Characterization of atrial fibrillation linked mutations using the Nav1.5 knock-out of atrial cardiomyocytes derived from iPSCs.

Abstract

Atrial fibrillation (AF) is the most common cardiac arrhythmia diagnosed and affects more than 33 million on people word wide. AF increases the risk of heart failure and myocardial infarction. Some studies carried out on HEK293 cells and murine models have shown that SCN5A mutations are linked to AF. However, the mechanism by which NaV1.5 mutations generate cardiac arrhythmias is still to be elucidated. Human induced pluripotent stem cells (hiPSCs) hold great promise to model NaV1.5-linked AF mutations and recapitulate the functional changes. Previously, our laboratory established an NaV1.5 knock-out (KO) iPSC line with CRISPR/Cas9 technology. In this study, atrial cardiomyocytes (iPSC-aCMs) were differentiated from the Control and NaV1.5 KO iPSC lines. We aim to characterise the atrial statue of the iPSC-aCMs, and then, use the model to express two AF Nav1.5-linked mutations (K1493R and M1875T). To verify that NaV1.5 protein expression was suppressed, immunocytofluorescence and Western blot analysis were performed. Action potentials (APs) recordings in current-clamp mode showed that APs from the NaV1.5 KO iPSC-aCMs exhibited a significant decrease of the AP overshoot and the upstroke velocity. Voltage-clamp analysis in NaV1.5/K1493R-transfected KO iPSC-aCMs showed a shift of the steady-state activation toward hyperpolarized voltage while the NaV1.5/M1875T expression provoked an increase of the Na+ current density and a shift of the steady-state inactivation toward depolarized voltage. These alterations increase the window current which indicate a channel gain-of-function. The AP recordings showed a decrease of the depolarization threshold corresponding to a cellular excitability increase. 61 and 80 % of NaV1.5/K1493R and /M1875T-transfected iPSC-aCMs respectively, exhibited arrhythmic events such as early and delayed afterdepolarizations (EADs and DADs). Our results confirm that the gain-of-function by shifting of the activation and inactivation properties is the cause of AF.