A consistent arrhythmogenic trait in Brugada syndrome cellular phenotype

Abstract

To the 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; Tables S1 and S2) with a familial history of SCD or syn-cope 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 car-diomyocytes (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 (Fig-ure 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 This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. 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 two BrS, and the non-BrS lines (Figure 2E). Concordantly, reduction in I Na density was detected in these same lines (Figure 2F-H). 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. Similarly, reduction in I Ca,L channel protein expression and current density were not a common trait of BrS hiPSC-CMs (Figure 2I-L, Figure S3B; Table 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 afterdepolariza-tions (EADs) were observed in 39-70% 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, Figure S5). Thereby, the high EAD occurrence in ventricular-like hiPSC-CMs was associated with the presence of a BrS Clin. Transl. Med. 2021;11:e413. wileyonlinelibrary.com/journal/ctm2 1 of 7 https://doi.