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+].