Ivabradine

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).

Episodic rhythmicity was evoked by bath application of dopamine hydrochloride (50 μM; Sigma-Aldrich). We blocked the I_Pump with ouabain (100 nM–1 μM; Tocris), and I_h – producing hyperpolarization-activated cyclic nucleotide (HCN) channels with ZD 7288 (30 – 50 μM; Tocris) or with ivabradine (1 nM – 100 μM; Sigma-Aldrich). I_Pump was potentiated by monensin [monensin sodium salt, 2 μM dissolved in ethanol (0.03%), Sigma-Aldrich, M5273]. All pharmacological agents were prepared following solubility guidelines specified by their respective vendors. We were careful to ensure that volumes of drugs prepared in dimethyl sulfoxide (DMSO) did not exceed 0.04% (vol/vol) concentration in working solutions.

Oxaliplatin (OXAL; Eloxatin^®, trans-1-diaminocyclohexane oxaliplatinum, C₈H₁₄N₂O₄Pt, (PubChem CID: 43805)) was acquired from Sanofi-Aventis (New York, NY, USA); ivabradine, protopine (4,6,7,14-tetrahydro-5-methylbis[1,3]benzodioxolo[4,5-c:5′,6′-g]azecin-13(5H)-one) was from Sigma-Aldrich (St. Louis, MO, USA); and dexmedetomidine (DEX) was from Abbott Laboratories (Abbott Park, IL, USA). All culture media, horse serum, fetal calf or bovine serum, L-glutamine, and trypsin/EDTA were obtained from Invitrogen (Carlsbad, CA, USA), unless otherwise indicated, while other chemicals, including EGTA, HEPES, LaCl₃, aspartic acid, and N-methyl-D-glucamine⁺ (NMDG⁺), were of the highest purity and analytical grade.
The composition of normal Tyrode’s solution used in this study was as follows (in mM): NaCl 136.5, KCl 5.4, CaCl₂ 1.8, MgCl₂ 0.53, glucose 5.5, and HEPES-NaOH buffer 5.5 (pH 7.4). To record I_h or I_MEP, we filled the patch electrode with the following solution (composition in mM): K-aspartate 130, KCl 20, KH₂PO₄ 1, MgCl₂ 1, Na₂ATP 3, Na₂GTP 0.1, EGTA 0.1, and HEPES-KOH buffer 5 (pH 7.2). The medium or solution was commonly filtered using a 0.22 μm pore filter.

In effort to identify the mechanism of SNAG-mGluR2 signaling in RGCs, we applied either 5 µM LY341495 (Tocris) or 300 nM Tertiapin-Q (Abcam) or 1 mM barium (Sigma) or 500 nM linopirdine (Sigma) or 50 µM ivabradine (Sigma) into the MEA recording bath. Additionally, concentrations of 150 uM pertussis toxin (PTX) were injected into the eye, 24 h later the eyes were removed and recorded from using the procedures described above. For wt retina expressing SNAP-mGluR2, the addition of 50µM L-AP4 (Sigma), and and 1 µM ACET (Tocris) was applied to the perfusion in order to block photoreceptor mediated responses.

A total of 100 mM of Ivabradine (Sigma), 100 mg/mL of puromycin (Sigma), 50 mM of sodium phenylbutyrate (4‐PBA; Santa Cruz Biotechnology) and 50 mg/mL of doxycycline hyclate (Santa Cruz Biotechnology) were dissolved in double‐distilled water. A total of 50 μM of Paclitaxel (Selleckchem) was dissolved in DMSO. Water and/or saline were used as solvent control in in vitro and in vivo experiments. Also, 10 mM of Inositol 1,4,5‐Trisphosphate (407137; Sigma) in water was used. A sum of 1 mM of Fura‐2‐AM (Invitrogen) stock solution in DMSO was prepared and 10 mg/mL of Hoechst33342 was purchased from ThermoFisher.

To evaluate the effects of ivabradine (IVB, Sigma-Aldrich) on the spontaneous network activity, we increased its concentration by direct injection into the culture medium. A wide administration scale (300 nM–30 µM) with significant points in the logarithmic scale was chosen to quantify the effects on the neuronal activity. For each concentration, the electrophysiological activity was measured for 10 min. Since the increasing concentrations of IVB were sequentially applied to the culture by directly pipetting the drug solution into the medium, we discarded the first two minutes of each phase to avoid observing mechanical effects due to the administration of the compound or seeing the transient effect due to diffusion processes. Therefore, the analyses were performed on 8-min recordings for each phase (i.e., IVB concentration).

To record firing rate and analyze autonomic response a multi-electrode array (MEA) system (Multichannel Systems, Reutlingen, Germany) was used. PCC were plated on laminin/fibronectin-coated MEAs (60MEA200/30iR-Ti, Multichannel Systems) and cultured for 3 days to achieve attachment to the electrodes. Electrical signals (extracellular field potentials) were recorded in Medium 199 (Sigma-Aldrich) supplemented with 1 mM CaCl₂ media at 37 °C and sampled at 20 kHz with the MEA_Rack software (Multichannel Systems). Signals were analyzed using Spike2 software (v7, Cambridge Electronic Design, Cambridge, UK). PCC underwent β-adrenergic (1 μM isoproterenol; Sigma-Aldrich) and muscarinic (1 μM carbachol; Sigma-Aldrich) challenge to study the autonomic capacity to modulate firing rate (n = 6, each). Moreover, different concentrations of ivabradine (1, 3, 50 μM; Sigma-Aldrich) were applied to assess effects of I_f blockage (n = 6, each).