Brugada Syndrome (BrS) is a rare inherited cardiac arrhythmia disorder that predisposes young adults with structurally normal hearts to ventricular fibrillation and sudden cardiac death. Loss-of-function mutations in SCN5A, encoding the cardiac sodium channel Nav1.5, are the main genetic cause, accounting for 20–25% of cases.. Recent genome-wide association studies and integrative epigenomic analyses have uncovered several common and rare variants associated with BrS risk that map to regulatory regions active during early cardiac development, including transcription factors essential for cardiac morphogenesis, such as TBX5, GATA4, ZFPM2 and IRX3/IRX5. These data suggest a developmental component in BrS pathogenesis.

In vitro cardiac differentiation of human induced pluripotent stem cells (hiPSCs) recapitulates early stages of cardiogenesis, offering a relevant model to study potential developmental defects in BrS. Here, we investigate whether BrS, in the SCN5A haploinsufficiency, alters hiPSC-derived cardiomyocytes differentiation at the single-cell level.

We performed single-cell RNA sequencing (scRNA-seq) at Day 30 of 2D cardiac differentiation on a control line and a BrS hiPSC lines carrying a rare SCN5A heterozygous mutation leading to haploinsufficiency.

The 14 cell clusters identified were annotated using a 7 post-conception week human embryonic heart reference. Five clusters were annotated as cardiomyocytes, each displaying >90% cardiomyocyte identity with high prediction scores and specific expression of the five markers MLIP, MYO18B, TRDN, MYOM1, and POPDC2. Four clusters were annotated as ventricular (only by predictive annotation), whereas one cluster was annotated as atrial (expressing NR2F2, TBX5, CACNA1D, EPHA4) and excluded for further analysis as BrS affects ventricular compartments.

Among ventricular cardiomyocytes, we identified a mature signature characterized by expression of MYL2, ANKRD1, and CRYAB, and an immature signature defined by expression of IRX1, IRX2, and GRAMD1B. Accordingly, MYH7/MYH6 expression ratio was increased in the mature signature as compared to the immature signature. The proportion of immature cardiomyocytes was significantly higher in the BrS sample (14.5%) compared to the control (0.6%)

Altogether, these data suggest that cell maturation process is altered in BrS ventricular cardiomyocytes of BrS patient with SCN5A haploinsufficiency. These datat reinforces the hypothesis that BrS affects cardiac developmement.