of branch length, the
conduction delay for the HZ was always longer than for
the WT. For branch lengths of 20, 10, 7 and 5 myocytes,
increasing g
to
led to a progressively increasing conduction
delay, until conduction block occurred. The prolongation
of the conduction delay was much more prominent for the
HZ and the level of g
to
at which block occurred was consider-
ably lower. While, for both genotypes, the g
to
level at which
block occurred was almost the same for branch lengths of 20
and 10 myocytes, this g
to
level was larger for shorter
branches. Only when branches were much shorter (two
Figure 4 Recovery from inactivation and onset of inactivation. (A) Recovery from inactivation recorded at 378C (protocol in inset) was characterized by the time
to half recovery (Table 1). Data were fitted using mono-exponentia l functions (solid lines). Averaged values and the number of cells used are presented in Table 1.
(B) Simulated recovery from inactivation, using the same protocol as in A.(C) Onset of inactivation is accelerated for C1850S at 378C. t values were obtained by
fitting the decaying phase of individual current traces (Figure 2A) with a mono-exponential function and plotted vs. V
m
; n = 7 and 5 cells for WT and C1850S,
respectively; *P , 0.05, **P , 0.01, ***P , 0.001. (D) Corresponding values of t for the simulated I
Na
. These values were obtained using the same protocols
and analyses as in C.
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cells) than the space constant, conduction did not fail and
the conduction delay remained small for both the WT and
the HZ.
Thus, successful conduction in the branched HZ strand is
strongly dependent on I
to
and branch length. This contrasts
with the minimal dependence of the velocity of continuous
conduction on I
to
: as illustrated in Figure 6D, increasing g
to
from 0 to 4 mS/mF slowed conduction by a few percent only.
When the local conduction delay across the branch point
was longer than 1 ms, it provided enough time for I
to
to
activate in the cells immediately before the branch point.
The activated I
to
then acted to diminish the amount of
current available to depolarize the cells beyond the
branch point to threshold. When g
to
was increased, it there-
fore resulted in a prolongation of the conduction delay and
precipitated block. In the presence of mutant I
Na
character-
ized by accelerated inactivation, the marked diminution of
I
Na
after 1
–
2ms(Figure 2) further exacerbated these altera-
tions of conduction.
In Figure 6E, the (g
to
,[K
þ
]
o
) parameter space was explored
to identify the combinations of g
to
and [K
þ
]
o
leading to con-
duction block in the HZ but not in the WT (light grey area).
In this series of simulations, branches were 20 myocytes
long. [K
þ
]
o
was varied from 2.5 to 12.0 mmol/L in steps of
0.5 mmol/L, and, for each tested [K
þ
]
o
, the critical value
of g
to
at which conduction block occurred was assessed
with a precision of 0.005 mS/mF. Conduction block in the
HZ but not in the WT was obtained in a wide region in the
parameter space, delimited by bell-shaped boundaries.
Furthermore, for all [K
þ
]
o
, there was a several fold differ-
ence between WT and HZ regarding the critical levels of
g
to
at which block occurred. A qualitatively similar differ-
ence was observed for branches 10, 7, and 5 myocytes
long (data not shown). These findings therefore suggest
that in discontinuous cardiac tissue, mutations of I
Na
exhibit-
ing biophysical characteristics similar to those of the C1850S
mutant result in cardiac excitation that is definitely more
prone to slow conduction and conduction block, the key
ingredients of re-entrant arrhythmogenesis.
21
4. Discussion
In this study, we investigated the biophysical characteristics
of a novel SCN5A BrS mutation, C1850S, at physiological
temperature. In addition, we performed AP and conduction
simulations using the LRd model to investigate the
Figure 5 Investigation of the action potential (AP) phenotype in single cell simulations. (A) Boundaries between the spike and dome and premature repolariza-
tion phenotypes in the ([K
þ
]
o
, g
to
) parameter space for the WT, HZ and C1850S genotypes. Combinations of [K
þ
]
o
and g
to
below the corresponding curves produce
a spike and dome phenotype and combinations above the curves lead to loss of the dome and premature repolarization. The points labelled a
–
d correspond to the
APs shown in B.(B) APs for the three genotypes at a constant [K
þ
]
o
(4 mmol/L) but at increasing levels of g
to
.a:g
to
= 1.450 mS/mF; b: g
to
= 1.515 mS/mF; c: g
to
=
1.535 mS/mF; d: g
to
= 1.600 mS/mF. ( C ) Trains of 30 APs elicited at 1 Hz following a resting period of 1 min, for the three genotypes. [K
þ
]
o
= 4 mmol/L and g
to
=
1.315 mS/mF. Asterisks indic ate premature repolarization. (D) Boundaries between the spike and dome and premature repolarization phenotypes in the ([K
þ
]
o
,
g
to
) parameter space for the three genotypes, for trains of 30 action potentials elicited at 1 Hz. Combinations of [K
þ
]
o
and g
to
above the corresponding curves
lead to loss of the dome and premature repolarization in at least one AP. (E) Same as D, but for trains of 60 APs elicited at 2 Hz.
S. Petitprez et al.500
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mechanisms underlying the arrhythmogenic alterations of
repolarization and conduction. C1850S channels generated
smaller peak I
Na
, and many of the Na
v
1.5 steady-state and
time-dependent inactivation properties were altered.
These alterations significantly reduce I
Na
during the early
phases of the AP. The simulations suggest that at the hetero-
zygous state, the mutation can depress conduction in myo-
cardium exhibiting sites of current-to-load mismatch, as
exemplified in our study by a myocyte strand releasing
branches.
Figure 6 Conduction in a discontinuous structure is more prone to block in the presence of the C1850S mutation. (A) Schematic of discontinuous cardiac tissue
(top) and its representa tion using the Luo-Rudy model as a strand releasing two branches (bottom). Gray areas represent inexcitable clefts (e.g. connective tissue
in fibrosis) between scarcely connected parallel fibres of cardiac myocytes. (B) Simulated action potentials (V
m
), WT and mutant I
Na
as well as I
to
for the wild-type
(WT) and heterozygote (HZ) genotypes. Branches were 20 myocytes long in this simulation. Data are shown for the 10 cells proximal and distal to the branch
point. The bold traces correspond to the cell at the branch point. g
to
= 0.5 mS/mF and [K
þ
]
o
= 4 mmol/L. ( C ) Conduction delay across the branch point for
[K
þ
]
o
= 4 mmol/L, as a function of g
to
for the WT and HZ genotypes. These simulations were conducted with branches 20, 10, 7, 5, and 2 myocytes long
(labels). Round symbols denote the occurrence of conduction block. (D) Conduction velocity in unbranched WT and HZ strands as a function of g
to
([K
þ
]
o
=
4 mmol/L). (E) Boundaries between successful conduction and conduction block in the ([K
þ
]
o
, g
to
) parameter space for the WT and HZ genotypes (branch
length: 20 cells). Combinations of [K
þ
]
o
and g
to
above the corresponding curves lead to conduction block (white area: successful conduction for both genotypes;
light grey area: successful conduction for the WT genotype but block for the HZ genotype; dark grey area: conduction block for both genotypes). The dot corre-
sponds to the simulation represented in B.
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4.1 Heterogeneity of the biophysical properties of
Brugada syndrome Na
v
1.5 channels
Thus far, the cardiac arrhythmias database
2
of the European
Society of Cardiology lists more than 90 SCN5A BrS
mutations. Among them, five are located in the intracellular
C-terminus of Na
v
1.5 (Figure 1B). Functional characteriz-
ation has been performed for only 23 mutations (three of
them are in the C-terminus).
23
The vast majority of these
mutations reduce the I
Na
mediated by Na
v
1.5. However,
the molecular mechanisms underlying this reduction are
very heterogeneous,
23
and most of the mutant channels
have a unique pattern of alterations. One can divide these
mechanisms into (1) alterations leading to a reduced I
Na
membrane density, and (2) changes in biophysical proper-
ties.
23
The mutation found in this BrS patient replaces
Cys-1850 by a Ser located in a highly conserved part of the
C-terminus of Na
v
1.5. It appears that the first 150 residues
of the C-terminus form a highly structured domain compris-
ing six a-helices.
24
Without more knowledge about the
detailed role of this domain, one cannot but speculate
about the consequences of this mutation. The biophysical
alterations induced by C1850S are mainly related to the I
Na
fast inactivation process. The negative shift of the
steady-state availability curve and faster onset of fast
inactivation are both consistent with a ‘stabilization’ of
inactivated states (fast and slow/intermediate) of mutant
channels. Altogether, these findings suggest that Ser-1850,
by enhancing the inactivation process, contributes to a
‘loss-of-function’ of mutant channels. Our simulations
suggest that acceleration of inactivation alone (determined
by the rate
b
h
) can underlie both the acceleration of inacti-
vation and the shift of steady-state inactivation (by render-
ing inactivated states more stable) without influencing the
activation process.
4.2 Action potential and conduction alterations in
the Brugada syndrome
The molecular, cellular, and arrhythmogenic mechanisms
underlying the ECG alterations seen in BrS are still a
matter of debate.
1
According to the ‘repolarization disorder
hypothesis’, re-entrant arrhythmias are caused by a hetero-
geneous loss of the AP epicardial dome leading to epicardial
dispersion of refractoriness, which forms the substrate for
reentry. According to the ‘conduction disorder hypothesis’,
the typical ECG signs can be explained by slow conduction
and activation delays in the right ventricle (in particular in
the right ventricular outflow tract).
Our computational results support the conduction dis-
order hypothesis. On the one hand, loss of the dome was
more prone to occur in the presence of mutant I
Na
in
single cells. However, the level of I
to
at which abrupt repo-
larization occurred was only minimally smaller (1%) for the
HZ compared with the WT. On the other hand, in discontinu-
ous tissue, conduction delays and block were markedly
potentiated by the presence of mutant I
Na
. Interestingly,
the levels of I
to
conductance at which these manifestations
occurred were strikingly different from those necessary to
result in premature repolarization. Increasing g
to
to
1.3 mS/mF or more was necessary to result in a loss of the
dome in the HZ cell. However, in the branched HZ tissue,
a prominent prolongation of the branching-induced local
conduction delay and conduction block were already
observed at g
to
levels ,0.6 mS/mF for branches consisting
of five myocytes or more. In the original formulation of
right ventricular I
to
by Dumaine et al.,
12
a value of
1.1 mS/mF was used. In human right ventricular tissue,
17
maximal peak I
to
was measured to be 9.8 pA/pF (at
þ60 mV at 368C), which translates, assuming a reversal
potential of 290 mV and a maximal channel open prob-
ability of 0.25, to 0.25 mS/mF. Our study thus suggests
that I
to
-mediated premature repolarization would require
a considerable increase in I
to
density, while conduction
alterations in discontinuous tissue could occur at I
to
levels
comparable with those measured previously. In view of the
recent findings indicating that structural abnormalities
may be involved in the pathogenesis of BrS,
19,20
we
therefore propose the ‘conduction disorder in discontinuous
tissue hypothesis’ as a further mechanism that should
deserve a particular attention.
4.3 Role of I
to
in discontinuous conduction
It has been shown
21
that the L-type calcium current (I
Ca,L
)
greatly contributes to the success of conduction when con-
duction is characterized by local conduction delays that
extend beyond I
Na
inactivation. In such situations, suppres-
sion of I
Ca,L
can precipitate conduction block. Because the
time to peak of I
Ca,L
and I
to
is very similar, our study indi-
cates that I
to
modulates discontinuous conduction via an
analogous mechanism with an opposed polarity. Thus, an
increased I
to
density may precipitate block across a discon-
tinuous structure, while blocking I
to
may result in a recovery
from conduction block. Moreover, our ‘conduction disorder
in discontinuous tissue’ concept is in line with the recent
finding that a BrS ECG pattern can also be caused by
mutations causing loss of function of I
Ca,L
.
25
4.4 Effects of extracellular [K
1
]
Previous work has shown that both hypo- and hyperkalemia
can exacerbate the ECG phenotype of BrS.
15,16
In our simu-
lations, we observed a biphasic dependence on [K
þ
]
o
of the
level of g
to
leading to conduction block in the branching
strand (Figure 6E). This biphasic dependence is reminiscent
of the well-known biphasic dependence of conduction vel-
ocity (CV) on [K
þ
]
o
, which was investigated computationally
in detail by Shaw and Rudy.
26
As shown in their work, when
the resting membrane is hyperpolarized by a decrease of
[K
þ
]
o
, CV decreases because the charge necessary to bring
the membrane to threshold increases. However, during
elevation of [K
þ
]
o
, CV initially increases (supernormal con-
duction) until K
þ
-induced membrane depolarization results
in an increasing fraction of inactivated I
Na
, leading then to
conduction slowing. In the setting of a current-to-load mis-
match, as exemplified by tissue branching, this consider-
ation regarding the charge necessary to reach threshold
(load) vs. the available I
Na
also explains the biphasic beha-
viour of conduction block in the branching strand. Thus,
our results are in agreement with the notion that changes
in [K
þ
]
o
in both directions may exacerbate the clinical mani-
festations of carriers of mutations leading to a loss of I
Na
function. Moreover, it could be hypothesized that, among
other factors, variations in plasma [K
þ
]
o
may play a role in
the transient nature of the BrS ECGs.
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4.5 Study limitations
The clinical manifestations of BrS are rate-dependent.
18
Because of the computational cost of the LRd model, we
did not investigate the rate-dependence of conduction
characteristics in the branching strands and left the rate-
dependent aspects of BrS during long term pacing of single
cells out of our study. The simulations of conduction there-
fore suffer from the limitation that a single stimulus was
applied instead of continuous pacing until intracellular ion
concentrations have reached their true steady state. As
shown in this study, the AP phenotype and conduction
block in discontinuous tissue critically depend on the
balance between inward and outward currents during early
repolarization. Furthermore, the rate-dependence of the
AP and of conduction is governed by very intricate
dynamic interactions between repolarization, recovery of
I
Na
from inactivation and intracellular Na
þ
and Ca
2þ
over-
load at rapid pacing rates.
10,27
Pacing at different rates or
during different periods of time will therefore affect the
availability of I
Na
, I
Ca,L
, and I
to
during the early phases of
the AP and thus the quantitative outcome of the simulations
in comparison to an isolated stimulus.
BecausenumerousmembranetransportersrelevanttoNa
þ
and K
þ
homeostasis are not incorporated into the LRd model
(e.g. the Na
þ
/glucose symport and the Na
þ
/H
þ
exchanger), it
cannot be absolutely guaranteed that the intracellular ion
concentrations in the model exactly match those in the
human heart in vivo. One can nevertheless speculate that
at a given heart rate, [Na
þ
]
i
might be lower in the presence
of the mutation because less Na
þ
enters the cell during the
AP upstroke. This would increase the driving force for I
Na
.
While this increase might partially compensate for the I
Na
loss due to the mutation, it appears however unlikely that
the extent of this increase would suffice to fully offset the
loss of I
Na
that we observed experimentally.
In addition, since the patient also showed a BrS ECG at
normal temperature, we did not test for effects of higher
temperature on the biophysical characteristics of mutant
cells.
4.6 Conclusions
In conclusion, these results confirm that mutations of the C-
terminal domain of Na
v
1.5 significantly alter the inactivation
properties of the channel, and support the notion that con-
duction alterations in discontinuous tissue may be involved
in the pathogenesis of BrS. Finally, the computational ana-
lyses allowed us to formulate clinically-relevant hypotheses
regarding the cellular basis of arrhythmogenesis in the
context of BrS, which deserve further investigations.
Supplementary material
Supplementary material is available at Cardiovascular
Research online.
Conflict of interest: none declared.
Funding
Supported by grants of the Swiss National Science Foundation
(PP00-110638/1 to HA and 3100A0-100285 to J.P.K.), CardioMet
Center, Fondations Vaudoise de Cardiologie, Rita et Richard
Barme
´,
Carlsberg Foundation to T.J., and Swiss Heart Foundation
and L.&Th. La Roche Foundation (Basel) to D.K.
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