Carbamylcholine

After overnight culture, the islets were rinsed twice with a M-KRB supplemented with 3.3 mM glucose and 0.1% BSA. In the perifusion experiments, 30 pre-incubated islets were transferred to each of the perifusion chambers [37 (link)]. The experiments were designed as follows: (1) 10-min pre-perifusion at 3.3 mM glucose; (2) 20-min perifusion at 3.3 mM glucose with/without ISV (10⁻⁷ M); (3) 40-min wash-out at 3.3 mM glucose; (4) 20-min perifusion at 16.7 mM glucose with/without ISV (10⁻⁷ M); (5) 40-min wash-out at 3.3 mM glucose; (6) 20-min perifusion at 16.7 mM glucose with 0.1 mmol/L carbamylcholine (Sigma). The flow rate was 75 μL/min. Samples were collected every 2 min.

The electrophysiological properties of the cardiomyocytes derived from iPS cells were evaluated using a microelectrode array (MEA) recording system (Multichannel Systems, Reutlingen, Germany). Beating cell clusters at day 10 post-plating were transferred onto MEA plates coated with 0.1% gelatin and 10 µg/mL fibronectin. Responsiveness to pharmacological agents was determined 4–6 days later at 37 °C in Krebs-Ringer buffer (composition in mM: 125 NaCl, 5 KCl, 1 Na₂HPO₄, 1 MgSO₄, 20 HEPES, 5.5 glucose, 2 CaCl₂; pH 7.4). The responsiveness of cells to isoproterenol hydrochloride (1–1000 nM, Sigma-Aldrich) and carbamylcholine (1–1000 nM, Sigma-Aldrich) was tested. Each cell cluster was treated with all drugs in random order and cells were allowed to recover to their baseline contraction in fresh Krebs-Ringer buffer in between drug treatments. Extracellular field potentials were recorded at baseline and 2 min after addition of drugs. Data were analysed offline with MC Rack version 4.3.5 software for beating rate, RR interval and extracellular field potential duration (FPD) as previously described^{35 (link),36 (link)}. FPD measurements were normalized (corrected FPD, cFPD) with the Bazzet correction formula: cFPD = FPD/√(RR interval).

Extracellular field potential recording of the beating colonies was performed using the microelectrode array (MEA) recording system (Multichannel Systems, Reutlingen, Germany). Beating EBs at day 10 postplating were transferred onto MEA plates precoated with 0.1% gelatin and 10 μg/mL fibronectin. Responsiveness to isoproterenol hydrochloride (1–100 nM, Sigma-Aldrich) and carbamylcholine (1–100 nM, Sigma-Aldrich) was determined 4–6 days later at 37°C in Krebs-Ringer buffer (composition in mM: 125 NaCl, 5 KCl, 1 Na2HPO4, 1 MgSO4, 20 HEPES, 5.5 glucose, and 2 CaCl2; pH 7.4). Each cell cluster was treated with all drugs in random order, and cells were allowed to recover to their baseline contraction in fresh Krebs-Ringer buffer in between drug treatments. Extracellular field potentials were recorded at baseline and 2 minutes after the addition of drugs. Data were analyzed offline with MC Rack version 4.3.5 software for the beating rate, RR interval, and extracellular field potential duration (FPD) as previously described [7 (link), 44 (link), 46 (link)]. FPD measurements were normalized (corrected FPD, cFPD) with the Bazzet correction formula: cFPD = FPD/√(RR interval).