Electrophysiological Characterization of hiPSC-CMs

Whole-cell action potentials (APs) were recorded with patch-clamp technique, as previously-described (8 (link)). Cultured hiPSC-CMs were dissociated using TrypLE and plated as single cells on glass cover slips coated with Matrigel. Cells were placed in a RC-26C recording chamber (Warner) and mounted onto an inverted microscope (Nikon). The chamber was continuously perfused with warm (35–37°C) extracellular solution of following composition: (mM) 150 NaC1, 5.4 KCl, 1.8 CaCl₂, 1.0 MgCl₂, 1.0 Na pyruvate, 15 HEPES, and 15 glucose; pH was adjusted to 7.4 with NaOH. Glass micropipettes (2–3 MΩ tip resistance) were fabricated from standard wall borosilicate glass capillary tubes (Sutter BF 100-50-10) and filled with the following intracellular solution: 120 KCl, 1.0 MgCl₂, 10 HEPES, 10 EGTA, and 3 Mg-ATP; pH was adjusted to 7.2 with KOH. Single beating cardiomyocytes were selected and APs were recorded in whole-cell current clamp mode using an EPC-10 patch-clamp amplifier (HEKA). External solution containing 0.1% DMSO (vehicle) was applied to establish the baseline. Then, cells were treated with TKI solution containing imatinib, axitinib, nilotinib, or vandetanib (LC Labs). Data were acquired using PatchMaster software (HEKA), digitized at 1.0 kHz and analyzed using FitMaster (HEKA), Igor Pro (Wave Metrics), and Prism 5 (GraphPad). For recordings on differentiated ventricular-like hiPSC-CMs, the maximum diastolic potential (MDP) of single cardiomyocytes varied from −70 mV to −50 mV, action potential amplitude (APA) was greater than 90 mV, and action potential duration (APD)₉₀/APD₅₀ was less than 1.20. Only cardiomyocytes satisfying aforementioned criteria were ventricular-like cardiomyocytes and selected for assessing the effects of TKIs. Baseline APs were recorded for 3 minutes before application of drug and at 3, 5 and 10 minutes while keeping a continuous perfusion with drug. In a separate series of experiments, drugs were added for 2 hours at 37°C before patching. Average responses of N=10 APs were analyzed per treatment. Significant APD₉₀ prolongation is defined as >10% change in APD₉₀.