Multi-electrode array recordings
Extracellular field potentials were recorded from hiPSC-CM monolayers
using the Maestro-APEX MEA system and AxIS software v2.5.1.10 software
(Axion Biosystems, Atlanta, GA, USA) as previously described (16).
Dissociated hiPSC-CM were spotted into Matrigel® matrix coated wells of
E-STIM+ Classic 48 well MEA plates (Axion Biosystems) at a density of
80,000 cells per well, using a robotic Maestro-APEX MEA system (Axion
Biosystems, Atlanta, GA, USA). HiPSC-CM were initially maintained for 2
days in MEM alpha media (Gibco) supplemented with 10% foetal bovine
serum (Sigma-Aldrich), before switching to RPMI 1640 medium (Thermo
Fisher) containing B-27™ supplement (Gibco, Thermo Fisher), supplemented
to 1mM extracellular calcium concentration, for 20-30 days prior to
recording. Extracellular field potentials were recorded from hiPSC-CM
monolayers using the Maestro-APEX MEA system and AxIS software v2.5.1.10
software (Axion Biosystems, Atlanta, GA, USA). Monolayers, maintained at
37°C and 5% CO2, were paced at 1 Hz and signals digitized at 12.5 kHz.
Field potential durations were measured from the peak depolarization
spike to the peak of the repolarization wave, using the CiPA analysis
tool (Axion Biosystems, Atlanta, GA, USA). After 5 minutes of pacing
equilibration, signals were recorded for 2-5 minutes under baseline
conditions, after acute addition of drug (<2 hours), and
following 24 hours and 48 hours in the presence of hydroxychloroquine
alone (10 µM, nominal concentration) or in combination with azithromycin
(5 µM, nominal concentration). Only wells showing properly paced signals
at all time points were included in the analysis with field potential
durations being measured from an average of 30 stable beast from a
single electrode in each well. For MEA experiments examining the effect
of kalaemic variation, cells were switched to Tyrode’s solution 2 hours
prior to recording (containing in mM: 140 NaCl, 5.4 or 3 KCl, 1.8
CaCl2, 1 MgCl2, 10 HEPES, 10 Glucose,
adjusted with NaOH to pH 7.4) to allow for manipulation of
[K+].