gain of function mutation in scn5a causes ventricular arrhythmias and early onset atrial fibrillation

So far, only one mutation in SCN5A-International Journal of Cardiology xxx 2017 xxx–xxx Abbreviations: SCN5A, the gene encoding the α-subunit of the cardiac sodium channel; Na V 1.5, car

Gain-of-function mutation in SCN5A causes ventricular arrhythmias and

early onset atrial fibrillation ☆

Krystien V Lievea,1, Arie O Verkerka,b,1, Svitlana Podliesnaa, Christian van der Werfa, Michael W Tanckc,

Arthur A.M Wildea,e, Elisabeth M Loddera,⁎

a Heart Center, Department of Clinical and Experimental Cardiology, Academic Medical Center, Amsterdam, The Netherlands

b Department of Anatomy, Embryology and Physiology, Academic Medical Center, Amsterdam, The Netherlands

c

Department of Clinical Epidemiology, Biostatistics and Bioinformatics, Academic Medical Center, Amsterdam, The Netherlands

d

Department of Cardiology, Westfriesgasthuis, Hoorn, The Netherlands

e

Princess Al-Jawhara Al-Brahim Centre of Excellence in Research of Hereditary Disorders, Jeddah, Saudi Arabia

a b s t r a c t

a r t i c l e i n f o

Article history:

Received 7 November 2016

Accepted 24 January 2017

Available online xxxx

associated with a broad spectrum of inherited cardiac arrhythmia disorders The purpose of this study was to identify the genetic and functional determinants underlying a Dutch family that presented with a combined phe-notype of ventricular arrhythmias with a likely adrenergic component, either in isolation or in combination with

a mildly decreased heart function and early onset (b55 years) atrial fibrillation

Methods and results: We performed next generation sequencing in the proband of a two-generation Dutch family and demonstrated a novel missense mutation in SCN5A-(p.M1851V) which co-segregated with the clinical phe-notype in the family We functionally evaluated the putative genetic defect by patch clamp electrophysiological studies in human embryonic kidney cells transfected with mutant or wild-type Nav1.5 The current inactivation was slower and recovery from inactivation was faster in SCN5A-M1851V channels The voltage dependence of inactivation was shifted towards more positive potentials and consequently, a larger TTX-sensitive window cur-rent was observed in SCN5A-M1851V channels Furthermore, a higher upstroke velocity was observed for the SCN5A-M1851V channels, while the depolarization voltage was more negative, both indicating increased excit-ability

Conclusions: This mutation leads to a gaof-function mechanism based on increased channel availability and

with mutations in SCN5A

creativecommons.org/licenses/by/4.0/)

Keywords:

Genetics

Inherited channelopathies

Atrial fibrillation

Ventricular ectopy

SCN5A mutation

Arrhythmia

1 Introduction Mutations in SCN5A encoding the main voltage-gated sodium chan-nelα-subunit in the heart (NaV1.5)[1]have been associated with a spectrum of cardiac arrhythmias including congenital long QT syn-drome[2], Brugada syndrome[3], sick sinus syndrome[4,5], progressive cardiac conduction defect[6], atrialfibrillation (AF)[7], and more re-cently, multifocal ectopic Purkinje-related premature contraction (MEPPC) [8,9] NaV1.5 channels initiate the action potential in cardiomyocytes by inducing a fast depolarizing inward current and thereby play an essential role in cardiac conduction[10]

MEPPC, a recently identified novel SCN5A-related channelopathy, is characterized by frequent premature ventricular contractions (PVCs) arising from the Purkinje system that occur at rest and that are sup-pressed at high heart rates So far, only one mutation in

SCN5A-International Journal of Cardiology xxx (2017) xxx–xxx

Abbreviations: SCN5A, the gene encoding the α-subunit of the cardiac sodium channel;

Na V 1.5, cardiac sodium channel; AF, atrial fibrillation; AP, action potential; ECG,

electrocar-diogram; HEK, human embryonic kidney cell; I Na , voltage gated sodium current; MRI,

magnetic resonance imaging; NSVT, non sustained ventricular tachycardia; PVC,

prema-ture ventricular contraction; TTE, transthoracic echocardiogram; WT, wild type; MT,

mutant.

☆ All authors take responsibility for all aspects of the reliability and freedom from bias of

the data presented and their discussed interpretation.

⁎ Corresponding author at: Department of Experimental Cardiology, Academic Medical

Center, University of Amsterdam, Meibergdreef 15, Room K2-110, PO Box 22660, 1100DD

Amsterdam, The Netherlands.

E-mail address: E.M.Lodder@amc.uva.nl (E.M Lodder).

1

These authors contributed equally.

http://dx.doi.org/10.1016/j.ijcard.2017.01.113

0167-5273/© 2017 The Authors Published by Elsevier Ireland Ltd This is an open access article under the CC BY license ( http://creativecommons.org/licenses/by/4.0/ ).

Contents lists available atScienceDirect

International Journal of Cardiology

j o u r n a l h o m e p a g e :w w w e l s e v i e r c o m / l o c a t e / i j c a r d

(p.R222Q)[8,9]has been associated with the MEPPC phenotype

Anoth-er SCN5A mutation, p.I141V, was recently associated with

predominant-ly exercise-induced ventricular arrhythmias[11] On a cellular level,

both mutations (i.e p.R222Q and p.I141V) lead to a gain-of-function

due to an increased window current of NaV1.5

Here, we present a third mutation with gain-of-function effects on

NaV1.5 in a family with an MEPPC-like phenotype combined with a

like-ly important adrenergic component

2 Methods

2.1 Family evaluation

Outpatient charts were reviewed on medical history, 12-lead ECG,

Holter monitoring, exercise testing and cardiac imaging data Peripheral

blood was drawn for genomic deoxyribonucleic acid (DNA) extraction

following standard procedures Informed consent was obtained from

all participating family members The study complied with the

Declara-tion of Helsinki

2.2 Mutation analysis

A panel of the genes that have previously been associated with

dif-ferent cardiomyopathies and catecholaminergic polymorphic

ventricu-lar tachycardia were sequenced by next-generation sequencing (NGS)

in the proband (individual III.4 InFig 1) NGS was complemented

with Sanger sequencing to ensure coverage of all exons and

exon-intron boundaries The following genes were included in the panel:

HCN4, ACTC1, ACTN2, ANKRD1, BAG3, CALR3, CAV3, CRYAB, CSRP3,

CTNNA3, DES, DSC2, DSG2, DSP, EMD, FHL1, GLA, JPH2, JUP, LAMA4,

LAMP2, LDB3, LMNA, MIB1, MYBPC3, MYH6, MYH7, MYL2, MYL3, MYOZ2,

MYPN, NEXN, PKP2, PLN, PRDM16, PRKAG2, RBM20, SCN5A, TAZ, TCAP,

TMEM43, TNNC1, TNNI3, TNNT2, TPM1, TTR, VCL and RYR2 NGS was

per-formed on an Illumina HiSeq2000 (Illumina, San Diego, California) using

the paired end 2 × 100 bp method Variants with an allele frequency

of N1% in reference databases (GoNL (12), ExAC (http://exac

broadinstitute.org), dbSNP137 (www.ncbi.nlm.nih.gov/SNP) and ESP (http://evs.gs.washington.edu/EVS)), were considered to have a benign effect and were therefore excluded from further analysis Other genetic variants were retained and tested for segregation with the phenotype in the family by Sanger sequencing

2.3 DNA constructs, mutagenesis, HEK cell culture and transfection The c.5551AN G point mutation (p M1851V) was introduced into the wild type SCN5A (in a bicistronic GFP vector)[12]by site directed mutagenesis using Quick Change XL kit (Agilent Technologies, Santa Clara, USA) using standard procedures

HEK-293A cells were cultured in DMEM (21969-035) (Gibco) sup-plemented with 10% FBS (Biowest), penicillin-streptomycin (Gibco) andL-glutamine (Gibco) in a 5% CO2incubator at 37 °C Cells were transfected at 70% confluency in 25 cm culture flasks with the wild-type or the mutant NaV1.5 construct (0.2μg) together with a β1-subunit (SCN1B) construct[13](0.2μg) using lipofectamine (Invitrogen, Carlsbad, USA) Gene-transfer was monitored by means of green fluo-rescence from the SCN5A-GFP bicistronic vector Patch clamp experi-ments were performed onfluorescent cells 2 days after transfection 2.4 Electrophysiology

2.4.1 Data acquisition The sodium current (INa) and action potential (AP) upstroke velocity (dV/dt) were measured in the whole-cell configuration of the patch-clamp technique using an Axopatch 200B amplifier (Molecular Devices Corporation, Sunnyvale, CA, USA) or a custom-made amplifier, capable

of fast switching between voltage clamp (VC) and current clamp (CC) modes[14] Voltage control, data acquisition, and analysis were accom-plished using custom software Signals were low-passfiltered with a cut-off frequency of 5 kHz and digitized at 20 kHz and 40 kHz for INa

and AP upstrokes, respectively Series resistance was compensated by

≥80% Cell membrane capacitance (Cm) was calculated by dividing the time constant of the decay of the capacitive transient after a−5 mV

Fig 1 Pedigree of the family Squares/circles indicate male/female respectively Open symbols indicate unaffected persons Symbols with a slash indicate deceased persons Grey color indicates undetermined phenotype The proband (III.4) is indicated by the arrow SCN5A-p.M1851V mutation carriers are indicated by a plus sign, non-carriers with a minus sign and persons in which carriership is unknown with a question mark Solid left upper quarter: early onset atrial fibrillation Solid right upper quarter: exercise-induced ventricular arrhythmia Solid left lower quarter: decreased left ventricular function Solid right lower quarter: non-exercise induced ventricular arrhythmia The numbers below the persons (i.e II.2) represent the identification of the family members used in the text.

Please cite this article as: K.V Lieve, et al., Gain-of-function mutation in SCN5A causes ventricular arrhythmias and early onset atrialfibrillation, Int

J Cardiol (2017),http://dx.doi.org/10.1016/j.ijcard.2017.01.113

voltage step from−40 mV by the series resistance Peak INawas

mea-sured at room temperature with patch pipettes (borosilicate glass;

re-sistance≈2.0 MΩ) containing (in mM): 10 CsCl, 110 CsF, 10 NaF, 11

EGTA, 1.0 CaCl2, 1.0 MgCl2, 2.0 Na2ATP, 10 HEPES, pH 7.2 (CsOH) Bath

solution contained (in mM): 20 NaCl, 120 CsCl, 1.8 CaCl2, 1.2 MgCl2,

11.0 glucose, 5.0 HEPES; pH 7.4 (CsOH) AP upstrokes and sustained

and window INacurrents were measured at 37 °C Patch solution was

similar to the INameasurements; bath solution contained (in mM):

140 NaCl, 10 CsCl, 1.8 CaCl2, 1.0 MgCl2, 5.5 glucose, 5.0 HEPES, pH 7.4

(NaOH)

2.4.2 Conventional VC and alternating VC/CC experiments

The current density of the peak INa, voltage dependence of

(in)acti-vation, recovery from inacti(in)acti-vation, and sustained and window currents

were determined using the voltage protocols as described below The

holding potential was−120 mV, except in the protocols for recovery

from inactivation and the AP upstrokes measurements In the latter

ex-periments we chose a holding potential of−85 mV, a value close to the

resting membrane potential of working cardiomyocytes In alternating

VC/CC experiments, dV/dt was measured by switching for 20 ms to

the CC mode of the patch clamp amplifier Noteworthy, HEK cells

dis-play fast depolarizations (in the present study named AP upstrokes)

upon switching from VC to CC mode AP upstrokes were elicited by

1.2× threshold current pulses through the patch pipette Maximal

up-stroke velocity (dV/dtmax) during VC/CC, offline corrected for the

contri-bution of stimulus current, served as an indicator of available INa

Peak INawas defined as the difference between peak and

steady-state current The sustained as well as the window INacurrent were

measured as the current sensitive for 30μM TTX To determine the

acti-vation characteristics of INa, current-voltage (I-V) curves were corrected

for differences in driving force and normalized to maximum peak

cur-rent Steady-state activation and inactivation curves werefit using the

Boltzmann eq I/Imax= A / {1.0 + exp[(V1/2− V) / k]} to determine

V1/2(membrane potential for the half-maximal (in)activation) and

the slope factor k Recovery from inactivation was analyzed by

fitting a double-exponential function to the data to obtain the time

con-stants of the fast and the slow components of recovery from

inactiva-tion: I/Imax= Af× [1.0− exp(−t / τf)] + As× [1.0− exp(−t / τs)],

where t is the recovery time interval,τfandττsthe time constants

of the fast and slow components, and Afand Asthe fractions of the

fast and slow components, respectively The time course of current

inactivation wasfitted by a double-exponential equation: I/Imax=

Af× exp(−t / τf) + As× exp(−t / τs), where Afand Asare the fractions

of the fast and slow inactivation components, andτfandτsare the time

constants of the fast and slow inactivating components, respectively

2.5 Statistics

Data are expressed as mean ± standard error of the mean (SEM)

Values were considered significantly different if p b 0.05 in unpaired

t-test or in Two-Way Repeated Measures of Analysis of Variance

(Two-Way Repeated Measures ANOVA) followed by pairwise comparison

using the Student-Newman-Keuls test

3 Results

3.1 Clinical phenotype of the family

The patient characteristics of the affected family members are

shown inTable 1 The proband (Fig 1, III.4) presented at the outpatient

cardiology clinic at the age of 16 with chest pain and palpitations, most

often occurring during exercise Her baseline ECG showed sinus

tachy-cardia Holter monitoring was performed and revealed monomorphic

PVCs during daytime, including non-sustained ventricular tachycardia

(NSVT) starting from a heart rate of 96 bpm, and no ventricular

arrhyth-mia during sleep (Table 1) During exercise testing, polymorphic PVCs Tab

Patient #

symptoms (years) Symptoms before diagnosis

diagnosis (years) Resting heart rate Atrial fibrillation (age

Average HR

Total supra-ventricular ectopies

Longest Salvo

Decompensated heart

Palpitations, near-syncope

Not applicable

occurred at a heart rate of 137 bpm, including PVCs bigeminy PVCs

dis-appeared during maximal exercise and returned in the recovery phase

of the exercise test Transthoracic echocardiography (TTE) showed a

normal left ventricular ejection fraction (LVEF) and mild mitral valve

prolapse Subsequently, cardiac magnetic resonance imaging showed a

mildly dilated left ventricle with a diminished LVEF of 44% The patient

was prescribed propranolol 80 mg daily (1.6 mg/kg/day) An TTE

re-peated one year later showed a normalized cardiac function

The patient's family history was positive for diverse cardiac

arrhyth-mias at the paternal side (see below for details,Table 1) The father of

the proband (II.6) was evaluated for palpitations triggered by exercise

at the age of 40 years Exercise testing revealed paroxysmal AF and

the patient was discharged from cardiology follow-up Cardiac

evalua-tion for cascade screening twelve years later showed frequent PVCs on

his baseline ECG Subsequent Holter monitoring and exercise testing

re-vealed self-terminating episodes of paroxysmal AF and NSVT that

oc-curred mostly during daytime During exercise testing the number

and complexity of PVCs increased at higher workloads TTE showed a

normal left ventricular function and normal left atrial volumes Medical

treatment with propranolol 160 mg daily (2.3 mg/kg/day) was initiated

The eldest brother of the proband (III.1) had palpitations both at rest

and during exercise, and near-syncope Baseline ECG showed

prema-ture atrial contractions and PVCs TTE showed a normal left ventricular

function and an enlarged left atrial volume On exercise testing sinus

ar-rhythmia and polymorphic PVCs were noted from the start of the

exer-cise test (heart rate of 80 bpm) and lasted throughout the recovery

phase (PVCs, bigeminal PVCs, couplets and NSVT,Fig 2) Another

broth-er (III.2) showed ventricular arrhythmias during exbroth-ercise testing that

increased in frequency and complexity at higher workloads to

bigemi-nal PVCs TTE was normal The third brother (III.3) had NSVT on Holter

monitoring and isolated PVCs during exercise testing (onset at heart

rate of 98 bpm) TTE showed normal cardiac function The paternal

grandfather of the proband died at the age of 66 He suffered from

un-known cardiac complaints Patient II.2 was admitted to the hospital at

the age of 42 years with decompensated heart failure due to preexistent

AF After cardioversion sinus rhythm was restored and the LVEF

normal-ized However, at the age of 52 years, polymorphic VES and NSVTs were

noted during Holter monitoring; TTE and coronary angiography were

performed and were both normal This patient is currently treated

with sotalol 240 mg daily (2.6 mg/kg/day) Patient II.5 was initially

diag-nosed with paroxysmal AF at the age of 35 years which later progressed

to permanent AF Holter monitoring showed PVCs and couplets and he

is being treated with bisoprolol 10 mg daily (0.12 mg/kg/day) TTE

showed mild mitral valve prolapse with normal left atrial volumes

and a normal LVEF Exercise testing in patient II.4 revealed PVCs and

couplets (onset at heart rate of 91 bpm), premature atrial contractions

and self-terminating short episodes of paroxysmal AF that were absent

at rest Cardiac evaluation was normal for II.1 and no medical details

were available for II.3

In summary, the family was affected with early-onset severe,

exer-cise induced and non-exerexer-cise induced ventricular arrhythmias in

com-bination with early onset (b55 years) AF in the setting of normal left

atrial volumes and mild cardiac remodeling

3.2 Genotyping

Considering the atypical phenotype (consisting of exercise induced

arrhythmias with mild cardiac remodeling) of the family we tested a

panel of 48 cardiomyopathy associated genes and the ryanodine

recep-tor 2 (RYR2) using NGS in the proband This screen yielded one rare

var-iant in SCN5A (NM_001099404.1), c 5551AN G, predicted to result in

the substitution of a methionine residue with a valine residue in the

C-terminal domain of the protein (p.M1851V, suppl Fig 1A) This

muta-tion was absent inN2000 index patients (in house data) and in N60,000

control individuals from the NHLBI Exome Sequencing Project (http://

evs.gs.washington.edu/EVS/) and ExAC (http://exac.broadinstitute

org) The affected amino acid is modestly conserved across species (see suppl Fig 1B) Sanger sequencing in family members

demonstrat-ed co-segregation of this variant with the phenotype in the family (Fig 1)

3.3 Biophysical characterization of INa

Finally, we characterized the effects of the p.M1851V SCN5A muta-tion on INafunction First, the current density and gating properties of

WT and p.M1851V were assessed.Fig 3A, top, shows representative

INaactivated by 50-ms depolarizing voltage clamp steps of 5 mV incre-ment Typical INacurrent starts to activate around−60 mV, peaks around−30 mV, and subsequently decreases in amplitude due to the reduction in Na+driving force.Fig 3B shows average data for the current-voltage (I-V) relationships The current density, the INa ampli-tude divided by the Cm, did not differ significantly between WT and mutant currents The typical examples inFig 3A suggest that the speed of current inactivation is slower in M1851V channels

Fig 3A, bottom panel, summarizes the average fast and slow time constants of INainactivation Indeed,τfastis significantly higher which indicates a slowed current inactivation.Fig 3C shows the average (in)-activation relationships The curves of the voltage dependence of activa-tion of mutant and wildtype channels were overlapping indicating that

it was not affected by the p.M1851V SCN5A mutation Voltage-dependency of inactivation was measured using a two-pulse protocol where a 500-ms conditioning prepulse to membrane potentials be-tween−120 and 0 mV, to induce steady-state inactivation, was

follow-ed by a 50-ms test pulse (Fig 3C, inset) Voltage-dependency of inactivation was significantly shifted towards more positive potentials

in the M1851V channels

Recovery from INainactivation was measured using a two-pulse pro-tocol, where a 1-s conditioning prepulse (P1) to−20 mV (to inactivate

Na+channels) was followed by a test pulse (P2) after a variable recov-ery interval ranging between 1 and 1000 ms at a recovrecov-ery potential of

−85 mV (see inset ofFig 3D) The peak amplitudes in response to P2 were normalized to the peak amplitudes at P1 and plotted versus the interpulse interval The resulting curve was fitted with a double-exponential function to obtain the time constants and fractions of the fast and the slow components of recovery from inactivation Both aver-ageτfastandτslowwere significantly lower in M1851V channels indicat-ing faster recovery from inactivation (Fig 3D)

Secondly, we characterized sustained and window INacurrents in

WT and M1851V channels The slower speed on INa inactivation (Fig 3A) may have implications for sustained currents, while the posi-tive shift in voltage-dependency of inactivation (Fig 3C) may result in changes in window currents The sustained INacurrent was assessed during a 300-ms depolarizing voltage steps from −120 mV to

−20 mV and defined as the current sensitive for 30 μM TTX.Fig 3E top, left, shows typical TTX-sensitive currents;Fig 3E top, right, summa-rize the average TTX-sensitive sustained current The density of the sustained current was not affected in the M1851V channels The win-dow currents were measured as the TTX-sensitive current during a 200-ms depolarizing ramp from−100 to −20 mV from a −120 mV holding potential (Fig 3E top left, inset) The window current is signi fi-cantly larger in M1851V channels (Fig 3E bottom panel)

Thirdly, we assessed AP upstroke velocities in HEK cells transfected with either WT or M1851V channels, as a positive shift in the voltage-dependency of inactivation (Fig 3C) will result in a larger availability

of channels at the resting membrane potential (around−85 mV) of cardiomyocytes and thus may lead to enhanced upstroke velocities Noteworthy, HEK cells display fast depolarizations upon switching from VC (voltage clamp) to CC (current clamp) mode due to the sodium channel activation[14]and reflects thus a more dynamic, physiological condition then when INacharacteristics are measured atfixed mem-brane potentials in VC.Fig 3F shows typical upstrokes and theirfirst de-rivatives measured upon switching from−85 mV in VC to current Please cite this article as: K.V Lieve, et al., Gain-of-function mutation in SCN5A causes ventricular arrhythmias and early onset atrialfibrillation, Int

J Cardiol (2017),http://dx.doi.org/10.1016/j.ijcard.2017.01.113

Fig 2 ECGs at rest (A) and during exercise (B and C) of subject III.1 A: sinus rhythm at rest B: appearance of first premature ventricular contraction during exercise C: non-sustained ventricular tachycardia during exercise.

clamp for 20 ms The dashed line indicates the depolarization due the

applied stimulus current alone The arrow indicates the threshold for

the upstroke and was measured at a 4-mV difference between the linear

stimulus current and INa-drive voltage change The average maximal

up-stroke velocity was significantly higher and the threshold voltage was

significantly more negative by M1851V channels (Fig 3F)

4 Discussion

We present a relatively large Dutch family with supraventricular

ar-rhythmias including AF, and ventricular arar-rhythmias, including

poly-morphic NSVT, with a likely important adrenergic component,

although we cannot exclude that increased heart rate only is the

triggering factor Genetic studies identified a novel SCN5A mutation,

p.M1851V, co-segregating with the phenotype in the family

Subse-quent electrophysiological studies demonstrated a faster recovery

from inactivation and a positive shift in voltage dependency of

inactiva-tion in SCN5A-p.M1851V channels leading to increased sodium channel

availability and increased window current, explaining the phenotype in

the family Thesefindings provide further insight into the broad

spec-trum of cardiac arrhythmias caused by mutations in SCN5A

Complete co-segregation of the SCN5A-p.M1851V mutation with the

phenotype in this family in combination with its absence inN120,000

control alleles (ExAC)[15]and the evolutionary conservation of the

amino acid support causality of this mutation Moreover, the

gain-of-function defect we uncovered in electrophysiological studies is concor-dant with the clinical presentation in the family Of note, the functional effects we uncovered for SCN5A-p.M1851V are similar to those

previous-ly described for SCN5A-p.R222Q and SCN5A-p.I141V, which are both as-sociated with a clinical phenotype that strongly overlaps with that of the current family[8,9,11]

A number of mechanistic links can be laid by comparing the various biophysical defects of the M1851V channel to the clinical phenotypes observed in the family Firstly, we found a slower inactivation of INa

(Fig 3), which, however, did not result in an increase of the sustained

INa(Fig 3E) The latter is in agreement with the absence of prolonged QTc changes (Table 1)

Secondly, M1851V channels have a more positive V1/2of voltage de-pendency of inactivation (Fig 3C) This may have two important impli-cations for INafunction, i.e., an increase in the window current and a larger availability of channels at the resting membrane potential (−85 mV) of cardiomyocytes Indeed, the TTX-sensitive current during

a depolarizing RAMP was larger in M1851V channels (Fig 3E), while the

AP upstroke velocity was significantly increased (Fig 3F) This is in con-cordance with the observed atrial and ventricular arrhythmias in the p.M1851V carriers

Thirdly, M1851V channels have a faster recovery from inactivation (Fig 3D), indicating a greater availability of INaat higher heart rate compared to wild-type channels This faster recovery from inactivation

in the p.M1851V mutation carriers may contribute to a larger

Fig 3 Current density and gating properties of WT and M1851V channels A, top: Typical examples of peak sodium current (I Na ) in response to 50-ms depolarizing voltage steps from

−120 mV For protocol, see inset of panels B; cycle length was 5-s A, bottom: Average fast and slow time constants of I Na inactivation Note logarithmic ordinate scale τ fast is significantly higher indicating a slowed current inactivation B: Average current-voltage (I-V) relationships C: Average steady-state (in)activation curves Inset: voltage clamp for inactivation The solid lines are the Boltzmann fit to the average data Voltage-dependency of inactivation was significantly shifted towards more positive potentials in M1851V channels (−81.4 ± 1.7 mV (WT) vs −71.9 ± 1.5 mV (M1851V)), without changes in k D: Average recovery from I Na inactivation on a logarithmic time scale assessed with interpulse interval of 1–1000 ms Solid lines are double-exponential fits to the average data Both average τ fast (65 ± 6 ms (WT) vs 37 ± 6 ms (M1851V)) and τ slow (1073 ± 324 ms (WT) vs

253 ± 53 ms (M1851V)) are significantly lower in M1851V channels indicating faster recovery from inactivation E, top left: typical TTX-sensitive currents activated during a 300-ms depolarizing voltage steps from −120 mV to −20 mV E, top right: average density of the TTX-sensitive sustained current measured during the last 50-ms of the depolarizing voltage step Current density was not affected in the M1851V channels E, bottom: Average I Na window measured during a 200 ms depolarizing ramp from −100 to −20 mV from a

−120 mV holding potential (inset) The window current is significantly larger in M1851V channels Asterisks denote p b 0.05 F, top left: Upstrokes in HEK cells with WT or M1851V channels Typical upstrokes measured upon switching from −85 mV in voltage (VC) to current clamp for 20 ms The dashed line indicated the depolarization due the applied stimulus current alone The arrow indicated the threshold for the upstroke and was measured at a 4-mV difference between the linear stimulus current and I Na -drive voltage change F, bottom left: first derivatives of the upstrokes depicted in panel F top left F, top right: Average upstroke velocities F, bottom right: average threshold voltages Asterisks denote p b 0.05.

Please cite this article as: K.V Lieve, et al., Gain-of-function mutation in SCN5A causes ventricular arrhythmias and early onset atrialfibrillation, Int

J Cardiol (2017),http://dx.doi.org/10.1016/j.ijcard.2017.01.113

susceptibility for atrial arrhythmias eventually resulting in AF in all

sec-ond generation carriers

Fourthly, we observed a more negative threshold for upstroke

gen-eration for M1851V channels (Fig 3F) Atfirst glance this contrasts the

finding that V1/2of INaactivation was unaffected as found in VC

experi-ments (Fig 3C) We think, however, that the more negative threshold

for upstroke generation is observed due to dynamic and more

close-to-physiological behavior of sodium channels Likely the shift occurs

due to a combination of increased channel availability in combination

with increased window current, which makes it more easy to depolarize

the membrane potential at negative potentials

Gain-of-function mutations in SCN5A can lead to a spectrum of

inherited cardiac arrhythmias[15] Three previously published

muta-tions in SCN5A show considerable overlap with the p.M1851V described

here on the phenotypical level and/or the biophysical level (suppl

Table 1)[8,9,11] The p.R222Q mutation in SCN5A associated with

MEPPC is characterized by the occurrence of frequent PVCs at rest

orig-inating from various foci along the fascicular Purkinje system Even

though none of the p.M1851V mutation carriers underwent an

electro-physiological study, which would provide prove of the origin of the

ven-tricular arrhythmias, the configuration of the PVCs may suggest an

origin from the Purkinje system The p.R222Q mutation carriers also

presented with atrial tachyarrhythmias and mild left ventricular

dys-function due to frequent PVCs In contrast to the phenotype observed

in the M1851V mutation carriers, the p.R222Q carriers had PVCs that

oc-curred mostly during rest and were suppressed by higher heart rates

such as exercise Furthermore, an extremely high PVC burden often

leading to a decreased cardiac function was observed in the p.R222Q

mutation carriers On an electrophysiological level, the p.R222Q

muta-tion leads to an increased window current by affecting the voltage

de-pendence of both activation and inactivation of the NaV1.5 channel,

while the p.M1851V mutation only affects the voltage dependence of

activation

Swan et al described a large multigenerational Finnish family with

the SCN5A-p.I141V mutation and a cardiac phenotype with

predomi-nantly exercise-induced ventricular arrhythmias[11] The voltage

de-pendence of activation of SCN5A-p.I141V was shifted towards more

negative potentials leading to an increase and shift of the window

cur-rent A third mutation, SCN5A-p.K1493R, with similar

electrophysiolog-ical properties to our mutation has also been described[16] This

mutation was reported in a male suffering from AF since the age of 50

and his mother who suffered from AF from the age of 63[16] However,

limited details were provided on the cardiac phenotype of these two

pa-tients and no exercise testing was performed

Altogether a total of four mutations (including ours) are now

de-scribed leading to an increase of window current of the cardiac sodium

channel NaV1.5 without causing congenital long QT syndrome The

clin-ical phenotype associated with these mutations encompasses both

sup-raventricular and ventricular arrhythmias that occur at rest and increase

in severity with exercise in most cases The observed left ventricular

dysfunction in the index patient is most likely secondary to frequent

PVCs

In conclusion, we here provide further evidence that SCN5A

muta-tions with a gain-of-function defect stemming from an increased

win-dow current cause MEPPC, exercise-induced ventricular arrhythmias

and early onset supraventricular arrhythmias

Funding sources

We acknowledge the support from the Netherlands CardioVascular

Research Initiative: the Dutch Heart Foundation, Dutch Federation of

University Medical Centres, the Netherlands Organisation for Health

Re-search and Development and the Royal Netherlands Academy of

Sci-ences (CVON 2010-12 PREDICT) to KL, CRB, AAMW and EML; the

E-Rare Joint Transnational Call for Proposals 2015 "Improve CPVT” to

AAMW; and the Netherlands Organization for Scientific Research (VICI project 016.150.610) to CRB

Conflicts of interest The authors report no relationships that could be construed as a con-flict of interest

Acknowledgements

We thank J.R Andela for her assistance in the echocardiographic measurements

Appendix A Supplementary data Supplementary data to this article can be found online athttp://dx doi.org/10.1016/j.ijcard.2017.01.113

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