Effects of Antiarrhythmic Drugs on hERG Gating in Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes From a Patient With Short QT Syndrome Type 1

Aims: The short QT syndrome type 1 (SQT1) is linked to hERG channel mutations (e.g., N588K). Drug effects on hERG channel gating kinetics in SQT1-cells have not been investigated. Methods: This study used hiPSC-CMs of a healthy donor and a SQT1-patient carrying the N588K mutation and patch clamp to examine the drug effects on hERG channel gating kinetics. Results: Ajmaline, amiodarone, ivabradine, flecainide, quinidine, mexiletine and ranolazine inhibited the hERG channel current (IKr) less strongly in hiPSC-CMs from the SQTS1-patient (SQT1-hiPSC-CMs) comparing with cells from the healthy donor (donor-hiPSC-CMs). Quinidine and mexiletine reduced, but ajmaline, amiodarone, ivabradine and ranolazine increased the time to peak of IKr similarly in SQT1-hiPSC-CMs and donor-hiPSC-CMs. Although regarding the shift of activation and inactivation curves, tested drugs showed differential effects in donor- and SQT1-hiPSC-CMs, quinidine, ajmaline, ivabradine and mexiletine but not amiodarone, flecainide and ranolazine reduced the window current in SQT1-hiPSC-CMs. Quinidine, ajmaline, ivabradine and mexiletine differentially changed the time constant of recovery from inactivation, but all of them increased the time constant of deactivation in SQT1-hiPSC-CMs. Conclusion: The window current-reducing and deactivation-slowing effects may be important for the antiarrhythmic effect of ajmaline, ivabradine, quinidine and mexiletine in SQT1-cells. This information may be helpful for selecting drugs for treating SQT1-patients with hERG channel mutation.


Somatic cell isolation and primary culture
Human fibroblast cultures were derived from skin punch biopsies of a healthy donor and patient with SQTS type 1 carrying p.N588K in hERG channel. Skin punch biopsy (3-4 mm 10 ng/ml bFGF (Peprotech, #100-18B), 100 U/ml penicillin and 100 µg/ml streptomycin (Thermo Fisher Scientific, #15140122) at 37°C with 5% CO2 atmosphere. Medium was changed every other day. Fibroblasts before passage 3 (p3) was used for generation of iPS cells.
Medium was changed every other day with HFBM supplemented with 500 µM sodium butyrate.

At day 7 post transduction/transfection, cells were replated in various dilutions in
Matrigel-coated 6-well plates in HFBM supplemented with 500 µM sodium butyrate and 2 µM Thiazovivin. From day 8, medium was changed to E8 medium (Thermo Fisher Scientific, #A1517001) supplemented with 500 µM sodium butyrate (day 8-day 11) with daily medium change. Cells were monitored for morphology change and appearance of colonies (typically after 2-3 weeks). Individual colonies with iPSC-like morphology were picked mechanically in Matrigel-coated 12-well plates in E8 medium supplemented with 2 µM Thiazovivin. Newly established iPSC lines were passaged with Versene solution (Thermo Fisher Scientific, #15040066) and cultured in E8 medium supplemented with 2 µM Thiazovivin on the first day after passaging in Matrigel-coated plates for at least ten passages before being used for pluripotency characterization and differentiation experiments.

Characterization of hiPSCs
To examine pluripotent characteristics of generated hiPSCs, cell morphology,

Generation of hiPSC-CMs
Frozen aliquots of hiPSCs were thawed and cultured without feeder cells and differentiated into hiPSC-CMs as described with some modifications (2).

Thawing and culture of hiPSCs
To thaw iPS, the following steps were performed.
One day before thawing cells, a T-25 flask coated with Matrigel was put in the incubator.
Mix medium (13 ml E8 medium + 6.5 ul from 10 mM stock solution of Rock inhibitor) was prepared. 2.5ml mix medium was added in the T-25 flask and put into incubator.
Frozen hiPS cells were taken out from liquid N2 tank and thawed in hand until only small lumps of ice exist. Then, the cell suspension was transferred to 50 ml Falcon prepared with 5 ml of mixed medium. The cell suspension was centrifuged at 250 x g (1200 rpm), 4min, 20 ° C. The supernatant was discarded and 2.5ml Mix medium was added. After 4x pipetting up and down, the cell suspension was transferred into the T-25 with 2.5 ml mix medium. The T-25 stayed into incubator until the next day.

Differentiation
On the next day, the medium in T-25 flask was changed to E8 medium without ROCK inhibitor. Then the E8 medium was changed every two days. 2 to 4 days later, when cells reached 85-95% confluence, the differentiation was started.
First, cells were treated with EDTA and dissolved in E8 Medium. Then, the cells were counted and plated on Matrigel / Geltrex coated plates (optimal cell density for plating is cell line dependent; 15.000 cell/cm2 -26.000 cells/cm2 = 150.000-260.000 cells per 6-well). Lastly, E8 medium was added to final volume (3ml per 6-well with 5µM ROCK inhibitor on the first day. The plate was shaken and incubated at 37°C with 5% CO2.
Day -x to -1: Daily medium change with E8 Medium was performed. I: inhibition. A: activation. N: no effect. NA: no analysis.