BackgroundAtrial fibrillation (AF) poses a major risk for heart failure, myocardial infarction, and stroke. Several studies have linked SCN5A variants to AF, but their precise mechanistic contribution remains unclear. Human induced pluripotent stem cells (hiPSCs) provide a promising platform for modeling SCN5A-linked AF variants and their functional alterations. ObjectiveThe purpose of this study was to assess the electrophysiological impact of 3 three AF-linked SCN5A variants (K1493R, M1875T, N1986K) identified in 3 unrelated individuals. MethodsCRISPR-Cas9 was used to generate a new hiPSC line in which NaV1.5 was knocked out. Following differentiation into specific atrial cardiomyocyte by using retinoic acid, the adult wild-type (WT) and SCN5A variants were introduced into the NaV1.5 knockout (KO) line through transfection. Subsequent analysis including molecular biology, optical mapping, and electrophysiology were performed. ResultsThe absence of NaV1.5 channels altered the expression of key cardiac genes. NaV1.5 KO atrial-like cardiomyocytes derived from human induced pluripotent stem cells displayed slower conduction velocities, altered action potential (AP) parameters, and impaired calcium transient propagation. The transfection of the WT channel restored sodium current density and AP characteristics. Among the AF variants, 1 induced a loss of function (N1986K) while the other 2 induced a gain of function in NaV1.5 channel activity. Cellular excitability alterations and early afterdepolarizations were observed in AF variants. ConclusionOur findings suggest that distinct alterations in NaV1.5 channel properties may trigger atrial hyperexcitability and arrhythmogenic activity in AF. Our KO model offers an innovative approach for investigating SCN5A variants in a human cardiac environment.