E 4031

E-4031 and Dofetilide were obtained from Sigma-Aldrich. E-4031 stock solution (10 mM) was prepared by dissolving in distilled H₂O; Dofetilide was dissolved in DMSO to make a 10mM stock. Hearts were dissected from both Wt^CS and sei^ts1 mutant flies, allowed to equilibrate for 30 min in oxygenated AHL and were then filmed for 30sec (T₀). The hemolymph was replaced with AHL containing either 1μM E-4031 or 1 μM Dofetilide. For E4032 experiments a second set of hearts were filmed using AHL as the “vehicle” and for Dofetilide an equivalent amount of DMSO without drug was added to the AHL. Hearts were filmed again for 30sec following a 15 min exposure to either drug or vehicle.

A single mold of microtissues was mounted on a temperature-controlled chamber (Dual Automatic Temperature Controller TC-344B, Warner Instrument) to maintain 35 ± 1 °C and bathed with Tyrode’s solution containing (in mM) 140 NaCl, 5.1 KCl, 1 MgCl₂, 1 CaCl₂, 0.33 NaH₂PO₄, 5 HEPES, and 7.5 glucose. Microtissues were stimulated with a platinum field stimulation electrode (Supplemental Fig. 7, Myopacer EP field stimulator, IonOptix, Milton, MA). The test compounds including E4031, 4-AP, BayK8644, ISO were purchased from Sigma Aldrich and dissolved in 100% DMSO to prepare 0.01–0.5 mM stock solutions. BPA was dissolved in 10% ethanol stock solution and diluted in Tyrode solution to the final concentration. Microtissues were exposed to vehicle (DMSO or ethanol) and the indicated concentrations of test compounds for 20 min, and action potentials were measured as described above.

Vernakalant was from Sigma-Aldrich (Taufkirchen, Germany). The drug was applied to a cell by a perfusion pipette, from low to high concentrations. The used concentrations were determined according to previous or our preliminary studies in hiPSC-CMs. E-4031, chromanol 293B, nifedipine, NiCl₂, glibenclamide, niflunic acid, lidocaine and dihydroouabain were from Sigma-Aldrich, 4-AP from RBI, apamin from Alomone Labs, tetrodotoxin (TTX) from Carl Roth (Karlsruhe, Germany). E-4031, NiCl₂, TTX, 4-AP, apamin, niflumic acid and dihydrooubain were dissolved in H₂O. nifedipine, and chromanol 293B were dissolved in DMSO, lidocaine in ethanol. Stock solutions were kept at −20 °C.

The ability of cardiovascular construct to conduct electrical signal was analyzed using the MEA system (Multi Channel Systems MCS GmbH, Reutlingen, Germany). The MEA platforms (8 × 8 standard MEAs or 6-well MEAs) were first hydrophilized with FBS and then coated with 0.1% gelatin type A (Sigma-Aldrich).
Field potentials were recorded at day 10 or 18 (Fig. 1) at 37 °C, and signals were recorded for 2 min. The sampling frequency was 20 kHz. Field potentials were recorded during spontaneous baseline beating, and with 1 µM adrenaline (Sigma-Aldrich) or 300 nM E-4031 (Sigma-Aldrich), which were incubated 2 min before measurements. Drugs were diluted and measurements performed in EB 5% medium. The field potentials were recorded with MC_Rack v.4.5.7 software (Multi Channel Systems MCS GmbH). Signals were analyzed with Cardiomyocyte MEA Data Analysis (CardioMDA) software (Pradhapan et al. 2013 (link)).

Disopyramide is from SigmaAldrich. The drug was applied to a cell sequentially from low to high concentrations by a perfusion pipette. The tested concentrations were selected according to previous or our preliminary studies in hiPSC-CMs. E-4031, chromanol 293B, nifedipine, NiCl₂, niflunic acid, lidocaine, and dihydroouabain are from Sigma Aldrich, 4-AP from RBI, apamin from Alomone Labs, TTX from Carl Roth. E-4031, NiCl₂, TTX, 4-AP, apamin, niflumic acid, and dihydrooubain were dissolved in H₂O. nifedipine, and chromanol 293B were dissolved in DMSO, lidocaine in ethanol. Stock solutions were kept at -20 °C.