Functional Characterisation of the Rare SCN5A p.E1225K Variant, Segregating in a Brugada Syndrome Familial Case, in Human Cardiomyocytes from Pluripotent Stem Cells

Figures

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  Figure 1. Graphical overview of the experimental work. The red asterisk (*) indicates the mutation on the Nav1.5 channel.

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  Figure 2. Pedigree structure and main features of the rare SCN5A detected variant. (A) In the pedigree, the arrow indicates the proband (proband’s age: two years later the first sudden syncope) and circles and squares indicate females and males, respectively. The genotype characterization of the family is depicted with an empty symbol (wild-type mother) and black/white symbol (mutated members). (B) The electropherogram of the mutated (mut) SCN5A gene sequence in the proband is reported and compared to the wild-type (wt) sequence; arrows indicate the nucleotide substitution c.3673G>A leading to the p.E1225K mutation. Reverse strands are reported. (C) The evolutionary conservation of Nav1.5-E1225 residue among different species (UniProtKB/Swiss-Prot). (D) Schematic representation of Nav 1.5 protein. The p.E1225K mutation was localized in the extracellular loop between segments 1 and 2 of the III repeat of the protein.

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  Figure 3. Overexpression of p.E1225K-Nav1.5 leads to reduced peak sodium currents in PSC-CMs. (A) Lentiviral vectors expressing the C-terminal GFP-tagged SCN5A (wild-type and carrying the p.E1225K variant) used to produce the lentiviral particles for overexpression. (B–D) Voltage-gated sodium currents in PSC-CMs. (B) Left panel: examples of Na+ current (INa) traces recorded in CMs overexpressing SCN5A-WT (top—WT GFP) and SCN5A-E1225K (bottom—MUT GFP) (Scale bar, 1 ms, 50 pA/pF). Right panel: I-V curves constructed from average peak sodium current density as a function of voltage command measured in WT GFP-CMs and MUT GFP-CMs, showing a significant reduction in the latter. WT GFP-CMs: n = 18; MUT GFP-CMs: n = 18. (C) Voltage dependences: steady-state activation (WT GFP-CMs: n = 18; MUT GFP-CMs: n = 18)/inactivation curves (WT GFP-CMs: n= 18; MUT GFP-CMs: n = 16). All values are reported as mean ± SD. * p < 0.05, ** p < 0.005 (unpaired t-test). Data are relative to four independent cell differentiations. (D) Dot plot of peak sodium current densities, measured at −30 mV, in MUT GFP-CMs (56.53 ± 33.96 pA/pF) relative to WT GFP-CMs (171.73 ± 57.83 pA/pF). Average current density values are…