Temperature controller

The 35-mm plates containing CM-mESCs were transferred to the stage of an inverted microscope (Nikon). Action potentials were recorded as described previously [17 (link)]. CM-mESC preparations were superfused with Tyrode’s solution containing 150.8 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl₂, 1.0 mM MgCl₂, 11.0 mM d-glucose and 10.0 mM HEPES (pH 7.4 adjusted with NaOH). The superfusion was carried out at 37.0 ± 0.5 °C using a temperature controller (Harvard Apparatus) saturated with oxygen at a perfusion flow rate of 0.5 ml/minute (Miniplus 3). Transmembrane potential was recorded using glass microelectrodes (40–80 MΩ DC resistance) filled with 2.7 M KCl connected to a microelectrode amplifier (MultiClamp 700B; Molecular Devices, USA). Amplified signals were digitized (1440 Digidata A/D interface) and stored on a computer for future analysis using LabChart 7.3 software (ADInstruments). The following parameters were analyzed: resting membrane potential (RMP), action potential amplitude (APA) and action potential duration at 30, 50, 70 and 90% repolarization (APD30, APD50, APD70 and APD90, respectively) for 10 consecutive action potentials from each cell (n = 8).

35-mm plates with 1 × 105 iPSCs derived cardiomyocytes were transferred to a recording chamber as described previously (Silva Dos Santos et al., 2018 (link)). Action potentials were recorded as described elsewhere (José et al., 2017 (link)). CM preparations were superfused with Tyrode’s solution containing (in mM): 150.8 NaCl, 5.4 KCl, 1.8 CaCl2, 1.0 MgCl2, 11.0 d-glucose, 10.0 HEPES (pH 7.4 adjusted with NaOH) saturated with oxygen at a perfusion flow rate of 0.5 ml/min (Miniplus 3, Gilson, Middleton, WI, United States ) and 37.0 ± 0.5 °C using Temperature Controller (Harvard Apparatus, Holliston, MA, United States ). Transmembrane potential was recorded using glass microelectrodes (40–80 MΩ DC resistance) filled with 2.7 M KCl connected to a Microelectrode Amplifier (MultiClamp 700B, Molecular Devices, San Jose, CA, United States ). Amplified signals were digitized (1,440 digidata A/D interface, Axon Instruments, Molecular Devices, San Jose, CA, United States ) and stored in a computer for later analysis using LabChart 7.3 software (ADInstruments, Bella Vista, NSW, Australia). The following parameters were analyzed: resting potential, maximum upstroke velocity, and action potential duration at 90% (APD90) repolarization, from three consecutive action potentials from each cell.