Simultaneous optical recording of action potentials and calcium transients in cardiac single cells differentiated from type 1 CPVT-iPS cells

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  FIGURE 1. Establishment of a system for simultaneous recording of action potentials (AP) and Ca2+ transients using the membrane potential-sensitive dye FluoVolt (FV) and calcium indicator Calbryte 590 (CB590) and analysis of cardiomyocytes differentiated from control human induced pluripotent stem cells (hiPSC-CMs) using this system. (A) Schematic diagram of the simultaneous recording system. Two excitation wavelengths of LED light are attenuated to 1/16 intensity by an ND filter and wavelength-limited through a dual bandpass filter before being simultaneously irradiated onto cells stained with the fluorescent dyes. The emitted light at two wavelengths passes through a dichroic filter, is split into two by a beam splitter, and is simultaneously captured by a single electron multiplying CCD (EMCCD) camera through separate bandpass filters. (B) Screenshot during recording. Recorded CB590 and FV signals in hiPSC-CMs are on the left and right sides, respectively. Regions of interest (ROIs) were obtained by encircling blinking cells. (C) Representative waveform diagram showing membrane potential and Ca2+ transient simultaneously recorded from a ventricular-like cell with normal action potential…

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  FIGURE 2. Analysis of cardiomyocytes differentiated from type 1 catecholaminergic polymorphic ventricular tachycardia (CPVT) patient-derived human induced pluripotent stem cells (CPVT1 hiPSC-CMs) using the system to record action potentials (APs) and Ca2+ transients simultaneously. (A) The waveform of Cell 1 in Supplementary Figure S2a,b. The fluorescence intensity was expressed relative to the minimum value, 0, and the maximum value, 1. Calcium double peaks and ventricular-like AP with normal AP duration (APD) are depicted. (B) The waveform of Cell 3 in Supplementary Figure S2a,b. The fluorescent intensity was expressed relative to the minimum value, 0, and the maximum value, 1. Calcium oscillation and ventricular-like AP with normal APD are depicted. (C) The left and right pie charts show the subtype classification based on the action potential waveforms of CPVT1 hiPSC-CMs and the classification of Ca2+ transients in ventricular-like cells with normal APD, respectively (n = 338). Among the subtypes, most were ventricular-like CMs with normal APD (93%). In their calcium transients, calcium oscillations accounted for a relatively large proportion, with calcium plateau abnormalities only…

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  FIGURE 3. Effects of approved clinical drugs on CPVT1 hiPSC-CMs. (A) The upper and lower panels show concentration-dependent changes in action potentials and Ca2+ transients induced by nonselective β-blocker carvedilol in two CPVT1 hiPSC-derived ventricular-like cells exhibiting calcium oscillation. Carvedilol at 0.3 µM did not improve calcium abnormalities. At 1 μM, while calcium abnormalities tended to improve (p = 0.0152), some cells showed a shortening of the plateau phase. At 3 μM, while calcium abnormalities were improved (p = 0.0476), some cells stopped beating. (B) Analysis of the rate of improvement of Ca2+ transient abnormality by carvedilol (n = 5). (C) Concentration-dependent changes in action potentials and Ca2+ transients induced by a class 1C antiarrhythmic drug, flecainide, in two CPVT1 hiPSC-derived ventricular-like cells, exhibiting calcium oscillation in the upper panel and calcium double peaks in the lower panel. Some cells showed improvement at 1 μM, while another showed a semi-response at 10 µM. (D) Analysis of the rate of improvement of Ca2+ transient abnormality by flecainide at 1 µM (n = 5) (p = 0.545) and 10 µM (n = 4) (p = 0.545). Cells that stopped beating were…

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  FIGURE 4. Effects of a RyR2 modulator, JTV519, and a Ca2+/calmodulin-dependent serine-threonine protein kinase II (CaMKII) inhibitor, KN-93, on CPVT1 hiPSC-CMs. (A) JTV519 (K201)-induced changes in action potentials and Ca2+ transients in two CPVT1 hiPSC-derived ventricular-like cells. The upper and lower panels show cells with calcium oscillation and triple peaks before drug additions, respectively. Although Ca2+ transient abnormalities tended to improve as the concentration increased, some cells showed a change in action potential to atrial-like at 3 µM. (B) Analysis of the rate of improvement of Ca2+ transient abnormality by JTV519 (K201) (n = 8). No significant difference at 0.3 (p = 0.350) or 1 µM (p = 0.350). Although a significant difference was observed at 3 µM (p = 0.0230), 80% of the cells displayed atrial-like action potential waveforms. (C) KN-93-induced changes in action potentials and Ca2+ transients in two CPVT1 hiPSC-derived ventricular-like cells. The upper and lower panels show cells with calcium oscillation and triple peaks before drug additions, respectively. Ca2+ transient abnormalities tended to improve as the concentration increased, but some cells showed asystole or…