IJCA-24482; No of Pages International Journal of Cardiology xxx (2017) xxx–xxx Contents lists available at ScienceDirect International Journal of Cardiology journal homepage: www.elsevier.com/locate/ijcard Gain-of-function mutation in SCN5A causes ventricular arrhythmias and early onset atrial fibrillation☆ Krystien V Lieve a,1, Arie O Verkerk a,b,1, Svitlana Podliesna a, Christian van der Werf a, Michael W Tanck c, Nynke Hofman a, Paul F van Bergen d, Leander Beekman a, Connie R Bezzina a, Arthur A.M Wilde a,e, Elisabeth M Lodder a,⁎ a Heart Center, Department of Clinical and Experimental Cardiology, Academic Medical Center, Amsterdam, The Netherlands 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 b a r t i c l e i n f o Article history: Received November 2016 Accepted 24 January 2017 Available online xxxx Keywords: Genetics Inherited channelopathies Atrial fibrillation Ventricular ectopy SCN5A mutation Arrhythmia a b s t r a c t Background: Mutations in SCN5A, the gene encoding the α-subunit of the cardiac sodium channel (NaV1.5), are 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 phenotype 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 phenotype 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 current 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 excitability Conclusions: This mutation leads to a gain-of-function mechanism based on increased channel availability and increased window current, fitting the observed clinical phenotype of (likely adrenergic-induced) ventricular arrhythmias and atrial fibrillation These findings further expand the range of cardiac arrhythmias associated with mutations in SCN5A © 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/) Introduction Abbreviations: SCN5A, the gene encoding the α-subunit of the cardiac sodium channel; NaV1.5, cardiac sodium channel; AF, atrial fibrillation; AP, action potential; ECG, electrocardiogram; HEK, human embryonic kidney cell; INa, voltage gated sodium current; MRI, magnetic resonance imaging; NSVT, non sustained ventricular tachycardia; PVC, premature 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) These authors contributed equally Mutations in SCN5A encoding the main voltage-gated sodium channel α-subunit in the heart (NaV1.5) [1] have been associated with a spectrum of cardiac arrhythmias including congenital long QT syndrome [2], Brugada syndrome [3], sick sinus syndrome [4,5], progressive cardiac conduction defect [6], atrial fibrillation (AF) [7], and more recently, 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 suppressed at high heart rates So far, only one mutation in SCN5A- 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/) Please cite this article as: K.V Lieve, et al., Gain-of-function mutation in SCN5A causes ventricular arrhythmias and early onset atrial fibrillation, Int J Cardiol (2017), http://dx.doi.org/10.1016/j.ijcard.2017.01.113 K.V Lieve et al / International Journal of Cardiology xxx (2017) xxx–xxx (p.R222Q) [8,9] has been associated with the MEPPC phenotype Another SCN5A mutation, p.I141V, was recently associated with predominantly 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 likely important adrenergic component 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 Declaration of Helsinki 2.2 Mutation analysis A panel of the genes that have previously been associated with different cardiomyopathies and catecholaminergic polymorphic ventricular tachycardia were sequenced by next-generation sequencing (NGS) in the proband (individual III.4 In Fig 1) NGS was complemented with Sanger sequencing to ensure coverage of all exons and exonintron 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 performed on an Illumina HiSeq2000 (Illumina, San Diego, California) using the paired end × 100 bp method Variants with an allele frequency of N 1% 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.5551A N 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) supplemented with 10% FBS (Biowest), penicillin-streptomycin (Gibco) and L-glutamine (Gibco) in a 5% CO2 incubator at 37 °C Cells were transfected at 70% confluency in 25 cm culture flasks with the wildtype or the mutant NaV1.5 construct (0.2 μg) together with a β1subunit (SCN1B) construct [13] (0.2 μg) using lipofectamine (Invitrogen, Carlsbad, USA) Gene-transfer was monitored by means of green fluorescence from the SCN5A-GFP bicistronic vector Patch clamp experiments were performed on fluorescent cells 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 patchclamp 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 accomplished using custom software Signals were low-pass filtered with a cut-off frequency of 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 − mV Fig 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 atrial fibrillation, Int J Cardiol (2017), http://dx.doi.org/10.1016/j.ijcard.2017.01.113 Rest and exercise Monomorphic 849 12.6 ml/m2 660 83 150 HR, heart rate; LA, left Atrium; BSA, body surface area; PVC, premature ventricular contraction; n/a, not available 52 433 90 160 No 125 16 Palpitations III.4 t1:12 Female Not applicable 16 III.3 t1:11 Male n/a III.2 t1:10 Male 55 35 40 n/a Female Male Male Male II.4 II.5 II.6 III.1 t1:6 t1:7 t1:8 t1:9 t1:13 Rest and exercise 21 81 No 139 106 407 64 145 87 22 77 Monomorphic Exercise induced Polymorphic 34,1 ml (BSA n/a) n/a 3103 90 169 45 383 99 151 No 63 82 61 56 52 52 27 25 2 Rest and exercise n/a Rest and exercise Rest and exercise Polymorphic n/a Polymorphic Polymorphic 514 469 13,104 5928 ml/m2 ml/m2 ml/m2 ml/m2 19.5 18,9 17.1 49,5 772 n/a 9101 551 67 63 91 65 140 n/a 224 139 53 43 61 43 372 373 n/a 343 100 98 n/a 80 96 – 120 124 n/a Polymorphic 3203 18 ml/m2 3771 67 123 42 396 103 160 Paroxysmal or persistant (42) Paroxysmal (55) Paroxysmal (40) Paroxysmal (40) No 70 58 Decompensated heart failure Palpitations – None Palpitations, near-syncope Feels extra heart beat None 42 II.2 t1:5 Gender Patient # t1:1 t1:2 Table Patient characteristics 3.1 Clinical phenotype of the family t1:3 t1:4 Results Age at first symptoms (years) Symptoms before diagnosis Age at diagnosis (years) Data are expressed as mean ± standard error of the mean (SEM) Values were considered significantly different if p b 0.05 in unpaired ttest or in Two-Way Repeated Measures of Analysis of Variance (TwoWay Repeated Measures ANOVA) followed by pairwise comparison using the Student-Newman-Keuls test Male 2.5 Statistics The patient characteristics of the affected family members are shown in Table 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 tachycardia 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 arrhythmia during sleep (Table 1) During exercise testing, polymorphic PVCs Total PVCs in 24 h on Holter Resting heart rate Atrial fibrillation (age onset) PR QRS QTc Min HR Holter Max HR Holter Average HR Holter Total supra-ventricular ectopies in 24 h on Holter 2.4.2 Conventional VC and alternating VC/CC experiments The current density of the peak INa, voltage dependence of (in)activation, recovery from inactivation, 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 experiments 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 display 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 upstroke velocity (dV/dtmax) during VC/CC, offline corrected for the contribution of stimulus current, served as an indicator of available INa Peak INa was defined as the difference between peak and steadystate current The sustained as well as the window INa current were measured as the current sensitive for 30 μM TTX To determine the activation characteristics of INa, current-voltage (I-V) curves were corrected for differences in driving force and normalized to maximum peak current Steady-state activation and inactivation curves were fit 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 constants of the fast and the slow components of recovery from inactivation: I/Imax = Af × [1.0 − exp(− t / τf)] + As × [1.0 − exp(− t / τs)], where t is the recovery time interval, τf and ττs the time constants of the fast and slow components, and Af and As the fractions of the fast and slow components, respectively The time course of current inactivation was fitted by a double-exponential equation: I/Imax = Af × exp(−t / τf) + As × exp(−t / τs), where Af and As are the fractions of the fast and slow inactivation components, and τf and τs are the time constants of the fast and slow inactivating components, respectively LA volume PVC morphology on exercise test or Holter PVCs at rest or during exercise Longest Salvo of PVC voltage step from −40 mV by the series resistance Peak INa was measured at room temperature with patch pipettes (borosilicate glass; resistance ≈ 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 INa currents were measured at 37 °C Patch solution was similar to the INa measurements; 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) 3 K.V Lieve et al / International Journal of Cardiology xxx (2017) xxx–xxx Please cite this article as: K.V Lieve, et al., Gain-of-function mutation in SCN5A causes ventricular arrhythmias and early onset atrial fibrillation, Int J Cardiol (2017), http://dx.doi.org/10.1016/j.ijcard.2017.01.113 K.V Lieve et al / International Journal of Cardiology xxx (2017) xxx–xxx occurred at a heart rate of 137 bpm, including PVCs bigeminy PVCs disappeared 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 repeated one year later showed a normalized cardiac function The patient's family history was positive for diverse cardiac arrhythmias 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 evaluation for cascade screening twelve years later showed frequent PVCs on his baseline ECG Subsequent Holter monitoring and exercise testing revealed self-terminating episodes of paroxysmal AF and NSVT that occurred 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 premature atrial contractions and PVCs TTE showed a normal left ventricular function and an enlarged left atrial volume On exercise testing sinus arrhythmia and polymorphic PVCs were noted from the start of the exercise test (heart rate of 80 bpm) and lasted throughout the recovery phase (PVCs, bigeminal PVCs, couplets and NSVT, Fig 2) Another brother (III.2) showed ventricular arrhythmias during exercise testing that increased in frequency and complexity at higher workloads to bigeminal 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 unknown 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 normalized 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 diagnosed 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, exercise induced and non-exercise induced ventricular arrhythmias in combination with early onset (b 55 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 receptor (RYR2) using NGS in the proband This screen yielded one rare variant in SCN5A (NM_001099404.1), c 5551A N 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 mutation was absent in N 2000 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 demonstrated 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 mutation on INa function First, the current density and gating properties of WT and p.M1851V were assessed Fig 3A, top, shows representative INa activated by 50-ms depolarizing voltage clamp steps of mV increment Typical INa current 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 amplitude divided by the Cm, did not differ significantly between WT and mutant currents The typical examples in Fig 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 INa inactivation Indeed, τfast is significantly higher which indicates a slowed current inactivation Fig 3C shows the average (in)activation relationships The curves of the voltage dependence of activation of mutant and wildtype channels were overlapping indicating that it was not affected by the p.M1851V SCN5A mutation Voltagedependency of inactivation was measured using a two-pulse protocol where a 500-ms conditioning prepulse to membrane potentials between −120 and mV, to induce steady-state inactivation, was followed 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 INa inactivation was measured using a two-pulse protocol, where a 1-s conditioning prepulse (P1) to −20 mV (to inactivate Na+ channels) was followed by a test pulse (P2) after a variable recovery interval ranging between and 1000 ms at a recovery potential of −85 mV (see inset of Fig 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 doubleexponential function to obtain the time constants and fractions of the fast and the slow components of recovery from inactivation Both average τfast and τslow were significantly lower in M1851V channels indicating faster recovery from inactivation (Fig 3D) Secondly, we characterized sustained and window INa currents in WT and M1851V channels The slower speed on INa inactivation (Fig 3A) may have implications for sustained currents, while the positive shift in voltage-dependency of inactivation (Fig 3C) may result in changes in window currents The sustained INa current 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, summarize the average TTX-sensitive sustained current The density of the sustained current was not affected in the M1851V channels The window 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 significantly 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 voltagedependency 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 INa characteristics are measured at fixed membrane potentials in VC Fig 3F shows typical upstrokes and their first derivatives 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 atrial fibrillation, Int J Cardiol (2017), http://dx.doi.org/10.1016/j.ijcard.2017.01.113 K.V Lieve et al / International Journal of Cardiology xxx (2017) xxx–xxx Fig 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 Please cite this article as: K.V Lieve, et al., Gain-of-function mutation in SCN5A causes ventricular arrhythmias and early onset atrial fibrillation, Int J Cardiol (2017), http://dx.doi.org/10.1016/j.ijcard.2017.01.113 K.V Lieve et al / International Journal of Cardiology xxx (2017) xxx–xxx Fig Current density and gating properties of WT and M1851V channels A, top: Typical examples of peak sodium current (INa) 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 INa inactivation Note logarithmic ordinate scale τfast 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 INa 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 τfast (65 ± ms (WT) vs 37 ± ms (M1851V)) and τslow (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 INa 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 INa-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 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 upstroke velocity was significantly higher and the threshold voltage was significantly more negative by M1851V channels (Fig 3F) Discussion We present a relatively large Dutch family with supraventricular arrhythmias including AF, and ventricular arrhythmias, including polymorphic 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 Subsequent electrophysiological studies demonstrated a faster recovery from inactivation and a positive shift in voltage dependency of inactivation in SCN5A-p.M1851V channels leading to increased sodium channel availability and increased window current, explaining the phenotype in the family These findings provide further insight into the broad spectrum 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 in N120,000 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 concordant with the clinical presentation in the family Of note, the functional effects we uncovered for SCN5A-p.M1851V are similar to those previously described for SCN5A-p.R222Q and SCN5A-p.I141V, which are both associated 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 V1/2 of voltage dependency of inactivation (Fig 3C) This may have two important implications for INa function, 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 concordance 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 INa at higher heart rate compared to wild-type channels This faster recovery from inactivation in the p.M1851V mutation carriers may contribute to a larger Please cite this article as: K.V Lieve, et al., Gain-of-function mutation in SCN5A causes ventricular arrhythmias and early onset atrial fibrillation, Int J Cardiol (2017), http://dx.doi.org/10.1016/j.ijcard.2017.01.113 K.V Lieve et al / International Journal of Cardiology xxx (2017) xxx–xxx susceptibility for atrial arrhythmias eventually resulting in AF in all second generation carriers Fourthly, we observed a more negative threshold for upstroke generation for M1851V channels (Fig 3F) At first glance this contrasts the finding that V1/2 of INa activation was unaffected as found in VC experiments (Fig 3C) We think, however, that the more negative threshold for upstroke generation is observed due to dynamic and more closeto-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 mutations 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 originating from various foci along the fascicular Purkinje system Even though none of the p.M1851V mutation carriers underwent an electrophysiological study, which would provide prove of the origin of the ventricular 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 dysfunction due to frequent PVCs In contrast to the phenotype observed in the M1851V mutation carriers, the p.R222Q carriers had PVCs that occurred 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 mutation leads to an increased window current by affecting the voltage dependence 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 predominantly exercise-induced ventricular arrhythmias [11] The voltage dependence of activation of SCN5A-p.I141V was shifted towards more negative potentials leading to an increase and shift of the window current A third mutation, SCN5A-p.K1493R, with similar electrophysiological 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 patients and no exercise testing was performed Altogether a total of four mutations (including ours) are now described leading to an increase of window current of the cardiac sodium channel NaV1.5 without causing congenital long QT syndrome The clinical phenotype associated with these mutations encompasses both supraventricular 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 mutations with a gain-of-function defect stemming from an increased window 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 Research and Development and the Royal Netherlands Academy of Sciences (CVON 2010-12 PREDICT) to KL, CRB, AAMW and EML; the ERare 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 conflict of 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Please cite this article as: K.V Lieve, et al., Gain- of- function mutation in SCN5A causes ventricular arrhythmias and early onset atrial. .. exercise induced and non-exercise induced ventricular arrhythmias in combination with early onset (b 55 years) AF in the setting of normal left atrial volumes and mild cardiac remodeling 3.2 Genotyping