International Journal of Molecular Sciences

Article Functional Characterization of Two Novel in SCN5A Associated with Brugada Syndrome Identified in Italian Patients

Cristina Balla 1,† , Elena Conte 2,†, Rita Selvatici 3 , Renè Massimiliano Marsano 4 , Andrea Gerbino 5 , Marianna Farnè 3, Rikard Blunck 6 , Francesco Vitali 1 , Annarita Armaroli 3, Alessandro Brieda 1 , Antonella Liantonio 2, Annamaria De Luca 2 , Alessandra Ferlini 3, Claudio Rapezzi 1,7, Matteo Bertini 1 , Francesca Gualandi 3,*,† and Paola Imbrici 2,*,†

1 Cardiological Center, University of Ferrara, 44121 Ferrara, Italy; [email protected] (C.B.); [email protected] (F.V.); [email protected] (A.B.); [email protected] (C.R.); [email protected] (M.B.) 2 Department of Pharmacy-Drug Sciences, University of Bari “Aldo Moro”, 70125 Bari, Italy; [email protected] (E.C.); [email protected] (A.L.); [email protected] (A.D.L.) 3 Unit of Medical Genetics, Department of Medical Sciences, University of Ferrara, 44121 Ferrara, Italy; [email protected] (R.S.); [email protected] (M.F.); [email protected] (A.A.); [email protected] (A.F.) 4 Department of Biology, University of Bari “Aldo Moro”, 70125 Bari, Italy; [email protected] 5 Department of Biosciences, Biotechnologies and Biopharmaceutics, University of Bari “Aldo Moro”,  70125 Bari, Italy; [email protected]  6 Department of Physics, Université de Montréal, Montréal, QC H3C 3J7, Canada; [email protected] 7 Maria Cecilia Hospital, GVM Care & Research, 48033 Cotignola, Italy Citation: Balla, C.; Conte, E.; * Correspondence: [email protected] (F.G.); [email protected] (P.I.) Selvatici, R.; Marsano, R.M.; Gerbino, † Equally contributing. A.; Farnè, M.; Blunck, R.; Vitali, F.; Armaroli, A.; Brieda, A.; et al. Abstract: Background. Brugada syndrome (BrS) is an autosomal dominantly inherited cardiac Functional Characterization of Two disease characterized by “coved type” ST-segment elevation in the right precordial leads, high Novel Mutations in SCN5A susceptibility to ventricular and a family history of sudden cardiac death. The SCN5A Associated with Brugada Syndrome , encoding for the cardiac voltage-gated Nav1.5, accounts for ~20–30% of BrS Identified in Italian Patients. Int. J. Mol. Sci. 2021, 22, 6513. https:// cases and is considered clinically relevant. Methods. Here, we describe the clinical findings of two doi.org/10.3390/ijms22126513 Italian families affected by BrS and provide the functional characterization of two novel SCN5A mutations, the missense variant Pro1310Leu and the in-frame insertion Gly1687_Ile1688insGlyArg. Academic Editor: Carlo Pappone Results. Despite being clinically different, both patients have a family history of sudden cardiac death and had history of arrhythmic events. The Pro1310Leu significantly reduced peak sodium Received: 12 May 2021 current density without affecting channel membrane localization. Changes in the gating properties of Accepted: 14 June 2021 expressed Pro1310Leu channel likely account for the loss-of-function phenotype. On the other hand, Published: 17 June 2021 Gly1687_Ile1688insGlyArg channel, identified in a female patient, yielded a nearly undetectable sodium current. Following mexiletine incubation, the Gly1687_Ile1688insGlyArg channel showed Publisher’s Note: MDPI stays neutral detectable, albeit very small, currents and biophysical properties similar to those of the Nav1.5 with regard to jurisdictional claims in wild-type channel. Conclusions. Overall, our results suggest that the degree of loss-of-function published maps and institutional affil- shown by the two Nav1.5 mutant channels correlates with the aggressive clinical phenotype of iations. the two probands. This genotype-phenotype correlation is fundamental to set out appropriate therapeutical intervention.

Keywords: Brugada syndrome; SCN5A; electrophysiology; Na+ current Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and 1. Introduction conditions of the Creative Commons Attribution (CC BY) license (https:// Brugada syndrome (BrS) is an autosomal dominantly inherited cardiac arrhythmia re- creativecommons.org/licenses/by/ sponsible for 4–12% of all sudden cardiac deaths (SCD) in patients without overt structural 4.0/). cardiac abnormalities [1]. The diagnosis is based on the electrocardiogram (ECG) type 1

Int. J. Mol. Sci. 2021, 22, 6513. https://doi.org/10.3390/ijms22126513 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2021, 22, 6513 2 of 17

pattern that is characterized by a distinct coved-type ST-segment elevation with negative in the right precordial leads. Sodium channel blockers are used as additional diag- nostic tools to unmask asymptomatic patients and are contraindicated in BrS [2]. or SCD often occur during rest or sleep and are usually due to polymorphic (VT), which can degenerate in some patients into ventricular fibrillation (VF). BrS occurs predominantly in males of >40 years of age with a male:female ratio of 9:1 [3]. To date, mutations in 25 different have been linked to BrS, 18 of which encoding subunits and 7 encoding regulatory proteins, including SCN5A, SCN10A, SCN1B, PKP2, RANGRF, TRPM4, and several calcium and potassium channels genes [4–6]. In about 20–30% of BrS probands, mutations have been found in SCN5A, encoding for the cardiac voltage-gated sodium channel Nav1.5 [1]. Nav1.5 consists of a pore-forming α-subunit composed of four homologous domains (DI–IV), each containing six transmem- brane segments (S1–S6), with S1–S4 forming the voltage sensor domain (VS) and the S5 and S6 the pore module (PM) [7]. Most SCN5A mutations are missense, are localized to the transmembrane segments of the channel and cause different degrees of loss-of-function (LoF) either by impairing trafficking or by modifying Nav1.5 gating properties [8–14]. LoF can be worsened at higher temperatures for some SCN5A mutations, accordingly with the trigger effect of in affected patients [15]. Implantable cardioverter defibrillator (ICD) is the first line therapy in BrS high-risk patients; the ablation of the right ventricular outflow tract is recently gaining consideration [16]. , a class Ia antiarrhythmic drug, and , a β adrenoreceptor agonist, have been proven useful in patients with contraindication for ICD and on suppression of VT/VF in some BrS patients [17]. One major concern in the pathogenesis and management of BrS is that patients can present with an array of clinical manifestations, ranging from asymptomatic (the vast majority) to high-risk clinical course, even within the same family. Complex genotypes and inheritance patterns have been described in BrS patients, probably accounting for the reported variable expressivity [18]. Many of the mutations identified in other genes have been found in single families and are only responsible for 5% of the BrS cases; therefore, their role in BrS remains to be investigated [6]. SCN5A mutations are, however, likely responsible for a minority of cases and many patients lack a genetic diagnosis. Digenic inheritance has also been reported, which underlines mutation load as a pathogenic basis for BrS [19]. Thus, whereas in some families BrS can be considered a monogenic disease, in others this disease has an oligogenic origin and BrS phenotypes likely result from the interplay of rare and common multiple variants and can partially overlap those of a cardiomyopathy [20,21]. The poor genotype–phenotype correlation and the lack of validated predictors of cardiac risk make the therapeutic management of BrS difficult, especially for asymptomatic patients and those at low risk (drug-induced patients). In this setting, the identification of a SCN5A mutation, family segregation analysis and functional studies can contribute to prognostic risk stratification and provide additional information to clinic to address the proper management of affected families [22]. Here, we describe in depths two Italian BrS families carrying two novel mutations in SCN5A, Pro1310Leu, and Gly1687_Ile1688insGlyArg, and provide the functional char- acterization of mutant channel to understand the likelihood of pathogenicity and draw a genotype-phenotype association.

2. Results 2.1. Clinical and Genetic Analysis We identified two novel SCN5A mutations in Italian BrS patients. The missense variation c.3929C>T (Pro1310Leu, named P1310L throughout the text) in SCN5A exon 22 in patient I and the in-frame insertion c.5058_5059insGGCCGC (Gly1687_Ile1688insGlyArg, named Ins1687GR throughout the text) in SCN5A exon 28 in patient II. Both variants are novel and not found in gnomAD exomes and genomes (MAF = 0). In silico prediction tools define P1310L as likely pathogenic and the Ins1687GR insertion as of uncertain significance. Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 3 of 18

2. Results 2.1. Clinical and Genetic Analysis We identified two novel SCN5A mutations in Italian BrS patients. The missense var- iation c.3929C>T (Pro1310Leu, named P1310L throughout the text) in SCN5A exon 22 in patient I and the in-frame insertion c.5058_5059insGGCCGC (Gly1687_Ile1688insGlyArg, named Ins1687GR throughout the text) in SCN5A exon 28 in patient II. Both variants are novel and not found in gnomAD exomes and genomes (MAF = 0). In silico prediction tools define P1310L as likely pathogenic and the Ins1687GR insertion as of uncertain signifi- cance. Patient I. The missense variant P1310L was identified in a 64 years-old man who pre- sented a type I Brugada pattern on ECG at the age of 56 (III-1; Figure 1A,B). He had a positive family history because the proband’s brother died suddenly at 51 years of age (Figure 1A). He received an ICD in primary prevention of SCD. During the follow up, he developed multiple repetitive ventricular treated by the ICD. Due to the re-

Int. J. Mol. Sci. 2021, 22, 6513 current arrhythmias, he underwent a successful epicardial radiofrequency ablation.3 of 17 Asymptomatic cases from family I did not perform the genetic analysis due to non-com- pliance, except for proband’s son who resulted negative for the P1310L variation. Patient II. In family II, the in-frame six nucleotides insertion Ins1687GR was searched exclusivelyPatient in I. the The two missense subjects variant manifesting P1310L symptoms was identified and/or in Br aS 64 ECG, years-old considering man who the uncertainpresented significanc a type I Brugadae of the patternvariation on at ECG the attime the of age genetic of 56 counselling (III-1; Figure. The1A,B). Ins1687GR He had insertiona positive was family iden historytified in because a 57 years the-old proband’s female resuscitated brother died from suddenly cardiac at arrest 51 years at the of age of(Figure 28 during1A). He post received-partum an (II ICD-1; Figure in primary 1C,D) prevention. She received of SCD. an ICD During in secondary the follow prevention up, he de- ofveloped SCD. The multiple proband’s repetitive mother ventricular died suddenly arrhythmias at the treated age of by 38 the years. ICD. The Due mutation to the recurrent segre- gatesarrhythmias, in the 36 he years underwent-old proband’s a successful son who epicardial showed radiofrequency a spontaneous ablation. type 1 BrS Asymptomatic pattern. He didcases not from have family any symptom I did not performor arrhythmic the genetic event analysis and received due to an non-compliance, ICD in primary except preven- for tionproband’s. The proband’s son who resultedson also negativecarries the for His558Arg the P1310L benign variation. polymorphism in SCN5A [23].

A C

I I 1 1 II II 1 1 2

BrS III 1 2 III 2 1 SCD IV IV syncope B D

Figure 1. Pedigree and ECG of the BrS families. (A) Pedigree of family of patient I (III-1) carrying the missense variation Figurec.3929C>T 1. Pedigree (Pro1310Leu) and ECG in SCN5A of the.BrS The families. genetic analysis (A) Pedigree was performed of family of only patient in proband’s I (III-1) carrying son who the resulted missense negative variation for c.3929C>Tthe Pro1310Leu (Pro1310Leu) variation. in I-SCN5A1: SCD .at The 87 geneticyears; II analysis-1: SCD at was 78 performedyears; III-2: only SCD in at proband’s 51 years. ( sonB) ECG who of resulted patient negative I shows forBrugada the Pro1310Leu type 1 pattern variation. in V1-V2 I-1: recorded SCD at at 87 standard years; II-1: intercostal SCD at space. 78 years; (C) Pedigree III-2: SCD of atfamily 51 years. of patient (B) ECG II (II- of1) patientcarrying I showsGly1687_Ile1688insGlyArg Brugada type 1 pattern in inheterozygosis V1-V2 recorded in SCN5A at standard. Her intercostal son (III-1) space. carries (C both) Pedigree the maternal of family variation of patient and II (II-1) the carryingHis558Arg Gly1687_Ile1688insGlyArg benign polymorphism in inSCN5A heterozygosis. The Ins1687GR in SCN5A insertion. Her sonwas (III-1) searched carries exclusively both the in maternal the two variationsubjects man- and theifesting His558Arg symptoms benign and/or polymorphism BrS ECG pattern in SCN5A, in light. The of Ins1687GRthe uncertain insertion significanc wase searched of the variation. exclusively I-1: SCD in the at two 38 years; subjects II- manifesting symptoms and/or BrS ECG pattern, in light of the uncertain significance of the variation. I-1: SCD at 38 years; II-2: SCD at 50 years; III-2: syncope and negative drug-induced testing for BrS. (D) ECG of patient II shows Brugada type 1 pattern in V1-V2 recorded at second intercostal space.

Patient II. In family II, the in-frame six nucleotides insertion Ins1687GR was searched exclusively in the two subjects manifesting symptoms and/or BrS ECG, considering the uncertain significance of the variation at the time of genetic counselling. The Ins1687GR insertion was identified in a 57 years-old female resuscitated from at the age of 28 during post-partum (II-1; Figure1C,D). She received an ICD in secondary prevention of SCD. The proband’s mother died suddenly at the age of 38 years. The mutation segregates in the 36 years-old proband’s son who showed a spontaneous type 1 BrS pattern. He did not have any symptom or arrhythmic event and received an ICD in primary prevention. The proband’s son also carries the His558Arg benign polymorphism in SCN5A [23].

2.2. Functional Characterization of P1310L Mutant Channels We generated a model of human Nav1.5 built upon the 3D structure of the recently de- termined cryo-electron microscopy structure of rat Nav1.5 α-subunit [24]. P1310L consists Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 4 of 18

2: SCD at 50 years; III-2: syncope and negative drug-induced testing for BrS. (D) ECG of patient II shows Brugada type 1 pattern in V1-V2 recorded at second intercostal space.

Int. J. Mol. Sci. 20212.2., 22, 6513Functional Characterization of P1310L Mutant Channels 4 of 17 We generated a model of human Nav1.5 built upon the 3D structure of the recently determined cryo-electron microscopy structure of rat Nav1.5 -subunit [24]. P1310L con- sists in the substitution of a proline with a leucine at S4 in DIII of the Nav1.5 channel, a position that isin conserved the substitution within of the a proline family with of Nav1.x a leucine channels at S4 in (Figure DIII of thes 2A Nav1.5,B and channel, S1) a position [7,24]. that is conserved within the family of Nav1.x channels (Figure2A,B and Figure S1) [7,24].

A B

C D

Figure 2. LocalizationFigure 2. Localization of the BrS mutations of the BrS in mutations the Nav1.5 in channelthe Nav1.5 modeled channel on modeled the 3D structure on the 3D of thestructure rat Nav1.5 of α-subunit (pdb: 6UZ3).the Lateral rat Nav1. (A) and5 -subunit top (B) view(pdb: of 6UZ3). Nav1.5 Lateral channel (A) showing and top the(B) P1310Lview of mutationNav1.5 channel depicted showing in purple. the Lateral (C) and top (D)P1310L view of mutation the Nav1.5 depicted channel in showing purple. theLateral Ins1687GR (C) and insertion top (D) view in purple. of the The Nav1.5 DEKA channel residues showing are shown in red. the Ins1687GR insertion in purple. The DEKA residues are shown in red. To test whether P1310L mutation was responsible for BrS in the affected patient, To test whetherwe recorded P1310L sodium mutation currents was responsible throughthe for patchBrS in clampthe affected technique patient, from we HEK 293 cells recorded sodiumexpressing currents either through Nav1.5 the patch WT or clamp mutant technique channels. from Figure HEK3A 293 shows cells representativeexpress- sodium ing either Nav1.5currents WT or elicited mutant by channels. WT and P1310L Figure channels. 3A shows Typical representative WT sodium sodium currents cur- start to activate rents elicited byat WT−60 and mV, P1310L peak at channels.−45 mV Typical and decrease WT sodium in amplitude currents at start more to depolarized activate potentials at −60mV, peakdue at − to45mV a reduction and decrease in driving in amplitude force. As at shown more depolarized in the IV plot potentials in Figure due3B, peak current to a reduction densityin driving was force. significantly As shown reduced in the IV by plot more inthan Figure 3-fold 3B, peak in P1310L current channels density compared with was significantlyWT reduced (Table1). by more than 3-fold in P1310L channels compared with WT (Table 1). We then determined whether modifications of the voltage-dependent activation and inactivation by P1310L mutation might contribute to the reduced activity of P1310L chan- nels and be of pathogenic relevance for BrS. As shown in Figure4A, the midpoint activation voltage for P1310L channels was shifted by +15 mV towards positive potentials with re- spect to WT, increasing the degree of depolarization required for activation (Table1). The voltage-dependence of fast inactivation was instead slightly shifted by +7 mV towards more positive voltages for P1310L channels relative to WT (Figure4B). P1310L channels also showed a faster recovery from fast inactivation with respect to WT (Figure4C; Table1). Current decay measured at −30 mV was instead not modified by the mutation (Table1). The small positive shift in the voltage-dependence of fast inactivation and accelerated recovery from inactivation would argue for an increase in P1310L channel availability leading to a mixed phenotype. Actually, among the voltage sensor mutations identified to date, the very close R1309H mutation, was associated with an overlapping phenotype [25]. Int. J. Mol. Sci. 2021, 22, 6513 5 of 17

We then determined whether P1310L may affect sustained and window currents. The analysis of the area beneath the intersection of activation and steady-state inactivation curves demonstrated that P1310L decreased the amplitude of the window current by 40% compared with Nav1.5 WT (Figure4D). P1310L also reduced the density of the sustained current measured at −30 mV (Figure4E; Table1). Nav1.5 sodium channels can form dimers upon direct interaction between α-subunits, which suggests the possibility that dominant-negative effect exerted by mutant on WT subunit may occur in the heterozygous patient [26,27]. The co-expression of equal amount of WT and P1310L cDNAs gave rise to sodium currents that were similar to the calculated sum of those carried by WT and mutant expressed alone (Figure3C). In addition, the analyses of the biophysical properties measured for the currents evoked by WT+P1310L Int. J. Mol. Sci. 2021, 22, x FOR PEERchannels REVIEW suggested a predominant LoF defect for WT+P1310L channels compared5 with of 18

Nav1.5 WT (Figure4A–E).

A B WT 1microg Nav1.5 WT 0

20 ms -100 +70 mV P1310L 1microg n=21

−100 mV (pA/pF) -200 Na −150 mV I v Nav1.5 WT -300 P1310L WT+PL (0.5+0.5 microg) P1310L WT+P1310L -400 -80 -40 0 40 80 C Voltage (mV) Nav1.5 P1310L Nav1.5+P1310L calculated Current (3g) (3g) (1.5g+1.5g) sum 0 Nav1.5 WT+P1310L 2nA -100 *

5ms

(pA/pF) (pA/pF) -200

Na Na I

-300

-400

Figure 3. Current density of Nav1.5 WT, P1310L and WT+P1310L channels expressed in HEK 293 cells. (A) Representative sodiumFigure current3. Current traces density from of cells Nav1.5 transfected WT, P1310L with and Nav1.5 WT+P1310L WT (3 µ g),channels P1310L expressed1 (3 µg), and 2in HEK Nav1.5+P1310L 2933 cells. (4A (1.5) Representativeµg +1.5 µg) cDNAs.sodium The current voltage traces protocol from iscells shown transfected in the inset. with ( BNav1.5) IV plot WT showing (3 µg), theP1310L mean (3 current µg), and density Nav1.5+P1310L of Nav1.5 WT, (1.5 P1310L µg +1.5 and µg) cDNAs. The voltage protocol is shown in the inset. (B) IV plot showing the mean current density of Nav1.5 WT, P1310L Nav1.5+P1310L channels as a function of membrane potential. (C) Bar graph showing the mean current density measured and Nav1.5+P1310L channels as a function of membrane potential. (C) Bar graph showing the mean current density meas- at −30mV for the indicated channels. Data are mean ± SE; n = 11–20 cells. * p < 0.05 for P1310L channels compared with ured at −30mV for the indicated channels. Data are mean ± SE; n = 11–20 cells. * p < 0.05 for P1310L channels compared Nav1.5with Nav1.5 WT. WT.

Int. J. Mol. Sci. 2021, 22, 6513 6 of 17

Table 1. Current density and biophysical parameters of Nav1.5 WT and BrS mutant P1310L and Ins1687GR channels.

Sustained Channel Peak Current Voltage Dependent Voltage Dependent Fast Time Constant Recovery from Current Type Density Activation Inactivation of Inactivation Inactivation Density τ , ms τ , ms −30 mV, −30 mV, pA/pF V , mV k, mV V , mV k, mV −30 mV, ms fast slow h h (A1%) (A2%) 100 ms, pA/pF 1.2 ± 0.1 −286 ± 53 −49.6 ± 0.6 −86.7 ± 0.5 0.86 ± 0.07 6.7 ± 1.1 13.7 ± 1.9 Nav1.5 WT 6.0 ± 0.4 9.9 ± 0.4 (87%) n = 11 n = 6 n = 18 n = 14 (13%) n = 20 n = 12 0.78 ± 0.2 * −46 ± 10 * −34.7 ± 0.6 * −78.5 ± 0.4 * 1.02 ± 0.09 2.4 ± 1.2 * 9.8 ± 1.5 P1310L 8.7 ± 0.6 7.0 ± 0.3 (78%) n = 20 n = 19 n = 15 n = 18 (22%) n = 16 n = 8 WT+ −146 ± 23 −42.0 ± 0.5 −84.0 ± 0.6 1.06 ± 0.17 1.5 ± 0.6 (83%) 4.7 ± 1.2 9.7 ± 2.4 9.2 ± 0.8 10.0 ± 1.0 P1310L n = 9 n = 9 n = 15 n = 11 n = 10 (17%) n = 13 −11.1 ± 1.5 * Ins1687GR //////// n = 21 1.6 ± 0.4 WT+ −153 ± 52 −52.5 ± 0.4 −88.0 ± 1.0 0.99 ± 0.12 8.0 ± 3.1 10.8 ± 3.6 5.1 ± 0.3 9.3 ± 0.7 (88%) Ins1687GR n = 7 n = 6 n = 6 n = 6 (12%) n = 6 n = 6 * p < 0.05 for mutant channels compared with Nav1.5 WT. Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 7 of 18

We then determined whether modifications of the voltage-dependent activation and inactivation by P1310L mutation might contribute to the reduced activity of P1310L chan- nels and be of pathogenic relevance for BrS. As shown in Figure 4A, the midpoint activa- tion voltage for P1310L channels was shifted by +15 mV towards positive potentials with respect to WT, increasing the degree of depolarization required for activation (Table 1). The voltage-dependence of fast inactivation was instead slightly shifted by +7 mV towards more positive voltages for P1310L channels relative to WT (Figure 4B). P1310L channels also showed a faster recovery from fast inactivation with respect to WT (Figure 4C; Table 1). Current decay measured at −30 mV was instead not modified by the mutation (Table 1). The small positive shift in the voltage-dependence of fast inactivation and accelerated recovery from inactivation would argue for an increase in P1310L channel availability leading to a mixed phenotype. Actually, among the voltage sensor mutations identified to date, the very close R1309H mutation, was associated with an overlapping phenotype [25]. We then determined whether P1310L may affect sustained and window currents. The analysis of the area beneath the intersection of activation and steady-state inactivation curves demonstrated that P1310L decreased the amplitude of the window current by 40% compared with Nav1.5 WT (Figure 4D). P1310L also reduced the density of the sustained current measured at −30 mV (Figure 4E; Table 1). Nav1.5 sodium channels can form dimers upon direct interaction between -subu- nits, which suggests the possibility that dominant-negative effect exerted by mutant on WT subunit may occur in the heterozygous patient [26,27]. The co-expression of equal amount of WT and P1310L cDNAs gave rise to sodium currents that were similar to the Int. J. Mol. Sci. 2021, 22, 6513 calculated sum of those carried by WT and mutant expressed alone (Figure 3C). In addi-7 of 17 tion, the analyses of the biophysical properties measured for the currents evoked by WT+P1310L channels suggested a predominant LoF defect for WT+P1310L channels com- pared with Nav1.5 WT (Figure 4A–E).

A B y = m1/(1+exp((m0-m2)/m3)) v Nav1.5 WT G/Gmax WT Value Error 1 WT ssi P1310L m1 0.99188 0.0095526 P1310L 1 WT+P1310L m2 -86.748 0.51075 WT+P1310L m3 9.9498 0.43191 0.8 0.8 Chisq 0.0032372 NA y = 0.05+m1/(1+exp(-(m0-m2)/... R 0.99935 NA y = 0.04+m1/(1+exp(-(m0-m2)/... Value Error Value Error y = m1/(1+exp((m0-m2)/m3)) 0.6 m1 0.91979 0.01277 0.6 m1 0.93147 0.014507 Value Error PL ssi max m2 -49.953 0.60074 m2 -34.757 0.6576 m1 0.98319 0.0067829 m3 5.6501 0.51988 m3 8.7008 0.54907 m2 -78.457 0.34838 Chisq 0.03202 NA G/G 0.4 20 ms 0.4 Chisq 0.018757 NA m3 6.9064 0.30157 R 0.99618 NA +70 mV R 0.99745100ms 20msNA −20 mV Chisq 0.0025948 NA R 0.9995 NA 0.2

Normalised current current Normalised 0.2 −150 mV y = m1/(1+exp((m0-m2)/m3)) −100 mV y = 0.02+m1/(1+exp(-(m0-m2)/... Value Error WT+P1310L ssi Value Error −150 mV m1 1.0001 0.0089623 m1 0.96054 0.011928 0 0 m2 -84.192 0.49701 m2 -42.773 0.59611 -100 -80 -60 -40 -20 0 20 -140 -120 -100 -80 -60 -40 -20 m3 10.602 0.41941 m3 9.5504 0.50058 Chisq 0.0028739 NA Chisq 0.01553 NA Voltage (mV) Voltage (mV) R 0.99939 NA R 0.99799 NA

Nav1.5 WT C P1310L E D integral window current current -30mV 100ms P1310L WT+P1310Ly = m1+m2*exp(-(m0/m3)) + m4...

0.1 Value Error G/Gmax WT 2 P1310L16 WT ssi 1 m1 1.0062 0.0045774 WT+P1310L P1310L ssi y = m1/(1+exp(-(m0-m2)/m3)) WT+P1310L ssi Value Error

m2 -0.73464 0.058966 y = m1/(1+exp((m0-m2)/m3)) m1 0.97241 0.01335 max Value Error m2 -50.911 0.6255 m3 1.196 0.13776 m1 0.99318 0.012008 m3 6.3193 0.54006

0.05 m2 -86.69 0.65199 Chisq 0.035498 NA G/G Nav1.5 m4 -0.41393 0.067107 m3 10.503 0.54885 R 0.99605 NA 0.8 1.5 Chisq 0.0048348 NA 12R 0.999 NA m5 6.7036 1.0659 y = m1/(1+exp((m0-m2)/m3)) y = m1/(1+exp(-(m0-m2)/m3)) Value Error Value Error m1 0.98055 0.017228 Chisq 0.001393 NAcurrent Normalized m1 0.98584 0.0052317 0 m2 -79.857 0.2687 m2 -35.701 0.77623 R 0.99967 NA m3 7.1745 0.23235 m3 9.7452 0.63948 -100 -80 -60 -40 -20 Chisq 0.001471 NA Chisq 0.024811 NA 0.6 (pA/pF) R 0.99679 NA R 0.99972 NA peak Voltage (mV) y = m1/(1+exp((m0-m2)/m3)) y = m1/(1+exp(-(m0-m2)/m3)) /I 1 8 Value Error Value Error y = m1 + m2*exp(-(m0/m3))m1 0.99708 0.020205 + ... m1 1.0134 0.019541 WT+P1310L m2 -89.335 1.0506 m2 -41.434 0.9035 y = m1 + m2*exp(-(m0/m3)) + ... m3 10.105 0.88435 m3 9.2808 0.75912

final Value Error Chisq 0.013145 NA Chisq 0.04105 NA I 0.4

−30 mV −30 mV Value Error m1 R0.997940.99715 NA 0.010614R 0.99507 NA sustained

m1 0.99089 0.0046208 m2 -0.98064 0.52643

Window current current Window Na

0.5 I m2 -0.97942 0.23093 m3 4 1.5248 0.59876 0.2 −160ms m3 0.77925 0.151 m4 -0.20105 0.54923 m4 -0.27455 0.25116 100 ms m5 4.7585 7.6629 m5 2.3587 1.2391 Chisq 0.0080808 NA 0 0 Chisq 0.0018295 NA R 00.99831 NA 0.1 1 10 100 1000 Nav1.5 P1310L WT+R 0.99942 NA Nav1.5 P1310L WT+ P1310L P1310L Interpulse interval (ms)

1 2 3 Figure 4. Biophysical properties of Nav1.5 WT, P1310L, and WT+P1310L channels expressed in HEK 2931 cells.2 (A)3 Voltage- dependence of activation. (B) Voltage-dependence of fast inactivation. (C) Recovery from fast inactivation. Voltage protocols are indicated in the insets and detailed in the Supplementary Materials and Methods. (D) Bar graph showing window current measure for the indicated channels. The inset shows the triangular area under the overlapping point of the activation and inactivation curves of the respective channels. (E) Bar graph showing the amplitude of the sustained current measured at the end of a 100 ms pulse at −30 mV for the indicated channels. Data are mean ± SE; n = 6–20 cells.

2.3. Functional Characterization of Ins1687GR Mutant Channels The mutation Ins1687GR consists in two amino acids, Gly and Arg, being inserted at position 1687 of the S5-P loop of DIV of the Nav1.5 channel. As for P1310L, Ins1687GR mutation occurs at a conserved and functionally relevant pore region within the family of Nav1.x channels (Figure2C,D and Figure S1) [ 7,24]. Figure5A shows representative current traces recorded from cells expressing Ins1687GR channels. Ins1687GR insertion caused a complete loss of Nav1.5 function reducing the sodium current density by more than 25-fold (Figure5A,B). Unfortunately, the very small current level recorded from Ins1687GR channels hampered any further biophysical analysis of the mutant channel alone. The co-expression of equal amount of WT and Ins1687GR cDNAs gave rise to sodium currents that were similar to the calculated sum of those carried by WT and mutant expressed alone (Figure5C) and with biophysical properties similar to those of Nav1.5 WT (Figure6A–E; Table1).

2.4. Cellular Localization of Nav1.5 WT, P1310L and Ins1687GR Mutant Channels Reduction in current density could be due to dysfunctional channel gating or impaired trafficking of an otherwise functional channel or both. We therefore performed confocal microscopy analysis to assess the impact of P1310L and Ins1687GR mutations on the localization of Nav1.5 channels in HEK 293 cells. We used wheat germ agglutinin (WGA) AlexaFluor™555 to stain cells plasma membrane and evaluated the co-localization with Int. J. Mol. Sci. 2021, 22, 6513 8 of 17

Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 8 of 18

the anti-Nav fluorescence. As shown in Figure7A, Nav1.5 WT was mainly expressed at the plasma membrane as shown by the high degree of co-localization with WGA-555. Despite Figure 4. Biophysical propertiesproducing of Nav1.5 smaller WT, P1310L sodium, and currents, WT+P1310L P1310L channels showed expressed membrane in HEK expression 293 cells. (A very) Voltage similar- to dependence of activation. (Bthat) Voltage of Nav1.5-dependence WT channels of fast inactivation. (Figure7A). (C In) Recovery addition, from the totalfast inactivation. protein amount Voltage was proto- similar cols are indicated in the insetsfor and WT detailed and P1310L in the channels Supplementa expressedry Materials in HEK and 293 Methods. cells (Figure (D) Bar7 B,C).graph Similarly, showing window Ins1687GR current measure for the indicatedchannels channels. appeared The inset correctly shows inserted the triangular at the area plasma under membrane the overlapping and point quantitative of the activa- analysis tion and inactivation curvesrevealed of the respective similar total channels. protein (E) expression Bar graph showing level for the Ins1687GR amplitude and of the WT sustained (Figure7 A–C).current measured at the end of a 100 ms pulse at −30 mV for the indicated channels. Data are mean ± SE; n = 6–20 cells. 2.5. Effect of Mexiletine Incubation on Nav1.5 WT and Ins1687GR Mutant Channels 2.3. Functional Characterization of Ins1687GR Mutant Channels Mexiletine is a class Ia antiarrhythmic drug that reversibly blocks Nav1.5 channels by bindingThe mutation to a local Ins1687GR anesthetic consists binding in sitetwo includingamino acids the, Gly S6 DIVand Arg, [7]. Incubationbeing inserted with at highposition concentrations 1687 of the ofS5 mexiletine-P loop of hasDIV been of the shown Nav1.5 to increasechannel. membraneAs for P1310L, expression Ins1687GR and currentmutation of occurs both Nav1.5 at a conserved WT and and some functionally BrS mutated relevant channels pore [28 region,29]. Thus, within to the increase family the of membraneNav1.x channels expression (Figure ands current2C,D and amplitude S1) [7,24 of]. Ins1687GR Figure 5A mutant shows channels,representative we incubated current HEKtraces 293 recorded cells expressing from cells expressing Nav1.5 WT Ins1687GR and Ins1687GR channels. channels Ins1687GR for 24 insertion h with caused 300 µM a mexiletine.complete loss The of drugNav1.5 was function washed reducing out prior the to sodium recording current sodium density currents. by more A than total 25 of- 300foldµ (FigureM mexiletine 5A,B). caused Unfortunately, a ~30% increase the very of small Nav1.5 current WT current level recorded density and from did Ins1687GR not affect activationchannels ham andpered inactivation any further properties biophysical (Figure analysis8B–E; Table of the2). mutant Similarly, channel mexiletine alone. 300 Theµ coM- increasedexpression the of currentequal amount amplitude of WT of Ins1687GRand Ins1687GR channels cDNAs by 7-foldgave rise compared to sodium with currents that of non-treatedthat were similar channels to the (Figure calculated8B; Table sum2). of This those allowed carried the by evaluationWT and mutant of the expressed biophysics alone of mutant(Figure channels.5C) and with No significant biophysical change properties was observed similar to in those the activation of Nav1.5 and WT fast (Figure inactivation 6A–E; propertiesTable 1). of mutant channels with respect to Nav1.5 WT channels (Table2).

InsGR curr density A WT 1microg B WT+InsGR 0 Nav1.5 WT -100

20 ms +70 mV

(pA/pF) -200

Na I −100 mV v Nav1.5 WT −150 mV -300 Ins1687GR WT+Ins1687GR -400 Ins1687GR -100 -80 -60 -40 -20 0 20 40 60 Voltage (mV)

C Nav1.5+ Nav1.5 Ins1687GR Ins1687GR calculated Current (1.5g+1.5g) 0 (3g) (3g) sum Nav1.5 WT+Ins1687GR * 2nA -100

5ms

(pA/pF) (pA/pF) -200

Na I -300

-400

Figure 5. Current density of Nav1.5WT, Ins1687GR, and WT+Ins1687GR channels expressed in HEK 293 cells. (A ) Repre- sentativeFigure 5. sodiumCurrent currentdensity tracesof Nav1.5WT, from cells Ins1687GR transfected, and with WT+Ins1687GR Nav1.5 WT (3 channelsµ1 g), Ins1687GR expressed2 (33µ ing) HEK and 4 Nav1.5+Ins1687GR293 cells. (A) Repre- (sentative1.5 µg +1.5 sodiumµg) cDNAs. current The traces voltage from protocol cells transfect is showned inwith the Nav1.5 inset. (B WT) IV (3 plot µg) showing, Ins1687GR the mean (3 µg) current and Nav1.5+Ins1687GR density of Nav1.5 WT,(1.5 Ins1687GRµg +1.5 µg) and cDNAs. WT+Ins1687GR The voltage channels protocolas is ashown function in the of membraneinset. (B) IV potential. plot showing (C) Bar the graph mean showing current density the mean of currentNav1.5 densityWT, Ins1687GR measured and at WT+Ins1687GR -30mV for the indicated channels channels.as a function Data of membrane are mean ± potSE;ential.n = 11–21(C) Bar cells. graph * p showing< 0.05 for the Ins1687GR mean cur- rent density measured at -30mV for the indicated channels. Data are mean ± SE; n = 11–21 cells. * p < 0.05 for Ins1687GR channels compared with Nav1.5 WT. channels compared with Nav1.5 WT.

Int. J. Mol. Sci. 2021, 22, 6513 9 of 17 Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 9 of 18

A v Nav1.5 WT B WT+Ins1687GR G/Gmax WT WT SSI 1 WT+InsGR 1 WT+InsGR 0.8 y = 0.05+m1/(1+exp(-(m0-m2)/...0.8 Value Error m1 0.91979 0.01277 0.6 m20.6 -49.953 0.60074 max m3 5.6501 0.51988 Chisq 0.03202 NA 20 ms R 0.99618 NA

G/G 0.4 +70 mV 0.4 100ms 20ms y = 0.05+m1/(1+exp(-(m0-m2)/... −20 mV Value Error 0.2 y = m1/(1+exp((m0-m2)/m3)) −100 mV current Normalised m10.2 0.91473 0.013492 −150 mV y = m1/(1+exp((m0-m2)/m3)) Value Error −150 mV m2 -52.956 0.39804 Value Error y = 0.05+m1/(1+exp(-(m0-m2)/...m1 0.99188 0.0095526 m3 2.3219 0.32494 m1 0.97124 0.016051 Value Errorm2 -86.748 0.51075 0 Chisq 0 0.025588 NA m2 -88.783 0.8414 m1 0.90801 0.013428m3 9.9498 0.43191 -100 -80 -60 -40 -20 0 20 R 0.99607-140 -120NA-100 -80 -60 -40 -20 m3 9.3236 0.71339 m2 -53.003 0.41568Chisq 0.0032372 NA Chisq 0.0091818 NA Voltage (mV) m3 2.2793 0.33828R 0.99935 CurrentNA density -30mV 100msR 0.99811 NA Voltage (mV) y = 0.05+m1/(1+exp(-(m0-m2)/...Chisq 0.030021 NA C D integral window currentE R Value0.99549 ErrorNA m1 0.8996 0.016528 Nav1.5 WT m2 -53.061 0.5124 2 Nav1.5WT WT+Ins1687GR 16 1 m3 2.2278 0.41514 0.1 y = m1/(1+exp(-(m0-m2)/m3)) G/Gmax WT y = m1/(1+exp(-(m0-m2)/m3)) Value Error WT+InsGR Chisq Value0.045659Error NA m1 0.97241 0.01335 WT ssi m1 0.96745 0.015243 m2 -50.911 0.6255 m2 -63.412 0.45763 WT+InsGR m3 6.3193 0.54006 Rm3 2.80760.993050.39533 NA Chisq 0.035498 NA Chisq 0.031746 NA

max R 0.99605 NA 0.8 R 0.99512 NA 1.5 WT+InsGR 0.05 12y = m1/(1+exp((m0-m2)/m3))

G/G Value Error m1 0.99318 0.012008 y = m1/(1+exp((m0-m2)/m3)) m2 -86.69 0.65199 Value Error m3 10.503 0.54885 m1 0.97124 0.016051 Chisq 0.0048348 NA m2 -88.784 0.84141 R 0.999 NA (pA/pF) m3 9.3239 0.7134 0.6 current Normalized Chisq 0.0091818 NA

peak 0 R 0.99811 NA

/I 1 -100 -80 -60 -40 -20 8 Voltage (mV)

y = m1+m2*exp(-(m0/m3)) + m4... final I 0.4 −30 mV −30 mV y = m1+m2*exp(-(m0/m3)) + m4... Value Error sustained Value Error

m1 1.0051 0.004073

Na Window current current Window

m1 0.98179I 0.011115 0.5m2 -0.72291 0.058325 4 m2 -1.0851 0.21496 0.2 −160ms m3 1.171 0.1357 m3 2.0342 0.42811 m4 -0.42671 0.06678 m4 -0.14428 0.23057 100 ms m5 6.4673 0.96842 m5 8.8394 11.698 0 Chisq0 0.0014257 NA 0 Chisq 0.0088448 NA 0.1 1 10 100 1000 RNav1.50.99968 WT+ NA Nav1.5 WT+ Ins1687GR R 0.99825 NA Ins1687GR Interpulse interval (ms)

FigureFigure 6. 6. BiophysicalBiophysical properties properties of ofNav1.5WT Nav1.5WT and and WT+Ins1687GR WT+Ins1687GR1 channels2 channels expressed expressed in HEK in HEK 293 cells. 293 cells. (A) Voltage (A) Voltage--de- 1 2 pendencedependence of activation. of activation. (B) (VoltageB) Voltage-dependence-dependence of fast of fast inactivation. inactivation. (C ()C Recovery) Recovery from from fast fast inactivation. inactivation. Voltage Voltage protocols protocols areare indicated indicated in in the the insets insets a andnd detailed detailed in in the the Supplementary Supplementary Materials Materials and and Methods. Methods. (D (D) )Bar Bar graph graph showing showing window window currentcurrent measure measure for for the the indicated indicated channels. channels. The inset shows thethe triangulartriangular areaarea under under the the overlapping overlapping point point of of the the activation activa- tion and inactivation curves of the respective channels. (E) Bar graph showing the amplitude of the sustained current and inactivation curves of the respective channels. (E) Bar graph showing the amplitude of the sustained current measured measured at the end of a 100 ms pulse at −30 mV for the indicated channels. Data are mean ± SE; n = 6–20 cells. at the end of a 100 ms pulse at −30 mV for the indicated channels. Data are mean ± SE; n = 6–20 cells. 2.4. Cellular Localization of Nav1.5 WT, P1310L and Ins1687GR Mutant Channels Table 2. Current density and biophysicalReduction parameters in current of Nav1.5density WT could and BrS be mutantdue to Ins1687GR dysfunctional channels channel expressed gating in HEK or im- 293 cells after 24 h incubationpaired with trafficking mexiletine 300of µanM. otherwise functional channel or both. We therefore performed confocal microscopy analysis to assess the impact of P1310L and Ins1687GR mutations on Channel Type Current Voltage Dependent Voltage Dependent Time Constant Recovery from + Mexiletine Density the localization of Nav1.5 channels in HEK 293 cells.of Inactivation We used wheat germ agglutinin 300 µM Activation Inactivation Inactivation (WGA) AlexaFluor™555 to stain cells plasma membrane and evaluated the co-localization − τ τ 30 mV, V , mV V , mV −30 mV, ms fast, ms slow, ms pA/pF with theh anti-Navk, fluorescence. mV h As shown ink, mVFigure 7A, Nav1.5 WT was(A1%) mainly expre(A2%)ssed at the plasma membrane as shown by the high degree of co-localization with WGA-555. − ± − ± − ± ± 2.1 ± 0.1 ± Nav1.5 WT 388 78 51.9 0.4 ± 91.0 0.5 ± 1.17 0.13 22 4 n = 6 Despiten = producing 6 4.4 smaller0.3 sodiumn = 6 currents,9.7 P1310L0.4 showedn = 6 membrane(83%) expression(17%) very similar to that of Nav1.5 WT channels (Figure 7A). In addition, the totaln = protein 6 amount ± −72 ± 16 * was− 49.7similar± 0.5 for WT6.0 ±and0.5 P1310L−91.0 channels± 0.4 8.2expressed± 0.4 in 1.20HEK± 0.20293 cells 1.99(Figure0.1 7B,C20). Simi-± 4 Ins1687GR n n n n (73%) = 8 larly, Ins1687GR= 6 channels appeared= 7 correctly inserted at the= plasma 6 membranen = 6 and(27%) quan- titative analysis* p < 0.05 for revealed mutant channels similar compared total protein with Nav1.5 expression WT. level for Ins1687GR and WT (Fig- ure 7A–C).

Int. J. Mol. Sci. 22 Int. J. Mol. Sci. 2021, 22, x FOR 2021PEER, REVIEW, 6513 10 of 18 10 of 17

A Nav1.5 WGA Merge

Nav1.5 WT

P1310L

Ins1687GR

B C

3

2 Nav1.5 290 kDa

1 Total protein density protein Total -Act 45 kDa 0

.5 R L 1 G 0 7 1 v 8 3 a 6 1 N 1 P s In

Figure 7. PlasmaFigure membrane 7. Plasma localization membrane of localization Nav1.5 WT, of P1310L Nav1.5 and WT, Ins1687GR P1310L and channels. Ins1687GR (A) Immunofluorescence channels. (A) Im- confocal microscopy analysismunofluorescence of the Nav1.5 confocal WT, P1310L microscopy and Ins1687GR analysis of channels the Nav1.5 (green WT, signal; P1310L left) and and Ins1687GR plasma membrane chan- marker WGA-555 (rednels signal, (green middle) signal; inleft) HEK and 293 plasma cells membrane grown on marker coverslips. WGA The-555 overlay (red signal, column middle reports) in HEK the co-localization 293 of the two fluorescencecells grown signalson coverslips. (yellow, The right). overlay WGA, column Wheat reports Germ the Agglutinin. co-localization The of horizontal the two fluorescence bar indicates 10 µm. signals (yellow, right). WGA, Wheat Germ Agglutinin. The horizontal bar indicates 10 μm. (B) (B) Immunoblotting analysis of Nav1.5 WT, Ins1687GR and P1310L channels expressed in HEK 293 cells. The position Immunoblotting analysis of Nav1.5 WT, Ins1687GR and P1310L channels expressed in HEK 293 of molecular weight markers is at the right of the blots. Expressions of β-actin are displayed as controls for the loaded cells. The position of molecular weight markers is at the right of the blots. Expressions of -actin protein amounts.are displayed (C) Bar graph as controls showing for the loaded total protein protein expression amounts. ( levelC) Bar for graph the indicated showing channel.the total protein For total protein expression, densityexpression was standardizedlevel for the indicated as the ratio channel. of the Nav1.5For total signal protein to theexpression, cognate βdensity-actin signal.was standardized Quantitative analysis was performedas fromthe ratio 3 independent of the Nav1.5 experiments. signal to the cognate -actin signal. Quantitative analysis was performed from 3 independent experiments.

2.5. Effect of Mexiletine Incubation on Nav1.5 WT and Ins1687GR Mutant Channels Mexiletine is a class Ia antiarrhythmic drug that reversibly blocks Nav1.5 channels by binding to a binding site including the S6 DIV [7]. Incubation with high concentrations of mexiletine has been shown to increase membrane expression and cur- rent of both Nav1.5 WT and some BrS mutated channels [28,29]. Thus, to increase the membrane expression and current amplitude of Ins1687GR mutant channels, we incu- bated HEK 293 cells expressing Nav1.5 WT and Ins1687GR channels for 24 h with 300 μM mexiletine. The drug was washed out prior to recording sodium currents. A total of 300

Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 11 of 18

μMCTRL mexiletine caused a ~30% increase of Nav1.5 WT current density and did not affect activation and inactivation24h properties (Figure 8B–E; Table 2). Similarly, mexiletine 300 μM increased the current amplitude of Ins1687GR channels by 7-fold compared with that of Int. J. Mol. Sci. 2021, 22, 6513 non-treated channels (Figure 8B; Table 2). This allowed the evaluation of the biophysics11 of 17 hClC-1 A531V of mutant channels. No significant change was observed in the activation and fast inacti- vation properties of mutant channels with respect to Nav1.5 WT channels (Table 2).

insGR

A MEX 300M B WT 24h 0 InsGR mex 300 media maggio ottobre 24h WT mex 300 -100

-200 NFAIns1687GR 50 μM

Dose range used in vivo (pA/pF) Na (7-124 μM) I -300 Nav1.5 WT Nav1.5 WT+MEX 300M -400 1nA Ins1687GR Ins1687GR+MEX 300M 10ms -500 -100 -80 -60 -40 -20 0 20 40 60 Voltage (mV)

WT Nav1.5 WT SSI C D E InsGR Mex 300 y = m1+m2*exp(-(m0/m3))+m4*e... Value Error 1 1 1 m1 1.0046 0.0050828 Nav1.5 Mex 300 WT mex 300 m2 -0.75974 0.060354 m3 1.3271 0.1626 WT+MEX 300 g/gmax 0.8 0.8 0.8 m4 -0.3639 0.070855 wt m5 7.3378 1.395 Chisq 0.0011176 NA InsGR+Mex 300microM

0.6 0.6 0.6 R 0.99965 NA

peak max

InsGR+mex 300+100 /I y = m1/(1+exp((m0-m2)/m3)) y = m1+m2*exp(-(m0/m3)) + m4...

y = m1/(1+exp((m0-m2)/m3)) y = m1+m2*exp(-(m0/m3))+m4*e...

final G/G

I Value Error Value Error 0.4 0.4 0.4 Value Error Value Error y = 0.04+m1/(1+exp(-(m0-m2)/... y = 0.04+m1/(1+exp(-(m0-m2)/...m1 0.99436 0.0091824 m1 1.0191 0.0088599 y = 0.05+m1/(1+exp(-(m0-m2)/... m1 0.99188 0.0095526 m1 1.0089 0.029225 Value Error m2 Value-91.002 Error0.46487 m2 -0.3077 0.012935 Value Error m2 -86.748 0.51075 m2 -0.97944 0.029024 0.2 m1 0.93315 0.011012 m1 m30.95634 9.72230.0118450.39175 m3 2.0223 0.15007 m3 50.149 5.7375 Normalised current current Normalised 0.2 m1 0.93236 0.0083892 0.2 m3 9.9498 0.43191 m2 -51.94 0.4079 m2 Chisq-49.6480.00268390.46721 NA m4 -0.83338 0.014509 m2 -46.411 0.36393 Chisq 0.0032372 NA m4 -0.2073 0.027369 m3 4.4415 0.35315 m3 R6.07010.999450.40036 NA m5 2.0759 0.090834 m3 7.0144 0.31019 R 0.99935 NA m5 79.001 34.223 Chisq 0.016662 NA Chisq 0.015136 NA Chisq 0.0010015 NA 0 0 Chisq 0.0075859 NA 0 y = m1/(1+exp((m0-m2)/m3)) Chisq 0.0045835 NA R 0.99773 NA R 0.99799 NA R 0.99971 NA -100 -80 -60 -40 -20 0 20 -140 -120R -1000.99891 -80 NA-60 -40 -20 0.1 1 10Value 100Error 1000 R 0.99871 NA m1 0.99379 0.0091717 Voltage (mV) Voltage (mV) Interpulsem2 interval-91.007 0.44069 (ms) m3 8.1908 0.37599 Chisq 0.0031586 NA R 0.9994 NA FigureFigure 8.8.Effect Effect ofof 24h24h incubation incubation withwith300 300µ μMM mexiletine mexiletine on on Nav1.5 Nav1.5 WT WT and and Ins1687GR Ins1687GR channels. channels. (A(A)) Representative Representative sodiumsodium current current traces traces from from cells cells expressing expressing Nav1.5 Nav1.5 WT WT and and Ins1687GR Ins1687GR channels channels in control in control solution solution and after and 24after h incubation 24 h incu- withbation 300 withµM 300 mexiletine. μM mexiletine. Mexiletine, Mexiletine, was washed was washed out prior out to prior recording to recording sodium sodium currents. currents. (B) IV plot(B) IV showing plot showing the mean the currentmean current density density of Nav1.5 of Nav1.5 WT and WT Ins1687GRand Ins1687GR channels channels as a as function a function of membraneof membrane potential potential before before and and after after 300 300µ μMM mexiletinemexiletine incubation. incubation. (C (C) Voltage-dependence) Voltage-dependence of of activation activation for for Nav1.5 Nav1.5 WT WT and and Ins1687GR Ins1687GR channels channels in control in control solution solution and afterand 300afterµ 300M mexiletine μM mexiletine incubation. incubation. (D) Voltage-dependence (D) Voltage-dependence of fast of inactivation fast inactivation for Nav1.5 for Nav1.5 WT and WT Ins1687GR and Ins1687GR channels chan- in nels in control solution and after 300 μM mexiletine incubation. (E) Recovery from fast inactivation for Nav1.5 WT and control solution and after 300 µM mexiletine incubation. (E) Recovery from fast inactivation for Nav1.5 WT and Ins1687GR Ins1687GR channels before and after 300 μM mexiletine incubation. Data are mean ± SE; n = 6–8 cells. channels before and after 300 µM mexiletine incubation. Data are mean ± SE; n = 6–8 cells. Table 2. Current density and biophysical parameters of Nav1.5 WT and BrS mutant Ins1687GR channels expressed in HEK 293 cells after 24h incub3.ation Discussion with mexiletine 300 μM. 3.1. Genotype-Phenotype Correlation Channel Type Current Voltage Dependent Voltage Dependent Time Constant of Recovery from Mutations in SCN5A, encoding for the cardiac voltage-gated sodium channel Nav1.5, + Mexiletine 300 µM Density Activation Inactivation Inactivation Inactivation represent a frequent cause of BrS and, for this reason, SCN5A screening commonly follows −30 mV, fast, ms slow, ms Vh, mV k, mV Vh, mV k, mV SCN5A−30 mV, ms pA/pF clinical diagnosis in affected patients [6]. However, is a highly polymorphic(A1%) gene(A2%) SCN5A and 2-5% of healthy subjects of different ethnicity carry missense 2.1 ±variants 0.1 of un- −388 ± 78 −51.9 ± 0.4 −91.0 ± 0.5 1.17 ± 0.13 22 ± 4 Nav1.5 WT known significance4.4 [±30 0.3]. This adds to the variable9.7 ± 0.4 expressivity and incomplete(83%) n = 6 n = 6 n = 6 n = 6 (17%) reported for BrS patients, that, even in the presence of a SCN5A mutation,n = may 6 challenge the causative effect of the identified genetic variant. Thus, despite discriminating1.99 ± 0.1 between −72 ± 16 * −49.7 ± 0.5 6.0 ± 0.5 −91.0 ± 0.4 8.2 ± 0.4 1.20 ± 0.20 20 ± 4 Ins1687GR pathogenic mutations and harmless variants is of critical importance for(73%) the therapeutic n = 8 n = 6 n = 7 n = 6 (27%) management of genotype-positive BrS patients, the interpretation of the geneticn = 6 test results is often* p < hard. 0.05 for In mutant this context, channels functional comparedin with vitro Nav1.5studies, WT addressing. the impact of the muta- tion on channel function, represent a powerful tool for clarifying pathogenicity and risk stratification and for exploring pharmacological approaches based on mutations defects. Here, we describe the functional characterization of two previously unreported SCN5A mutations occurring in BrS families, classified as with an uncertain pathogenic role by in silico tools. Our results confirm that loss of Nav1.5 activity is one of the molecular mechanisms underlying BrS symptoms and agree with the observation that the diagnosis of SCN5A mutations is associated with an elevated risk of major arrhythmic events in both Int. J. Mol. Sci. 2021, 22, 6513 12 of 17

Asian and Caucasian populations [17,31,32]. More specifically, P1310L and WT+P1310L channels showed reduced macroscopic current density due to altered channel gating, being P1310L channel membrane expression preserved. The positively shifted voltage-dependent activation of both P1310L and WT+P1310L channels appears to principally accounts for the reduced open probability of the channels at physiological potentials and correlates with the position of the P1310L mutation at S4 in DIII, an important region for the voltage-sensitive gating of the channel (Figure2A,B). As clarified by the recently solved 3D structure of rat Nav1.5 (pdb: 6UZ3) [24], during depolarization, the VS of the four domains moves the sliding helix S4 quickly outward to activate the channel and this voltage-dependent conformational change is transferred to the pore domain through the S4-S5 linker. Fast inactivation, occurring in cardiac channels within a few milliseconds of their activation, is promoted by the interaction of an isoleucine–phenylalanine–methionine (IFM) motif, located in the DIII-DIV linker, with amino acid residues in the S4-S5 linkers in DIII and DIV and in the intracellular ends of the S5 and S6 segments of DIV. The replacement of a rigid, hydrogen-bond acceptor proline with the branched-chain amino acid leucine at position 1310 of S4 DIII, between R1309 and R1312, might alter both the conformational changes required for the voltage sensor movement and the normal coupling of activation to fast inactivation [24]. The LoF defect of the channel is in agreement with the BrS phenotype of the proband who received an ICD in primary prevention and during follow up developed an arrhythmic storm treated with epicardial transcatheter ablation. The insertion of the two amino acids glycine and arginine at the highly conserved pore region (DIV P loop; [24]) of the Nav1.5 channels reduced the sodium current density without affecting the amount of protein expressed at the , with respect to WT. In agreement with previous findings, mexiletine was able to increase, albeit modestly, sodium currents carried by Ins1687GR channels, which seem to activate and fast inactivate similarly to Nav1.5 WT. The Ins1687GR insertion places the positively charged arginine side chain at the entrance of the electronegative ion conduction pathway in DIV through an apparently structurally normal Nav1.5 protein (Figure2C,D). In turn, we could assume a reduction of sodium conductance due to a lower local Na+ concentration at the pore entry. Other pore mutations associated with BrS have been shown to drastically impact Nav1.5 channel function and a similar hypothesis has been postulated for the mutations T1711R in DIV and G1422R in DIII [11–14,24]. In a very close site, the D1690N mutation, identified in a BrS patient, produced a similar marked reduction in sodium current density [23]. Despite the notion that women are somehow protected from BrS and are less represented in the BrS population, the severe biophysical profile displayed by the pore Ins1687GR mutation may give reason to the clinical phenotype shown by the female carrier who had a major arrhythmic event during post-partum and lost her mother prematurely. Published data suggest that sex hormones might play a role in the phenotypic manifestations of BrS, although the basis for gender-related differences is not yet fully understood [33]. The Ins1687GR mutation segregates in the proband’s son, who also presents the SCN5A H558R variant and received an ICD in primary prevention. The benign H558R polymorphism is present in 20% of the population and has been shown to be protective against the BrS phenotype [34,35]. Whether the presence of the additional SCN5A polymorphism might contribute to the asymptomatic clinical course of the proband’s son remains to be established.

3.2. Pathophysiology Nav1.5 channels form different macromolecular complexes at different microdomains in cardiomyocytes and carry the inward current responsible for the main component of the initial upstroke of the action potential [36,37]. The loss of inward current and conse- quent increase of outward potassium current may generate a “transmural dispersion of repolarization” between right epicardium and , which can facilitate and explain reentry-based arrhythmias [38,39]. Alternatively, the reduction of sodium current, by impairing phase 0 of the action potential, may slow the electrical conduction Int. J. Mol. Sci. 2021, 22, 6513 13 of 17

through the right ventricle outflow tract, causing action potential re-entry and abnormal rhythm [38,39]. In addition, it is now increasingly recognized that alterations in Nav1.5 may affect both ionic and non-ionic processes that alone or in concert contribute significantly to arrhythmogenesis and mechanical dysfunction and likely explain the BrS spectrum of clinical phenotypes [40,41]. These cellular processes, including an altered inter- action with neighboring proteins, developing with different features in different patients, may also contribute to the clinical characteristics of Ins1687GR and P1310L families. In conclusion, our results confirm the pathogenicity of two novel mutations identified in families with BrS and major arrhythmic events. The identification and functional assess- ment of novel SCN5A variants in cell lines and further exploration of the multifunctional effects of Nav1.5 in patients’ iPSCs-derived cardiomyocytes, are essential steps for under- standing BrS pathogenesis, for a better risk stratification of BrS patients and for developing personalized therapeutic interventions [42].

4. Materials and Methods 4.1. Clinical and Genetic Analysis Patients with spontaneous type I BrS pattern with or without previous symptoms were evaluated at the Cardiogenetic Clinic—University Hospital S. Anna—Ferrara. All patients underwent standard 12 leads ECG with V1-V2 at 2nd and 3rd intercostal space, Holter monitoring, and two-dimensional and three-dimensional echocardiography. Writ- ten informed consent for was obtained (local ethical committee approval 26/7/2012, P. 7/2012). DNA was isolated from the peripheral blood by standard meth- ods.20 NGS analysis was performed using a custom panel in patient I (Pro1310Leu) and in the proband’s son. A commercial gene panel PED MASTR Plus (Agilent, Santa Clara, CA, USA) was used for patient II and for the proband’s son (Gly1687_Ile1688insGlyArg) [19]. Runs were performed on a MiSeq-Dx sequencer. Data were analyzed and filtered us- ing Sophia Genetics DDM software (https://dropgen.sophiagenetics.com; licence from 9 January 2018).

4.2. Mutagenesis and Nav1.5 Channel Expression Mutations were introduced into the plasmid pRcCMV-hNav1.5 using the QuickchangeTM site-directed mutagenesis kit (Agilent, Santa Clara, CA, USA) [43]. HEK 293 cells were tran- siently transfected in 100-mm dishes with WT (3 µg) or mutant (3 µg) pRcCMV-hNav1.5 and pCD8-IRES-hβ1 (1.5 µg) expressing the sodium channel auxiliary β1 subunit and the CD8 receptor gene reporter, using the calcium phosphate precipitation method. The complete coding region of the cDNA was sequenced to exclude polymerase errors. The transfected cells were identified by microbeads coated with anti-CD8 antibodies (Dyn- abeads M-450 CD8; Dynal, Great Neck, NY, USA) and were used for electrophysiological recordings. For co-expression experiments, equal amount of WT and mutant channels cDNAs (1.5 µg + 1.5 µg) were transfected together with pCD8-IRES-hβ1 (1.5 µg) and currents compared with those generated by transfecting Nav1.5 WT cDNA alone (3 µg).

4.3. Electrophysiology Whole-cell sodium currents were recorded at room temperature from HEK 293 cells 48 h after transfection using an Axopatch 200A amplifier, a Digidata 1550B digitizer, and the pClamp 10.6 software (Molecular Devices, San Jose, CA, USA). Current signals were filtered at 5 kHz and sampled at 10 kHz. Patch-clamp pipettes were pulled from borosilicate glass using a vertical puller (Narishige, London, UK) to a resistance of 3–4 MΩ. The internal solution contained (in mM): NaCl 10, CsF 120, CsCl 10, EGTA/CsOH 5, HEPES 5 (pH 7.2 with CsOH). The external solution contained (in mM): NaCl 150, KCl 4, CaCl2 2, MgCl2 1, glucose 5, HEPES 5 (pH 7.4 with NaOH) [43]. Detailed protocols are described in Supplementary Materials and Methods. Int. J. Mol. Sci. 2021, 22, 6513 14 of 17

4.4. Homology Modeling A homology model of Nav1.5 was built from the crystal structure of the ratNav1.5 (PDB 6UZ3) [25]. Residues that differed or were missing in the ratNav1.5 as compared with the human Nav1.5 were replaced and modeled, respectively, using Modeller 9.25.49,50. We introduced the mutations P1310L and Ins1687GR in the respective domain in the Nav1.5 α-subunit. The resulting channel was introduced into a POPE: POPC: PSPS membrane (3:2:1) using CHARMM-GUI11-13 and was equilibrated using NAMD 2.14.14 [44,45].

4.5. Immunocytochemistry HEK 293 cells were grown on glass coverslips, transfected with cDNAs encoding Nav1.5 WT or mutant channels and β1 subunit, and subjected to immunofluorescence 48h after transfection. Transfection was performed using Lipofectamine™ 2000 reagent (ThermoFisher Scientific, Waltham, MA, USA). Transfected HEK 293 cells were fixed and permeabilized in PFA with 0.1% Triton X-100 for 20 min at room temperature. After washes with PBS, cells were blocked in saturation buffer (1% bovine serum albumin in PBS) for 30 min at room temperature (RT) and incubated with a mouse monoclonal anti- sodium channel pan primary antibody (1:1000; S8809; Sigma-Aldrich Merck Life Science, Milano, Italy) for 2 h at RT in blocking buffer. After three washes in PBS cells were incubated with 488 Alexafluor-conjugated secondary antibodies (ThermoFisher Scientific, Waltham, MA, USA) for 1 h at RT. Plasma membrane was stained with wheat germ agglutinin, Alexa Fluor™ 555 Conjugate (WGA-555; ThermoFisher Scientific, Waltham, MA, USA) in Hank’s balanced salt solution (HBSS) for 30 min at 37 ◦C at a concentration of 5 µg/mL. Cell coverslips were mounted on glass microscopy slides and observed on a Nikon Eclipse TE 2000-U fluorescent microscope equipped with a 40X/1.30 N.A. fluor objective (Nikon Corporation, Tokyo, Japan) and a spinning-disk confocal setup (Crisel Instruments, Rome, Italy). Nav1.5 fluorescence was excited with a green laser (SVL-473- 0200 at 473 nm of excitation wavelength) and recorded at 520 nm emission wavelength. Wheat germ agglutinin AlexaFluor™ 555 fluorescence was excited using a mercury lamp light, selecting an excitation at 555 nm on the excitation filter wheel and emission at 585 nm on the emission filter wheel. Excitation light was projected through 1000 pinholes (Ø 70 µm) using a CREST CARVII™ spinning disk. Images were collected by a Photometrics Cool Snap HQ camera (1392 × 1040 imaging pixels) and digitalized with the MetaMorph® software (Molecular Devices, San Jose, CA, USA).

4.6. Western Blot Analysis To measure total Nav1.5 protein expression, HEK 293 cells were transfected with Nav1.5 WT and mutants cDNAs. 24h after transfection, cells were harvested in 200 µL of cold RIPA buffer (20 mM Tris-HCl, 150 mM NaCl, 1.5% Non-idet P-40, 100 mM sodium orthovanadate and a protease inhibitor cocktail) and placed for 10 min in ice. To complete cell lysis, suspensions were passed through a syringe with a needle for 10 times. After 15 min in ice, cell lysates were centrifuged at 14,000 rpm for 30 min at 4 ◦C and supernatant was collected. Total protein amounts were quantified by using a BCA protein assay kit (Bio-Rad, Hercules, CA, USA). Total proteins were separated on a 7.5% SDSPAGE and transferred onto nitrocellulose membrane for 1 h at 200 mA (SemiDry transferblot, Bio-Rad Laboratories, Segrate (MI), Italy). Membrane was blocked for 1 h with 0.2 M Tris-HCl, 1.5 M NaCl and pH 7.4 buffer (TBS) containing 5% non-fat dry milk and 0.5% Tween- 20 and was incubated overnight at 4 ◦C with a mouse monoclonal anti-sodium channel pan primary antibody (S8809; Sigma-Aldrich Merck Life Science, Milano, Italy) diluted 1:400 and monoclonal mouse anti-Actin (Sc-47778, Santa Cruz Biotechnology, Dallas, TX, USA) diluted 1:300 with TBS containing 5% non-fat dry milk. After three washes with TBS containing 0.5% Tween-20 (TTBS), membrane was incubated for 1 h with goat anti- mouse IgG conjugated to horseradish peroxidase (Biorad Laboratories, Segrate (MI), Italy). Membrane was then washed with TTBS, developed with a chemiluminescent substrate (Clarity Western ECL Substrate; Bio-Rad Laboratories, Segrate (MI), Italy), and visualized Int. J. Mol. Sci. 2021, 22, 6513 15 of 17

on a Chemidoc imaging system (Bio-Rad Laboratories, Segrate (MI), Italy). Western blots were quantified with Image Lab software (Bio-Rad Laboratories, Segrate (MI), Italy), which allows the chemiluminescence detection of each experimental protein band to obtain the absolute signal intensity automatically adjusted by subtracting the local background.

4.7. Statistical Analysis Statistical analysis was performed using Student’s t-test, with p < 0.05 or less con- sidered as significant. Results are reported as mean ± SEM from the indicated number of cells.

Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/ijms22126513/s1, Supplementary Materials and Methods; Figure S1: Amino acids alignment of Nav1.x proteins highlighting the position of the P1310L and Ins1687GR BrS mutations; Figure S2: (A) Immunoblotting analysis of Nav1.5 WT, Ins1687GR and P1310L channels expressed in HEK 293 cells as in Figure7B. The positions of molecular weight markers are at the right of the blots. Expressions of β-actin are displayed as controls for the loaded protein amounts. (B) Stain-free blot image, corresponding to the total protein load, of the immunoblot reported in (A); Supplementary References. Author Contributions: Conceptualization, P.I., F.G., C.B. and M.B.; molecular biology, R.M.M.; confocal microscopy, A.G.; Western blot, E.C.; molecular dynamic simulations, R.B.; genetic analysis, F.G., R.S., M.F. and A.A.; clinical analysis, C.B., M.B., A.B. and F.V.; patch clamp experiments, P.I., E.C. and A.L.; writing—original draft preparation, P.I., F.G., C.B. and M.B.; review and editing, A.D.L., A.F. and C.R. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by PRIN 2017, grant 2017 to A.L., University of Bari “Aldo Moro”, Fondi Ateneo 2017-2018 to P.I. Institutional Review Board Statement: The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Ethics Committee of University Hospital S. Anna— Ferrara (26/7/2012, P. 7/2012). Informed Consent Statement: Written informed consent for genetic testing was obtained (local ethical committee approval 26/7/2012, P. 7/2012). Data Availability Statement: The data presented in this study are available on request from the corresponding author. Genetic data have been submitted to LOVD at https://databases.lovd.nl/. Acknowledgments: We would like to thank Celine Marionneau and Isabelle Deschenes for kindly providing SCN5A clones. Conflicts of Interest: The authors declare no conflict of interest.

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