In vitro cardiotoxicity of methadone and metabolites in hiPSC-CM using the MEA

The MEA system of Multi Channel System (MCS GmbH, Ruetlingen, Germany) combined with Pluricyte^® Cardiomyocytes was used to detect the cardiotoxicity of methadone, and the metabolites EDDP and EMDP. The Pluricyte^® Cardiomyocytes were thawed and seeded on the six-well MEA chips (60-6well MEA200/30iR-Ti-tcr, MCS GmbH) according to the manufacturer’s protocol. Briefly, each well of the MEA chips was precoated with 50 μg/ml fibronectin for 3 h in the incubator at 37 °C with 5% CO₂. The fibronectin coating solution was aspirated before seeding. Cells were thawed in the incubator at 37 °C for exactly 4 min and carefully transferred to a 50-ml tube. The original vial was rinsed with serum free Pluricyte^® Cardiomyocyte Medium and added drop-wise to the tube containing the cardiomyocytes. Subsequently, cell counting was manually performed, using 20 μl of obtained homogenous cell suspension in a Buerker-Tuerk Counting Chamber (Marienfeld Superior GmbH & Co. KG, Lauda-Königshofen, Germany) and at the same time the remaining cells were centrifuged at 300g for 3 min. Then, the supernatant was removed and medium was drop-wisely added to reach the aimed concentration of cells in the suspension (10⁴ cells/μl). 2-μl cell suspension per well was placed on the six-well MEA chips in a density of 10⁴ cells/μl. After 3-h incubation (37 °C, 5% CO₂), 200 μl of medium was filled into each well of the MEA chips which were subsequently incubated at 37 °C with 5% CO₂ and refreshed with medium every 2 days.

At 7–8 days after seeding, MEA chips were placed on the headstage of a MEA2100-system (MCS GmbH) integrated with the chamber providing a stable atmosphere (37 °C, 5% CO₂) to record the extracellular field potential (Fig. 2) of spontaneous beating hiPSC-CM. After an equilibration time of 20 min, half of the medium (100 μl) in each well was replaced by culture medium containing 0.2% (v/v) DMSO to reach a final concentration of 0.1% (v/v) DMSO, which was used as baseline condition. Subsequently, the model compounds were tested in separate wells, and each test compound was cumulatively added to the well with increasing concentrations in the same way (Harris et al. 2013; Nozaki et al. 2017; Ando et al. 2017). At each concentration, the extracellular field potential was recorded for 1 min after 10-min exposure. Stock solutions of model compounds were prepared in DMSO and further diluted in Pluricyte^® Cardiomyocyte medium to make exposure medium with the final concentration of 0.1% (v/v) DMSO. The following concentrations were tested, 0.01, 0.03, 0.1, 0.2, 0.3, 0.4, 1, 3, 10, 30 µM (methadone), 0.01, 0.03, 0.1, 0.3, 1, 3, 10, 30 µM (EDDP) and 0.1, 0.3, 1, 3, 10, 30 µM (EMDP), at which no cytotoxicity was observed (data not shown). The test concentrations of methadone were based on reported human methadone plasma concentrations that were observed after oral methadone treatment. Same test concentrations were chosen for EDDP and EMDP, which enables definition of concentration-dependent curves for EDDP and EMDP that allow potency comparison.

Typical extracellular field potential waveform consisting of a rapid upstroke corresponding to depolarization, a slow wave/plateau and a repolarization peak. Signals were generated under the baseline condition (0.1% (v/v) DMSO) in Pluricyte cardiomyocytes cultured in six-well MEA-chips measured by the MEA2100-System platform of MCS

One well of 0.1% (v/v) DMSO on each MEA chip was used as the vehicle control well and run at the same time as the compound exposure wells to correct for time- and DMSO-dependent effects on the field potential. A detailed exposure scheme can be found in Fig. S1. Data were collected using Cardio 2D software (MCS GmbH) with a sample frequency of 10 kHz and a 0.1–3.5-kHz band-pass filter.

After exposure, MEA data generated from the electrodes showing stable baseline field potential with clearly visible depolarization (peak amplitude ≥ 200 μV) and repolarization peaks (peak amplitude ≥ 20 μV) (Ando et al. 2017; Sala et al. 2017) were selected for further analysis using Multiwell-Analyzer software Version 1.5.1.0 (MCS GmbH). Field potential duration (FPD) was defined as duration between the beginning of the sodium spike and the repolarizing peak (Fig. 2). RR-intervals were defined as the duration between two depolarization peaks (Fig. 2). The FPD and RR-interval were measured as the average of at least 30 beats from 1-min recording at each concentration of the test compound. In addition, the Fridericia formula (Eq. 1) was applied to correct for the effect of beat rate on FPD (Vandenberk et al. 2016) as widely used in other MEA studies (Ando et al. 2017; Kitaguchi et al. 2017):

In this formula, the FPD and RR-interval are expressed in seconds. Data were collected from at least three independent experiments (4–8 wells, 26–38 electrodes), using a new vial of cells (all from the same batch) at each independent experiment. In vitro cardiotoxic effects are expressed as relative percentage of FPDc compared to the FPDc results obtained for the baseline condition [0.1% (v/v) DMSO] and further corrected for the time- and DMSO-dependent effects by subtracting the response of 0.1% (v/v) DMSO obtained from the corresponding time-matched vehicle control well. The concentrations inducing irregularities in the field potential trace (Fig. S2) were also noted. Such irregularities included arrhythmia-type changes in the waveform, a flattened unclear second peak and/or beating arrest (Asakura et al. 2015; Kitaguchi et al. 2017; Nakamura et al. 2014; Zwartsen et al. 2019). Concentrations inducing these irregularities were excluded from the FPD analysis since the FPD and RR-interval could not be determined.

Effective concentrations expressed as the FPDc (% to the baseline control) were determined using the benchmark dose (BMD) approach which was performed as described in the “Evaluation of the PBK modeling-based reverse dosimetry approach”. The concentration–response curves were plotted with GraphPad Prism 5.0 using the four-parameters logistic fit (GraphPad Software Inc., San Diego, USA). Each data point is presented as the mean value of at least three independent experiments ± standard deviation (SD). Statistical significance of the changes in response of cells exposed to the compound compared to the solvent control was analyzed by one-way ANOVA followed by post Dunnett test. Values of p < 0.05 were regarded as statistically significant (p < 0.05: *, p < 0.01: ** and p < 0.001: ***). Statistical analysis was performed by GraphPad Prism 5.0 (GraphPad Software Inc.).