Flecainide

Ivabradine, flecainide, amiodarone, mexiletine, quinidine, and ranolazine were from Sigma, ajmaline from MP Biomedicals. The tested concentrations were selected according to literatures and our previous studies in hiPSC-CMs (Wu et al., 2004 (link); El-Battrawy et al., 2018 (link); Salvage et al., 2018 (link); Zhao et al., 2019 (link)). Our previous studies showed that 10 µM quinidine, 10 µM Ivabradine, 30 µM ajmaline, 100 µM mexiletine, prolonged APD, 10 µM amiodarone inhibited I_Kr (El-Battrawy et al., 2018 (link); Zhao et al., 2019 (link)). ranolazine (5–30 µM) significantly reduced episodes of EADs and VT produced by ATX-II (Wu et al., 2004 (link)). flecainide of 3–30 µM was shown to inhibit Na and K channel currents (Salvage et al., 2018 (link)). Therefore, the concentrations of 10 µM quinidine, 30 µM ajmaline, 10 µM amiodarone, 10 µM Ivabradine, 30 µM flecainide, 100 µM mexiletine and 30 µM ranolazine were chosen for the study. Of note, all these drugs can affect other channel currents besides hERG channel currents (Supplementary Table 1).

In total, 32 isolated, perfused heart preparations were used in this study, in order to examine electrophysiological effects produced by Flecainide, quinidine, lidocaine, and mexiletine (8 experiments in each group). For precise dosing, drug infusions were performed at a rate of 0.3 ml/min using a calibrated infusion pump, while perfusing the hearts with protein-free saline solution at a constant coronary flow rate (see above). Flecainide (1.5 μM), quinidine (5 μM), lidocaine (5 μM), and mexiletine (5 μM) (all from Sigma-Aldrich, Germany) were infused over 30 min, at concentrations close to the maximum free (i.e. protein-unbound) therapeutic plasma levels determined in cardiac patients [6 (link), 25 (link)–30 (link)] (S1 Table).

In total, 30 heart preparations were used in this study, in order to examine effects produced by Dofetilide, quinidine, procainamide, and flecainide (7–8 experiments in each study group). For precise dosing, drug infusions were performed at a rate of 0.3 ml/min using a calibrated infusion pump, while perfusing the hearts with protein-free saline solution at a constant coronary flow rate (see above). Dofetilide (10 nM), quinidine (5 μM), procainamide (10 μM), and flecainide (1.5 μM) (all from Sigma-Aldrich, Germany) were infused over 30 min, at concentrations close to the maximum free (i.e. protein-unbound) therapeutic plasma levels [22 (link)–25 (link)] (S1 Table).

All procedures were performed in licensed institutional premises under a UK Home Office project licence approved by a university ethics review board, under the UK Animals (Scientific Procedures) Act (1986), and conforming to European Parliament Directive 2010/63/EU. 3.5- to 11.5-month-old wild-type (WT, n = 22) and RyR2-P2328S (RyR2^S/S, n = 23) inbred 129/Sv mice (Harlan, UK) were kept in plastic cages at room temperature in 12-h light/dark cycles. Mice had free access to sterile rodent chow and water. All chemical agents were purchased from Sigma-Aldrich (Poole, UK) except where otherwise indicated, with effects of flecainide studied at concentrations of 1 and 5 μm and dantrolene Na at 10 μm.

For sodium currents (I_Na), whole-cell patch-clamp recordings were obtained as described previously.⁷ (link)^,8 (link) I_Na was elicited in 100-ms steps to –30 mV from holding potentials of –100 to –70 mV. Currents were measured before and after propafenone (300 nM or 1 μM; Sigma-Aldrich, Gillingham, UK), dronedarone (5 and 10 μM; Sigma-Aldrich, Gillingham, UK), and flecainide (1 μM; Sigma-Aldrich, Gillingham, UK). These concentrations are consistent with those previously reported in the literature.⁷ (link)^,9 (link)^,10 (link) Cells were paced at 1 Hz while the drugs were introduced to the perfusate.
Action potentials (APs) were recorded at 36°–37°C in the whole-cell current-clamp configuration and triggered by 2-ms current injections (1.5 nA). AP trains were stimulated at 1 Hz. APs were recorded at RMPs from –90 to –65 mV by varying the background current injection. Action potential amplitude (APA) and peak AP phase 0 upstroke velocity (dV·dt^-1) were analyzed using modified algorithms from ElectroMap.¹¹ (link)

Dissociated spontaneously beating CMs were seeded on a coverslip and loaded with 4-µM Fluo-4 AM (Thermo Fisher Scientific). An HEPES-based medium, consisting of 137-mM NaCl, 5-mM KCl, 0.44-mM KH₂PO₄, 20-mM HEPES, 4.2-mM NaHCO₃, 5-mM D-glucose, 2-mM CaCl₂, 1.2-mM MgCl₂, and 1-Na pyruvate (pH was adjusted to 7.4 with NaOH), was used for Ca²⁺ imaging measurements at 36 °C ± 1 °C. The Ca²⁺ kinetics were photographed using an Axio Observer.A1 microscope and an Objective Fluar 20x/0.75 M27 (both Carl Zeiss Microscopy GmbH, Göttingen, Germany). Images were obtained using an ANDOR iXon3 885 EM-CCD camera (Andor Technology, Belfast, Northern Ireland) synchronized with Lambda DG-4 Plus (Sutter Instrument, California, USA) wavelength switcher, ZEISS Filter set 69 (Carl Zeiss Microscopy GmbH, Göttingen, Germany), and ZEN 2 blue edition software (Carl Zeiss Microscopy GmbH, Göttingen, Germany). In addition to baseline measurements, the effects of 1-µM adrenaline (Sigma-Aldrich) and 1-µM flecainide (Sigma-Aldrich), used to induce clinical phenotypes in the hiPSC-CMs, were studied. For the Ca²⁺ analysis, the regions of interest were selected from spontaneously beating cells, and background noise was subtracted before data processing. All recordings lasted at least 30 s, with a sampling rate of 20 to 30 Hz.

Real-time circadian bioluminescence monitoring was conducted by using adult mouse ear fibroblast cells isolated from Per2::LucSV knock-in mice by replacement of the 3′-UTR with an SV40 late poly(A) sequence as described [59 (link)]. Cells were grown to confluency on 35 mm dishes in Dulbecco’s Modified Eagle’s Medium (DMEM) medium supplemented with 10% Fetal Bovine Serum (FBS) and 1% penicillin/streptomycin, and subsequently synchronized with 200 nM dexamethasone (Dex) for 1.5 h or forskolin (Fsk, 1 or 5 µM) for 1 h [37 (link),41 (link),59 (link)]. Moricizine (Sigma–Aldrich or Santa Cruz) was then added at concentrations of 0 (vehicle control), 0.3, 1, 3, 10, 20, and 30 µM, or Flecainide (Sigma-Aldrich) at 3, 10, and 30 µM, along with luciferin-containing recording media. The dishes were then tightly sealed with vacuum grease and placed in a LumiCycle luminometer (Actimetrics) for continuous bioluminescence monitoring over six days. The data were detrended using a first-order polynomial, then best-fit to a sine wave estimated by a Levenberg–Marquardt algorithm for measurement of circadian parameters in the LumiCycle data analysis program (Actimetrics).