Fluovolt

iPSC-CMs were cultured on 35-mm glass-bottom dishes (MatTek, Ashland, MA; P35G-1.5-10-C) that were precoated with Matrigel matrix at 37°C, 5% CO₂. For imaging, cells were incubated at 37°C, 5% CO₂ for 20 minutes in Tyrode solution containing a volage-sensitive fluorescent dye, FluoVolt, that responds to changes in membrane potential (Thermo Fisher Scientific; F10488). The cells were then washed 3 times in fresh Tyrode solution. During imaging, the dishes were kept in a heated 37°C stage-top environment chamber supplied with 5% CO₂. Imaging of voltage-indicated cellular APD was taken under a ×40 water objective using a Nikon Eclipse Ti light microscope (Nikon, Tokyo, Japan). Time-lapse videos of multiple, individual beating iPSC-CMs, paced at 1 Hz were recorded at a speed of 20 ms per frame for 20 seconds at 10% LED power. Single regions of interest were selected for every beating iPSC-CM captured in the recordings. The raw data were exported to Excel software (Microsoft, Redmond, WA) and then analyzed with an “in-lab” developed Excel-based program.¹¹

Voltage experiments were conducted on day 11. The microtissues were washed in serum-free medium consisting of DMEM (Gibco, Paislay, UK) supplemented with 10 mM D-Galactose (Sigma) and 1 mM sodium pyruvate (Sigma). The microtissues were loaded with voltage-sensitive dye: 0.1% FluoVolt and 1% PowerLoad (ThermoFisher, Waltham, WA, USA) in the above listed serum-free medium for 25 min at 37 °C 5% CO₂. The voltage-sensitive dye was removed by washing in serum-free medium, and the multiwell plate was placed in an environmentally controlled stage incubator (37 °C, 5% CO₂, >75% humidity) in the CellOPTIQ^® platform (Clyde Biosciences, BioCity Scotland) 30 min before experimentation. The fluorescent signal was recorded with an excitation at 470 nm, and emitted light was collected from the entire microtissue using a 10× Fluor objective and the intensity recorded by a photomultiplier tube at 510–560 nm at 10 kHz. A 15 s recording was taken of each microtissue. Offline analysis was performed using CellOPTIQ^®.

Coverslips containing iPSC-CM were transferred to a 37 ± 0.5°C heated chamber with bath solution containing (in mmol/L): CaCl₂ 2, Glucose 10, HEPES 10, KCl 4, MgCl₂ 1, NaCl 140; pH = 7.35 adjusted with NaOH. Cells were loaded with 0.1 × FluoVolt (Thermo Scientific; 20 min loading). Intact cells were electrically field-stimulated at 1 Hz with 3–5 ms bipolar pulses at voltages ∼25% above the contraction threshold (normally between 10 and 30 V). AP were recorded optically at λ_ex = 470 nm and λ_em = 535 nm. Three AP from each iPSC-CM were ensemble averaged during offline analysis of AP parameters with Clampfit 10.7 (Molecular Devices, CA, USA).³²

hiPSC-CMs were seeded at a density of approximately 150.000 per Geltrex-coated coverslip, which facilitated the formation of cell clusters. Coverslips were incubated with Powerload and FluoVolt (ThermoFischer F10488, 1:1000, Waltham, MA, USA) in complete RMPI medium at 37 °C for 20 min. Action potentials were recorded at 37 °C, during which the coverslips were placed in a bath solution containing (mM): NaCl (130), KCl (4), CaCl₂ (1.8), MgCl₂ (1.2), NaHCO₃ (18), HEPES (10), and glucose (10), with a pH of 7.4. Fluorescent dye signals were recorded on a custom-built microscope (Cairn Research, Faversham, UK) using a 10× objective. Blue light was filtered using a 482/35 excitation filter and projected onto the objective with a 515-nm dichroic mirror. Fluorescent signals were captured via a 514 long-pass emission filter by a high-speed camera (Andor Zyla 5.5.CL3, Oxford Instruments, Abingdon, UK). Analysis of the data was performed using Fiji and Peaks, a custom-written Matlab script (DOI: 10.17605/OSF.IO/86UFE). APD of spontaneously beating hiPSC-CMs was determined at 90% repolarization and then adjusted for the beating rate using a modified Fredericia’s correction: APDcorrected = APD/(∛(60/BPM).

When loading FluoVolt (FV; Thermo Fisher Scientific), the medium was exchanged with modified Tyrode's solution and FV (0.1% volume). Pluronic surfactants were not used in this study. Twenty minutes after loading, the medium was refreshed with modified Tyrode's solution and used in each assay.

The voltage dye staining was performed according to the manufacturer’s protocol (FluoVolt™, Thermo Fisher Scientific). Five microliters of FluoVolt^TM dye was diluted in 50 µL PowerLoad and then suspended in 5 mL of high glucose DMEM (Sigma-Aldrich) preheated to 37 °C. Then, 200 µL of the dye solution was applied to the well containing SOTRs for 15 min. After removing the dye solution, the tissues were washed with DMEM twice.