Optical Recording of Action Potentials in Human Induced Pluripotent Stem Cell-Derived Cardiac Single Cells and Monolayers Generated from Long QT Syndrome Type 1 Patients

Figures

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  Figure 1. Type1 long QT syndrome family background and cardiac differentiation from human iPSCs. (a) Family pedigree. The squares indicate males and the circles indicate females. Closed symbols mark patients confirmed by their DNA sequences. Hexagrams mark members who have a syncope history. The QTc values of three patients before taking a beta-blocker are stated. (b) ECG of II-2 in Figure 1(a) before the patient started taking a beta-blocker. (c) Sanger sequencing of the three patients and one control. (d) Schematic figure of KCNQ1 protein. The black circle indicates the mutation site within the transmembrane region. The lower side locates intracellular. (e) Outline of the cardiac differentiation. Lower, representative shapes of beating EBs. Scale bar, 200 μm.

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  Figure 2. Patch clamp analysis of cardiomyocytes from control- and LQT-iPSC lines. (a) Representative APs of 1 Hz paced a control-iPSC-CM and an LQT-iPSC-CM from II-2 in Figure 1(a). Ten consecutive waves are shown. (b) MDP, APA, and APD90 from cardiomyocytes derived from the six lines: 201B7 (n = 6), 409B2 (n = 5), 692D2 (n = 6), LQT1A1 (n = 5), LQT1B1 (n = 5), and LQT1C1 (n = 7). Data are represented as means ± SEM; ∗∗ p < 0.005. (c) Representative current traces from control- and LQT1-iPSC-CMs. Upper, the protocol in current clamp recording. Middle, representative traces before and after perfusion with 3R4S-chromanol 293B (30 μmol/l). Lower, 3R4S-chromanol 293B-subtraction. (d) I-V plots of IKs at the end of the depolarizing step. 692D2 (control) (n = 3), LQT1B1 (n = 3); ∗ p < 0.05.

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  Figure 3. AP measurements of single cells using a membrane potential dye. (a) Examples of AP waves from control (409B2) measured by the dye. ROIs (1)–(5) are ventricular type. ROI (5) (green wave) shows long APD. (b) Examples of AP waves from LQT1B1. ROIs (1)–(4) are ventricular type with long APD. ROI (4) (purple wave) shows EADs. ROI (5) (green wave) is nodal type. (c) Proportion of subtypes in 3 control- and 3 LQT1-iPSC-CMs: 201B7 (n = 128), 409B2 (n = 41), 692D2 (n = 92), LQT1A1 (n = 192), LQT1B1 (n = 101), and LQT1C1 (n = 202). Ventricular-type cells predominate in all six lines. (d) Representative EADs in a ventricular-type cell from LQT1B1. The EADs were defined as over 20% of the AP amplitude. Arrows show EADs. (e) Occurrence ratio of EADs in ventricular cardiomyocytes from the six lines. The number of ventricular cells was used as the denominator; ∗ p < 0.05.

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  Figure 4. AP measurements in a cardiomyocyte monolayer. Data are represented as means ± SEM. (a) An example phase contrast image of the monolayer culture. Scale bar, 500 μm. (b) Representative AP of a paced control-iPSC-CM monolayer. APD90 was calculated from the average of 10 consecutive waves. (c) APD90 from 3 control- and 3 LQT-iPSC-CM monolayers: 201B7 (n = 3), 409B2 (n = 3), 692D2 (n = 3), LQT1A1 (n = 3), LQT1B1 (n = 3), and LQT1C1 (n = 3). LQT-iPSC-CMs showed longer APD than controls; ∗∗ p < 0.005.

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  Figure 5. AP measurements in cardiomyocyte monolayers using a high-throughput plate reader. (a) Representative AP waves on a 96-well plate. Pacing at 1 Hz starts 10 s after recording. (b) Cisapride prolonged APD90 of both control- and LQT1-iPSC-CMs; 692D2 (control) (n = 3), LQT1B1 (n = 5). (c) Cisapride prolonged APD90 of both control- and LQT1-iPSC-CMs with 100 nM isoproterenol; 692D2 (n = 5), LQT1B1 (n = 3). (d) Erythromycin prolonged APD90 of both control- and LQT1-iPSC-CMs with 100 nM isoproterenol; 692D2 (n = 4), LQT1B1 (n = 3). LQT-iPSC-CM monolayers in 300 nM and 1000 nM cisapride were not synchronized at 1 Hz pacing. Data are represented as means ± SEM; ∗∗ p < 0.005, ∗ p < 0.05.