1 2 3 4 Q4 Q3 5 6 7 8 9 10 11 12Q5 Q1 13 14 15 16 17 18Q6 19 20 21 Q7 22 23 24 25 26 27 28 29 30 31 32Q8 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54

A truncating SCN5A mutation combined with genetic variability causes sick sinus syndrome and early atrial fibrillation Azza Ziyadeh-Isleem, MD, PhD,1*† Jérôme Clatot, PhD,1*†‡ Sabine Duchatelet, PhD,*† Estelle Gandjbakhch, MD, PhD,*†§ Isabelle Denjoy, MD,*†║ Françoise Hidden-Lucet, MD,*†§ Stéphane Hatem, MD, PhD,*† Isabelle Deschênes, PhD,‡ Alain Coulombe, PhD,*† Nathalie Neyroud, PhD,2*† Pascale Guicheney, PharmD, PhD2*† From the *INSERM, UMR_S1166, Paris, France, †Sorbonne Universités, UPMC Univ Paris 06, UMR_S1166, Institute of Cardiometabolism and Nutrition (ICAN), Paris, France, ‡Heart and Vascular Research Center, MetroHealth Campus, Case Western Reserve University, Cleveland, Ohio, §AP-HP, Hôpital Pitié-Salpêtrière, Département de Cardiologie, Paris, France and ║AP-HP, Hôpital Bichat, Département de Cardiologie, Centre de Référence des Maladies Cardiaques Héréditaires, Paris, France. BACKGROUND Mutations in the SCN5A gene, encoding the α subunit of the cardiac Naþ channel, Nav1.5, can result in several life-threatening arrhythmias. OBJECTIVE To characterize a distal truncating SCN5A mutation, R1860Gfs*12, identified in a family with different phenotypes including sick sinus syndrome, atrial fibrillation (AF), atrial flutter, and atrioventricular block. METHODS Patch-clamp and biochemical analyses were performed in human embryonic kidney 293 cells transfected with wild-type (WT) and/or mutant channels. RESULTS The mutant channel expressed alone caused a 70% reduction in inward sodium current (INa) density compared to WT currents, which was consistent with its partial proteasomal degradation. It also led to a negative shift of steady-state inactivation and to a persistent current. When mimicking the heterozygous state of the patients by coexpressing WT and R1860Gfs*12 channels, the biophysical properties of INa were still altered and the mutant channel α subunits still interacted with the WT channels. Since the proband developed paroxysmal AF at a young age, we screened 17 polymorphisms associated with AF risk in this family and showed that the proband carries at-risk polymorphisms upstream of PITX2, a gene widely associated with AF

development. In addition, when mimicking the difference in resting membrane potentials between cardiac atria and ventricles in human embryonic kidney 293 cells or when using computer model simulations, R1860Gfs*12 induced a more drastic decrease in INa Q9 at the atrial potential. CONCLUSION We have identified a distal truncated SCN5A mutant associated with gain- and loss-of-function effects, leading to sick sinus syndrome and atrial arrhythmias. A constitutively higher susceptibility to arrhythmias of atrial tissues and genetic variability could explain the complex phenotype observed in this family. KEYWORDS Arrhythmia; Atrial fibrillation; Nav1.5; SCN5A; Sodium; PITX2; Polymorphism; SNP ABBREVIATIONS AF ¼ atrial fibrillation; AP ¼ action potential; BrS ¼ Brugada syndrome; GFP ¼ green fluorescent protein; HEK293 ¼ human embryonic kidney 293; HP ¼ holding potential; INa ¼ inward sodium current; LQTS ¼ long QT syndrome; SNP ¼ single nucleotide polymorphism; SSS ¼ sick sinus syndrome; τf and τs ¼ fast and slow time constants of inward sodium current inactivation; TTX ¼ tetrodotoxine; WT ¼ wild type (Heart Rhythm 2014;0:-2–9) I 2014 Heart Rhythm Society. Published by Elsevier Inc. All rights reserved.

Introduction This work was supported by INSERM, the Université Pierre et Marie Curie, the Agence Nationale de la Recherche (ANR-09-GENO-003-CaRNaC), the French Ministry of Health (PHRC AOR04070, P040411), and NIH (R01 HL094450). Address reprint requests and correspondence: Dr Pascale Guicheney, INSERM, UMR_S 1166, Faculté de médecine Pierre et Marie Curie, 91, boulevard de l'Hôpital, F-75013 Paris, France. E-mail address: [email protected]. 1 Dr Ziyadeh-Isleem and Dr Clatot contributed equally to this work. 2 Dr Neyroud and Dr Guicheney contributed equally to this work.

The coordinated activity of multiple ion channels tightly controls the generation and propagation of cardiac action potentials (APs).1 Mutations in the SCN5A gene, encoding the α subunit of the cardiac sodium channel, Nav1.5, have been involved in numerous inherited cardiac arrhythmias including long QT syndrome (LQTS), Brugada syndrome (BrS), and rare cases of sick sinus syndrome (SSS) and atrial fibrillation (AF).2 Atrial arrhythmias are being increasingly diagnosed in patients with BrS (incidence of 6%–38%)3 as

1547-5271/$-see front matter B 2014 Heart Rhythm Society. Published by Elsevier Inc. All rights reserved.

http://dx.doi.org/10.1016/j.hrthm.2014.02.021

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2 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81Q10 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111Q11 112 113 114 115 116 117Q12 118 119Q13 120 121 122

well as LQTS.4 Originally, various SCN5A-related arrhythmias were considered separate clinical entities with distinct phenotypical characteristics. Recently, a wide spectrum of mixed disease phenotypes was reported in these arrhythmias, referred to as an overlap syndrome of cardiac Naþ channelopathy.2 The reasons why the same SCN5A mutation can result in different phenotypes remain unknown, but it raises the possibility that the disease expressivity is affected by altered biophysical properties and genetic modifiers.5 In this study, we characterized the Nav1.5 C-terminal truncating mutation R1860Gfs*12 identified in a family presenting with a complex clinical picture of SSS and AF or atrial flutter. The heterologous expression of the mutant channels alone or with wild-type (WT) channels led to a reduction in inward sodium current (INa) density, a persistent Naþ current, and a drastic alteration of the inactivation properties. Interestingly, because of the constitutively different resting membrane potentials in atrial and ventricular tissues, the atrium of the patients might be more susceptible to the altered biophysical properties of the mutant channels and, thus, more prompt to develop arrhythmias. Moreover, the proband carries at-risk polymorphisms upstream of PITX2, a gene widely associated with AF development. Altogether, our results could explain the mixed clinical phenotype of this family.

Methods Patient Blood samples were obtained after signed written informed consent for genetic analyses and after approval by the local ethics committee. The study was conducted according to the principles of the Helsinki Declaration.

Mutation and single nucleotide polymorphism analysis Genomic DNA was isolated from leukocytes according to standard procedures. Screening for mutations was performed by genomic DNA amplification of all exons and splice junctions of several genes responsible for arrhythmias (see the Online Supplemental Methods). We also genotyped single nucleotide polymorphisms (SNPs) associated with AF6,7 (Online Supplemental Table 1). The polymerase chain reaction products were directly sequenced with the Big Dye Terminator v.3.1 kit on an ABI PRISM 3730 automated DNA sequencer (Applied Biosystems, Carlsbad, CA, USA). Variants were identified by visual inspection of the sequences with Seqscape software (Applied Biosystems).

Heart Rhythm, Vol 0, No 0, Month 2014 XL Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA, USA) according to the manufacturer’s instructions and verified by sequencing. The plasmid pIRES2-acGFP1-FHF1B (C-terminal His6) was the gift of Dr G.S. Pitt (Durham, NC).

Human embryonic kidney 293 cell culture and transfection Human embryonic kidney 293 (HEK293) cells were transfected with jetPEI (Polyplus Transfection, Illkirch, France) according to the manufacturer’s instructions. For patch-clamp recordings, HEK293 cells were transfected with pcDNA3.1GFP-hH1a WT or mutant in 35-mm well dishes, with a total of 0.6 mg of plasmid per 35-mm well dish. To mimic the heterozygous state of the patient, cells were cotransfected with 0.3 mg of pcDNA3.1-hH1a (no tag) WT (50% WT) and 0.3 mg of pcDNA3.1-GFP-hH1a mutant (50% mutant). For biochemical analysis, cells were plated in 25-cm² flasks and transfected with 2 mg of pcDNA3.1-GFP-hH1a WT or mutant.

Electrophysiological recordings Patch-clamp recordings were carried out in the whole-cell configuration at room temperature ( 221C). Solutions for patch-clamp recordings are described in the Online Supplemental Methods. Ionic currents were recorded with the amplifier Axopatch 200B (Axon Instruments, Foster City, CA, USA). Patch pipettes (Corning Kovar Sealing code 7052, WPI) had resistances of 1.5–2.5 MΩ when filled with pipette medium. Currents were filtered at 10 kHz (3 dB, 8pole low-pass Bessel filter) and digitized at 50 kHz (NI PCI6251, National Instruments, Austin, TX). Data were acquired and analyzed with ELPHY2 software (developed by G. Sadoc, CNRS, Gif-sur-Yvette, France). Current-voltage relationships and steady-state inactivation-Vm relationships were used as reported previously.8 Data for the activation-Vm and steady-state inactivation-Vm relationships of INa were fitted to the Boltzmann equation as reported previously.8 The putative involvement of a persistent Naþ current was assessed by using 100 mM tetrodotoxine (TTX). Currents were increased from a holding potential (HP) of 120 to 20 mV in 500-ms steps. The percentage of persistent current was calculated by dividing the TTX-sensitive INa amplitude determined at the end of the pulse by the peak INa amplitude obtained before TTX application. Computer simulations of atrial and ventricular membrane APs were performed by using Oxsoft Heart model (Version 4.8, Oxsoft Ltd, Oxford, England) as described in the Online Supplemental Methods.

SCN5A cDNA cloning and mutagenesis Plasmids pcDNA3.1-hH1a (no tag) and pcDNA3.1-GFPhH1a (N-terminal green fluorescent protein (GFP)) were the gift of Dr H. Abriel (Bern, Switzerland). The plasmid pRcCMV-Flag-SCN5A (N-terminal Flag) was the gift of Dr N. Makita (Nagasaki, Japan). All these plasmids contain the hH1a isoform of SCN5A. The Nav1.5 mutant R1860Gfs*12 was prepared by using the QuikChange II

Protein extraction and Western blot They were performed as reported previously.8 Primary antibodies used were as follows: rabbit anti-GFP (1:2000, Torrey Pines Biolabs, Houston, TX, USA), mouse anti-Flag (1:500, Sigma, St Louis, MO, USA), and rabbit anti–glyceraldehyde 3-phosphate dehydrogenase (1:2000, Abcam, Cambridge, UK). Total protein signals were first normalized

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Ziyadeh-Isleem et al 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195Q14 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 P 219 R 220 I 221 N 222 T 223 & W 224 E 225 B 226 4 227 C 228 / 229 F 230 P O 231 232 233 234 235 236

Effects of a Nav1.5 Truncating Mutation

to glyceraldehyde 3-phosphate dehydrogenase levels and then to WT levels.

Co-immunoprecipitation Co-immunoprecipitation experiments were performed as described in the Online Supplemental Methods. Briefly, total cell lysates were incubated with Dynabeads (Dynal, Oslo, Norway) cross-linked to anti-GFP antibody (Torrey Pines Biolabs) for 2 hours at room temperature. Western blots were revealed by using either rabbit anti-GFP (Torrey Pines Biolabs) or mouse anti-Flag (Sigma) antibodies.

Statistical analysis Data are presented as mean ⫾ standard error of the mean. Statistical significance was estimated with SigmaPlot software, San Jose, CA by using the Student t test or analysis of variance, as appropriate. A P value of o.05 was considered significant.

3

Results Identification of a truncating mutation in SCN5A The proband (III.2) was a 26-year-old woman diagnosed at the age of 13 years with severe SSS associated with severe sinus bradycardia, symptomatic sinus pauses, and syncope (Figures 1A and 1B), which required a pacemaker implantation. At the age of 20 years, paroxysmal AF was documented in the memories of the pacemaker. She secondarily developed a first-degree atrioventricular block (PR interval 280 ms; QRS duration 80 ms; corrected QT interval 360 ms). Her father (II.2) was diagnosed at the age of 27 years with an asymptomatic sinus node dysfunction. At the age of 42 years, he had 1 episode of atrial flutter after jogging and was treated by cardioversion. Moreover, he developed firstdegree atrioventricular block with left anterior hemiblock (PR interval 240 ms; QRS duration 80 ms; QT interval 400 ms) (Figures 1A and 1C, a and b). The proband’s uncle (II.1) was diagnosed with SSS, which required a pacemaker

Figure 1 Pedigree and ECG recordings of the family. A: Pedigree of the family. The proband (III.2) is indicated by an arrow. Squares represent males; circles represent females; and þ and  signs represent mutation carriers and noncarriers, respectively. The haplotypes for 5 single nucleotide polymorphisms located upstream of the PITX2 gene are indicated under each subject. At-risk alleles are written in red and protective alleles in blue. B: ECG recordings of the proband. The pacemaker spikes are indicated by arrows. The proband’s ECG showed prolongation of the PR interval, which is a sign of first-degree atrioventricular block. C: ECG recordings of the father (II.2), showing (a) the atrial flutter that he developed at the age of 42 years and (b) his normal sinus rhythm after cardioversion with left-axis deviation. (c) and (d) ECG recordings of II-2 during the ajmaline test (1 mg/kg over 5 min) he underwent at the age of 64 years. At basal condition (panel c, time 0), the patient had a regular sinus rhythm with a heart rate of 60 beats/min, a prolongation of the PR interval of 240 ms, and a QRS duration of 120 ms. After the injection of ajmaline (panel d, time 7 min), bradycardia was observed (heart rate 40 beats/min) with a prolongation of the PR interval (300 ms) and the QRS duration (240 ms) but without a sign of BrS. Rapid sodium-bicarbonate infusion was then started immediately. AV ¼ atrioventricular; ECG ¼ electrocardiographic; SSS ¼ sick sinus syndrome.

237 238 239 240 241 F1242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293

4 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347Q21 348 349 350

implantation, at the age of 30 years. None displayed spontaneous Brugada electrocardiographic pattern or developed ventricular arrhythmias. The proband’s father underwent an ajmaline challenge that excluded BrS electrocardiographic pattern (Figure 1C, c and d). It induced bradycardia (40 beats/min) with a severe prolongation of the PR interval (300 ms) and the QRS duration (240 ms). In addition, sinus node dysfunction with junctional escape rhythm and supraventricular premature contractions were observed (Online Supplemental Figure 1). Echocardiograms ruled out structural heart disease except for mild atrial dilatation in the proband’s father. The mother and the proband’s sister were asymptomatic (Figure 1A). We sequenced the coding regions of 14 genes involved in cardiac arrhythmias and identified a unique mutation in SCN5A, a deletion of 1 bp (A) at the position 5578 in exon 28 (c.5578delA). This deletion induced a frameshift mutation, p.R1860Gfs*12, which changed the amino acid arginine at position 1860 to glycine followed by 10 frameshift amino acids before a premature stop codon (Online Supplemental Figure 2). The proband and her father and uncle carried this mutation, whereas her mother and sister did not (Figure 1A). This variant has never been described and is absent from publicly available databases. Suspecting the possible contribution of additional genetic factors for AF development, we genotyped the family members for 17 SNPs that alter AF susceptibility (Online Supplemental Table 1). Interestingly, the proband, who experienced severe SSS and early-onset AF, is the only

Heart Rhythm, Vol 0, No 0, Month 2014 mutation carrier to have 1 at-risk allele of rs6817105 and rs2200733 located upstream of the PITX2 gene, which she received from her mother (Figure 1A and Online Supplemental Table 1). In addition, the father had 2 copies of the protective allele of rs3853445, another SNP located upstream of the PITX2 gene, while the proband carried only 1 copy (Figure 1A and Online Supplemental Table 1). The multimarker risk score for AF based on combined rs2200733, rs17570669, and rs3853445 genotypes9 was higher in the proband (1.74) than in her father (o1) and uncle (o1).

The R1860Gfs*12 mutation produced a loss- and gain of function of Nav1.5

Naþ currents were recorded in HEK293 cells 36 hours after transfection with WT or mutant channels. INa traces and current-voltage relationships are shown in Figure 2. Peak current densities and P values are given in Table 1. R1860Gfs*12 alone showed a drastic reduction in INa density compared to WT channels (Figures 2A and 2B). Furthermore, the biophysical characteristics of the Cterminal mutant channels were impaired (Table 1 and Figures 2C and 2D). Indeed, the V1/2 of activation for R1860Gfs*12 was shifted by 7.1 mV and the V1/2 of inactivation was shifted by 24.5 mV compared to WT channels. No change in recovery from inactivation was observed (Online Supplemental Figure 3). Fast and slow time constants of inactivation (τf and τs) were

Figure 2 Biophysical characterization of Nav1.5 channels. A: Representative families of Naþ current traces of WT, R1860Gfs*12, and WT þ R1860Gfs*12 channels. B: Current-voltage relationships. The current density of the R1860Gfs*12 channel is significantly reduced compared to WT and WT þ R1860Gfs *12 channels. C: Activation-Vm relationship for the R1860Gfs*12 channel was shifted to more positive potentials compared to WT and WT þ R1860Gfs*12 channels. D: Steady-state inactivation-Vm relationships for R1860Gfs*12 and WT þ R1860Gfs*12 channels were shifted to negative potentials compared to WT channels. Numbers of cells and the results of the statistical analysis are given in Table 1. WT ¼ wild type.

351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 F2368 T1369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407

***

* 6.3 ⫾ 0.5 0.07 5.9 ⫾0 .4

Data are presented as mean ⫾ standard of error of the mean. Peak current density was calculated at 20 mV. INa ¼ inward sodium current; k ¼ inverse slope factor; τf and τs ¼ fast and slow time constants of inward sodium current inactivation; V1/2 ¼ half activation or inactivation value; WT ¼ wild type. 1/2 WT þ 1/2 R1860Gfs*12 indicates that cells were co-transfected with the WT and the mutant channels in a 1:1 ratio. *Pr0.05, **Pr0.005 and ***Pr0.001 when compared to cells transfected with the WT channel alone

WT

280.8 ⫾ 22.2 (n ¼ 20) 80.3 ⫾ 16.4 Q23 R1860Gfs*12 (n¼13) 237.0 ⫾ 22.5 Q24 ½ WTþ ½ R1860Gfs*12 (n ¼ 14)

0.19

***

44.2 ⫾ 1.6 (n ¼ 17) 37.1⫾1.4 (n ¼ 12) 40.8 ⫾ 2.0 (n ¼ 12)

0.19

0.21 **

7.0 ⫾ 0.4

6.1 ⫾ 0.6

84.5 ⫾ 1.9 (n ¼ 12) 108.1 ⫾ 1.8 (n ¼ 8) 101.1⫾1.7 (n ¼ 11)

***

6.3 ⫾ 0.4 ***

8.1 ⫾ 0.6

*

0.48 ⫾ 0.02 (n ¼ 15) 1.27 ⫾ 0.05 (n ¼ 22) 0.69 ⫾ 0.05 (n ¼ 17)

***

***

2.54 ⫾ 0.30 (n ¼ 15) 9.80 ⫾ 0.80 (n ¼ 22) 4.55 ⫾ 0.40 (n ¼ 17)

P τs (ms) P τf (ms) P k (mV) P V1/2 (mV) Naþ channels

Peak current density (pA/pF)

P

V1/2 (mV)

P

k (mV)

P

Inactivation Activation

Biophysical and kinetic properties of INa from WT and mutant channels Table 1

408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464

Effects of a Nav1.5 Truncating Mutation ***

Ziyadeh-Isleem et al

5 significantly increased compared to WT channels (Table 1 and Figures 3A–3C). The presence of a TTX-sensitive persistent Naþ current was assessed by using 100 μM TTX (Figure 3D and Online Supplemental Figure 4). The mutant channel generated a persistent current of 4% of the peak current.

Coexpression of WT and R1860Gfs*12 channels also led to a loss- and gain of function of Nav1.5 To mimic the heterozygous state of the patient, cells were cotransfected with the WT channel (no tag) and the R1860Gfs*12 mutant channel (GFP tagged) in a 1:1 ratio. In cotransfected cells, peak current densities were slightly reduced (Figures 2A and 2B), but not significantly different from those transfected with the WT alone, as were the V1/2 of activation. In contrast, the V1/2 of steady-state inactivation remained significantly shifted by 16.6 mV compared to cells transfected with the WT alone (Table 1 and Figures 2C and 2D). Moreover, τf and τs were significantly increased at the heterozygous state (Table 1 and Figures 3A and 3B), and the persistent current was still present (Table 1, Figure 3D, and Online Supplemental Figure 4). Since the steady-state inactivation of the mutant channels alone or coexpressed with WT channels were drastically shifted, we measured INa successively from an HP of 86 and 83 mV, mimicking the ventricular vs atrial resting membrane potentials.10,11 Interestingly, we showed that the 3-mV difference in HPs was sufficient to enhance the reduction in INa density due to the mutation in the atrium compared to the ventricle (Figure 3E). Indeed, the reduction in INa density was 17% for WT channels, 25% for WT þ R1860Gfs*12, and 40% for R1860Gfs*12 at an HP of 83 mV vs 86 mV. Differences between WT and heterozygous channel characteristics were included into computer model simulations of single atrial and ventricular cell membrane APs (see the Online Supplemental Methods). The AP maximum upstroke velocity of the heterozygous state exhibited a significant decrease in the atrial (68%) compared to the ventricular (33%) myocyte, as well as a marked lengthening of the AP duration in the atria (Figure 4).

The R1860Gfs*12 mutant was partially degraded by the ubiquitin-proteasome system Western blots of total lysates from transfected HEK293 cells showed a significant decrease of  70% in the total protein expression of the mutant channels as compared to the WT channels in accordance with the reduction in INa density (Figure 5A). Moreover, incubation of the cells with the 26Ssubunit ubiquitin-proteasome inhibitor MG132 significantly increased the mutant total expression, suggesting a proteasomal degradation of the R1860Gfs*12 mutant (Figure 5A).

The R1860Gfs*12 mutant interacted with WT α subunits We have previously demonstrated through co-immunoprecipitation that Nav1.5 α subunits interact with each other.8

465 F3466

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Heart Rhythm, Vol 0, No 0, Month 2014

Figure 3 Kinetics of INa characterization of Nav1.5 channels. A and B: Fast and slow time constants (τf, and τs, respectively) for WT, R1860Gfs*12, and WT þ R1860Gfs*12 channels were plotted against the test potential. R1860Gfs*12 and WT þ R1860Gfs*12 channels showed an overall slowing of the fast and slow rates of inactivation compared to the WT channel. C: Three superimposed representative INa traces corresponding to the above conditions. The R1860Gfs*12 and the WT þ R1860Gfs*12 current traces were normalized to the peak of the WT current (E1200 pA). D: Ratios of the persistent Naþ current amplitude (end of pulse) over the peak INa amplitude for WT, WT þ R1860Gfs*12, and R1860Gfs*12 channels, reported in percentage. E: Ratios of the INa loss between HPs of 86 and 83 mV for WT, WTþ R1860Gfs*12, and R1860Gfs*12. Peak current density was calculated at 20 mV. *P r .05, **P r .01, *** P r .001, and n is the number of tested cells. INa ¼ inward sodium current; WT ¼ wild type.

To assess whether the R1860Gfs*12 truncating mutant still interacted with the WT channel, we performed coimmunoprecipitation in HEK293 cells transfected with either Flag-WT þ GFP-WT or Flag-WT þ GFP-R1860Gfs*12 and with Flag-WT alone as a negative control. Immunoprecipitations were performed with the anti-GFP antibody, and Western blots were revealed with the anti-Flag antibody. In cells expressing Flag-WT þ GFP-WT or Flag-WT þ GFPR1860Gfs*12, a specific band at the expected size of FlagWT was present (Figure 5B), indicating that the WT and the R1860Gfs*12 truncated α subunits interacted with each other. To test whether the R1860Gfs*12 α subunits could interact with each other, co-immunoprecipitation was performed in HEK293 cells transfected with Flag-R1860Gfs*12 þ GFP-R1860Gfs*12. Figure 5B shows that the interaction between truncated α subunits still occurred. Thus, our results suggested that the last 156 amino acids of the C-terminal domain of Nav1.5 were not essential for the interaction between Nav1.5 α subunits.

Discussion We functionally characterized a novel frameshift deletion in SCN5A inducing the most distal truncation of the C terminus of Nav1.5 reported so far, which was identified in a family

with early SSS and atrial arrhythmias. We report that the R1860Gfs*12 mutant showed a loss- and gain-of-function phenotype, which, combined with the variable genetic background of the mutation carriers, may explain the patients’ clinical characteristics.

Functional characteristics of R1860Gfs*12 channels Mutations in SCN5A are known to cause various types of arrhythmias including BrS, LQTS, sinus node dysfunction, AF, and overlap syndrome.2 In general, SCN5A mutations leading to a decrease in INa (loss of function) have been associated with conduction slowing and BrS. Gain-offunction mutations in SCN5A are typical of LQTS. Nevertheless, numerous reports have shown that a single Nav1.5 mutation may induce various combinations of clinical phenotypes.2 Here, the R1860Gfs*12 mutant channel expressed alone was mostly degraded, causing a severe reduction in INa density. Moreover, it induced a 7-mV positive shift of activation, which could delay the AP, and a dramatic 25-mV shift of the steady-state inactivation, which is likely due to the location of the mutation within a region involved in the inactivation of Nav1.5 and would decrease INa and affect the AP velocity. In addition, we showed an inactivation defect delaying the fast and slow inactivation. This loss-of-function phenotype was associated

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Ziyadeh-Isleem et al 636 637 638 639 640 641 642 643 644 645 646 647 648 649 650 651 652 653 654 655 656 657 658 659 660 661 P 662 R I 663 N 664 T 665 & 666 W 667 E 668 B 669 4 C 670 / 671 F 672 P 673 O 674 675 676 677Q22 678 679 680 681 682 683Q19 684 685 686 687 688 689 690 691 692

Effects of a Nav1.5 Truncating Mutation

7 some BrS SCN5A mutations through the retention of WT channels by mutant channels.8 Here, we showed that the truncated R1860Gfs*12 mutant channels were still interacting with the WT channels. This result suggests that the last 156 amino acids of the C-terminal domain of Nav1.5 are not the main site for the α-subunit interaction. In addition, the significant negative shift of the steady-state inactivation in cells coexpressing mutant and WT channels suggests that Nav1.5 α subunits also function as oligomers. Alternatively, the C terminus of Nav1.5 is a site of interactions with several proteins that have important roles not only in channel function but also in the stabilization of the channels within the plasma membrane.18 We verified that the truncated R1860Gfs*12 channel did not interact with FHF1B (see the Online Supplemental Methods and Online Supplemental Figure 5). A similar loss of interaction with partners such as SAP97 and syntrophin could contribute to a loss of stability and expression of the truncated channels at the cell membrane.18

R1860Gfs*12 gain- and loss-of-function characteristics underlie a complex clinical phenotype

Figure 4 Computer model simulations of single atrial cell and ventricular cell membrane action potentials. Computed AP time courses and expanded timescale inset exhibiting first time derivatives (dV/dt) of the AP upstroke in the atrium (A) and in the ventricle (B). Computed dV/dt time courses exhibit a larger decrease in [dV/dt]max for the “heterozygous atrium” condition than for WT (68 V/s vs 212 V/s, respectively) and a smaller decrease for the “heterozygous ventricle” condition than for WT (147 V/s vs 220 V/s, respectively). AP ¼ action potential; WT ¼ wild type.

with gain-of-function characteristics since the mutant produced a significant persistent Naþ current. When coexpressing this mutant with WT channels in order to mimic the heterozygosity of the patients, the steady-state inactivation was also importantly shifted to the left and a persistent current was still present. These parameters have been shown to be a substrate for AF by causing repolarization failure, early afterdepolarizations, and delayed afterdepolarizations.12–14 Moreover, regarding the important role of Nav1.5 channels in the sinoatrial node function,15,16 the negative shift of inactivation and the persistent current could account for the sinus node dysfunction in this family.17 We have recently shown that Nav1.5 α subunits were able to oligomerize, leading to the dominant-negative effect of

Since Nav1.5 channels are expressed in both the atrium and the ventricle, it is unclear why the R1860Gfs*12 mutation carriers showed only atrial but no ventricular arrhythmias. One hypothesis is that the constitutively different electrophysiological properties of atrial and ventricular cells might be an underlying cause. Indeed, it has been shown that the resting membrane potential is more depolarized10,11 and the INa density is more important with a negative shift in the steady-state inactivation in the atrium compared to the ventricle.10 Moreover, atrial cells are more readily excitable than ventricular cells.11 This suggests that the atrium is constitutively more susceptible to develop arrhythmias than is the ventricle. This led us to explore the R1860Gfs*12 mutant current density at the atrial vs the ventricular resting membrane potential. Here, we showed that a 3-mV difference in resting membrane potentials led to a significant decrease in the peak current by 40% in the atria vs the ventricle for the mutant alone and by 25% when the mutant was coexpressed with WT channels. These results were confirmed by computer model simulations, showing that the coexpression of mutant and WT channels led to a significant decrease in the maximum upstroke velocity in the atrial compared to the ventricular myocytes as well as a significant increase in the AP duration. Therefore, this SCN5A mutation could have different tissue consequences and could contribute to the predominance of an atrial phenotype in this family. A very few C-terminal truncating mutations have been identified in Nav1.5,19,20 but their functional consequence have not been studied, except for the C-terminal truncating mutation L1821fs*10. This mutation has been detected in a patient with a complex clinical phenotype of congenital SSS, cardiac conduction disorder with recurrent monomorphic ventricular tachycardia, but no BrS.20 Functional

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Figure 5 Biochemical analysis. HEK cells were transfected with GFP-Nav1.5 (panel A) and with GFP-Nav1.5 and/or Flag-Nav1.5 (panel B). Data are represented in arbitrary units. Glyceraldehyde 3-phosphate dehydrogenase was used as total protein loading control. A: Western blots of total cell lysates show that the R1860Gfs*12 protein quantity is significantly lower than the WT protein quantity. Incubation of transfected cells with MG132 prevented the degradation of the C-terminal mutant. ***P r .001. B: Co-immunoprecipitation of Nav1.5 α subunits tagged with either Flag or GFP. Immunoprecipitation was performed with the anti-GFP antibody, and Western blot was revealed with the anti-Flag antibody. These results demonstrated an interaction between R1860Gfs*12 α subunits and between WT and R1860Gfs*12 α subunits. IP ¼ immunoprecipitation; WT ¼ wild type.

characterization of this mutant evidenced biophysical properties similar to the ones of R1860Gfs*12, displaying a gainand loss-of-function phenotype. Nevertheless, Tan et al20 reported only the absence of a dominant-negative effect of the mutant on the WT channel INa density whereas we observed an alteration of the inactivation biophysical parameters and the presence of a persistent current when R1860Gfs*12 was coexpressed with WT channels. Altogether, these observations suggest that other parameters, such as additional genetic factors, might affect the clinical expression of the R1860Gfs*12 mutation.

Genetic background of the R1860Gfs*12 mutation carriers The early occurrence of sinus node dysfunction in the proband at the age of 13 years, followed by AF at the age of 20 years was suggestive of a specific genetic background

in this patient. Thus, we explored the possibility that polymorphisms associated with AF could modulate the phenotypic expression of the R1860Gfs*12 mutation. Interestingly, the proband is the only mutation carrier who received the PITX2 at-risk alleles for AF (rs6817105 and rs2200733).6,7 Furthermore, she received only 1 PITX2 protective allele (rs3853445)9 while her father had 2 copies. The role of the PITX2 transcription factor has been reported in the development of the sinus node and pulmonary vein myocardium,21 which is an important site for AF initiation. Among the numerous variants associated with AF, the 4q25 variants upstream of the PITX2 gene have the largest effect in early and late AF.6,7 Reduced PITX2c expression, the main cardiac isoform, was found in patients with AF and was shown to induce atrial electrical and structural remodeling in a murine model with a reduction in mRNA and Nav1.5 protein levels.21 Thus, it is tempting to speculate that the presence of 4q25 variants could induce early atrial

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Effects of a Nav1.5 Truncating Mutation

morphological changes and further loss of function of Nav1.5 channels in the atrium of the proband and may thus account for the occurrence of severe SSS with AF at a young age as compared to her father who had asymptomatic SSS and her uncle who developed SSS later in his life. Of course, additional factors may contribute to the clinical phenotypes.

Conclusion We showed that a C-terminal truncated Nav1.5 α subunit interacts with WT α subunits. Their coexpression induced only a mild reduction in INa density but a marked shift in inactivation and gave rise to a persistent current, which was compatible with SSS and AF. The mutant biophysical defects associated with the difference in resting membrane potentials between the atrium and the ventricle combined with genetic factors might explain the predominantly atrial expression of this mutant. A better knowledge of the functional effects of mutations in SCN5A and the genetic background of the mutation carriers should help in the future to explain the large phenotypical variability of the sodium channelopathies.

Appendix Supporting information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.hrthm. 2014.02.021.

References 1. Roden DM, Balser JR, George AL Jr, Anderson ME. Cardiac ion channels. Annu Rev Physiol 2002;64:431–475. 2. Remme CA. Cardiac sodium channelopathy associated with SCN5A mutations: electrophysiological, molecular and genetic aspects. J Physiol 2013;591: 4099–4116. 3. Francis J, Antzelevitch C. Atrial fibrillation and Brugada syndrome. J Am Coll Cardiol 2008;51:1149–1153. 4. Johnson JN, Tester DJ, Perry J, Salisbury BA, Reed CR, Ackerman MJ. Prevalence of early-onset atrial fibrillation in congenital long QT syndrome. Heart Rhythm 2008;5:704–709. 5. Scicluna BP, Wilde AAM, Wilde AW, Bezzina CR. The primary arrhythmia syndromes: same mutation, different manifestations: are we starting to understand why? J Cardiovasc Electrophysiol 2008;19:445–452.

9 6. Gudbjartsson DF, Arnar DO, Helgadottir A, et al. Variants conferring risk of atrial fibrillation on chromosome 4q25. Nature 2007;448:353–357. 7. Ellinor PT, Lunetta KL, Albert CM, et al. Meta-analysis identifies six new susceptibility loci for atrial fibrillation. Nat Genet 2012;44:670–675. 8. Clatot J, Ziyadeh-Isleem A, Maugenre S, Denjoy I, Liu H, Dilanian G, Hatem SN, Deschênes I, Coulombe A, Guicheney P, Neyroud N. Dominant-negative effect of SCN5A N-terminal mutations through the interaction of Nav1.5 α-subunits. Cardiovasc Res 2012;96:53–63. 9. Lubitz SA, Sinner MF, Lunetta KL, et al. Independent susceptibility markers for atrial fibrillation on chromosome 4q25. Circulation 2010;122:976–984. 10. Burashnikov A, Di Diego JM, Zygmunt AC, Belardinelli L, Antzelevitch C. Atrium-selective sodium channel block as a strategy for suppression of atrial fibrillation: differences in sodium channel inactivation between atria and ventricles and the role of ranolazine. Circulation 2007;116:1449–1457. 11. Golod DA, Kumar R, Joyner RW. Determinants of action potential initiation in isolated rabbit atrial and ventricular myocytes. Am J Physiol 1998;274: H1902–H1913. 12. Lemoine MD, Duverger JE, Naud P, Chartier D, Qi XY, Comtois P, Fabritz L, Kirchhof P, Nattel S. Arrhythmogenic left atrial cellular electrophysiology in a murine genetic long QT syndrome model. Cardiovasc Res 2011;92:67–74. 13. Song Y, Shryock JC, Belardinelli L. An increase of late sodium current induces delayed afterdepolarizations and sustained triggered activity in atrial myocytes. Am J Physiol Heart Circ Physiol 2008;294:H2031–H2039. 14. Sossalla S, Kallmeyer B, Wagner S, Mazur M, Maurer U, Toischer K, Schmitto JD, Seipelt R, Schö ndube FA, Hasenfuss G, Belardinelli L, Maier LS. Altered Naþ currents in atrial fibrillation: effects of ranolazine on arrhythmias and contractility in human atrial myocardium. J Am Coll Cardiol 2010;55: 2330–2342. 15. Dobrzynski H, Boyett MR, Anderson RH. New insights into pacemaker activity: promoting understanding of sick sinus syndrome. Circulation 2007;115: 1921–1932. 16. Lei M, Goddard C, Liu J, et al. Sinus node dysfunction following targeted disruption of the murine cardiac sodium channel gene Scn5a. J Physiol 2005;567: 387–400. 17. Lei M, Huang CL-H, Zhang Y. Genetic Naþ channelopathies and sinus node dysfunction. Prog Biophys Mol Biol 2008;98:171–178. 18. Shy D, Gillet L, Abriel H. Cardiac sodium channel Na(V)1.5 distribution in myocytes via interacting proteins: the multiple pool model. Biochim Biophys Acta 2013;1833:886–894. 19. Kapplinger JD, Tester DJ, Alders M, et al. An international compendium of mutations in the SCN5A-encoded cardiac sodium channel in patients referred for Brugada syndrome genetic testing. Heart Rhythm 2010;7:33–46. 20. Tan B-H, Iturralde-Torres P, Medeiros-Domingo A, Nava S, Tester DJ, Valdivia CR, Tusié-Luna T, Ackerman MJ, Makielski JC. A novel C-terminal truncation SCN5A mutation from a patient with sick sinus syndrome, conduction disorder and ventricular tachycardia. Cardiovasc Res 2007;76:409–417. 21. Franco D, Christoffels VM, Campione M. Homeobox transcription factor Pitx2: the rise of an asymmetry gene in cardiogenesis and arrhythmogenesis. Trends Cardiovasc Med 2014;24:23–31.

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A truncating SCN5A mutation combined with genetic variability causes sick sinus syndrome and early atrial fibrillation.

Mutations in the SCN5A gene, encoding the α subunit of the cardiac Na(+) channel, Nav1.5, can result in several life-threatening arrhythmias...
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