Although the Na V1.5 α subunit acts as a full-fledged Na + channel in vivo, it is part of a large multiprotein complex. However, these expression and computational models have limitations. The use of computational simulations to explore how Na V1.5 variants affect cardiac excitability by simulating AP firings has also proven useful for understanding their clinical impact. The biophysical parameters of mutated Na + channels obtained from electrophysiological recordings in these expression systems have been extrapolated to deduce their effect on cardiac function. These heterologous expression systems remain in wide use today. They provided a platform for characterizing the biophysical properties of Na + channels and their variants. Subsequently, cell lines such as human embryonic kidney (HEK293) cells and Chinese hamster ovary (CHO) cells were used. The first studies were performed in vitro using Xenopus oocytes 3.
Most of the functional studies on specific SCN5A mutations have relied on heterologous expression systems.
Over the past 25 years, numerous SCN5A mutations have been associated with arrhythmic disorders, including congenital long QT syndrome type 3 (LQT3), Brugada syndrome (BrS), atrial fibrillation (AFib), progressive cardiac conduction defect (PCCD), sinus node dysfunction (SND), sudden infant death syndrome (SIDS), and dilated cardiomyopathy. Its biophysical properties contribute to controlling phase zero of APs and their durations (APDs) 1, and define the speed of propagation (conduction velocity) within the heart 2, 3. Na V1.5 plays a vital role in triggering and shaping cardiac action potentials (APs). The human SCN5A gene located on chromosome 3p21 encodes the voltage-gated sodium channel (VGSC) α subunit, the predominant Na + channel in the heart. The expression of Na V1.5/delQKP, a long QT type 3 (LQT3) variant, in the Na V1.5 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. A wild-type (WT) Na V1.5 channel expressed by transient transfection in the KO iPSC-CMs restored Na + channel expression and AP properties. Voltage optical mapping recordings revealed that the conduction velocity Ca 2+ transient waves propagation velocities were slow. The action potentials (APs) exhibited a reduction in their amplitude and in their maximal rate of rise.
The Na V1.5 KO iPSC-CMs exhibited an organized contractile apparatus, spontaneous contractile activity, and electrophysiological recordings confirmed the major reduction in total Na + currents. We develop a homozygous Na V1.5 KO iPSC line able to differentiate into cardiomyocytes with CRISPR/Cas9 tool. We aim to knockout (KO) Na V1.5, the cardiac sodium channel, in a healthy human iPSC line, characterize the model and then, use it to express variants of Na V1.5. However, this approach involve cell reprogramming from somatic tissue biopsies or genomic editing in healthy iPSCs for every mutation found and to be investigated. Cardiomyocytes derived from patient-specific induced pluripotent stem cells (iPSC-CMs) successfully reproduce the mechanisms of several channelopathies.
0 kommentar(er)
