R222Q SCN5A Mutation Is Associated With Reversible Ventricular Ectopy and Dilated Cardiomyopathy

Article (PDF Available)inJournal of the American College of Cardiology 60(16) · October 2012with94 Reads
DOI: 10.1016/j.jacc.2012.05.050
Abstract
Objectives The goal of this study was to characterize a variant in the SCN5A gene that encodes the alpha-subunit of the cardiac sodium channel, Nav1.5, which was identified in 1 large kindred with dilated cardiomyopathy (DCM) and multiple arrhythmias, including premature ventricular complexes (PVCs). Background Treatment guidelines for familial DCM are based on conventional heart failure therapies, and no gene-based interventions have been established. Methods Family members underwent clinical evaluation and screening of the SCN5A and LMNA genes. Cellular electrophysiology and computational modeling were used to determine the functional consequences of the mutant Nav1.5 protein. Results An R222Q missense variant located in a Nav1.5 voltage-sensing domain was identified in affected family members. Patch-clamp studies showed that R222Q Nav1.5 did not alter sodium channel current density, but did left shift steady-state parameters of activation and inactivation. Using a voltage ramp protocol, normalized current responses of R222Q channels were of earlier onset and greater magnitude than wild-type channels. Action potential modeling using Purkinje fiber and ventricular cell models suggested that rate-dependent ectopy of Purkinje fiber origin is the predominant ventricular effect of the R222Q variant and a potential cause of DCM. In R222Q carriers, there were only modest responses to heart failure therapies, but PVCs and DCM were substantially reduced by amiodarone or flecainide, which are drugs that have sodium channel-blocking properties. Conclusions The R222Q SCN5A variant has an activating effect on sodium channel function and is associated with reversible ventricular ectopy and DCM. Elucidation of the genetic basis of familial DCM can enable effective gene-targeted therapy to be implemented. (J Am Coll Cardiol 2012;60:1566-73) (c) 2012 by the American College of Cardiology Foundation
R222Q
SCN5A
Mutation Is Associated With Reversible
Ventricular Ectopy and Dilated Cardiomyopathy
Stefan A. Mann, PHD,* M. Leticia Castro, BMEDSCI(HONS),* Monique Ohanian, BMEDSCI(HONS),*
Guanglan Guo, P
HD,* Poonam Zodgekar, MSW, GRADDIPGENCOUNS,* Angela Sheu, MB, BS,*
Kathryn Stockhammer, BS
C,GRADDIPGENCOUNS,* Tina Thompson, BNURS,†
David Playford, MB, BS, P
HD,‡ Rajesh Subbiah, MB, BS, PHD,§ Dennis Kuchar, MD,§
Anu Aggarwal, MB, BS, P
HD,† Jamie I. Vandenberg, MB, BS, PHD,* Diane Fatkin, MD*§
Darlinghurst, New South Wales; Parkville, Victoria; Armadale, Western Australia;
and Kensington, New South Wales, Australia
Objectives Our aim was to characterize a variant in the SCN5A gene that encodes the alpha-subunit of the cardiac sodium
channel, Nav1.5, which was identified in 1 large kindred with dilated cardiomyopathy (DCM) and multiple ar-
rhythmias, including premature ventricular complexes (PVCs).
Background Treatment guidelines for familial DCM are based on conventional heart failure therapies, and no gene-based
interventions have been established.
Methods Family members underwent clinical evaluation and screening of the SCN5A and LMNA genes. Cellular electrophysiol-
ogy and computational modeling were used to determine the functional consequences of the mutant Nav1.5 protein.
Results An R222Q missense variant located in a Nav1.5 voltage-sensing domain was identified in affected family mem-
bers. Patch-clamp studies showed that R222Q Nav1.5 did not alter sodium channel current density, but did left
shift steady-state parameters of activation and inactivation. Using a voltage ramp protocol, normalized current
responses of R222Q channels were of earlier onset and greater magnitude than wild-type channels. Action po-
tential modeling using Purkinje fiber and ventricular cell models suggested that rate-dependent ectopy of Pur-
kinje fiber origin is the predominant ventricular effect of the R222Q variant and a potential cause of DCM. In
R222Q carriers, there were only modest responses to heart failure therapies, but PVCs and DCM were substan-
tially reduced by amiodarone or flecainide, which are drugs that have sodium channel-blocking properties.
Conclusions The R222Q SCN5A variant has an activating effect on sodium channel function and is associated with reversible ven-
tricular ectopy and DCM. Elucidation of the genetic basis of familial DCM can enable effective gene-targeted therapy
to be implemented. (J Am Coll Cardiol 2012;xx:xxx) © 2012 by the American College of Cardiology Foundation
Dilated cardiomyopathy (DCM) is associated with significant
morbidity and mortality, and is caused by inherited gene
variants in a substantial proportion of cases. Familial DCM is
clinically variable and genetically heterogeneous, with at least
40 disease genes reported to date (1). Identification of the
genetic basis of DCM provides an opportunity for early
diagnosis and preventative intervention in genotype-positive
family members. However, the current reality is that mutations
in most of the known disease genes are uncommon, and the
molecular defects underpinning DCM in the majority of
families (70%) are unknown (1,2). Moreover, no effective
disease gene-targeted therapies have been established for clin-
ical use.
Mutations in the SCN5A gene that encodes the cardiac
sodium (Na
) channel alpha-subunit, Nav1.5, cause a variety
of arrhythmic disorders, including long QT syndrome, Bru-
gada syndrome, ventricular tachycardia, sick sinus syndrome,
atrial standstill, conduction system abnormalities (CD), and atrial
fibrillation (AF). SCN5A mutations have also been associated with
DCM that is typically preceded by a prodrome of CD or AF, a
similar phenotype to that observed with mutations in the LMNA
gene, which encodes nuclear lamin A/C (3–5).
From the *Molecular Cardiology and Biophysics Division, Victor Chang Cardiac
Research Institute, Darlinghurst, New South Wales, Australia; †Clinical Genetics
Service, Royal Melbourne Hospital, Parkville, Victoria, Australia; ‡Armadale Health
Service, Galliers Private Hospital, Armadale, Western Australia, Australia; §Cardi-
ology Department, St. Vincent’s Hospital, Darlinghurst, New South Wales, Austra-
lia; and the Faculty of Medicine, University of New South Wales, Kensington, New
South Wales, Australia. This work was supported by the National Health and
Medical Research Council of Australia, Canberra (Grant numbers 354400, 404808,
459401, 573732). The authors have reported that they do not have any relationships
relevant to the contents of this paper to disclose.
Manuscript received November 9, 2011; revised manuscript received April 26,
2012, accepted May 1, 2012.
Journal of the American College of Cardiology Vol. xx, No. x, 2012
© 2012 by the American College of Cardiology Foundation ISSN 0735-1097/$36.00
Published by Elsevier Inc. http://dx.doi.org/10.1016/j.jacc.2012.05.050
A large Caucasian kindred with a
clinical diagnosis of DCM and CD
was referred to our laboratory for
molecular genetics analysis. Detailed
phenotype evaluation demonstrated
that multiple electrocardiographic
(ECG) abnormalities were present.
In particular, frequent polymorphic
ventricular ectopy was a prominent
and early manifestation, raising the
possibility of a causal relationship
with the development of DCM. We
re-sequenced the SCN5A and
LMNA genes and identified a
heterozygous missense R222Q
SCN5A variant that segregated with
disease status in the kindred. Func-
tional characterization of the
R222Q variant showed an activating
effect on cardiac Na
channel func
-
tion, and in a ventricular cell model,
these effects were predicted to in-
duce premature ventricular com-
plexes (PVCs) predominantly of Purkinje fiber cell origin. The
availability of genotype results changed the medical management
of affected family members, and administration of drugs with Na
channel-blocking properties enabled both ventricular ectopy and
DCM to be reversed.
Methods
Clinical evaluation. Informed written consent was ob-
tained from all participants, and the study protocol was
approved by the institutional Human Research Ethics
Committee. The proband and participating family members
16 years of age were evaluated by history and physical
examination, 12-lead ECG, and transthoracic echocardiog-
raphy. The results of additional cardiac investigations per-
formed for clinical indications, including Holter monitor
and electrophysiology studies (EPS), were obtained from
medical records. One hundred unrelated healthy Caucasian
volunteers comprised the control group.
Genetic analysis. Genomic DNA was isolated from pe-
ripheral blood samples. Protein-coding sequences of the
SCN5A and LMNA genes were amplified by polymerase
chain reaction and re-sequenced using an ABI PRISM 3730
DNA Analyzer (Applied Biosystems, Foster City, Califor-
nia). The R222Q SCN5A substitution results in loss of a
Hinf1 site and was independently confirmed by restriction
enzyme digestion.
Cellular electrophysiology and modeling. Chinese ham-
ster ovary (CHO) cells were transfected with cDNA clones
encoding wild-type (WT) and R222Q Nav1.5 plus WT
Nav
1. Cardiac Na
channel currents (I
Na
) were recorded
using conventional patch-clamp techniques. To investigate
the functional consequences of mutant channels, WT and
R222Q Nav1.5 channels were modeled using Hodgkin-
Huxley formalism as described (6), and WT and mutant
models were incorporated into Purkinje cell (6) and ventric-
ular cell (7) models (Online Appendix).
Results
Family phenotype. Forty-two individuals in 2 large kin-
dreds (Family HY) underwent clinical evaluation and ge-
netic testing (Fig. 1, Table 1). The family phenotype was
characterized by a high prevalence of atrial and ventricular
arrhythmias, with DCM occurring mainly in males. A
striking feature of the ECG tracings of family members
studied in sinus rhythm was the relative paucity of normally
conducted sinus beats, with the majority of beats being PVCs,
including narrow PVCs of probably high septal origin that had
varying morphology and axis, as well as wide PVCs of left and
right bundle branch type (Figs. 1B to 1D). Premature atrial
complexes (PACs) and accelerated junctional rhythms were
also seen. Six individuals had documented AF, and 3 individ-
uals received pacemakers for symptomatic bradycardia or com-
plete heart block in later adult life. EPS results were available
in 4 cases and uniformly showed multiple PVC foci in the left
and right ventricles, with no inducible ventricular arrhythmias.
Five individuals had prophylactic implantation of cardioverter-
defibrillators (ICDs).
Eight individuals had a diagnosis of DCM (Table 1). In 2
asymptomatic young males, DCM was detected only as a result
of family screening. A history of palpitations that predated
DCM diagnosis was elicited in all other cases. DCM was
present in 7 of the 10 genotype-positive males but in only 1 of
the 7 genotype-positive females. Myocardial fibrosis was ex-
cluded in 2 individuals with severe DCM by magnetic reso-
nance imaging (IV-27) and cardiac biopsy (IV-34), respec-
tively.
Identification of R222A SCN5A variant. The coding
regions of the SCN5A and LMNA genes were re-sequenced
in the proband’s DNA (III-10; Fig. 1A) and a heterozygous
665GA change in the SCN5A gene was identified that
alters the amino acid at codon 222 from arginine (R) to
glutamine (Q). The R222 residue is located in a voltage-
sensing S4 region of Nav1.5 and has high homology across
different members of the human Na
channel gene family
and different species (Online Fig. 1). Fourteen individuals
were genotype-positive, and 3 deceased individuals (II-5,
III-5, III-7) were obligate carriers. Sixteen of these 17 gene
variant carriers were clinically affected, with the exception
being a 56-year-old male (III-12) who had a normal ECG
and echocardiogram. R222Q is a recurrent rare variant
identified in 4 DCM families (8 –11). This variant was not
found in 200 control chromosomes (this study), in 506
control chromosomes in a previous report (8), or in the 1000
Genomes and dbSNP databases. None of the R222Q
carriers had the common SCN5A variant, H558R, which
has been shown to modify the functional effects of R222Q
in vitro (12).
Abbreviations
and Acronyms
AF atrial fibrillation
CD conduction system
abnormalities
CHO Chinese hamster
ovary
DCM dilated
cardiomyopathy
ECG electrocardiogram
EPS electrophysiology
studies
ICD implantable
cardioverter-defibrillator
I
Na
Na
channel current
Na
sodium
PAC premature atrial
complex
PVC premature
ventricular complex
WT wild-type
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Activating effect of R222A SCN5A on I
Na
activity.
Given its location in a voltage-sensor domain, the R222Q
variant is predicted to affect channel gating. CHO cells
expressing WT Nav1.5 or R222Q Nav1.5 had similar mean
current densities, but in R222Q channels, the midpoints of
the voltage-dependence of activation and inactivation were
left-shifted by 6.3 and 6.2 mV, respectively (Fig. 2A). Other
kinetic parameters were not altered (Online Figs. 2 and 3).
A consequence of the left-shifted activation and inactivation
curves was that the area beneath the intersection of these
curves (the “window” current) spanned a more negative
membrane potential area (Fig. 2A, inset). To determine the
effects of increased window current, we examined the re-
sponses of WT and R222Q channels to a ramp voltage-
protocol (13) analogous to the diastolic depolarization seen in
Purkinje fiber cells. During a voltage ramp, in which the
membrane potential was gradually changed between –120
and 40 mV at 0.16 mV/ms, the normalized current
response of the R222Q channels opened at more negative
potentials and had relatively greater peak current than that
observed in WT channels (Fig. 2B).
Purkinje fiber and ventricular cell modeling. To investi-
gate the functional consequences of having Na
channels
open at more negative potentials, we incorporated the
R222Q Nav1.5 properties into a mathematical model of a
Purkinje fiber cell (6). The original Stewart model displays
automaticity with a frequency just below 1 Hz. When paced
with 1-ms stimuli of 52 pA/pF at a frequency higher than
the intrinsic rate of the model, each stimulus is captured and
evokes an action potential. In contrast, the heterozygous
WT/R222Q model showed a higher rate of spontaneous
activity (79 beats/min), and pacing at 1 Hz resulted in
grossly disorganized action potential patterns (Fig. 2D).
Similar to the WT model, pacing at higher rates in the
R222Q model resulted in stimulus capture and a regular 1:1
relationship with each action potential (Fig. 2E). These
findings suggest that PVCs associated with the R222Q
variant may result from increased automaticity of Purkinje
fiber cells, and that these effects are rate dependent. To
simulate the effects of Na
channel-blocking drugs, decre
-
mental I
Na
densities were evaluated in WT and WT/
R222Q heterozygote model cells. A 33% reduction of I
Na
decreased the spontaneous rate of the WT/R222Q hetero-
zygote cell to 65 beats/min, and a 50% reduction decreased
the rate to 50 beats/min, which enabled the cell to regain
regular pacing with a 1-Hz stimulus (Fig. 2D). To deter-
mine whether the effects of R222Q Nav1.5 were specific for
Purkinje fiber cells, we also incorporated our modified Na
Figure 1 Phenotype Characteristics of R222Q SCN5A
(A) Family HY pedigree with the proband indicated by an arrow. Phenotypes are denoted as ventricular ectopy (solid symbols, left half), conduction abnormalities (solid
symbols, right half, upper quadrant), dilated cardiomyopathy (DCM) (solid symbols, right half, lower quadrant), unaffected (open symbols), unknown (gray symbols).
The presence () or absence (–) of the R222Q allele is shown. For untested obligate carriers, genotypes are shown within parentheses. (B) Electrocardiographic (ECG)
rhythm strip (lead II) from individual IV-16 showing P waves of varying morphologies and multiple narrow QRS complexes dissimilar to the dominant rhythm with varying
morphologies and axis. (C) ECG rhythm strip (lead V
6
) from individual IV-11 showing sinus rhythm, 2 normal axis left bundle branch block (LBBB)-type premature ventricu
-
lar complexes (PVCs) with different morphologies, and an accelerated junctional rhythm. (D) ECG rhythm strip (lead II) from IV-10 showing sinus rhythm, narrow right axis
PVCs, and wide LBBB-type PVCs with variable axis.
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Clinical Features of Genotype-Positive Family Members
Table 1 Clinical Features of Genotype-Positive Family Members
ID
Age (yrs)/
Sex
HR*
(beats/min)
PR
Interval (ms)
QT/QTc
(ms) CD Arrhythmias
PVCs/PSVCs
(% Total QRS)* Devices DCM Other
II-5 56/F† NA NA NA NA Nonspecified NA No NA Sudden death
II-9 88/M Paced NA NA Third-degree AVB,
RBBB
PVC, NSVT, PAC,
PmAF
NA PPM No
II-11 85/F Paced NA NA First-degree AVB,
LBBB
PVC, NSVT, JR,
PAC, PAF
NA PPM No HT, DM, IHD
III-5 59/M† 63 161 391/400 No PVC, PAF NA No NA Stroke†, IHD
III-6 60/M 72 NA 370/405 No PVC, PmAF 27,803 (27%)/NA No Yes Stroke
III-7 38/M† NA NA NA NA NA NA No Yes Heart failure†
III-10 62/F 76 180 380/428 No PVC, NSVT, PAC,
PAF
⫹⫹⫹ PPM No EPS: multifocal PVCs RVLV,
no inducible VT/VF
III-12 56/M 75 170 362/404 No No NA No No HT, DM
III-17 58/F 77 160 466/528 RBBB PVC 1,276 (1%)/138 (1%) No No HT, DM
IV-10 24/M 75 138 313/349 No PVC, NSVT, PAC 78,492 (58%)/12,566 (9%) ICD Yes
IV-11 25/F 78 100 376/428 No PVC, JR, PAC 395 (1%)/5,255 (4%) No No
IV-13 31/M 93 160 360/448 No PVC, NSVT, PAC,
PAF
20,600 (15%)/ ICD Yes
IV-16 21/F 94 160 304/401 No PVC, JR ⫹⫹⫹ No Yes
IV-21 41/F 112 139 348/475 No PVC, NSVT, PAC 58,000 (33%)/23,500 (13%) ICD No
IV-26 27/M 92 160 368/456 RBBB PVC, JR, PAC 2,911 (3%) 1,463 (1%) No Yes EPS: multifocal PVCs (mainly RV apex,
LVOT), no inducible VT/VF, frequent
JR, PACs with intermittent LBBB
aberrancy
IV-27 25/M 96 162 345/436 No PVC, NSVT, PAC 16,295 (12%)/18,812 (14%) ICD Yes EPS: multifocal PVCs (RVLV),
no inducible VT/VF
IV-34 30/M 123 156 336/482 No PVC, NSVT, PAC 48,000 (30%)/NA ICD Yes EPS: multifocal PVCs
*Data obtained from electrocardiographic (ECG) or Holter recordings before amiodarone or flecainide therapy. Assessment of ECG parameters was complicated by frequent ectopy and was based on isolated sinus beats in some cases. Daily premature ventricular complex
(PVC) numbers are estimates only due to difficulty differentiating between ventricular and supraventricular premature complexes; in some cases, PVC burden was qualitatively reported as “occasional” () or “very frequent” (⫹⫹⫹). †Death.
AVB atrioventricular conduction block; CD conduction system abnormalities; DCM dilated cardiomyopathy; DM diabetes mellitus; EPS electrophysiology studies, HR heart rate; HT hypertension; ICD implantable cardioverter-defibrillator; IHD ischemic
heart disease; JR accelerated junctional rhythm; LBBB left bundle branch block; LV left ventricle; LVOT left ventricular outflow tract; NA not available; NSVT nonsustained ventricular tachycardia; PAC premature atrial complex; PAF paroxysmal atrial
fibrillation; PmAF permanent atrial fibrillation; PPM permanent pacemaker; PSVC premature supraventricular complex; RBBB right bundle branch block; RV right ventricle; VT/VF ventricular tachycardia/ventricular fibrillation.
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channels into a ventricular cell model (7). There were no
significant differences between WT and homozygous
R222Q model cells when paced at rates between 2 and 0.5
Hz. We could, however, elicit a subtle difference by intro-
ducing an artificial current stimulus, analogous to a delayed
afterdepolarization, during the diastolic interval. A smaller
current stimulus could elicit a premature action potential in
the R222Q but not in WT cells (Online Fig. 4).
Phenotype-modifying interventions. The rate-dependence
predicted by the modeling studies was supported by clinical
observations in affected family members with PVC frequency
increased during periods of low heart rate at rest and at night,
and reduced by high heart rates during exercise. During EPS,
acute reductions in PVC numbers were seen following rapid
pacing (cycle length 450 ms), or intravenous administration
of isoproterenol, metoprolol (doses 5 mg), or flecainide. All
family members diagnosed with DCM were treated with
standard heart failure therapy, beta-blockers, and/or
angiotensin-converting enzyme inhibitors, but these drugs had
modest or no benefits (Figs. 3E and 3F, Online Table 1). In
contrast, addition of amiodarone (IV-10, IV-27) or flecainide
(IV-21, IV-34), drugs that have Na
channel-blocking prop
-
erties (14,15), resulted in a dramatic reduction of PVC num-
bers and recovery of normal left ventricular function in those
individuals with DCM (Figs. 3A to 3F, Online Table 1). The
effects on PVCs occurred early, after treatment was started,
whereas left ventricular remodeling changes occurred more
slowly and took approximately 6 months.
Discussion
R222Q is a recurrent SCN5A variant identified to date in 4
large kindreds with DCM. In this study, we found that the
clinical phenotype associated with R222Q SCN5A included
multiple arrhythmias, suggestive of enhanced automaticity
in the atrium and ventricles. Our data provided new
Figure 2 Electrophysiological Characteristics of R222Q SCN5A
(A) Both steady-state activation and steady-state inactivation curves in R222Q channels (red lines) are left-shifted in comparison to wild-type (WT; black lines) channels,
resulting in a left-shifted and increased window current (inset). (B) Averaged normalized current responses from WT (n 3) and R222Q (n 3) cells at different ramp
slopes. R222Q channels activate at more negative potentials and have larger normalized current amplitude compared with WT channels. (C) Simulated effects of WT or
R222Q sodium channels in a Purkinje cell model closely resemble experimental results seen in B. In heterozygous R222Q/WT cells, ectopic beats (asterisks) occur dur-
ing the diastolic interval during pacing at 1 Hz (D, middle tracing). Ectopic beats are suppressed with a 50% reduction in sodium (Na
) channel current (I
Na
), for exam
-
ple, by Na
channel blocking drugs (D, lower tracing) or during pacing at 2 Hz (E).
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perspectives on the biophysical properties of R222Q Nav1.5
and showed that it has activating effects on I
Na,
which in the
ventricle predispose to PVCs mainly of Purkinje fiber
origin. These findings raise interesting questions about the
cause of DCM and highlight the value of genotype infor-
mation as a guide to family management.
The high prevalence of PVCs was a prominent feature of
this family’s phenotype. PVCs are ectopic impulses that
arise from ventricular myocardium and can result from focal
sites with increased automaticity, re-entrant circuits that
occur in regions of heterogeneous conduction (e.g., border
zones between infarcted and normal tissue) or from trig-
gered activity due to early or late afterdepolarizations.
Although PVCs are considered benign in healthy adults
(16,17), PVCs that are frequent (60/h) or complex (mul-
tiple morphologies or repetitive patterns) have been associ-
ated with an increased long-term risk of malignant ventric-
ular arrhythmias and sudden death (17,18). PVCs have a
higher prevalence in patients with reduced ejection fraction,
and although often regarded as a complication of DCM,
there has been increasing recognition that a high burden of
PVCs may independently impair cardiac contraction and
Figure 3 Effects of Na
Channel-blocking Treatment
A 22-year-old male had frequent complex ventricular ectopy on ECG (A) and echocardiography showed severe DCM (C). He was started on heart failure therapy with
carvedilol 25 mg twice daily, ramipril 10 mg/day, and spironolactone 25 mg/day. After 4 months, he continued to have severe left ventricular dysfunction, and a Holter
monitor showed 70,000 PVCs, representing 58% of the daily number of QRS complexes. An implantable cardioverter-defibrillator (ICD) was implanted and amiodarone
100 mg was started. ECG performed 6 weeks later was normal (B), with only 22 PVCs (1% total QRS complexes) on 24-h monitoring. Follow-up echocardiography
showed improvement in ventricular function after 6 months (D). The effects of drug therapy were evaluated in those family members with DCM who had serial Holter and
echocardiographic studies. When compared with those on beta-blockers and/or angiotensin-converting enzyme inhibitors alone (n 4; red symbols, dashed lines), fam-
ily members (n 3; green symbols, solid lines) who received amiodarone (n 2) or flecainide (n 1) showed resolution of PVCs (E) and normalization of left ventricu-
lar ejection fraction (LVEF) (F). Abbreviations as in Figure 1.
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promote DCM (“ventricular ectopy-induced DCM”)
(19,20). The risk of developing DCM increases with PVC
burden, with a cutoff value of 24% estimated in 1 study
(20). This threshold level can be variable, however, and
lower numbers of PVCs may be sufficient to promote DCM
in individual cases. The precise mechanisms underpinning
ectopy-induced DCM are unclear, and may be related to
factors such as dyssynchronous myocardial contraction or
increased sympathetic nervous system activation (21).
PVCs of left and right ventricular origin have previously
been associated with SCN5A mutations (22). Patch-clamp
and modeling data in Family HY point to hyperexcitability
of Purkinje fiber cells due to increased I
Na
window current
as a mechanism for PVC genesis. This differs from gain-
of-function SCN5A mutations associated with long QT
syndrome 3 that disrupt fast inactivation of I
Na,
prolonging
the action potential duration and increasing the propensity
for early afterdepolarizations (15). In Family HY, there
were multiple PVC morphologies, with a high prevalence of
narrow complexes of probable high septal fascicular origin
(23), as well as wider complexes suggesting distal Purkinje
fiber or ventricular origin. These multiple morphologies
made estimation of the daily PVC numbers difficult to
assess on Holter monitor recordings because ventricular and
supraventricular complexes were not always readily distin-
guished. PVCs may also be confused with aberrantly con-
ducted PACs, which were demonstrated during EPS in 1
family member. Nevertheless, ECG and Holter tracings clearly
showed a massive number of PVCs, raising the possibility that
ventricular ectopy could have an important role in causing
DCM. Factors supporting ventricular ectopy-induced DCM
include the history of palpitations that preceded DCM in
many cases, and the dramatic reductions in PVC numbers
before improvements in contraction as a result of amiodarone
or flecainide therapy. PVCs are commonly seen in patients
with myopathic hearts, and it is equally plausible that at least
some of the PVCs, especially those of ventricular origin, might
have arisen as a consequence of DCM. Similar arguments can
be made for the contribution of AF, which may be a cause or
effect of DCM. Only 2 of the 6 family members with AF
developed DCM, and there was no clear history of AF
predating DCM in either case.
Altered I
Na
activity may directly impair ventricular func
-
tion independently of effects of PVCs or AF. Many of the
SCN5A variants associated with DCM reduce I
Na
density or
exhibit rate-dependent reductions of Na
current (12,24 –26).
Reductions in myocardial I
Na
have been observed in human
heart failure (27) and in transgenic mice with DCM due to
overexpression of the transcriptional repressor, Snail (28).
Previous cellular electrophysiology studies have shown that the
R222Q variant does not reduce I
Na
when expressed alone in
transfected cells (12), and our data are concordant with this.
R222Q Nav1.5 was reported to reduce I
Na
when co-expressed
with the common H558R SCN5A variant (12). However,
because none of the genotype-positive members of Family HY
were H558R carriers, the modifying effects of this variant were
not clinically relevant. Gain-of-function effects on I
Na
have
been seen with some DCM-causing SCN5A variants, includ-
ing the R814W variant, which increases window current (26),
analogous to our findings with R222Q. It has been proposed
that the increased window current results in augmented dia-
stolic Na
fluxes, and that this could promote DCM by
causing regional heterogeneity in myocardial impulse propaga-
tion or by altering intracellular Na
and Ca
2
homeostasis.
Although myocardial structural defects can contribute to
DCM associated with SCN5A variants (29), ventricular fibrosis
was excluded in 2 members of Family HY with severe DCM
who underwent magnetic resonance imaging and cardiac
biopsy, respectively.
An intriguing question in Family HY is why only half of
the genotype-positive individuals developed DCM. There
was a clear sex difference, with DCM occurring in 6 of the
7 living clinically affected males, but only in 1 of 6 females.
Sex differences in ion channel gene expression (30) and in
ECG parameters (31) have been observed, and may account
for differential susceptibility to ventricular arrhythmias.
From adolescence onwards, women have faster heart rates
and longer QTc intervals, with less intraventricular conduc-
tion disturbances, QRS prolongation, and QT dispersion
than men. Although these changes may increase the risk of
arrhythmias associated with long QT-causing SCN5A vari-
ants (32), they may be relatively protective in the setting of
the R222Q variant, which was predicted to have less
Purkinje fiber excitability at high heart rates.
The cause of the CD in a subset of family members is
unclear. Conduction system defects are often seen with loss-
of-function SCN5A mutations, but may occur with activating
SCN5A mutations (24,25,33). Age-related degenerative effects
and/or changes in Nav1.5 expression, as well as additional
genetic variants may be contributing factors.
Clinical guidelines for familial DCM recommend that
affected individuals receive standard pharmacological man-
agement as indicated by the severity of heart failure and its
complications. No specific gene-based treatment strategies
have been devised (34). Conventional heart failure therapies
were relatively ineffective, however, in members of Family
HY, whereas drugs with Na
channel-blocking properties
enabled the phenotype to be reversed. Amiodarone has
multiple actions, including effects on K
,Ca
2
, and Na
channels (14), and is often used to treat ventricular ectopy.
Its long-term use may not be tolerated, however, due to the
high risk of adverse effects (35). Flecainide is an alternative
option, because it is a more selective Na
channel blocker
with a lower side-effect profile overall, but this drug is
generally contraindicated in patients with reduced left ven-
tricular function due to potential proarrhythmic effects (35).
Flecainide was well-tolerated and markedly reduced PVC
numbers in 2 family members, 1 of whom had DCM and an
ICD. ICDs were implanted prophylactically in 5 family mem-
bers with frequent complex PVCs, but the availability of
phenotype-reversing drug treatment may obviate this need for
early ICD implantation. One prudent approach to manage-
7
JACC Vol. xx, No. x, 2012
Mann
et al.
Month 2012:xxx
SCN5A
Mutations and Familial Dilated Cardiomyopathy
ment of family members without ICDs would be to start with
amiodarone until DCM resolves, then switch to flecainide for
ongoing maintenance therapy. The responses to amiodarone or
flecainide therapy were quite remarkable, and the kindred
described here provided an exemplary case in which elucidation
of the genetic basis for familial forms of DCM can enable
effective disease-modifying therapy to be implemented.
Acknowledgments
The authors thank all of the physicians who contributed to the
cardiac evaluation of family members, and Robyn Otway,
Magdalena Soka, and Gunjan Trivedi, for laboratory assistance.
Reprints requests and correspondence: Dr. Diane Fatkin, Victor
Chang Cardiac Research Institute, Lowy Packer Building, 405
Liverpool St, PO Box 699, Darlinghurst NSW 2010, Australia.
E-mail: d.fatkin@victorchang.edu.au.
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ventricular ectopy.
APPENDIX
For an expanded Methods section, and supplemental tables and figures,
please see the online version of this article.
8 Mann
et al.
JACC Vol. xx, No. x, 2012
SCN5A
Mutations and Familial Dilated Cardiomyopathy
Month 2012:xxx
    • "These highly conserved R222 and R225 residues are located on the S4 segment of DI of the channel protein (Fig. 1). Although their clinical phenotypes share major similarities, highly divergent biophysical properties have also been observed (Table S1) (Bezzina et al., 2003; Cheng et al., 2010; Laurent et al., 2012; Mann et al., 2012; Nair et al., 2012). Indeed, the R222Q mutant channel exhibits gain of function characteristics, whereas the markedly lower current density observed for the R225W mutant channel leads to loss of function. "
    [Show abstract] [Hide abstract] ABSTRACT: The gating pore current, also called omega current, consists of a cation leak through the typically nonconductive voltage-sensor domain (VSD) of voltage-gated ion channels. Although the study of gating pore currents has refined our knowledge of the structure and the function of voltage-gated ion channels, their implication in cardiac disorders has not been established. Two Nav1.5 mutations (R222Q and R225W) located in the VSD are associated with atypical clinical phenotypes involving complex arrhythmias and dilated cardiomyopathy. Using the patch-clamp technique, in silico mutagenesis, and molecular dynamic simulations, we tested the hypothesis that these two mutations may generate gating pore currents, potentially accounting for their clinical phenotypes. Our findings suggest that the gating pore current generated by the R222Q and R225W mutations could constitute the underlying pathological mechanism that links Nav1.5 VSD mutations with human cardiac arrhythmias and dilatation of cardiac chambers. © 2015 Moreau et al.
    Full-text · Article · Feb 2015
    • "Our findings further suggest that the S4 segments in DIV may affect intermediate inactivation gating process and its stability of sodium channels. Moreover, there are a growing number of reports that mutations associated with SCN5A channelopathies resided in the voltage-sensor domain [10].For example, SCN5A mutations preferentially inclined to occur in the S4 segment in DCM patients, and among positive charge clusters acted as voltage sensor [35,36]. These mutations resulted in the functional defects of sodium channels with synthesis of a channel protein altered gating kinetics rather than reduced the current density [10,37]. "
    [Show abstract] [Hide abstract] ABSTRACT: Brugada syndrome (BrS) is an inherited arrhythmogenic syndrome leading to sudden cardiac death, partially associated with autosomal dominant mutations in SCN5A, which encodes the cardiac sodium channel alpha-subunit (Nav1.5). To date some SCN5A mutations related with BrS have been identified in voltage sensor of Nav1.5. Here, we describe a dominant missense mutation (R1629Q) localized in the fourth segment of domain IV region (DIV-S4) in a Chinese Han family. The mutation was identified by direct sequencing of SCN5A from the proband's DNA. Co-expression of Wild-type (WT) or R1629Q Nav1.5 channel and hβ1 subunit were achieved in human embryonic kidney cells by transient transfection. Sodium currents were recorded using whole cell patch-clamp protocols. No significant changes between WT and R1629Q currents were observed in current density or steady-state activation. However, hyperpolarized shift of steady-state inactivation curve was identified in cells expressing R1629Q channel (WT: V1/2 = -81.1 ± 1.3 mV, n = 13; R1629Q: V1/2 = -101.7 ± 1.2 mV, n = 18). Moreover, R1629Q channel showed enhanced intermediate inactivation and prolonged recovery time from inactivation. In summary, this study reveals that R1629Q mutation causes a distinct loss-of-function of the channel due to alter its electrophysiological characteristics, and facilitates our understanding of biophysical mechanisms of BrS.
    Full-text · Article · Oct 2013
    • "Mutations in the sodium channel gene, SCN5A, are found in patients with a variety of cardiac diseases, such as congenital long QT syndrome type 3 and Brugada syndrome (Wang et al., 1995a,b; Antzelevitch , 2001; Moric et al., 2003). Recent studies have associated mutations in SCN5A with dilated cardiomyopathy (Mcnair et al., 2004; Hesse et al., 2007; Mann et al., 2012). Many investigators have characterized naturally occurring SCN5A mutations, but little is known about the regulation of expression of Na v 1.5 in cardiac cells. "
    [Show abstract] [Hide abstract] ABSTRACT: The cardiac voltage-gated sodium channel, Nav1.5, plays a central role in cardiac excitability and impulse propagation and associates with the dystrophin multiprotein complex at the lateral membrane of cardiomyocytes. It was previously shown that Nav1.5 protein content and the sodium current (l Na) were both decreased in cardiomyocytes of dystrophin-deficient mdx (5cv) mice. In this study, wild-type and mdx (5cv) mice were treated for 7 days with the proteasome inhibitor MG132 (10 μg/Kg/24 h) using implanted osmotic mini pumps. MG132 rescued both the total amount of Nav1.5 protein and l Na but, unlike in previous studies, de novo expression of dystrophin was not observed in skeletal or cardiac muscle. This study suggests that the reduced expression of Nav1.5 in dystrophin-deficient cells is dependent on proteasomal degradation.
    Full-text · Article · Mar 2013
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