PhD Zeina R Al Sayed

PhD Mariam Jouni

MD-PhD Jean-Baptiste Gourraud

PhD Nadjet Belbachir

PhD Julien Barc

BSc Aurore Girardeau

PhD Virginie Forest

PhD c , Aude Derevier

MSc Anne Gaignerie

BSc Caroline Chariau

MSc Bastien Cimarosti

MSc Robin Canac

MSc Pierre Olchesqui

MSc Eric Charpentier

PhD Jean-Jacques Schott

PhD Richard Redon

PhD Isabelle Baró

MD-PhD Vincent Probst

PhD Flavien Charpentier

PhD Gildas Loussouarn

PhD Kazem Zibara

MD-PhD Guillaume Lamirault

MD-PhD Patricia Lemarchand
email: patricia.lemarchand@univ-nantes.fr

PhD Nathalie Gaborit
email: nathalie.gaborit@univ-nantes.fr

A consistent arrhythmogenic trait in Brugada syndrome cellular phenotype

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Dear Editor, Brugada syndrome (BrS) is an inherited arrhythmic disease predisposing to sudden cardiac death (SCD), characterized by a typical electrocardiogram pattern that includes a J point elevation with a coved type ST segment. 1 BrS is a complex genetic disease in which ~20% of patients carry rare variants in SCN5A gene whereas the others remain genetically unresolved. 2 Despite this genetic complexity, we hypothesize that a common cellular phenotypic trait is at the root of this specific BrS ECG pattern. In this study, we identified a phenotype that is common to human induced pluripotent stem cell-derived ventricular cardiomyocytes (hiPSC-CMs) generated from six Brugada patients with different genetic backgrounds. Our results unmasked a cellular arrhythmogenic phenotype combining gene expression and electrical abnormalities, including an increase in late sodium current.

Six patients affected by type I BrS (BrS1-6; Figure S1; Table S1; Table S2) with a familial history of SCD or syncope were selected, among whom two carry SCN5A variants (marked with a + symbol). An additional individual, not affected by BrS (Non-BrS), carrying the same SCN5A variant as BrS2 + , was also recruited, as well as four control (Ctrl) subjects. Somatic cells from all studied subjects were reprogrammed into hiPSC lines and differentiated into cardiomyocytes (Figure 1).

Transcriptional expression profiling identified 133 differentially expressed genes in BrS hiPSC-CMs (Figure 2A). Gene Set Enrichment Analyses showed that transcripts of transmembrane transporters and channels were significantly overrepresented (Figure 2B), including genes encoding sodium, calcium, and potassium channels (Figure 2C). High-throughput real-time RT-PCR, 3 on 96 genes related to cardiac electrical function (Table S3) identified 13 differentially expressed genes in BrS, in comparison to Ctrl and Non-BrS hiPSC-CMs (Figure 2D). Importantly, the expression of SCN5A, the main BrS culprit gene identified to-date, 4 remained unchanged, excluding SCN5A expression levels as a hallmark for BrS hiPSC-CM phenotype. Conversely, calcium and sodium transporters, playing important roles in membrane depolarization, were differentially expressed. Comparative analysis of hiPSC-CM electrophysiological functions investigated whether these modifications were a consistent trait of BrS phenotype at the cellular level.

Whereas decrease in sodium current is considered as the most frequently associated electrical alteration in BrS pathophysiology, 5,6 protein expression of Na v 1.5, encoded by SCN5A, was decreased in only 2 BrS, and the Non-BrS lines (Figure 2E). Concordantly, reduction in I Na density was detected in these same lines (Figure 2F-2H). This confirmed previous results, for BrS5 + , 7 and regarding BrS1 + , which carries an SCN5A rare variant, the reduction was confirmed using conventional transfection in COS-7 cells of this variant (Figure S2). Furthermore, the steady-state activation and inactivation gating properties were not modified in BrS hiPSC-CMs (Figure S3A; Table S4). Therefore, I Na reduction is not a common trait of BrS hiPSC-CMs and appears to be solely associated with the presence of variants affecting SCN5A expression or function. S4).

Global cellular electrophysiological phenotype was then evaluated with action potential (AP) recordings, but no AP basal parameters specifically segregated BrS hiPSC-CMs, and spontaneous beating frequencies did not differ between all cell lines (Figure S4). Noteworthy, ventricular-like AP analysis revealed an arrhythmic phenotype present mostly in BrS hiPSC-CMs, irrespective of their genetic background (Figure 3A). Early afterdepolarizations (EADs) were observed in 39% to 70% of all six BrS ventricular-like hiPSC-CMs versus only in 4% and 4.7% of Ctrl and Non-BrS hiPSC-CMs, respectively (Figure 3B, S5). Thereby, the high EAD occurrence in ventricular-like hiPSC-CMs was associated with the presence of a BrS phenotype in the investigated cell lines, but not to the presence of a variant in SCN5A.

The occurrence of EADs may be linked to an abnormally high density of depolarizing late sodium current (I Na,L ) during APs repolarizing phase. 8 Accordingly, BrS hiPSC-CMs presented with a higher density of I Na,L as compared to Ctrl and Non-BrS hiPSC-CMs (Figure 3C,3D). Moreover, an increase in I Na,L density was observed only in 6% and 12% of Ctrl and Non-BrS hiPSC-CMs respectively, in accordance with their low EAD occurrence, whereas increased I Na,L density was present in 50% to 85% of all BrS ventricular-like hiPSC-CMs, reminiscent of the high EAD occurrence (Figure 3B,3E). We then superfused ventricular-like BrS hiPSC-CMs during AP recording with GS-458967 (6-(4-(Trifluoromethoxy)phenyl)-3-(trifluoromethyl)- [1,2,4]triazolo [4,3-a]pyridine, which selectively blocks late sodium current), 9 causing full inhibition of I Na,L (Figure 3F), and found abolishment of EADs (Figure 3G,3H) and reduced APD90 dispersion (Figure 3I). Altogether, these data strongly suggested that the abnormal increase of I Na,L in BrS hiPSC-CMs is responsible for EADs.

Further strengthening the role of the I Na,L in the electrical cellular phenotype of BrS, when each ECG parameter was tested for its correlation with either I Na,L or I Na measured densities, only I Na,L density correlated significantly with one sole parameter, i.e., the J point elevation (Table S5).

To challenge the pathophysiological relevance of the ion current alterations identified in Non-BrS and BrS2 + hiPSC-CMs, we applied them to a mathematical human electrogram model, that allows visualizing transmural-like electrogram with a QRS-like complex, a ST-like segment, and a T-like wave (Figure 4A). 10 First, in accordance with BrS2 + patient's ECG, applying the alterations observed in peak I Na , I Ca,L and in I Na,L in BrS2 + hiPSC-CMs was sufficient to induce prolongation of the QRS-like complex, ST-like segment elevation and widening, and T-like wave inversion (Figure 4B). Then, sequential correction of each altered current in BrS2 + hiPSC-CMs was made (BrS2 + corrected ). Correction of I Na density led to QRS-like complex normalization; correction of I Ca,L density shortened the duration of the ST-like segment elevation and normalized the T-like wave orientation; and correction of I Na,L density led to reduction of ST-like segment amplitude towards normalization (Figure 4C, left to right). Overall, these In conclusion, in the present study, a particular cellular electrophysiological phenotype common to six out of six BrS hiPSC-CM lines with various genetic backgrounds has been unveiled. We showed that high EAD occurrence associates with an abnormal increase of I Na,L in all investigated BrS cell lines, and correlates with the corresponding patients' J point elevation on ECG. We focused on the ventricular cell type, at a single-cell level. Implementation of emerging phenotypic technologies, such as singlecell transcriptomics and cardiac tissue engineering, will allow investigation of the potential involvement of the other cardiac cell types in the disease phenotype and the role of specific cell-tocell interactions. Altogether, the obtained results open perspectives to better understand the ventricular arrhythmia occurrence in BrS and to identify a dedicated therapeutic approach to prevent the risk of SCD. In accordance with the "DFG Guidelines on the Handling of Research Data", the authors declare that all data supporting the findings of this study are available within the article and its supplementary information files or from the corresponding author upon reasonable request. The data set will be archived for at least 10 years after publication.

SUPPLEMENTAL MATERIAL

Supplemental methods:

Ethical Statement

The study was conducted according to the principles set forth under the Declaration of Helsinki (1989) and European guidelines for clinical and genetic research. Institutional review board approvals of the study were obtained before the initiation of patient enrollment. The study protocol was reviewed and approved by the regional "comité de protection des personnes" ethical committee (approval number:

2010-A01358-31). Regarding the patient-derived biological samples, signed informed consent allowing the experiments to be conducted has been received from all individuals. Any related health information was collected in compliance with applicable law/regulation and with any applicable policy of the ethics committee with jurisdiction over the biological sample collection. All biological samples and their related health information have been provided in coded form such that subjects cannot be identified directly. The provisions of French law, article L1110-4 of the Code de la santé publique, related to the privacy and confidentiality of information regarding patients, have been observed.

Study Population and design

BrS patients were enrolled according to the presence of a BrS ECG pattern (see below) and with a familial history of sudden death or syncope. Diagnosis of BrS was based on criteria from 2013 1 with the presence of a type-1 BrS ECG pattern, either spontaneous or induced by intravenous injection of class I antiarrhythmic drugs, in at least one right precordial lead (V1 or V2) positioned in the 2nd, 3rd or 4th intercostal space. Type-1 BrS ECG pattern was defined as a J point elevation higher than 0.2 mV, followed by a coved type ST segment elevation and ended with a negative T wave.

ECG analysis

Two physicians blinded to the clinical and genetic status reviewed all baseline ECGs. P wave, PQ interval, QRS, QT peak, QTend, QTc duration (corrected by Bazett formula), and Tpeak-Tend interval (TPE, time interval between the peak and the end of the T wave) were measured in D2, V1, V2 and V3.

S-wave duration and amplitude were additionally measured in D1. All measurements were performed using Image J software as previously described. 2,3 TPE in precordial lead, fragmented QRS, earlyrepolarization pattern and prominent R wave in lead aVR were determined as previously described. 4,5

Genetic analysis

Patients were screened for mutations in genes previously described in cardiomyopathies and inherited arrhythmias. 6,7 For this, genomic DNA of probands was extracted from peripheral blood lymphocytes by standard protocols. The DNA yields were assessed by measurements using Quant-IT™ dsDNA Assay Kit, Broad Range (Life Technologies, Q33130

SCN5A CACNA1C RANGRF KCNE1L GPD1L CACNB2 PKP2 KCNJ8 SCN1B CACNA2D1 FGF12 ABCC9 SCN2B KCNE3 SCN10A KCNH2 SCN3B KCND3 TRPM4 HCN4
Target enrichment and sequencing were performed as previously described. 7 First, 200 ng of gDNA sample were digested in eight different restriction reactions, each containing two restriction enzymes, to create a library of gDNA restriction fragments. These gDNA restriction fragments were hybridized to the HaloPlex probe capture library. Probes were designed to hybridize and circularize targeted DNA fragments. During the hybridization process, Illumina sequencing motifs including index sequences were incorporated into the targeted fragments.

The circularized target DNA biotinylated HaloPlex probe complexes were captured on magnetic streptavidin beads. We proceeded to a ligation reaction of the circularized complexes followed by an elution reaction before PCR amplification. The amplified target DNA was purified using AMPure XP bead (Beckman Coulter, A63881). To validate enrichment of target DNA in each library sample by microfluidics analysis, we used the 2200 TapeStation (Agilent Technologies, G2964AA), with D1K ScreenTape (Agilent Technologies, 5067-5361), and D1K Reagents (Agilent Technologies, 5067-5362).

We ensured that the majority of amplicons range from 175 to 625 bp. Finally, we quantified the library together with other libraries by qPCR using the KAPA Library Quantification Kit (Clinisciences, KK4854).

Libraries were pooled in an equimolar concentration and DNA was then denatured with NaOH. Finally, the library pool was diluted to a final concentration of 9 pM before proceeding to 100-bp paired-end Illumina sequencing on HiSeq1500.

Raw sequence reads were aligned to the human reference genome (GRCh37) using BWA-MEM (version 0.7.5a) after removing sequences corresponding to Illumina adapters with Cutadapt v1. Variants were considered as having a potential functional consequence if they were annotated with one or more of the following SO terms for at least one RefSeq transcript: "transcript_ablation" (SO:0001893), "splice_donor_variant" (SO:0001575), "splice_acceptor_variant" (SO:0001574), "stop_gained" (SO:0001587), "frameshift_variant" (SO:0001589), "stop_lost" (SO:0001578), "initiator_codon_variant" (SO:0001582), "inframe_insertion" (SO:0001821), "inframe_deletion" (SO:0001822), "missense_variant" (SO:0001583), "transcript_amplification" (SO:0001889). Loss-offunction variants (nonsense variants, frameshift variants and splice site variants) were defined by the following SO terms: "stop_gained", "frameshift_variant", "splice_donor_variant", or

"splice_acceptor_variant". The potential pathogenicity of variants was determined following the American College of Medical Genetics and Genomics (ACMG) guidelines using the CardioVAI tools from EnGenome. 9

Generation and validation of hiPSCs

BrS5 -hiPSCs were previously used in a study revealing the role of a variant in RRAD, which encodes RAD GTPase, in BrS. 10 BrS6 -hiPSC line, named iBrS1 in a previous study, was derived from a BrS patient with no defined causative variant. 6 Four different control hiPSC lines were used, including 2 studied previously. 10,11 BrS4 -cutaneous fibroblasts were reprogrammed using Stemgent mRNA reprogramming kit and pluriton medium. hiPSCs derived from dermal cells harvested from BrS1 + and BrS3 -as well as blood cells obtained from BrS2 + and his unaffected family relative (Non-BrS) were reprogrammed using Sendai virus method (Cytotune reprogramming Kit, Life technologies). Up to three clones of each hiPSC line were selected per patient. HiPSCs were maintained on matrigel-coated plates (0.05 mg/ml, BD Biosciences) with StemMACSTM iPS-Brew XF medium (Miltenyi Biotec). Pluripotency of each hiPSC clone was validated by qRT-PCR, immunostaining, and flow cytometry to verify the expression of endogenous pluripotent factors.

Validation of SCN5A rare variants and genome integrity

To validate SCN5A rare variants carried by BrS1 + , BrS2 + and Non-BrS, genomic DNA from corresponding hiPSC clones was extracted using NucleoSpin® Tissue kit (MACHEREYNAGEL). Variants were verified by sequencing. Single nucleotide polymorphism (SNP) analysis of all hiPSC clones compared to their parental skin fibroblast cells, was used to confirm genome integrity after reprogramming. DNA was extracted from somatic and hiPSC samples using the QIAGEN QiaAmp kit, according to the manufacturer's recommendations. The gDNA was quantified using a Nanodrop instrument. 200ng of gDNA were outsourced to Integragen Company (Evry, France) for karyotype analysis using HumanCore-24-v1 SNP arrays. This array contains over 300,000 probes distributed throughout the genome with a median coverage of one probe every 5700 bases. All genomic positions were based on Human Genome Build 37 (hg19

Differentiation of hiPSCs into cardiomyocytes

Cardiomyocytes were differentiated from hiPSCs using the established matrix sandwich method. 11 Briefly, when cells reached 90% confluence, an overlay of Growth Factor Reduced Matrigel (0.033 mg/ml, BD Corning) was added. Differentiation was initiated 24 h later by culturing the cells in RPMI1640 medium (Life Technologies) supplemented with B27 (without insulin, Life Technologies), 2 mM L-glutamine (Life Technologies), 1% NEAA (Life Technologies), 100 ng/mL Activin A (Miltenyi), and 10 ng/mL FGF2 for 24 hours. On the next day, the medium was replaced by RPMI1640 medium supplemented with B27 without insulin, 2 mM L-glutamine, 1% NEAA, 10 ng/mL BMP4 (Miltenyi), and 5 ng/mL FGF2 for 4 days. By day 5, cells were cultured in RPMI1640 medium supplemented with B27 complete (Life Technologies), 2 mM L-glutamine and 1% NEAA and changed every two days. Presence of beating cells was considered as the primary hallmark of a successful cardiomyocyte differentiation.

Transcript expression analysis

Quantitative RT-PCR: Total RNA samples were isolated using the NucleoSpin RNA kit (MACHEREY-NAGEL). One μg of tRNA was reverse transcribed using High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems) following the manufacturer's instructions. PCR amplification was performed using FAM labeled-TaqMan probes (Applied Biosystems) to verify the pluripotency of derived hiPSCs as compared to parental fibroblasts (OCT3/4 and NANOG). According to the ΔΔCt method, all data were normalized to ACTB and represented relative to controls.

TaqMan Low Density array (TLDA): TLDA studies were conducted using beating clusters of hiPSC-CMs obtained from 12 samples of Ctrl, 4 samples of Non-BrS and 14 samples of BrS hiPSC-CMs. One μg of RNA was reverse transcribed into cDNA using SuperScript IV Vilo Master Mix (Thermo Fisher Scientific).

TLDA probe selection covered gene families implicated in cardiac ion channel expression and regulation, and cardiomyocyte structure (Table S3). Genes with average Ct > 32 in all compared groups were considered undetectable and excluded from the analysis (SCN10A and ABCC8). Average Ct of remaining genes for each sample was used for data normalization. [12][13][14] 3' Sequencing RNA Profiling (3'SRP): 3'SRP protocol was performed according to Kilens et al. 15 Briefly, the libraries were prepared from 10 ng of total RNA. RNA samples were extracted from 26 BrS and 13 control hiPSC samples (a duplicate for each clone obtained at different cell passages) as well as their corresponding differentiated hiPSC-CMs. The mRNA poly(A) tail was tagged with universal adapters, well-specific barcodes and unique molecular identifiers (UMIs) during template-switching reverse

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transcriptase. Barcoded cDNAs from multiple samples were then pooled, amplified and tagmented using a transposon-fragmentation approach which enriches for 3'ends of cDNA. A library of 350-800 bp was run on an Illumina HiSeq 2500 using a HiSeq Rapid SBS Kit v2 (50 cycles; FC-402-4022) and a HiSeq Rapid PE Cluster Kit v2 (PE-402-4002). Read pairs used for analysis matched the following criteria: all sixteen bases of the first read had quality scores of at least 10 and the first six bases correspond exactly to a designed well-specific barcode. The second reads were aligned to RefSeq human mRNA sequences (hg19) using bwa version 0.7.17. Reads mapping to several transcripts of different genes or containing more than 3 mismatches with the reference sequences were filtered out from the analysis. Digital gene expression profiles were generated by counting the number of UMIs associated with each RefSeq genes, for each sample. R package DESeq2 (Bioconductor) was used to normalize gene expression, and detect differentially expressed genes. Sample correlation matrix was performed using R package ComplexHeatmap (Bioconductor). Cluster 3.0 software was used to perform the differentially expressed gene heatmap. Gene Set Enrichment Analysis (GSEA) was performed using GeneTrail2. Statistically significant categories within the GO Molecular Process were identified using Kolomogorov-Smirnov test and p values were adjusted using the Benjamini and Hochberg method.

Protein expression analysis

Beating clusters of hiPSC-CMs were lysed in buffer composed of 1% TritonX-100, 100 mM NaCl, 50 mM Tris-HCl, 1 mM EGTA, 1 mM Na3VO4, 50 mM NaF, 1 mM PMSF and protease inhibitors cocktail (P8340, Sigma-Aldrish). Protein quantification was then conducted using PierceTM BCA Protein Assay Kit (Thermo Fisher). The lysates were denatured for 5 min at 65°C in a mixture of NuPAGE® Sample

Reducing Agent (10X) and NuPAGE® LDS Sample Buffer (4X). 40 μg of each sample were loaded onto 12% precast polyacrylamide gels (Bio-Rad). After migration, the proteins were transferred onto Trans-Blot® TurboTM Nitrocellulose Transfer Packs (Bio-Rad). Membranes were saturated with 5% non-fat milk, then incubated with primary antibody (Anti-Na v 1.5: 14421S Cell Signaling; Anti-Cav1.2: AB5156 Sigma-Aldrich; Anti-TFRC:13-6890 Thermofisher) overnight followed by another incubation for 1 h with an adequate Horseradish peroxidase (HRP)-conjugated secondary antibody. Protein bands were detected using ECL detection system (Bio-Rad) and quantified using Image Lab software.

Immunostainings

To validate hiPSC pluripotency, hiPSC-CM differentiation into cardiac lineage as well as COS-7 cell expression, cells were dissociated and seeded onto 8-wells ibidi plates (Biovalley) coated with Matrigel (Corning). After 12 days following hiPSC-CM dissociation, and 24 h following COS-7 transfection, cultured cells were fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X100 and blocked with 1% BSA. Immunofluorescent stainings were performed using appropriate primary antibodies (Anti-TRA1-60: 14-8863-80 eBioscience™; Anti-OCT4: 14-5841-80 eBioscience™; Anti-troponin I: sc-

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15368 Santa Cruz; Anti-Na v 1.5: 14421S Cell Signaling) and their corresponding Alexa conjugated antibodies (Molecular Probes). DAPI was used for nuclear staining. Immunostainings were examined using an inverted epifluorescent microscope (Zeiss Axiovert 200M).

p.N1722D Na v 1.5 study in COS-7 cells

The point mutation p.N1722D (c.1859G>A) in SCN5A was introduced using mutated oligonucleotide extension (QuikChange II XL Site-Directed Mutagenesis Kit) in SCN5A isoform 2 cDNA (GenBank Acc.

Nb. NM_000335). The resulting plasmid was verified by complete sequencing of the cDNA insert.

African green monkey kidney-derived cells COS-7, were transiently transfected with 0. Borosilicate glass pipettes (2-3 MΩ of tip resistance) were filled with a solution containing (in mM):

125 K-Gluconate, 20 KCl, 5 NaCl, 5 HEPES; pH 7.2 (KOH) and 0.22 amphotericin-B. We first recorded spontaneous APs in order to determine, for each individual included in the study, the proportion of nodal-like, atrial-like and ventricular-like, as previously described. 11 Then to overcome limited I K1 contribution during hiPSC-CM AP, artificial I K1 was injected using dynamic patch-clamp. 16 Both cell stimulation and I K1 injection were realized using a custom-made software running on RT-Linux and an

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A/D converter (National Instrument PCI-6221) connected to the current command of the amplifier.

The AP parameters measured were the maximum diastolic potential (MDP), the maximum upstroke velocity of phase 0 depolarization (dV/dt max ), the AP amplitude and the AP duration at different levels of full repolarization, from 30% (APD30) to 90% (APD90), as previously described. 11 Following I K1 injection, at a peak outward density that sets the membrane potential between -80 to -85mV for all

Mathematical electrogram modeling

Right-ventricle electrogram was calculated as in Gima and Rudy, 17 by simulating a heterogeneous transmural wedge (right ventricular outflow tract, 1 Hz, 500th beat shown). This model aims at mimicking the global electrical activity of a row of 165 subendocardial, midmyocardial and subepicardial human ventricular cells. 18,19 In the original model used by Gima and Rudy, a slow inactivation gate (j) of the Na+ channel was added to include the property of slow recovery, 20 without modifying fast inactivation (realized with h gate). A few modifications were operated. Since this 'j' inactivation was originally as fast as 'h' inactivation, it prevents the appearance of a persistent current when altering the 'h' gate. So we slowed down this 'j' inactivation, but left the kinetics of recovery intact, by modifying the following equation: Since only a subset of hiPSC-CMs present the late Na + current, we performed simulations with the late Na + current only in midmyocardial cells, giving rise to a J point elevation followed by coved ST segment. 18 Since decreasing I Na by as much as 73-75% (BrS2 + and Non-BrS) was preventing conduction in the Gima and Rudy model, a slighter 30% decrease in I Na was applied.

If V >= -

Statistical analysis

Results are expressed as mean ± SEM. Patient #6 (BrS6 -) was a 42-year-old male, previously described by Veerman et al. 6 While he had a spontaneous BrS ECG pattern, he presented an unexplained syncope, and suffered from an outof-hospital cardiac arrest at night. Three of his relatives presented with a BrS phenotype after ajmaline administration.

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An additional Patient #7, not affected by Brugada syndrome (Non-BrS), was recruited. He was a 67-year-old male diagnosed with progressive cardiac conduction defect (PCCD). He was the nephew of patient BrS2 + . His ECG displayed broad PR interval and prolonged QRS duration, however, no ST segment elevation was detected even after challenge with flecainide.

Four control subjects were also included. One subject who was a relative of BrS5 -patient, 10 in whom BrS was excluded after sodium channel blocker challenge and three other unrelated healthy extra familial controls, including one previously described 11 and 2 others with different ethnicity. 21 Representative patient ECGs are presented in Figure S1. Description of patients and their corresponding ECG measurements are depicted in Table S1 and Table S2 respectively.

Genetic characterization

Genetic screening of the coding regions of 20 genes, selected according to the American College of Medical Genetics and Genomics (ACMG) guidelines, revealed SCN5A pathogenic variants in only two of the six BrS patients. BrS1 + carried a c.5164A>G missense rare variant, resulting in asparagine to aspartic acid substitution at position 1722 in the extracellular connecting loop, between segment 5 and 6 in domain IV forming the pore region of Na v 1.5 (p.N1722D). BrS2 + and his nephew (Non-BrS) affected by PCCD carried a 10bp duplication (c.1983-1993dup) in SCN5A creating a stop codon (p.A665G-fsX16). Both these variants had not been functionally investigated yet. BrS5 -patient presented a previously described rare genetic variant in the RRAD gene (p.R211H) 10 whereas no genetic variation was identified in BrS candidate genes in BrS3 -, BrS4 -or BrS6 -patients (Table S1).

Generation and characterization of hiPSCs and hiPSC-CMs

Somatic cells were obtained from all studied subjects and were reprogrammed into corresponding hiPSC lines. BrS5 -, BrS6 -and the control hiPSC lines have been previously characterized. 6,10,11,21 For each of the other 5 newly generated hiPSC lines (BrS1 to 4 and Non-BrS), up to three independent clones were amplified and characterized. SNP analysis verified that control and mutated hiPSC lines were free from any genomic aberrations compared to parental somatic cells (data not shown). The expression of the pluripotent stem cell markers was verified (Figure 1A, 1B and 1C).

Genetic screening confirmed that the lines arising from patients carrying SCN5A genetic variants, BrS1 + , BrS2 + and Non-BrS harbored the corresponding heterozygous rare variant (Figure 1D).

hiPSC differentiation process into cardiomyocytes was also validated at the transcriptional level. 3'SRPbased global transcriptomic analysis of control and BrS hiPSCs and hiPSC-CMs showed that cells clustered based on their stage (hiPSCs on one side and hiPSC-CMs on the other side) and independently from their genetic background (Figure 1E). Correlation analysis also showed that samples correlated according to their stage, with all hiPSC samples being correlated to other hiPSC samples and similarly for hiPSC-CM samples (Figure 1F). Finally, both Ctrl and BrS hiPSC-CMs presented a comparable global change in gene expression as compared to hiPSC stage (Figure 1G). Immunostaining analysis showed

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that striated troponin I, a cardiac and muscular specific cytoskeletal protein, was similarly present in all hiPSC-CM lines (Figure 1H). Spontaneous APs, in absence of I K1 injection, were classified based on MDP, dV/dt max , AP duration and morphology into nodal-like, atrial-like and ventricular-like, as previously described. 11 The proportion of each cell type was similar between all hiPSC-CM lines, with the ventricular-like type forming the majority of explored hiPSC-CMs (Figure 1I). Altogether, these data confirmed that Ctrl, BrS, and Non-BrS hiPSCs differentiated similarly into cardiomyocytes, and therefore a comparative electrophysiological analysis could be performed to unveil a potential common cellular phenotypic trait of BrS hiPSC-CMs.

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Response to decision letter

Responses to Editor

Editor: Please merge Supplemental Figures 2 and3 as ONE and add it into the text as Figure 4.

Response: Dear Editor, we have performed the figure modification accordingly, however, we numbered this new figure as Figure 1 and not Figure 4, as it is now the first described figure in the Letter.

Responses to Reviewer

Reviewer: Al Sayed and colleagues used a 2-dimentional patient-derived iPSC-CMs to collate data that reveal BrS molecular alterations and a story consequently, which is indeed important in understating the molecular mechanisms of the disease. Particularly, the genetic characterisation adds to the understanding. I would recommend the following minor comments:

Reviewer: 1) Define GS-458967 at first use.

Response:

We thank the Reviewer for pointing out this mistake, and clarified at the first use of GS-458967 that its full name is 6-(4-(Trifluoromethoxy)phenyl)-3-(trifluoromethyl)-[1,2,4]triazolo[4,3a]pyridine and that selectively blocks late sodium current. We added this explanation on page 3, paragraph 3, of the Letter.

Reviewer: 2) Increased triggered activity is associated with beating frequency. The Baseline-like arrhythmic events and average number of cells with increase beating frequency after baseline could be demonstrated.

Response:

We thank the Reviewer for this very relevant comment. Upon request, we performed a comparative analysis of spontaneous beating frequencies of hiPSC-CMs from all studied lines. We did not observe any statistical difference between the BrS lines and the Ctrl or Non-BrS lines (Figure S4E; p = ns; One-way Anova test). Therefore, in opposition to what we observed for the EAD occurrence (higher in BrS lines as compared to Ctrl and Non-BrS), abnormal beating frequency was not a common phenotype of the BrS lines. Therefore, the EAD phenotype could not be explained by an altered spontaneous beating frequency in BrS lines as compared to Ctrl and Non-BrS lines. This was added to the Letter on page 3, paragraph 2, and the new figure was added to the supplemental material (Figure S4E). Response: We thank the Reviewer for his suggestions. Concerning the transcriptomic data, to follow the Reviewer's comment, we annotated Figure 1E and Figure 2A highlighting all clones of each hiPSC line and changed the Figure legends accordingly. This shows that the clone-to-clone heterogeneity is lower than the cell line-to-cell line heterogeneity. Regarding the investigation of BrS hiPSC-CM clonespecific signature, unfortunately, the study was not designed nor powered to answer this specific question. In addition, the low number of patient cell lines available in this study might not have been sufficient to reliably describe clone-to-clone heterogeneity. Therefore, we respectfully believe that this is beyond the scope of the present letter. However, we thank the Reviewer for his comment, as it may trigger a future project aiming at unveiling heterogeneity among various BrS hiPSC lines, including at the transcriptomic level, with a potential link to the patients' phenotype.

Regarding the data on EADs, we displayed the percentage of ventricular cells with and without EADs, for each investigated clone, of each hiPSC line, showing good reproducibility of the data (both for the control and for the BrS cell lines). We added this figure to the supplementary material, as Figure S5.

Reviewer: 5) It would be difficult to some extent for non-specialist to follow that ''Furthermore, INa biophysical properties were not modified in BrS hiPSC-CMs (Figure S5A; Table S4)'' supports the argument that precedes it. This could be as a result of overly condensed writing. The authors should find a way to make the sentence more congruent to what is being said before it.

Response:

The Reviewer has a point and we apologize for the lack of clarity, due indeed to an oversimplification of the original sentences. By "INa biophysical properties", we meant the activation and inactivation kinetics of the sodium channels. We modified the paragraph to make it clearer and more meaningful: "Concordantly, reduction in INa density was detected in these same lines (Figure 2F-2H). This confirmed previous results, for BrS5+, and regarding BrS1+, which carries an SCN5A rare variant, the reduction was confirmed using conventional transfection in COS-7 cells of this variant (Figure S2). Furthermore, the steady-state activation and inactivation gating properties were not modified in BrS hiPSC-CMs (Figure S3A; Table S4)." (See page 2, paragraph 4).

Reviewer: 6) Refer to ''thereby, high EAD occurrence in ventricular-like hiPSC-CMs seemed to be associated with BrS, and not with the presence of SCN5A variant'' if ventricular-like hiPSC-CMs from patients with SCN5A variants had no AP triggering activity at all, it should be made clear.

Response: We realize from this Reviewer's comment, that this sentence was misleading. To sum up the findings, (1) triggered activities were observed in all studied hiPSC-CM lines, but with very different occurrences;

(2) all 6 BrS-derived cell lines, both the ones that carry variants in SCN5A and the ones that do not, displayed high occurrences of EADs (in 39% to 70% of the ventricular hiPSC-CMs) while (3) EAD occurrence was much lower in ventricular hiPSC-CMs of the Control cell lines (4%) and ( 4) EAD occurrence was also much lower in the "Non-BrS" cell line (4.7%), which carries a variant in SCN5A but is not affected by BrS. Using Fisher's exact test we confirmed the statistical significance of our findings. Therefore, the occurrence level of these triggered activities seemed to be associated to the presence of a BrS phenotype in the investigated cell lines, but not to the presence of a variant in SCN5A. We modified accordingly this part of the results on page 3, paragraph 2, of the Letter.

Reviewer: 7)

The phenotype investigated may be a product of complex interaction of multiple cell types, genetics, and the environment, and therefore iPSC-CMs may be an unsuitable model. Whether or not this view holds for the model could be expanded on in relation to the findings.

Response:

The Reviewer raises a critical point that should indeed be acknowledged in the study. We agree with the Reviewer that the use of hiPSC-CMs in the actual setting may not fully model the complex pathophysiology of BrS. First, as pointed out by the Reviewer the cardiac differentiation of hiPSCs generates a heterogeneous population of cardiac cell types that may all be involved in the BrS pathophysiology. In the present study, we focused on the ventricular cell type however, further studies will have to investigate the potential involvement of the other cell types in the disease phenotype. Second, the generated cells are not organized in layers or compartments and do not recapitulate the specific cell-to-cell interactions that take place in the cardiac tissue. Altogether, this, most probably, currently limits the complete understanding of the BrS mechanism. The implementation of emerging phenotypic technologies, such as single-cell transcriptomics and cardiac tissue engineering, should undoubtedly further expand the power of investigating BrS with the hiPSC technology. We added these notions to the conclusion of the Letter, on page 4, paragraph 2. characterized by a typical electrocardiogram pattern that includes a J point elevation with a coved type ST segment. 1 BrS is a complex genetic disease in which ~20% of patients carry rare variants in SCN5A gene whereas the others remain genetically unresolved. 2 Despite this genetic complexity, we hypothesize that a common cellular phenotypic trait is at the root of this specific BrS ECG pattern. In this study, we identified a phenotype that is common to human induced pluripotent stem cell-derived ventricular cardiomyocytes (hiPSC-CMs) generated from six Brugada patients with different genetic backgrounds. Our results unmasked a cellular arrhythmogenic phenotype combining gene expression and electrical abnormalities, including an increase in late sodium current.

Six patients affected by type I BrS (BrS1-6; Figure S1; Table S1; Table S2) with a familial history of SCD or syncope were selected, among whom two carry SCN5A variants (marked with a + symbol). An additional individual, not affected by BrS (Non-BrS), carrying the same SCN5A variant as BrS2 + , was also recruited, as well as four control (Ctrl) subjects. Somatic cells from all studied subjects were reprogrammed into hiPSC lines and differentiated into cardiomyocytes (Figure 1).

Transcriptional expression profiling identified 133 differentially expressed genes in BrS hiPSC-CMs (Figure 2A). Gene Set Enrichment Analyses showed that transcripts of transmembrane transporters and channels were significantly overrepresented (Figure 2B), including genes encoding sodium, calcium, and potassium channels (Figure 2C). High-throughput real-time RT-PCR, 3 on 96 genes related to cardiac electrical function (Table S3) identified 13 differentially expressed genes in BrS, in comparison to Ctrl and Non-BrS hiPSC-CMs (Figure 2D). Importantly, the expression of SCN5A, the main BrS culprit gene identified to-date, 4 remained unchanged, excluding SCN5A expression levels as a hallmark for BrS hiPSC-CM phenotype. Conversely, calcium and sodium transporters, playing important roles in membrane depolarization, were differentially expressed. Comparative analysis of hiPSC-CM electrophysiological functions investigated whether these modifications were a consistent trait of BrS phenotype at the cellular level.

Whereas decrease in sodium current is considered as the most frequently associated electrical alteration in BrS pathophysiology, 5,6 protein expression of Na v 1.5, encoded by SCN5A, was decreased in only 2 BrS, and the Non-BrS lines (Figure 2E). Concordantly, reduction in I Na density was detected in these same lines (Figure 2F-2H). This confirmed previous results, for BrS5 + , 7 and regarding BrS1 + , which carries an SCN5A rare variant, the reduction was confirmed using conventional transfection in COS-7 cells of this variant (Figure S2). Furthermore, the steady-state activation and inactivation gating properties were not modified in BrS hiPSC-CMs (Figure S3A; Table S4). Therefore, I Na reduction is not a common trait of BrS hiPSC-CMs and appears to be solely associated with the presence of variants affecting SCN5A expression or function. S4).

Global cellular electrophysiological phenotype was then evaluated with action potential (AP)

recordings, but no AP basal parameters specifically segregated BrS hiPSC-CMs, and spontaneous beating frequencies did not differ between all cell lines (Figure S4). Noteworthy, ventricular-like AP analysis revealed an arrhythmic phenotype present mostly in BrS hiPSC-CMs, irrespective of their genetic background (Figure 3A). Early afterdepolarizations (EADs) were observed in 39% to 70% of all six BrS ventricular-like hiPSC-CMs versus only in 4% and 4.7% of Ctrl and Non-BrS hiPSC-CMs, respectively (Figure 3B, S5). Thereby, the high EAD occurrence in ventricular-like hiPSC-CMs was associated with the presence of a BrS phenotype in the investigated cell lines, but not to the presence of a variant in SCN5A.

The occurrence of EADs may be linked to an abnormally high density of depolarizing late sodium current (I Na,L ) during APs repolarizing phase. 8 Accordingly, BrS hiPSC-CMs presented with a higher density of I Na,L as compared to Ctrl and Non-BrS hiPSC-CMs (Figure 3C,3D). Moreover, an increase in I Na,L density was observed only in 6% and 12% of Ctrl and Non-BrS hiPSC-CMs respectively, in accordance with their low EAD occurrence, whereas increased I Na,L density was present in 50% to 85% of all BrS ventricular-like hiPSC-CMs, reminiscent of the high EAD occurrence (Figure 3B,3E). We then superfused ventricular-like BrS hiPSC-CMs during AP recording with GS-458967 (6-(4-(Trifluoromethoxy)phenyl)-3-(trifluoromethyl)- [1,2,4]triazolo [4,3-a]pyridine, which selectively blocks late sodium current), 9 causing full inhibition of I Na,L (Figure 3F), and found abolishment of EADs (Figure 3G,3H) and reduced APD90 dispersion (Figure 3I). Altogether, these data strongly suggested that the abnormal increase of I Na,L in BrS hiPSC-CMs is responsible for EADs.

Further strengthening the role of the I Na,L in the electrical cellular phenotype of BrS, when each ECG parameter was tested for its correlation with either I Na,L or I Na measured densities, only I Na,L density correlated significantly with one sole parameter, i.e., the J point elevation (Table S5).

To challenge the pathophysiological relevance of the ion current alterations identified in Non-BrS and BrS2 + hiPSC-CMs, we applied them to a mathematical human electrogram model, that allows visualizing transmural-like electrogram with a QRS-like complex, a ST-like segment, and a T-like wave (Figure 4A). 10 First, in accordance with BrS2 + patient's ECG, applying the alterations observed in peak I Na , I Ca,L and in I Na,L in BrS2 + hiPSC-CMs was sufficient to induce prolongation of the QRS-like complex, ST-like segment elevation and widening, and T-like wave inversion (Figure 4B). Then, sequential correction of each altered current in BrS2 + hiPSC-CMs was made (BrS2 + corrected ). Correction of I Na density led to QRS-like complex normalization; correction of I Ca,L density shortened the duration of the ST-like segment elevation and normalized the T-like wave orientation; and correction of I Na,L density led to reduction of ST-like segment amplitude towards normalization (Figure 4C, left to right). Overall, these In conclusion, in the present study, a particular cellular electrophysiological phenotype common to six out of six BrS hiPSC-CM lines with various genetic backgrounds has been unveiled. We showed that high EAD occurrence associates with an abnormal increase of I Na,L in all investigated BrS cell lines, and correlates with the corresponding patients' J point elevation on ECG. We focused on the ventricular cell type, at a single-cell level. Implementation of emerging phenotypic technologies, such as singlecell transcriptomics and cardiac tissue engineering, will allow investigation of the potential involvement of the other cardiac cell types in the disease phenotype and the role of specific cell-tocell interactions. Altogether, the obtained results open perspectives to better understand the ventricular arrhythmia occurrence in BrS and to identify a dedicated therapeutic approach to prevent the risk of SCD. E. Percentage of cells presenting I Na,L density greater than the 97 th percentile value of I Na,L in the Ctrl hiPSC-CMs. ** p<0.01 and *** p<0.001 vs. control (Fisher's exact test). Indeed, an increase in I Na,L density was defined by values higher than the 97 th percentile of the Ctrl hiPSC-CMs.

F. Representative example of I Na,L current recorded in BrS hiPSC-CMs before (black) and after (red) application with GS-458967 (300 nM), a specific I Na,L inhibitor (inset: voltage protocol).

G. Representative AP recordings from control and BrS hiPSC-CMs obtained before and after GS-458967 application.

H. Percentage of cells with EADs before and after GS-458967 application. I. Poincaré plots showing APD 90 of each AP (n+1) vs. APD 90 of its preceding one, before and after GS-458967 application.

SUPPLEMENTAL MATERIAL

Supplemental methods:

Ethical Statement

The study was conducted according to the principles set forth under the Declaration of Helsinki (1989) and European guidelines for clinical and genetic research. Institutional review board approvals of the study were obtained before the initiation of patient enrollment. The study protocol was reviewed and approved by the regional "comité de protection des personnes" ethical committee (approval number: 2010-A01358-31). Regarding the patient-derived biological samples, signed informed consent allowing the experiments to be conducted has been received from all individuals. Any related health information was collected in compliance with applicable law/regulation and with any applicable policy of the ethics committee with jurisdiction over the biological sample collection. All biological samples and their related health information have been provided in coded form such that subjects cannot be identified directly. The provisions of French law, article L1110-4 of the Code de la santé publique, related to the privacy and confidentiality of information regarding patients, have been observed.

Study Population and design

BrS patients were enrolled according to the presence of a BrS ECG pattern (see below) and with a familial history of sudden death or syncope. Diagnosis of BrS was based on criteria from 2013 1 with the presence of a type-1 BrS ECG pattern, either spontaneous or induced by intravenous injection of class I antiarrhythmic drugs, in at least one right precordial lead (V1 or V2) positioned in the 2nd, 3rd or 4th intercostal space. Type-1 BrS ECG pattern was defined as a J point elevation higher than 0.2 mV, followed by a coved type ST segment elevation and ended with a negative T wave.

ECG analysis

Two physicians blinded to the clinical and genetic status reviewed all baseline ECGs. P wave, PQ interval, QRS, QT peak, QTend, QTc duration (corrected by Bazett formula), and Tpeak-Tend interval (TPE, time interval between the peak and the end of the T wave) were measured in D2, V1, V2 and V3. S-wave duration and amplitude were additionally measured in D1. All measurements were performed using Image J software as previously described. 2,3 TPE in precordial lead, fragmented QRS, earlyrepolarization pattern and prominent R wave in lead aVR were determined as previously described. 4,5

Genetic analysis

Patients were screened for mutations in genes previously described in cardiomyopathies and inherited arrhythmias. 6,7 For this, genomic DNA of probands was extracted from peripheral blood lymphocytes by standard protocols. The DNA yields were assessed by measurements using Quant-IT™ dsDNA Assay Kit, Broad Range (Life Technologies, Q33130). The purity of the DNA was assessed by DNA integrity was assessed by separation in E-Gel® 96 Agarose Gels, 1% (Life Technologies, G700801).

For multiplex amplification, we used the HaloPlex™ Target Enrichment System (Agilent Technologies, 1-500 kb, ILMFST, 96 reactions, G9901B, Protocol Version D.2 (November, 2012)). A custom HaloPlex™ design was used enabling high-throughput sequencing of the coding regions (exons ± 10 bp) of 20 genes previously associated with the BrS: 6,7

SCN5A CACNA1C RANGRF KCNE1L GPD1L CACNB2 PKP2 KCNJ8 SCN1B CACNA2D1 FGF12 ABCC9 SCN2B KCNE3 SCN10A KCNH2 SCN3B KCND3 TRPM4 HCN4
Target enrichment and sequencing were performed as previously described. 7 First, 200 ng of gDNA sample were digested in eight different restriction reactions, each containing two restriction enzymes, to create a library of gDNA restriction fragments. These gDNA restriction fragments were hybridized to the HaloPlex probe capture library. Probes were designed to hybridize and circularize targeted DNA fragments. During the hybridization process, Illumina sequencing motifs including index sequences were incorporated into the targeted fragments.

The circularized target DNA biotinylated HaloPlex probe complexes were captured on magnetic streptavidin beads. We proceeded to a ligation reaction of the circularized complexes followed by an elution reaction before PCR amplification. The amplified target DNA was purified using AMPure XP bead (Beckman Coulter, A63881). To validate enrichment of target DNA in each library sample by microfluidics analysis, we used the 2200 TapeStation (Agilent Technologies, G2964AA), with D1K ScreenTape (Agilent Technologies, 5067-5361), and D1K Reagents (Agilent Technologies, 5067-5362).

We ensured that the majority of amplicons range from 175 to 625 bp. Finally, we quantified the library together with other libraries by qPCR using the KAPA Library Quantification Kit (Clinisciences, KK4854).

Libraries were pooled in an equimolar concentration and DNA was then denatured with NaOH. Finally, the library pool was diluted to a final concentration of 9 pM before proceeding to 100-bp paired-end Illumina sequencing on HiSeq1500.

Raw sequence reads were aligned to the human reference genome (GRCh37) using BWA-MEM (version 0.7.5a) after removing sequences corresponding to Illumina adapters with Cutadapt v1. Variants were considered as having a potential functional consequence if they were annotated with one or more of the following SO terms for at least one RefSeq transcript: "transcript_ablation" (SO:0001893), "splice_donor_variant" (SO:0001575), "splice_acceptor_variant" (SO:0001574), "stop_gained" (SO:0001587), "frameshift_variant" (SO:0001589), "stop_lost" (SO:0001578), "initiator_codon_variant" (SO:0001582), "inframe_insertion" (SO:0001821), "inframe_deletion" (SO:0001822), "missense_variant" (SO:0001583), "transcript_amplification" (SO:0001889). Loss-offunction variants (nonsense variants, frameshift variants and splice site variants) were defined by the following SO terms: "stop_gained", "frameshift_variant", "splice_donor_variant", or

"splice_acceptor_variant". The potential pathogenicity of variants was determined following the American College of Medical Genetics and Genomics (ACMG) guidelines using the CardioVAI tools from EnGenome. 9

Generation and validation of hiPSCs

BrS5 -hiPSCs were previously used in a study revealing the role of a variant in RRAD, which encodes RAD GTPase, in BrS. 10 BrS6 -hiPSC line, named iBrS1 in a previous study, was derived from a BrS patient with no defined causative variant. 6 Four different control hiPSC lines were used, including 2 studied previously. 10,11 BrS4 -cutaneous fibroblasts were reprogrammed using Stemgent mRNA reprogramming kit and pluriton medium. hiPSCs derived from dermal cells harvested from BrS1 + and BrS3 -as well as blood cells obtained from BrS2 + and his unaffected family relative (Non-BrS) were reprogrammed using Sendai virus method (Cytotune reprogramming Kit, Life technologies). Up to three clones of each hiPSC line were selected per patient. HiPSCs were maintained on matrigel-coated plates (0.05 mg/ml, BD Biosciences) with StemMACSTM iPS-Brew XF medium (Miltenyi Biotec). Pluripotency of each hiPSC clone was validated by qRT-PCR, immunostaining, and flow cytometry to verify the expression of endogenous pluripotent factors.

Validation of SCN5A rare variants and genome integrity

To validate SCN5A rare variants carried by BrS1 + , BrS2 + and Non-BrS, genomic DNA from corresponding hiPSC clones was extracted using NucleoSpin® Tissue kit (MACHEREYNAGEL). Variants were verified by sequencing. Single nucleotide polymorphism (SNP) analysis of all hiPSC clones compared to their parental skin fibroblast cells, was used to confirm genome integrity after reprogramming. DNA was extracted from somatic and hiPSC samples using the QIAGEN QiaAmp kit, according to the manufacturer's recommendations. The gDNA was quantified using a Nanodrop instrument. 200ng of gDNA were outsourced to Integragen Company (Evry, France) for karyotype analysis using HumanCore-24-v1 SNP arrays. This array contains over 300,000 probes distributed throughout the genome with a median coverage of one probe every 5700 bases. All genomic positions were based on Human Genome Build 37 (hg19). Analysis was performed with GenomeStudio software. Chromosome abnormalities were determined by visual inspection of logR ratios and B-allele frequencies (BAF) values and

F o r R e v i e w O n l y
comparing parental cells with hiPSC-derived samples. LogR ratio, the ratio between observed and expected probe intensity, is informative of copy number variation (CNV, i.e. deletions/duplications), whereas BAF is informative of heterozygosity. SNP data were used to compute CNV. In particular, this type of chips allows detecting loss of heterozygosity (LOH), an important concern for hiPSCs, which is not detectable with classical CGH arrays.

Differentiation of hiPSCs into cardiomyocytes

Cardiomyocytes were differentiated from hiPSCs using the established matrix sandwich method. 11 Briefly, when cells reached 90% confluence, an overlay of Growth Factor Reduced Matrigel (0.033 mg/ml, BD Corning) was added. Differentiation was initiated 24 h later by culturing the cells in RPMI1640 medium (Life Technologies) supplemented with B27 (without insulin, Life Technologies), 2 mM L-glutamine (Life Technologies), 1% NEAA (Life Technologies), 100 ng/mL Activin A (Miltenyi), and 10 ng/mL FGF2 for 24 hours. On the next day, the medium was replaced by RPMI1640 medium supplemented with B27 without insulin, 2 mM L-glutamine, 1% NEAA, 10 ng/mL BMP4 (Miltenyi), and 5 ng/mL FGF2 for 4 days. By day 5, cells were cultured in RPMI1640 medium supplemented with B27 complete (Life Technologies), 2 mM L-glutamine and 1% NEAA and changed every two days. Presence of beating cells was considered as the primary hallmark of a successful cardiomyocyte differentiation.

Transcript expression analysis

Quantitative RT-PCR: Total RNA samples were isolated using the NucleoSpin RNA kit (MACHEREY-NAGEL). One μg of tRNA was reverse transcribed using High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems) following the manufacturer's instructions. PCR amplification was performed using FAM labeled-TaqMan probes (Applied Biosystems) to verify the pluripotency of derived hiPSCs as compared to parental fibroblasts (OCT3/4 and NANOG). According to the ΔΔCt method, all data were normalized to ACTB and represented relative to controls.

TaqMan Low Density array (TLDA): TLDA studies were conducted using beating clusters of hiPSC-CMs obtained from 12 samples of Ctrl, 4 samples of Non-BrS and 14 samples of BrS hiPSC-CMs. One μg of RNA was reverse transcribed into cDNA using SuperScript IV Vilo Master Mix (Thermo Fisher Scientific).

TLDA probe selection covered gene families implicated in cardiac ion channel expression and regulation, and cardiomyocyte structure (Table S3). Genes with average Ct > 32 in all compared groups were considered undetectable and excluded from the analysis (SCN10A and ABCC8). Average Ct of remaining genes for each sample was used for data normalization.

Protein expression analysis

Beating clusters of hiPSC-CMs were lysed in buffer composed of 1% TritonX-100, 100 mM NaCl, 50 mM Tris-HCl, 1 mM EGTA, 1 mM Na3VO4, 50 mM NaF, 1 mM PMSF and protease inhibitors cocktail (P8340, Sigma-Aldrish). Protein quantification was then conducted using PierceTM BCA Protein Assay Kit (Thermo Fisher). The lysates were denatured for 5 min at 65°C in a mixture of NuPAGE® Sample

Reducing Agent (10X) and NuPAGE® LDS Sample Buffer (4X). 40 μg of each sample were loaded onto 12% precast polyacrylamide gels (Bio-Rad). After migration, the proteins were transferred onto Trans-Blot® TurboTM Nitrocellulose Transfer Packs (Bio-Rad). Membranes were saturated with 5% non-fat milk, then incubated with primary antibody (Anti-Na v 1.5: 14421S Cell Signaling; Anti-Cav1.2: AB5156 Sigma-Aldrich; Anti-TFRC:13-6890 Thermofisher) overnight followed by another incubation for 1 h with an adequate Horseradish peroxidase (HRP)-conjugated secondary antibody. Protein bands were detected using ECL detection system (Bio-Rad) and quantified using Image Lab software.

Immunostainings

To validate hiPSC pluripotency, hiPSC-CM differentiation into cardiac lineage as well as COS-7 cell expression, cells were dissociated and seeded onto 8-wells ibidi plates (Biovalley) coated with Matrigel (Corning). After 12 days following hiPSC-CM dissociation, and 24 h following COS-7 transfection, cultured cells were fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X100 and blocked with 1% BSA. Immunofluorescent stainings were performed using appropriate primary antibodies (Anti-TRA1-60: 14-8863-80 eBioscience™; Anti-OCT4: 14-5841-80 eBioscience™; Anti-troponin I: sc-

p.N1722D Na v 1.5 study in COS-7 cells

The point mutation p.N1722D (c.1859G>A) in SCN5A was introduced using mutated oligonucleotide extension (QuikChange II XL Site-Directed Mutagenesis Kit) in SCN5A isoform 2 cDNA (GenBank Acc.

Nb. NM_000335). The resulting plasmid was verified by complete sequencing of the cDNA insert.

African green monkey kidney-derived cells COS-7, were transiently transfected with 0. Borosilicate glass pipettes (2-3 MΩ of tip resistance) were filled with a solution containing (in mM):

125 K-Gluconate, 20 KCl, 5 NaCl, 5 HEPES; pH 7.2 (KOH) and 0.22 amphotericin-B. We first recorded spontaneous APs in order to determine, for each individual included in the study, the proportion of nodal-like, atrial-like and ventricular-like, as previously described. 11 Then to overcome limited I K1 contribution during hiPSC-CM AP, artificial I K1 was injected using dynamic patch-clamp. 16 Both cell stimulation and I K1 injection were realized using a custom-made software running on RT-Linux and an The AP parameters measured were the maximum diastolic potential (MDP), the maximum upstroke velocity of phase 0 depolarization (dV/dt max ), the AP amplitude and the AP duration at different levels of full repolarization, from 30% (APD30) to 90% (APD90), as previously described. 11 subepicardial human ventricular cells. 18,19 In the original model used by Gima and Rudy, a slow inactivation gate (j) of the Na+ channel was added to include the property of slow recovery, 20 without modifying fast inactivation (realized with h gate). A few modifications were operated. Since this 'j' inactivation was originally as fast as 'h' inactivation, it prevents the appearance of a persistent current when altering the 'h' gate. So we slowed down this 'j' inactivation, but left the kinetics of recovery Since only a subset of hiPSC-CMs present the late Na + current, we performed simulations with the late Na + current only in midmyocardial cells, giving rise to a J point elevation followed by coved ST segment. 18 Since decreasing I Na by as much as 73-75% (BrS2 + and Non-BrS) was preventing conduction in the Gima and Rudy model, a slighter 30% decrease in I Na was applied.

Statistical analysis

Results are expressed as mean ± SEM. that striated troponin I, a cardiac and muscular specific cytoskeletal protein, was similarly present in all hiPSC-CM lines (Figure 1H). Spontaneous APs, in absence of I K1 injection, were classified based on MDP, dV/dt max , AP duration and morphology into nodal-like, atrial-like and ventricular-like, as previously described. 11 The proportion of each cell type was similar between all hiPSC-CM lines, with the ventricular-like type forming the majority of explored hiPSC-CMs (Figure 1I). Altogether, these data confirmed that Ctrl, BrS, and Non-BrS hiPSCs differentiated similarly into cardiomyocytes, and therefore a comparative electrophysiological analysis could be performed to unveil a potential common cellular phenotypic trait of BrS hiPSC-CMs. Each ECG parameter was tested for its correlation with either I Na,L or peak I Na density from the corresponding hiPSC-CMs. While the J point elevation correlated significantly with I Na,L density, it did not correlate with hiPSC-CM peak I Na density. Statistical significance threshold of r s was p < 0.05 (*).

  Similarly, reduction in I Ca,L channel protein expression and current density were not a common trait of BrS hiPSC-CMs (Figure2I-2L, FigureS3B; Table

  that depolarizing currents alterations can impact a multi-cellular electrogram model, mimicking BrS ECG phenotype.

Figure 1 .

 1 Figure 1. Pluripotency and SCN5A variant validation in hiPSCs, and characterization of derived cardiomyocytes. A. Transcript expression of pluripotency markers: NANOG and OCT3/4, in newly described hiPSCs as compared to fibroblasts (Fibro). B. Representative immunostainings of TRA1-60 (red) and OCT4 (green) in hiPSCs. C. Percentage of hiPSCs expressing SSEA4 and TRA1-60 evaluated by flow cytometry. D. Genomic sequence chromatograms validating (right) the 5164A>G SCN5A variant carried by BrS1 + and (left) the SCN5A 1983-1993 duplication carried by BrS2 + and Non-BrS, in the corresponding hiPSCs. E. Principal component analysis (PCA) of 39 hiPSC samples and their corresponding differentiated hiPSC-CMs, based on their expression pattern of 27106 analyzed transcripts (3'SRP data). All clones of each hiPSC lines are highlighted. F. Correlation matrix of hiPSCs and hiPSC-CMs expression profiles. Yellow and orange indicate high and low correlation, respectively. Samples were clustered using an ascending hierarchical method with Pearson as metric and ward.D2 linkage. G. Heatmap showing expression levels of 9661 differentially expressed genes between hiPSCs and hiPSC-CMs (same samples as in A). Genes were clustered using a hierarchical ascending method with an uncentered correlation metric and complete linkage. Yellow and blue indicate high and low levels respectively. H. Illustrative immunostainings of Troponin I (green) in hiPSC-CMs. Nuclei were stained with DAPI (blue). I. Percentages of nodal-like, atrial-like, and ventricular-like cells classified based on the analysis of spontaneous action potential recordings.

Figure 2 .

 2 Figure 2. Differential gene expression profiles and variations in I Na and I Ca,L , in BrS-hiPSC-CMs as compared to controls. A. Heatmap showing hierarchical clustering of expression profiles of 133 differentially expressed genes obtained by 3'SRP in control (Ctrl) and BrS hiPSC-CMs at day 28 of differentiation. A total of 27% were upregulated whereas 73% genes were downregulated in BrS hiPSC-CMs. Yellow and blue represent high and low expression levels, respectively. All clones of each hiPSC line are highlighted. B. Gene Set Enrichment Analysis (GSEA) of gene variations obtained by 3'SRP shows gene sets with statistically altered expression patterns. C. MindMap describing the transmembrane transporter activity alterations. D. Expression levels of differentially expressed genes identified using high-throughput TaqMan (TLDA) in BrS hiPSC-CMs (n=14), compared to control hiPSC-CMs (n=12), and in Non-BrS hiPSC-CMs (n=4) vs.

  BrS hiPSC-CMs. p-values: *, ** and *** or a, b and c: p<0.05, p<0.01 and p<0.001 vs. Ctrl or BrS, respectively (t-test). E. Representative immunoblots for Na v 1.5 and Transferrin receptor (TFRC) in hiPSC-CMs (left panel). Ratios of Na v 1.5 expression levels (right panel, Tukey plot, n=8). * p<0.05 vs. control (Mann-Whitney test). Na v 1.5 decrease in hiPSC-CMs from 3 subjects, BrS2 + and Non-BrS (both carrying a stop codon in SCN5A), as well as BrS5 -, harboring RRAD variant was observed. F. Representative superimposed I Na densities (inset: voltage-clamp protocol). Reduction was detected in BrS2 + , BrS5 -, and Non-BrS, as well as in BrS1 + hiPSC-CMs carrying the N1722D-SCN5A rare variant. G. Peak I Na densities measured in control (Ctrl), BrS and the non-affected carrier of SCN5A mutation (Non-BrS) hiPSC-CMs determined at -20 mV (Tukey plot). *** p<0.001 vs. control (Mann-Whitney test). H. Mean peak I Na densities (pA/pF) vs. membrane potential (V m ) recorded in hiPSC-CMs. ****, $$$$, #### and ^^^^ p<0.0001 vs. control for BrS1 + , BrS2 + , BrS5 -and Non-BrS, respectively (Two-way ANOVA with Bonferroni post-hoc test). I. Representative immunoblots for Ca v 1.2, the main pore-forming subunit of the cardiac L-type calcium channel, and Transferrin receptor (TFRC) in hiPSC-CMs (left panel). Ratios of Ca v 1.2 expression levels (right panel, Tukey plot, n=8). A decrease in Ca v 1.2 expression was solely observed in BrS5 -hiPSC-CMs, carrying a RRAD-variant. * p<0.05 vs. control (Mann-Whitney test). J. Representative superimposed I Ca,L densities (inset: voltage protocol). K. Peak I Ca,L densities measured in control (Ctrl), BrS and the non-affected carrier of SCN5A mutation (Non-BrS) hiPSC-CMs determined at 0 mV (Tukey plot). A decrease in I Ca,L was observed in BrS2 + , BrS4 - and, consistently with a previous description, in BrS5 -. 7 * p<0.05 and ** p<0.01 vs. control (Mann-Whitney test).L. Mean peak I Ca,L densities (pA/pF) vs. membrane potential (V m ) recorded in hiPSC-CMs. *, # and $ p<0.05, **, ## and $$ p<0.01 and ***, ### and $$$ p<0.001 vs. control for BrS1 + , BrS4 -and BrS5 -, respectively (Two-way ANOVA with Bonferroni post-hoc test).

Figure 3 .

 3 Figure 3. Increased early after depolarization (EAD) occurrence in all BrS ventricular-like hiPSC-CMs lines, linked to an increase in late sodium current. A. Representative AP recordings, showing EADs in BrS lines only. Representative ventricular-like AP when paced at 700 ms cycle length and when artificial I K1 was injected (dynamic current-clamp). APs are defined as ventricular-like when (APD 30 -APD 40 )/(APD 70 -APD 80 )>1.45. B. Percentage of ventricular-like hiPSC-CMs presenting at least 1 EAD, irrespective of the currentclamp conditions. * p<0.05, ** p<0.01 and *** p<0.001 vs. control (Fisher's exact test).C. Representative I Na,L recordings from hiPSC-CMs, before (black) and after (grey) TTX application (inset: voltage protocol).

  Na,L (TTX-sensitive current) densities at -10 mV. * p<0.05 and *** p<0.001 vs. Ctrl (Mann-Whitney test), and ## p<0.01 and ### p<0.001 vs. Non-BrS (Mann-Whitney test). E. Percentage of cells presenting I Na,L density greater than the 97 th percentile value of I Na,L in the Ctrl hiPSC-CMs. ** p<0.01 and *** p<0.001 vs. control (Fisher's exact test). Indeed, an increase in I Na,L density was defined by values higher than the 97 th percentile of the Ctrl hiPSC-CMs.F. Representative example of I Na,L current recorded in BrS hiPSC-CMs before (black) and after (red) application with GS-458967 (300 nM), a specific I Na,L inhibitor (inset: voltage protocol).G. Representative AP recordings from control and BrS hiPSC-CMs obtained before and after GS-458967 application.H. Percentage of cells with EADs before and after GS-458967 application. I. Poincaré plots showing APD 90 of each AP (n+1) vs. APD 90 of its preceding one, before and after GS-458967 application.

Figure 4 .

 4 Figure 4. Applying depolarizing ion current alterations from BrS hiPSC-CMs on an electrogram model to mimic BrS patient's ECG features. A. Top: The right-ventricle electrogram model simulates the global electrical activity of a transmural wedge comprising 60 subendocardial, 45 midmyocardial, and 60 subepicardial human ventricular cells. Bottom: Representative electrogram showing the Q-like, R-like, S-like and T-like waves. B. Ventricular transmural electrogram mathematical model of Ctrl (black), BrS2 + (pink) and Non-BrS (blue) illustrated based on hiPSC-CMs data of the relative variation in ion currents (I Na , I Ca,L and I Na,L ) mean amplitude as compared to Ctrl lines. In accordance with patient's ECGs, applying BrS2 + ionic current changes prolonged the QRS-like complex, elevated and widened the ST-like segment, and inversed the T-like wave; and applying I Na change identified in Non-BrS hiPSC-CMs only prolonged the QRS-like complex, similar to Non-BrS PCCD ECG. C. Each currents I Na , I Ca,L and I Na,L (from left to right) were sequentially corrected and the resulting electrograms are illustrated in green. Ctrl and BrS2 + electrograms are presented in black and pink respectively.

Figure 1 .Figure 2 .Figure 3 .Figure 4 .

 1234 Figure 1. Pluripotency and SCN5A variant validation in hiPSCs, and characterization of derived cardiomyocytes. 179x164mm (600 x 600 DPI)

4

 4 μg SCN5Aexpressing plasmid (wild type or mutant) together with 0.4 μg of Navβ1 subunit (SCN1B) plasmid for a 35-mm Petri dish. Transfections were performed using 4 μL JetPEI reagent (Polyplus Transfections, France) according to the manufacturer's instructions. Enhanced green fluorescent protein (eGFP)encoding plasmid (1.2 μg) was included to identify transfected cells. One day after transfection, cells were re-plated onto 35-mm Petri dishes for patch clamp experiments. Electrophysiological assessment Sodium current (I Na ) recordings in transfected COS-7 cells. Currents were recorded 2 days after transfection. Cells were superfused with a solution containing the following (in mM): 145 NaCl, 4 CsCl, 1 CaCl2, 1 MgCl2, 5 HEPES, and 5 glucose, pH=7.4 with NaOH. Patch pipettes were fabricated from borosilicate glass capillaries and had resistances between 1.5 and 2 MΩ when filled with pipette solution (in mM): 90 KCl, 45 K-Gluconate, 10 NaCl and 10 HEPES, pH=7.2 with CsOH. All recordings were made at room temperature (20°C-22°C), after capacitance and series resistance compensation, using an Axopatch 200B amplifier controlled by Axon pClamp 10.6 software through an A/D converter (Digidata 1440A). Data were analyzed using Clampfit 10.6 software (all Molecular Devices). Current-and voltage-clamp in hiPSC-CMs. Cardiomyocytes were dispersed as single cells around day 20 of differentiation, for 20 min in collagenase II (200 U/mL; Gibco) at 37°C. Ten to twelve days after dissociation, spontaneously beating cells were used for patch-clamp recordings. All experiments were conducted at 37°C. Data were collected from a minimum of 3 independent differentiations. Action potential (AP) recordings. Using amphotericin-B perforated-patch configuration, APs were acquired with the same amplifier and converter as above in hiPSC-CMs cells bathed in a Tyrode solution containing (in mM): 140 NaCl, 4 KCl, 1 CaCl2, 0.5 MgCl2, 10 glucose, 10 HEPES; pH 7.4 (NaOH).

4 ( 2 Na2ATP, 2 4 (

 4224 cells, action potential classification into ventricular type was assessed by (APD30-APD40)/(APD70-APD80)>1.45 reflecting the presence of a plateau phase. Cells were paced with a 1-ms 30-35 pA/pF stimulation pulse at 700 ms of cycle length. BrS being a ventricular arrhythmic disease, the analyses were focused on ventricular-like AP. Data from 7 consecutive APs were averaged. GS-458967 (Gilead Sciences) specifically inhibiting I Na,L , was solubilized in DMSO and used at 300 nM during AP recording in a solution containing (in mM): 140 NaCl, 4 KCl, 1 CaCl2, 0.5 MgCl2, 30 mannitol, 10 HEPES; pH 7.NaOH). Current recordings. I Na and I Ca,L measurements were recorded in the ruptured-patch configuration and low-pass filtered at, respectively, 10 KHz and 3 KHz using a VE-2 amplifier (Alembic Instrument, Qc, Canada). Cells were bathed using a Tyrode solution containing (in mM): 130 NaCl, 10 CsCl, 1.8 CaCl2, 1.2 MgCl2, 11 glucose and 5 HEPES; pH 7.4 (NaOH). Holding potentials were set to -80 mV and -100 mV, respectively. The series resistance was compensated. Current densities and gating properties were measured using appropriate voltage protocols shown in the relevant figures. After leak subtraction, current densities were calculated by dividing current amplitude by membrane capacitance. Voltagedependence of activation and inactivation curves were fitted with a Boltzmann function (y=[1+exp{-(V-V1/2)/K}]-1), where V1/2 is the half-maximal voltage of (in)activation and K is the slope factor. For transient sodium current (I Na ) recording, a local gravity microperfusion system allowed application of an extracellular solution containing (in mM): 20 NaCl, 110 CsCl, 1.8 CoCl2, 1.2 MgCl2, 30 mannitol and 5.0 HEPES; pH 7.4 (CsOH). The pipette solution contained (in mM): 3 NaCl, 133 CsCl, 2 MgCl2, TEACl, 10 EGTA, 5 HEPES; pH 7.2 (CsOH). Late sodium current (I Na,L ) was measured as a TTX-sensitive current (Tetrodotoxin Citrate, TOCRIS Bioscience) using an ascending voltage-ramp protocol. The same pipette solution as for peak I Na recording was used. The extracellular solution used in the local gravity microperfusion system had the following composition (in mM): 130 NaCl, 10 CsCl, 1.8 CoCl2, 1 MgCl2, 30 mannitol, 10 HEPES; pH 7.CsOH). TTX was used at a concentration of 0.03 mM. Calcium current (I Ca,L ) was recorded from cells perfused an extracellular solution containing (in mM): 160 TEACl, 5 CaCl2, 1 MgCl2, 1 MgCl2, 20 mannitol, 10 HEPES, and 0.01 TTX; pH 7.4 (CsOH). The pipette solution contained (in mM): 5 NaCl, 145 CsCl, 2 CaCl2, 5 EGTA, 5 MgATP, 10 HEPES; pH 7.2 (CsOH).

  50 beta_j=0.035*exp(+0.008*p->v)/(1.0+exp(-0.048*(p->v+77.329)). Thus, in the modified model, any incomplete fast inactivation (for instance, h parameter varies between 1 and 0.166 for BrS2 + , instead of 1 and zero for Ctrl) gives rise to a late Na + current similar to that observed in Figure 3C (see also Figure below) resulting in the following equation. Impact of incomplete inactivation on late sodium current in a simulated Ramp protocol. Ctrl: h parameter varies between 1 and 0.

  17

  BrS: h varies between 1 and 0.116. The ratio of maximal I Na,L current in BrS/Ctrl is 5.4, as in Figure 3C for BrS2 + . Noteworthy, this modification does not change inactivation properties of the Na + current in the WT model because fast inactivation (h) is not modified.

Figure S2 :Figure S2 :

 S2S2 Figure S2:

Figure

  Figure S3

Figure S4 .Figure S5 .

 S4S5 Figure S4:

  ECG patient characteristics. ECGs were performed in leads D1, D2, V1, V2, V3 and recordings used the following parameters: 25 mm/s, 0.1 mV/mm. RR interval duration (RR); S wave duration and amplitude (S); P wave duration (P); PR interval duration (PR); QRS duration (QRS); QT peak interval (QTp); QT end interval (QTe); Tpeak-to -Tend interval (TPE); J wave amplitude (J); Early Repolarization Pattern (ERP); Fragmented QRS according to Morita H et al. 22 (Frag); Concave aspect of the ST segment elevation (Concave aspect); Yes (y); No (n).

Reviewer: 3 )Response:Reviewer: 4 )

 34 The study nature, depth and statistical power of the samples may be insufficient to summit common cellular phenotypic singularities in feature as the basis of specific BrS ECG morphogenesis. I would suggest adjusting (playing down) the hypothesis to spot-on match the study findings and conclusion. BrS has been referred to as an overlap syndrome (channelopathy vs cardiomyopathy), and can result from a numerous diverse underlying pathophysiological mechanisms. We understand the Reviewer's concern and we have changed the conclusion accordingly: "In conclusion, in the present study, a particular cellular electrophysiological phenotype common to six out of six BrS hiPSC-CM lines with various genetic backgrounds has been unveiled." This change has been made on page 4, paragraph 2, of the Letter. The unique signature of gene expression and trigger potentials (EAD) of each hiPSC-CMs of the three clones selected per patient needs comment and could be added as a figure and in few lines to the body of the letter.

  trait in Brugada syndrome cellular phenotype Zeina R. Al Sayed, PhD a , Mariam Jouni, PhD a , Jean-Baptiste Gourraud, MD-PhD a,b , Nadjet Belbachir, PhD a , Julien Barc, PhD a , Aurore Girardeau, BSc a , Virginie Forest, PhD a , Aude Derevier, PhD c , Anne Gaignerie, MSc c , Caroline Chariau, BSc c , Bastien Cimarosti, MSc a , Robin Canac, MSc a , Pierre Olchesqui, MSc a , Eric Charpentier, MSc a , Jean-Jacques Schott, PhD a,b , Richard Redon, PhD a,b , Isabelle Baró, PhD a , Vincent Probst, MD-PhD a,b , Flavien Charpentier, PhD a,b , Gildas Loussouarn, PhD a , Kazem Zibara, PhD d , Guillaume Lamirault, MD-PhD a,b , Patricia Lemarchand, MD-PhD a,b* , Nathalie Gaborit, PhD a* . a. l'institut du thorax, INSERM, CNRS, UNIV Nantes, Nantes, France b. l'institut du thorax, CHU Nantes, Nantes, France c. Nantes Université, CHU Nantes, Inserm, CNRS, SFR Santé, Inserm UMS 016, CNRS UMS 3556, F-44000 Nantes, France d. Laboratory of stem cells, PRASE, Biology department, Faculty of Sciences, Lebanese University, Beirut, Lebanon. * co-corresponding authors Short title: Role of late sodium current in Brugada syndrome Address for correspondence: Nathalie GABORIT, PhD and Patricia LEMARCHAND, MD-PhD l'institut du thorax, Inserm UMR 1087, CNRS UMR 6291 IRS-UN, 8 quai Moncousu 44007 Nantes cedex 1, FranceE-mail: nathalie.gaborit@univ-nantes.fr and patricia.lemarchand@univ-nantes.fr BrS) is an inherited arrhythmic disease predisposing to sudden cardiac death (SCD),

  Similarly, reduction in I Ca,L channel protein expression and current density were not a common trait of BrS hiPSC-CMs (Figure2I-2L, FigureS3B; Table

  that depolarizing currents alterations can impact a multi-cellular electrogram model, mimicking BrS ECG phenotype.

Figure 1 .

 1 Figure legendsFigure 1. Pluripotency and SCN5A variant validation in hiPSCs, and characterization of derived cardiomyocytes. A. Transcript expression of pluripotency markers: NANOG and OCT3/4, in newly described hiPSCs as compared to fibroblasts (Fibro). B. Representative immunostainings of TRA1-60 (red) and OCT4 (green) in hiPSCs. C. Percentage of hiPSCs expressing SSEA4 and TRA1-60 evaluated by flow cytometry. D. Genomic sequence chromatograms validating (right) the 5164A>G SCN5A variant carried by BrS1 + and (left) the SCN5A 1983-1993 duplication carried by BrS2 + and Non-BrS, in the corresponding hiPSCs. E. Principal component analysis (PCA) of 39 hiPSC samples and their corresponding differentiated hiPSC-CMs, based on their expression pattern of 27106 analyzed transcripts (3'SRP data). All clones of each hiPSC lines are highlighted. F. Correlation matrix of hiPSCs and hiPSC-CMs expression profiles. Yellow and orange indicate high and low correlation, respectively. Samples were clustered using an ascending hierarchical method with Pearson as metric and ward.D2 linkage. G. Heatmap showing expression levels of 9661 differentially expressed genes between hiPSCs and hiPSC-CMs (same samples as in A). Genes were clustered using a hierarchical ascending method with an uncentered correlation metric and complete linkage. Yellow and blue indicate high and low levels respectively. H. Illustrative immunostainings of Troponin I (green) in hiPSC-CMs. Nuclei were stained with DAPI (blue). I. Percentages of nodal-like, atrial-like, and ventricular-like cells classified based on the analysis of spontaneous action potential recordings.

Figure 2 .

 2 Figure 2. Differential gene expression profiles and variations in I Na and I Ca,L , in BrS-hiPSC-CMs as compared to controls. A. Heatmap showing hierarchical clustering of expression profiles of 133 differentially expressed genes obtained by 3'SRP in control (Ctrl) and BrS hiPSC-CMs at day 28 of differentiation. A total of 27% were upregulated whereas 73% genes were downregulated in BrS hiPSC-CMs. Yellow and blue represent high and low expression levels, respectively. All clones of each hiPSC line are highlighted. B. Gene Set Enrichment Analysis (GSEA) of gene variations obtained by 3'SRP shows gene sets with statistically altered expression patterns. C. MindMap describing the transmembrane transporter activity alterations. D. Expression levels of differentially expressed genes identified using high-throughput TaqMan (TLDA) in BrS hiPSC-CMs (n=14), compared to control hiPSC-CMs (n=12), and in Non-BrS hiPSC-CMs (n=4) vs.

  BrS hiPSC-CMs. p-values: *, ** and *** or a, b and c: p<0.05, p<0.01 and p<0.001 vs. Ctrl or BrS, respectively (t-test). E. Representative immunoblots for Na v 1.5 and Transferrin receptor (TFRC) in hiPSC-CMs (left panel). Ratios of Na v 1.5 expression levels (right panel, Tukey plot, n=8). * p<0.05 vs. control (Mann-Whitney test). Na v 1.5 decrease in hiPSC-CMs from 3 subjects, BrS2 + and Non-BrS (both carrying a stop codon in SCN5A), as well as BrS5 -, harboring RRAD variant was observed. F. Representative superimposed I Na densities (inset: voltage-clamp protocol). Reduction was detected in BrS2 + , BrS5 -, and Non-BrS, as well as in BrS1 + hiPSC-CMs carrying the N1722D-SCN5A rare variant. G. Peak I Na densities in control (Ctrl), BrS and the non-affected carrier of SCN5A mutation (Non-BrS) hiPSC-CMs determined at -20 mV (Tukey plot). *** p<0.001 vs. control (Mann-Whitney test). H. Mean peak I Na densities (pA/pF) vs. membrane potential (V m ) recorded in hiPSC-CMs. ****, $$$$, #### and ^^^^ p<0.0001 vs. control for BrS1 + , BrS2 + , BrS5 -and Non-BrS, respectively (Two-way ANOVA with Bonferroni post-hoc test). I. Representative immunoblots for Ca v 1.2, the main pore-forming subunit of the cardiac L-type calcium channel, and Transferrin receptor (TFRC) in hiPSC-CMs (left panel). Ratios of Ca v 1.2 expression levels (right panel, Tukey plot, n=8). A decrease in Ca v 1.2 expression was solely observed in BrS5 -hiPSC-CMs, carrying a RRAD-variant. * p<0.05 vs. control (Mann-Whitney test). J. Representative superimposed I Ca,L densities (inset: voltage protocol). K. Peak I Ca,L densities measured in control (Ctrl), BrS and the non-affected carrier of SCN5A mutation (Non-BrS) hiPSC-CMs determined at 0 mV (Tukey plot). A decrease in I Ca,L was observed in BrS2 + , BrS4 - and, consistently with a previous description, in BrS5 -. 7 * p<0.05 and ** p<0.01 vs. control (Mann-Whitney test).L. Mean peak I Ca,L densities (pA/pF) vs. membrane potential (V m ) recorded in hiPSC-CMs. *, # and $ p<0.05, **, ## and $$ p<0.01 and ***, ### and $$$ p<0.001 vs. control for BrS1 + , BrS4 -and BrS5 -, respectively (Two-way ANOVA with Bonferroni post-hoc test).

Figure 3 .

 3 Figure 3. Increased early after depolarization (EAD) occurrence in all BrS ventricular-like hiPSC-CMs lines, linked to an increase in late sodium current. A. Representative AP recordings, showing EADs in BrS lines only. Representative ventricular-like AP when paced at 700 ms cycle length and when artificial I K1 was injected (dynamic current-clamp). APs are defined as ventricular-like when (APD 30 -APD 40 )/(APD 70 -APD 80 )>1.45. B. Percentage of ventricular-like hiPSC-CMs presenting at least 1 EAD, irrespective of the currentclamp conditions. * p<0.05, ** p<0.01 and *** p<0.001 vs. control (Fisher's exact test).C. Representative I Na,L recordings from hiPSC-CMs, before (black) and after (grey) TTX application (inset: voltage protocol).

  Na,L (TTX-sensitive current) densities at -10 mV. * p<0.05 and *** p<0.001 vs. Ctrl (Mann-Whitney test), and ## p<0.01 and ### p<0.001 vs. Non-BrS (Mann-Whitney test).

Figure 4 .

 4 Figure 4. Applying depolarizing ion current alterations from BrS hiPSC-CMs on an electrogram model to mimic BrS patient's ECG features. A. Top: The right-ventricle electrogram model simulates the global electrical activity of a transmural wedge comprising 60 subendocardial, 45 midmyocardial, and 60 subepicardial human ventricular cells. Bottom: Representative electrogram showing the Q-like, R-like, S-like and T-like waves. B. Ventricular transmural electrogram mathematical model of Ctrl (black), BrS2 + (pink) and Non-BrS (blue) illustrated based on hiPSC-CMs data of the relative variation in ion currents (I Na , I Ca,L and I Na,L ) mean amplitude as compared to Ctrl lines. In accordance with patient's ECGs, applying BrS2 + ionic current changes prolonged the QRS-like complex, elevated and widened the ST-like segment, and inversed the T-like wave; and applying I Na change identified in Non-BrS hiPSC-CMs only prolonged the QRS-like complex, similar to Non-BrS PCCD ECG. C. Each currents I Na , I Ca,L and I Na,L (from left to right) were sequentially corrected and the resulting electrograms are illustrated in green. Ctrl and BrS2 + electrograms are presented in black and pink respectively.

  trait in Brugada syndrome cellular phenotype Zeina R Al Sayed et al.

  260:280 and 260:230 ratios) using a Nanodrop instrument (Thermo Scientific).

  ; Anti-Na v 1.5: 14421S Cell Signaling) and their corresponding Alexa conjugated antibodies (Molecular Probes). DAPI was used for nuclear staining. Immunostainings were examined using an inverted epifluorescent microscope (Zeiss Axiovert 200M).

4

 4 μg SCN5Aexpressing plasmid (wild type or mutant) together with 0.4 μg of Navβ1 subunit (SCN1B) plasmid for a 35-mm Petri dish. Transfections were performed using 4 μL JetPEI reagent (Polyplus Transfections, France) according to the manufacturer's instructions. Enhanced green fluorescent protein (eGFP)encoding plasmid (1.2 μg) was included to identify transfected cells. One day after transfection, cells were re-plated onto 35-mm Petri dishes for patch clamp experiments. Electrophysiological assessment Sodium current (I Na ) recordings in transfected COS-7 cells. Currents were recorded 2 days after transfection. Cells were superfused with a solution containing the following (in mM): 145 NaCl, 4 CsCl, 1 CaCl2, 1 MgCl2, 5 HEPES, and 5 glucose, pH=7.4 with NaOH. Patch pipettes were fabricated from borosilicate glass capillaries and had resistances between 1.5 and 2 MΩ when filled with pipette solution (in mM): 90 KCl, 45 K-Gluconate, 10 NaCl and 10 HEPES, pH=7.2 with CsOH. All recordings were made at room temperature (20°C-22°C), after capacitance and series resistance compensation, using an Axopatch 200B amplifier controlled by Axon pClamp 10.6 software through an A/D converter (Digidata 1440A). Data were analyzed using Clampfit 10.6 software (all Molecular Devices). Current-and voltage-clamp in hiPSC-CMs. Cardiomyocytes were dispersed as single cells around day 20 of differentiation, for 20 min in collagenase II (200 U/mL; Gibco) at 37°C. Ten to twelve days after dissociation, spontaneously beating cells were used for patch-clamp recordings. All experiments were conducted at 37°C. Data were collected from a minimum of 3 independent differentiations. Action potential (AP) recordings. Using amphotericin-B perforated-patch configuration, APs were acquired with the same amplifier and converter as above in hiPSC-CMs cells bathed in a Tyrode solution containing (in mM): 140 NaCl, 4 KCl, 1 CaCl2, 0.5 MgCl2, 10 glucose, 10 HEPES; pH 7.4 (NaOH).

  converter (National Instrument PCI-6221) connected to the current command of the amplifier.

4 ( 2 Na2ATP, 2 4 (

 4224 Following I K1 injection, at a peak outward density that sets the membrane potential between -80 to -85mV for all cells, action potential classification into ventricular type was assessed by (APD30-APD40)/(APD70-APD80)>1.45 reflecting the presence of a plateau phase. Cells were paced with a 1-ms 30-35 pA/pF stimulation pulse at 700 ms of cycle length. BrS being a ventricular arrhythmic disease, the analyses were focused on ventricular-like AP. Data from 7 consecutive APs were averaged. GS-458967 (Gilead Sciences) specifically inhibiting I Na,L , was solubilized in DMSO and used at 300 nM during AP recording in a solution containing (in mM): 140 NaCl, 4 KCl, 1 CaCl2, 0.5 MgCl2, 30 mannitol, 10 HEPES; pH 7.NaOH).Current recordings. INa and I Ca,L measurements were recorded in the ruptured-patch configuration and low-pass filtered at, respectively, 10 KHz and 3 KHz using a VE-2 amplifier (Alembic Instrument, Qc, Canada). Cells were bathed using a Tyrode solution containing (in mM): 130 NaCl, 10 CsCl, 1.8 CaCl2, 1.2 MgCl2, 11 glucose and 5 HEPES; pH 7.4 (NaOH). Holding potentials were set to -80 mV and -100 mV, respectively. The series resistance was compensated. Current densities and gating properties were measured using appropriate voltage protocols shown in the relevant figures. After leak subtraction, current densities were calculated by dividing current amplitude by membrane capacitance. Voltagedependence of activation and inactivation curves were fitted with a Boltzmann function (y=[1+exp{-(V-V1/2)/K}]-1), where V1/2 is the half-maximal voltage of (in)activation and K is the slope factor. For transient sodium current (I Na ) recording, a local gravity microperfusion system allowed application of an extracellular solution containing (in mM): 20 NaCl, 110 CsCl, 1.8 CoCl2, 1.2 MgCl2, 30 mannitol and 5.0 HEPES; pH 7.4 (CsOH). The pipette solution contained (in mM): 3 NaCl, 133 CsCl, 2 MgCl2, TEACl, 10 EGTA, 5 HEPES; pH 7.2 (CsOH). Late sodium current (I Na,L ) was measured as a TTX-sensitive current (Tetrodotoxin Citrate, TOCRIS Bioscience) using an ascending voltage-ramp protocol. The same pipette solution as for peak I Na recording was used. The extracellular solution used in the local gravity microperfusion system had the following composition (in mM): 130 NaCl, 10 CsCl, 1.8 CoCl2, 1 MgCl2, 30 mannitol, 10 HEPES; pH 7.CsOH). TTX was used at a concentration of 0.03 mM. Calcium current (I Ca,L ) was recorded from cells perfused an extracellular solution containing (in mM): 160 TEACl, 5 CaCl2, 1 MgCl2, 1 MgCl2, 20 mannitol, 10 HEPES, and 0.01 TTX; pH 7.4 (CsOH). The pipette solution contained (in mM): 5 NaCl, 145 CsCl, 2 CaCl2, 5 EGTA, 5 MgATP, 10 HEPES; pH 7.2 (CsOH).

  electrogram was calculated as in Gima and Rudy,17 by simulating a heterogeneous transmural wedge (right ventricular outflow tract, 1 Hz, 500th beat shown). This model aims at mimicking the global electrical activity of a row of 165 subendocardial, midmyocardial and

  intact, by modifying the following equation: If V >= -50 beta_j=0.035*exp(+0.008*p->v)/(1.0+exp(-0.048*(p->v+77.329)). Thus, in the modified model, any incomplete fast inactivation (for instance, h parameter varies between 1 and 0.166 for BrS2 + , instead of 1 and zero for Ctrl) gives rise to a late Na + current similar to that observed in Figure 3C (see also Figure below) resulting in the following equation. Impact of incomplete inactivation on late sodium current in a simulated Ramp protocol. Ctrl: h parameter varies between 1 and 0.

17

 17

  BrS: h varies between 1 and 0.116. The ratio of maximal I Na,L current in BrS/Ctrl is 5.4, as in Figure 3C for BrS2 + . Noteworthy, this modification does not change inactivation properties of the Na + current in the WT model because fast inactivation (h) is not modified.

Figure S2 :

 S2 Figure S2:

  Figure S3

Figure S4 .Figure S5 .

 S4S5 Figure S4:

  Letter to Editor with previous submission number (CTM2-2021-01-0184) 20 TableS2. ECG patient characteristics.ECGs were performed in leads D1, D2, V1, V2, V3 and recordings used the following parameters: 25 mm/s, 0.1 mV/mm. RR interval duration (RR); S wave duration and amplitude (S); P wave duration (P); PR interval duration (PR); QRS duration (QRS); QT peak interval (QTp); QT end interval (QTe); Tpeak-to -Tend interval (TPE); J wave amplitude (J); Early Repolarization Pattern (ERP); Fragmented QRS according to Morita H et al.22 (Frag); Concave aspect of the ST segment elevation (Concave aspect); Yes (y); No (n).

  Comparisons were made by use of Mann-Whitney test, Student t-test, or two-way ANOVA with Bonferroni post-hoc test for repeated measures. Correlations were investigated based on correlation coefficient r s of Spearman.

	Supplemental results:
	Patient characteristics
	Six patients affected by type I BrS (BrS1-6) with a familial history of SCD or syncope were
	selected, among which two carrying SCN5A variants (marked with a + symbol in BrS1 + and BrS2 + ).
	Patient #1 (BrS1 + ) was a 35-year-old male with a history of recurrent near-syncope. He
	presented with a spontaneous BrS ECG pattern. After induction of ventricular fibrillations (VF) during
	electrophysiological study, an implantable cardioverter-defibrillator (ICD) was implanted with no
	recurrence of syncope or VF during a 14-year follow-up. Five of his relatives also exhibited a BrS ECG
	pattern.
	Patient #2 (BrS2
	F
	o r
	R
	e v i e w
	O
	n l
	y

Values of p < 0.05 were considered statistically significant. Statistical analyses were performed with GraphPad Prism software. + ) was an asymptomatic 55-year-old male whose brother died suddenly at age 38. He presented a spontaneous BrS ECG pattern with occurrence of VF during an electrophysiological study. An ICD was implanted with no occurrence of syncope or VF during a 13-year follow-up. Six of his relatives presented with a BrS ECG pattern including his son who exhibited one spontaneous episode of VF.

Patient #3 (BrS3 -) was a 35-year-old male who suffered from syncope at night and presented with a spontaneous BrS ECG pattern with VF occurrence during an electrophysiological study. Despite hydroquinidine administration, several episodes of VF were reported. Endocardial catheter ablation of premature ventricular beats was performed without further recurrences under hydroquinidine therapy. Two of his relatives presented with a BrS ECG pattern.

Patient #4 (BrS4 -) was a 44-year-old male who presented episodes of unexplained syncope at rest. Ajmaline test revealed a BrS ECG pattern. Electrophysiological study did not induce any arrhythmia. Due to familial history of SCD and recurrent syncope, an ICD was implanted without recurrence of syncope or VF during a 13-year follow-up. Type-1 BrS ECG was identified in 8 additional family members.

Patient #5 (BrS5 -) was a 41-year-old male, previously described by Belbachir et al,

10

who presented recurrent near-syncopes with palpitations and spontaneous BrS ECG pattern.

Electrophysiological study induced VF. An ICD was implanted with no recurrence of syncope or VF during a 16-year follow-up. Familial screening identified 6 relatives with a BrS ECG pattern and one with unexplained SCD at age 41.

Table S3 .

 S3 TLDA probe references and corresponding genes.

	gene symbol	protein symbol	Gene name	Assay ID	Category
	ABCC8	SUR1	ATP-binding cassette, sub-family C (CFTR/MRP), member 8	Hs01093761_m1	Potassium (K + ) channel
	ABCC9	SUR2	ATP-binding cassette, sub-family C (CFTR/MRP), member 9	Hs00245832_m1	Potassium (K + ) channel
	ATP1A3	Na/K-ATPase a3	ATPase, Na+/K+ transporting, alpha 3 polypeptide	Hs00958036_m1	Na + /K + ATPase
	ATP1B1	Na/K-ATPase b1	ATPase, Na+/K+ transporting, beta 1 polypeptide	Hs00426868_g1	Na + /K + ATPase
	ATP2A2	SERCA2	ATPase, Ca++ transporting, cardiac muscle, slow twitch 2	Hs00544877_m1	Calcium (Ca ++ ) regulator
	ATP2A3	SERCA3	ATPase, Ca++ transporting, ubiquitous	Hs00193090_m1	Calcium (Ca ++ ) regulator
	ATP2B4	Ca ATPase4	ATPase, Ca++ transporting, plasma membrane 4	Hs00608066_m1	Calcium (Ca ++ ) regulator
	CACNA1C	Cav1.2	calcium channel, voltage-dependent, L type, alpha 1C subunit	Hs00167681_m1	Calcium (Ca++) channel
	CACNA1D	Cav1.3	calcium channel, voltage-dependent, L type, alpha 1D subunit	Hs00167753_m1	Calcium (Ca++) channel
	CACNA1G	Cav3.1	calcium channel, voltage-dependent, alpha 1G subunit	Hs00367969_m1	Calcium (Ca++) channel
	CACNA1H	Cav3.2	calcium channel, voltage-dependent, alpha 1H subunit	Hs01103527_m1	Calcium (Ca++) channel
	CACNA2D1	Cava2d1	calcium channel, voltage-dependent, alpha 2/delta subunit 1	Hs00984856_m1	Calcium (Ca++) channel
	CACNA2D2	Cava2d2	calcium channel, voltage-dependent, alpha 2/delta subunit 2	Hs01021049_m1	Calcium (Ca++) channel
	CACNB2	Cavb2	calcium channel, voltage-dependent, beta 2 subunit	Hs01100744_m1	Calcium (Ca++) channel
	CALM1	CALM1	calmodulin 1 (phosphorylase kinase, delta)	Hs00300085_s1	Calcium (Ca ++ ) regulator
	CALM3	CALM3	calmodulin 3 (phosphorylase kinase, delta)	Hs00968732_g1	Calcium (Ca ++ ) regulator
	CASQ2	CASQ2	calsequestrin 2 (cardiac muscle)	Hs00154286_m1	Calcium (Ca ++ ) regulator
	GJA1	Cx43	gap junction protein, alpha 1, 43kDa (connexin 43)	Hs00748445_s1	Connexin
	GJA5	Cx40	gap junction protein, alpha 5, 40kDa (connexin 40)	Hs00270952_m1	Connexin
	GJA7	Cx45	gap junction protein, alpha 7, 45kDa (connexin 45)	Hs00271416_s1	Connexin
	GJD3	Cx30.2	gap Junction Protein, delta 3, 31.9kDa	Hs00987388_s1	Connexin
	HCN1	HCN1	hyperpolarization activated cyclic nucleotide-gated potassium channel 1	Hs01085412_m1	Cation channel
	HCN2	HCN2	hyperpolarization activated cyclic nucleotide-gated potassium channel 2	Hs00606903_m1	Cation channel
	HCN3	HCN3	hyperpolarization activated cyclic nucleotide-gated potassium channel 3	Hs00380018_m1	Cation channel
	HCN4	HCN4	hyperpolarization activated cyclic nucleotide-gated potassium channel 4	Hs00975492_m1	Cation channel
	ITPR1	ITPR1	inositol 1,4,5-triphosphate receptor, type 1	Hs00181881_m1	Calcium (Ca ++ ) regulator
	ITPR3	ITPR3	inositol 1,4,5-triphosphate receptor, type 3	Hs00609908_m1	Calcium (Ca ++ ) regulator
	KCNA2 KCNA4 KCNA5	Kv1.2 Kv1.4 Kv1.5	potassium voltage-gated channel, shaker-related subfamily, member 2 potassium voltage-gated channel, shaker-related subfamily, member 4 potassium voltage-gated channel, shaker-related subfamily, member 5 F	Hs00270656_s1 Hs00937357_s1 Hs00969279_s1	Potassium (K + ) channel Potassium (K + ) channel Potassium (K + ) channel
	KCNA7 KCNAB2 KCNAB3 KCNB1 KCNC4	Kv1.7 Kvb2 Kvb3 Kv2.1 Kv3.4	potassium voltage-gated channel, shaker-related subfamily, member 7 potassium voltage-gated channel, shaker-related subfamily, beta member 2 Hs00186308_m1 Hs00361015_m1 potassium voltage-gated channel, shaker-related subfamily, beta member 3 Hs01085073_m1 potassium voltage-gated channel, Shab-related subfamily, member 1 Hs00270657_m1 potassium voltage-gated channel, Shaw-related subfamily, member 4 Hs00428198_m1 o r	Potassium (K + ) channel Potassium (K + ) channel Potassium (K + ) channel Potassium (K + ) channel Potassium (K + ) channel
	KCND2	Kv4.2	potassium voltage-gated channel, Shal-related subfamily, member 2	Hs01054873_m1	Potassium (K + ) channel
	KCND3 KCNE1 KCNE1L KCNE2 KCNE3 KCNE4 KCNH2 KCNIP2 KCNJ11 KCNJ2 KCNJ3 KCNJ4 KCNJ5 KCNJ8 KCNK1 KCNK3 KCNK5 KCNQ1	Kv4.3 MinK MIRP4 MIRP1 MIRP2 MIRP3 hERG KChIP2 Kir6.2 Kir2.1 Kir3.1 Kir2.3 Kir3.4 Kir6.1 TWIK1 TASK1 TASK2 KvLQT1	potassium voltage-gated channel, Shal-related subfamily, member 3 potassium voltage-gated channel, Isk-related family, member 1 potassium voltage-gated channel, Isk-related family, member 1-like potassium voltage-gated channel, Isk-related family, member 2 R potassium voltage-gated channel, Isk-related family, member 3 potassium voltage-gated channel, Isk-related family, member 4 potassium voltage-gated channel, subfamily H (eag-related), member 2 Kv channel interacting protein 2 potassium inwardly-rectifying channel, subfamily J, member 11 potassium inwardly-rectifying channel, subfamily J, member 2 potassium inwardly-rectifying channel, subfamily J, member 3 potassium inwardly-rectifying channel, subfamily J, member 4 potassium inwardly-rectifying channel, subfamily J, member 5 potassium inwardly-rectifying channel, subfamily J, member 8 potassium channel, subfamily K, member 1 potassium channel, subfamily K, member 3 potassium channel, subfamily K, member 5 potassium voltage-gated channel, KQT-like subfamily, member 1 e v i e w Hs00542597_m1 Hs00264799_s1 Hs01085745_s1 Hs00270822_s1 Hs01921543_s1 Hs01851577_s1 Hs04234270_g1 Hs01552688_g1 Hs00265026_s1 Hs01876357_s1 Hs04334861_s1 Hs00705379_s1 Hs00168476_m1 Hs00958961_m1 Hs00158428_m1 Hs00605529_m1 Hs00186652_m1 Hs00923522_m1	Potassium (K + ) channel Potassium (K + ) channel Potassium (K + ) channel Potassium (K + ) channel Potassium (K + ) channel Potassium (K + ) channel Potassium (K + ) channel Potassium (K + ) channel Potassium (K + ) channel Potassium (K + ) channel Potassium (K + ) channel Potassium (K + ) channel Potassium (K + ) channel Potassium (K + ) channel Potassium (K + ) channel Potassium (K + ) channel Potassium (K + ) channel Potassium (K + ) channel
	NPPA NPPB PLN PPP3CA RYR2 SCN10A SCN1B SCN2B SCN3A	ANP BNP PLN CAM-PRP RYR2 Nav1.8 Navb1 Navb2 Nav1.3	natriuretic peptide precursor A natriuretic peptide precursor B phospholamban protein phosphatase 3 (formerly 2B), catalytic subunit, alpha isoform ryanodine receptor 2 (cardiac) O Hs00383230_g1 Hs01057466_g1 Hs01848144_s1 Hs00174223_m1 Hs00181461_m1 sodium channel, voltage-gated, type X, alpha Hs01045137_m1 sodium channel, voltage-gated, type I, beta Hs03987893_m1 sodium channel, voltage-gated, type II, beta Hs00394952_m1 sodium channel, voltage-gated, type III, alpha Hs00366913_m1 n l Signaling molecule Signaling molecule Calcium (Ca ++ ) regulator Calcium (Ca ++ ) regulator Calcium (Ca ++ ) regulator Sodium (Na + ) channel Sodium (Na + ) channel Sodium (Na + ) channel Sodium (Na + ) channel
	SCN3B SCN4A SCN4B	Navb3 Nav1.4 Navb4	sodium channel, voltage-gated, type III, beta sodium channel, voltage-gated, type IV, alpha sodium channel, voltage-gated, type IV, beta	Hs01024483_m1 Hs01109480_m1 Hs03681025_m1 y Sodium (Na + ) channel Sodium (Na + ) channel Sodium (Na + ) channel
	SCN5A	Nav1.5	sodium channel, voltage-gated, type V, alpha (long QT syndrome 3)	Hs00165693_m1	Sodium (Na + ) channel
	SCN7A	Nav2.1	sodium channel, voltage-gated, type VII, alpha	Hs00161546_m1	Sodium (Na + ) channel
	SCN9A	Nav1.7	sodium channel, voltage-gated, type IX, alpha	Hs00161567_m1	Sodium (Na + ) channel
	SLC8A1	NCX1	solute carrier family 8 (sodium/calcium exchanger), member 1	Hs01062258_m1	Calcium (Ca ++ ) regulator
	ANK2	ANKB	ankyrin 2	Hs00153998_m1	Cytoskeletal protein
	GATA3	GATA3	GATA Binding Protein 3	Hs00231122_m1	Transcription factor
	GATA4	GATA4	GATA Binding Protein 4	Hs00171403_m1	Transcription factor
	GATA5	GATA5	GATA Binding Protein 5	Hs00388359_m1	Transcription factor
	GATA6	GATA6	GATA Binding Protein 6	Hs00232018_m1	Transcription factor
	HEY2	HEY2	Hes Related Family BHLH Transcription Factor With YRPW Motif 2	Hs00232622_m1	Transcription factor
	IRX3	IRX3	iroquois homeobox 3	Hs01124217_g1	Transcription factor
	IRX4	IRX4	iroquois homeobox 4	Hs00212560_m1	Transcription factor
	IRX5	IRX5	iroquois homeobox 5	Hs04334749_m1	Transcription factor
	TBX2	TBX2	T-Box Transcription Factor 2	Hs00911929_m1	Transcription factor
	TBX3	TBX3	T-Box Transcription Factor 3	Hs00195612_m1	Transcription factor
	TBX5	TBX5	T-Box Transcription Factor 5	Hs00361155_m1	Transcription factor
	NKX2.5	NKX2-5	NK2 Homeobox 5	Hs00231763_m1	Transcription factor
	TNNI3	TNNI3	Troponin I3, Cardiac Type	Hs00165957_m1	Cytoskeletal protein
	TNNT2	TNNT2	Troponin T2, Cardiac Type	Hs00943911_m1	Cytoskeletal protein
	MYH6	MYH6	Myosin Heavy Chain 6	Hs01101425_m1	Cytoskeletal protein
	MYH7	MYH7	Myosin Heavy Chain 7	Hs01110632_m1	Cytoskeletal protein
	RRAD	RAD	RRAD, Ras Related Glycolysis Inhibitor And Calcium Channel Regulator	Hs00188163_m1	GTP-binding protein
	CLASP2	CLASP2	Cytoplasmic Linker Associated Protein 2	Hs00380556_m1	Cytoskeletal protein
	GPD1L	GPD1L	Glycerol-3-Phosphate Dehydrogenase 1 Like	Hs00380518_m1	Enzyme
	MYL7	MLC2A	Myosin Light Chain 7	Hs01085598_g1	Cytoskeletal protein
	MYL2	MLC2V	Myosin Light Chain 2	Hs00166405_m1	Cytoskeletal protein
	18S		Eukaryotic 18s rRNA	Hs99999901_s1	endogenous control
	B2M	B2M	Beta-2-Microglobulin	Hs00187842_m1	endogenous control
	ACTB	ACTB	actin beta	Hs99999903_m1	endogenous control
	RPL13A	RPL13A	Ribosomal Protein L13a	Hs04194366_g1	endogenous control

Table S4 .

 S4 Mean value ± SEM of I Na and I Ca,L activation and inactivation kinetics parameters in the different hiPSC-CMs lines. V1/2 and K represent voltage of half-maximum (in)activation and slope factor, respectively.

	F
	o r
	R
	e v i e w
	O
	n l
	y

Table S5 .

 S5 Correlation between ECG parameters and the corresponding hiPSC-CM sodium currents.

			Correlation coefficient r s of Spearman
			Late I Na	Peak I Na
		RR (ms)	0.48	-0.07
	D1	S duration (ms)	-0.55	0.00
		S amplitude (mm)	-0.10	0.26
		P (ms)	-0.47	0.01
		PR (ms)	-0.63	0.20
	D2	QRS (ms)	-0.55	-0.31
	V1	QTp (ms) QTe (ms) TPE (ms) J amplitude (mm) F o r -0.40 -0.32 -0.07 -0.81 *	-0.14 -0.14 -0.14 0.02
	V2 V3	TPE (ms) J amplitude (mm) TPE (ms) J amplitude (mm) TPE (ms)	0.11 -0.86 * R -0.33 0.23 -0.23 e v i e w	-0.14 0.14 -0.26 0.19 -0.31
			O
			n l
				y

Each ECG parameter was tested for its correlation with either I Na,L or peak I Na density from the corresponding hiPSC-CMs. While the J point elevation correlated significantly with I Na,L density, it did not correlate with hiPSC-CM peak I Na density. Statistical significance threshold of r s was p < 0.05 (*).

  transcriptase. Barcoded cDNAs from multiple samples were then pooled, amplified and tagmented using a transposon-fragmentation approach which enriches for 3'ends of cDNA. A library of 350-800 bp was run on an Illumina HiSeq 2500 using a HiSeq Rapid SBS Kit v2 (50 cycles; FC-402-4022) and a HiSeq Rapid PE Cluster Kit v2 (PE-402-4002). Read pairs used for analysis matched the following criteria: all sixteen bases of the first read had quality scores of at least 10 and the first six bases correspond exactly to a designed well-specific barcode. The second reads were aligned to RefSeq human mRNA sequences (hg19) using bwa version 0.7.17. Reads mapping to several transcripts of different genes or containing more than 3 mismatches with the reference sequences were filtered out from the analysis. Digital gene expression profiles were generated by counting the number of UMIs associated with each RefSeq genes, for each sample. R package DESeq2 (Bioconductor) was used to normalize gene expression, and detect differentially expressed genes. Sample correlation matrix was performed using R package ComplexHeatmap (Bioconductor). Cluster 3.0 software was used to perform the differentially expressed gene heatmap. Gene Set Enrichment Analysis (GSEA) was performed using GeneTrail2. Statistically significant categories within the GO Molecular Process were identified using Kolomogorov-Smirnov test and p values were adjusted using the Benjamini and

	F
	o r
	R
	Hochberg method.	w e i v e
		O
		n l
		y
		12-14
	3' Sequencing RNA Profiling (3'SRP): 3'SRP protocol was performed according to Kilens et al. 15 Briefly,
	the libraries were prepared from 10 ng of total RNA. RNA samples were extracted from 26 BrS and 13

control hiPSC samples (a duplicate for each clone obtained at different cell passages) as well as their corresponding differentiated hiPSC-CMs. The mRNA poly(A) tail was tagged with universal adapters, well-specific barcodes and unique molecular identifiers (UMIs) during template-switching reverse

  Comparisons were made by use of Mann-Whitney test, Student t-test, or two-way ANOVA with Bonferroni post-hoc test for repeated measures. Correlations were investigated based on correlation coefficient r s of Spearman. Values of p < 0.05 were considered statistically significant. Statistical analyses were performed with GraphPad Prism software.

	F F F
	o r o r o r
	R R R
	e v i e w e v i e w e v i e w
	O O O
	n l n l n l
	y y y

Table S5 .

 S5 Correlation between ECG parameters and the corresponding hiPSC-CM sodium currents.

			Correlation coefficient r s of Spearman
			Late I Na	Peak I Na
		RR (ms)	0.48	-0.07
	D1	S duration (ms)	-0.55	0.00
		S amplitude (mm)	-0.10	0.26
		P (ms)	-0.47	0.01
		PR (ms)	-0.63	0.20
	D2	QRS (ms)	-0.55	-0.31
		QTp (ms)	-0.40	-0.14
		QTe (ms)	-0.32	-0.14
		TPE (ms)	-0.07	-0.14
	V1	J amplitude (mm)	-0.81 *	0.02
		TPE (ms)	0.11	-0.14
	V2	J amplitude (mm)	-0.86 *	0.14
		TPE (ms)	-0.33	-0.26
	V3	J amplitude (mm)	0.23	0.19
		TPE (ms)	-0.23	-0.31

Clinical and Translational Medicine

Acknowledgements

The authors thank Dr. Connie Bezzina and Dr. Isabella Mengarelli for the gift of hiPSCs from BrS6patient, Dr. Pierre Lindenbaum and Dr. Stephanie Bonnaud for the Haloplex targeted capture and NGS experiments, and Adeline Goudal for her support in the variant annotation. Genomic and bioinformatics analysis, flow cytometry and iPSCs derivation were performed with the support of GenoBiRD (Biogenouest), CytoCell and iPS core facility of Nantes university, respectively. Finally, the authors are grateful to the patients and families who agreed to participate in our research.

Source of funding

This work was supported by grants from the Fondation pour la Recherche Médicale (DEQ20140329545), The National Research Agency ANR-14-CE10-0001-01 and HEART iPS ANR-15-CE14-0019-01, and La Fédération Française de Cardiologie. Dr. Nathalie Gaborit was laureate of fellowships from Fondation Lefoulon-Delalande and Marie Curie Actions, International Incoming Fellowship FP7-PEOPLE-2012-IIF [PIIF-GA-2012-331436]. Dr. Zeina R. Al-Sayed was supported by scholarships from the Lebanese University, Eiffel program of Excellence (Campus France), and The Fondation Genavie. Dr. Mariam Jouni was funded by a scholarship from the Association of Scientific Orientation and Specialization (ASOS) and by a grant from the Lebanese University to Dr. Kazem Zibara. Eric Charpentier was supported by Data-Santé (Région Pays de la Loire). Dr. Barc was supported by the H2020-MSCA-IF-2014. Acknowledgements The authors thank Dr. Connie Bezzina and Dr. Isabella Mengarelli for the gift of hiPSCs from BrS6patient, Dr. Pierre Lindenbaum and Dr. Stephanie Bonnaud for the Haloplex targeted capture and NGS experiments, and Adeline Goudal for her support in the variant annotation. Genomic and bioinformatics analysis, flow cytometry and iPSCs derivation were performed with the support of GenoBiRD (Biogenouest), CytoCell and iPS core facility of Nantes university, respectively. Finally, the authors are grateful to the patients and families who agreed to participate in our research.

Source of funding

This work was supported by grants from the Fondation pour la Recherche Médicale (DEQ20140329545), The National Research Agency ANR-14-CE10-0001-01 and HEART iPS ANR-15-CE14-0019-01, and La Fédération Française de Cardiologie. Dr. Nathalie Gaborit was laureate of fellowships from Fondation Lefoulon-Delalande and Marie Curie Actions, International Incoming Fellowship FP7-PEOPLE-2012-IIF [PIIF-GA-2012-331436]. Dr. Zeina R. Al-Sayed was supported by scholarships from the Lebanese University, Eiffel program of Excellence (Campus France), and The Fondation Genavie. Dr. Mariam Jouni was funded by a scholarship from the Association of Scientific Orientation and Specialization (ASOS) and by a grant from the Lebanese University to Dr. Kazem Zibara. Eric Charpentier was supported by Data-Santé (Région Pays de la Loire). Dr. Barc was supported by the H2020-MSCA-IF-2014.

Data availability statement.

In accordance with the "DFG Guidelines on the Handling of Research Data", the authors declare that all data supporting the findings of this study are available within the article and its supplementary information files or from the corresponding author upon reasonable request. The data set will be archived for at least 10 years after publication.

Conflict of Interest

None.

Supplemental figures:

Supplemental Tables:

Table S1. Patient description

Conflict of Interest

None.

Supplemental results:

Patient characteristics

Six patients affected by type I BrS (BrS1-6) with a familial history of SCD or syncope were selected, among which two carrying SCN5A variants (marked with a + symbol in BrS1 + and BrS2 + ).

Patient #1 (BrS1 + ) was a 35-year-old male with a history of recurrent near-syncope. He presented with a spontaneous BrS ECG pattern. After induction of ventricular fibrillations (VF) during electrophysiological study, an implantable cardioverter-defibrillator (ICD) was implanted with no recurrence of syncope or VF during a 14-year follow-up. Five of his relatives also exhibited a BrS ECG pattern.

Patient #2 (BrS2 + ) was an asymptomatic 55-year-old male whose brother died suddenly at age 38. He presented a spontaneous BrS ECG pattern with occurrence of VF during an electrophysiological study. An ICD was implanted with no occurrence of syncope or VF during a 13-year follow-up. Six of his relatives presented with a BrS ECG pattern including his son who exhibited one spontaneous episode of VF.

Patient #3 (BrS3 -) was a 35-year-old male who suffered from syncope at night and presented with a spontaneous BrS ECG pattern with VF occurrence during an electrophysiological study. Despite hydroquinidine administration, several episodes of VF were reported. Endocardial catheter ablation of premature ventricular beats was performed without further recurrences under hydroquinidine therapy. Two of his relatives presented with a BrS ECG pattern.

Patient #4 (BrS4 -) was a 44-year-old male who presented episodes of unexplained syncope at rest. Ajmaline test revealed a BrS ECG pattern. Electrophysiological study did not induce any arrhythmia. Due to familial history of SCD and recurrent syncope, an ICD was implanted without recurrence of syncope or VF during a 13-year follow-up. Type-1 BrS ECG was identified in 8 additional family members.

Patient #5 (BrS5 -) was a 41-year-old male, previously described by Belbachir et al, 10 who presented recurrent near-syncopes with palpitations and spontaneous BrS ECG pattern.

Electrophysiological study induced VF. An ICD was implanted with no recurrence of syncope or VF during a 16-year follow-up. Familial screening identified 6 relatives with a BrS ECG pattern and one with unexplained SCD at age 41.

Patient #6 (BrS6 -) was a 42-year-old male, previously described by Veerman et al. 6 While he had a spontaneous BrS ECG pattern, he presented an unexplained syncope, and suffered from an outof-hospital cardiac arrest at night. Three of his relatives presented with a BrS phenotype after ajmaline administration.

An additional Patient #7, not affected by Brugada syndrome (Non-BrS), was recruited. He was a 67-year-old male diagnosed with progressive cardiac conduction defect (PCCD). He was the nephew of patient BrS2 + . His ECG displayed broad PR interval and prolonged QRS duration, however, no ST segment elevation was detected even after challenge with flecainide.

Four control subjects were also included. One subject who was a relative of BrS5 -patient, 10 in whom BrS was excluded after sodium channel blocker challenge and three other unrelated healthy extra familial controls, including one previously described 11 and 2 others with different ethnicity. 21 Representative patient ECGs are presented in Figure S1. Description of patients and their corresponding ECG measurements are depicted in Table S1 and Table S2 respectively.

Genetic characterization

Genetic screening of the coding regions of 20 genes, selected according to the American College of Medical Genetics and Genomics (ACMG) guidelines, revealed SCN5A pathogenic variants in only two of the six BrS patients. BrS1 + carried a c.5164A>G missense rare variant, resulting in asparagine to aspartic acid substitution at position 1722 in the extracellular connecting loop, between segment 5 and 6 in domain IV forming the pore region of Na v 1.5 (p.N1722D). BrS2 + and his nephew (Non-BrS) affected by PCCD carried a 10bp duplication (c.1983-1993dup) in SCN5A creating a stop codon (p.A665G-fsX16). Both these variants had not been functionally investigated yet. BrS5 -patient presented a previously described rare genetic variant in the RRAD gene (p.R211H) 10 whereas no genetic variation was identified in BrS candidate genes in BrS3 -, BrS4 -or BrS6 -patients (Table S1).

Generation and characterization of hiPSCs and hiPSC-CMs

Somatic cells were obtained from all studied subjects and were reprogrammed into corresponding hiPSC lines. BrS5 -, BrS6 -and the control hiPSC lines have been previously characterized. 6,10,11,21 For each of the other 5 newly generated hiPSC lines (BrS1 to 4 and Non-BrS), up to three independent clones were amplified and characterized. SNP analysis verified that control and mutated hiPSC lines were free from any genomic aberrations compared to parental somatic cells (data not shown). The expression of the pluripotent stem cell markers was verified (Figure 1A, 1B and 1C).

Genetic screening confirmed that the lines arising from patients carrying SCN5A genetic variants, BrS1 + , BrS2 + and Non-BrS harbored the corresponding heterozygous rare variant (Figure 1D). hiPSC differentiation process into cardiomyocytes was also validated at the transcriptional level. 3'SRPbased global transcriptomic analysis of control and BrS hiPSCs and hiPSC-CMs showed that cells clustered based on their stage (hiPSCs on one side and hiPSC-CMs on the other side) and independently from their genetic background (Figure 1E). Correlation analysis also showed that samples correlated according to their stage, with all hiPSC samples being correlated to other hiPSC samples and similarly for hiPSC-CM samples (Figure 1F). Finally, both Ctrl and BrS hiPSC-CMs presented a comparable global change in gene expression as compared to hiPSC stage (Figure 1G). Immunostaining analysis showed

Supplemental Tables:

Table S1. Patient description