BPS2026 – Biophysical characterization of the Nav1.5/L828F variant across neonatal and adult contexts in a Nav1.5 iPSC-Ko model

Fetal arrhythmias are a serious condition that occur in 1 per 4,000 live births and may arise from various causes including genetic mutations. Mutations on SCN5A, the gene encoding Nav1.5, the cardiac sodium channel, have been linked to several cardiac disorders, including fetal arrhythmias. A novel and physiologically relevant strategy to study Nav1.5 variants involves the use of human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (CM) lacking endogenous Nav1.5 expression (Nav1.5 KO). This system provides a controlled human cardiac cellular context for precise evaluation of variant-specific effects. We investigated the Nav1.5/L828F variant, previously identified in a familial cohort with fetal arrhythmias. We compared the biophysical properties of both adult and neonatal Nav1.5 isoforms expressed in atrial and ventricular hiPSC-derived cardiomyocytes and we compared the biophysical properties of Nav1.5/WT adult and neonatal isoforms. The L828F variant in both adult and neonatal form of Nav1.5 was transfected into atrial and ventricular hiPS-CM. Electrophysiological properties were assessed using the whole cell patch clamp technique to record both sodium currents and action potentials. Activation of both adult and neonatal Nav1.5/L828F channels was shifted toward more hyperpolarized potentials, resulting in an increased window current and higher open probability. In the adult isoform, the L828F mutation additionally induced a faster inactivation and prolonged action potential duration. In the neonatal isoform, L828F increased current density and produced a hyperpolarizing shift of inactivation. The WT neonatal expressed a depolarized inactivation compared to the WT adult. The Nav1.5 KO hiPSC model provided valuable insights into the L828F variant, particularly by enabling direct comparison between the adult and neonatal isoforms. This unprecedented investigation highlights the Nav1.5 KO hiPSC model as a powerful platform to investigate Nav1.5 variants and their underlying mechanisms across developmental isoforms.