Na(V)1.5 knockout in iPSCs: a novel approach to study Na(V)1.5 variants in a human cardiomyocyte environment

Cardiomyocytes derived from patient-specific induced pluripotent stem cells (iPSC-CMs) successfully reproduce the mechanisms of several channelopathies. However, this approach involve cell reprogramming from somatic tissue biopsies or genomic editing in healthy iPSCs for every mutation found and to be investigated. We aim to knockout (KO) Na(V)1.5, the cardiac sodium channel, in a healthy human iPSC line, characterize the model and then, use it to express variants of Na(V)1.5. We develop a homozygous Na(V)1.5 KO iPSC line able to differentiate into cardiomyocytes with CRISPR/Cas9 tool. The Na(V)1.5 KO iPSC-CMs exhibited an organized contractile apparatus, spontaneous contractile activity, and electrophysiological recordings confirmed the major reduction in total Na(+) currents. The action potentials (APs) exhibited a reduction in their amplitude and in their maximal rate of rise. Voltage optical mapping recordings revealed that the conduction velocity Ca(2+) transient waves propagation velocities were slow. A wild-type (WT) Na(V)1.5 channel expressed by transient transfection in the KO iPSC-CMs restored Na(+) channel expression and AP properties. The expression of Na(V)1.5/delQKP, a long QT type 3 (LQT3) variant, in the Na(V)1.5 KO iPSC-CMs showed that dysfunctional Na(+) channels exhibited a persistent Na(+) current and caused prolonged AP duration that led to arrhythmic events, characteristics of LQT3.