a) Immunocytochemistry of human cardiomyocytes (CMs) generated from control (left) and TS iPSCs (right) using anti-α-Actinin antibodies (red). The nuclei (blue) are marked by Hoechst staining. Insets show high magnification images of the sarcomeres. Scale bar, 10 μm. b) Voltage-clamp recording of Ba²⁺ currents in control (black) and TS (red) CMs show a defect in voltage-dependent channel inactivation (VDI) following a voltage pulse from −90 to −10 mV. c) VDI in control and TS CMs 350 ms after the start of the pulse (**P<0.01; students T test). d) The IV relationship of TS (●) and control (○) Ca²⁺ currents (mean ± s.e.m.) are statistically identical. There were no significant differences in the peak amplitude of Ba²⁺ currents between control and TS CMs (data not shown). e) Ba²⁺ current in control and TS myocytes stimulated with a test pulse to +10 mV following a family of prepulses from −110 to +30 mV in 10 mV increments (raw traces in Supplementary Fig. 4c, control, n=23 cells in 4 lines; TS, n=19 in 4 lines, mean ± s.e.m.). f) Spontaneous action potentials (AP) in control and TS ventricular-like myocytes measured in current-clamp mode. Boxes show the regions indicated by underlines at an expanded time scale. Arrowheads show putative delayed afterdepolarizations (DADs). Dashed lines show 0 mV. g) AP duration in TS and control ventricular CMs. The expression of the ventricular marker, MLC2v, was confirmed with single-cell RT-PCR immediately after whole-cell patch recording (Supplementary Fig. 5). h) Putative DADs in TS and control ventricular-like cardiomyocytes (control, n=22 cells in 4 lines; TS, n=14 in 4 lines, mean ± s.e.m.). Statistical analyses were performed with Student’s t-test (**P<0.01).

a) Spontaneous Ca²⁺ transients in TS CMs before (black) and after treatment with 33.3 μM Ros (red) as well as after wash out (blue). Arrowheads show irregular Ca²⁺ elevations. b) Effects of Ros on the relative irregularity of the amplitude and timing of the spontaneous Ca²⁺ transients in TS CMs (n=8 cells in 2 lines, *P<0.05, **P<0.01). c) Ba²⁺ currents in TS CMs recorded in voltage-clamp mode before (black) during (red) and after (blue) treatment with 33.3 μM Ros. Ros promoted inactivation of currents in TS CMs. d) Effects of Ros on CaV1.2 VDI in CMs (n=5 cells in 2 lines, **P<0.01). e) Spontaneous APs recorded in current-clamp recording before, during and after treatment with Ros. Arrowheads show putative DADs. f) Ros prevented AP prolongation observed in TS CMs (n=8 cells in 2 lines, **P<0.01, mean ± s.e.m.).

a) Phase contrast images of iPSC line (9862-61) derived from a TS patient. Scale bar, 400 μm. b) Karyogram of TS iPSCs (7643-5). c) Images of spontaneously contracting embryoid bodies (EBs) generated from control (Con, left) and TS iPSCs (right). Scale bar, 100 μm. d) Examination of pluripotent and cardiac gene expression using RT-PCR with primer sets for pluripotent gene (NANOG), cardiac markers (β-MHC and ANP), CaV1.2 channels (exon 8 and 8a) and house keeping gene (β-actin). e) Relative motion of contracting control and TS EBs. Arrowheads show missing contractions. f) Contraction rate of TS and control EBs (control, n=85 EBs in 5 lines; TS, n=113 in 5 lines, mean ± s.e.m.). g) Fraction of TS and control EBs showing arrhythmic contractions (control, n=5 lines, 85 EBs; TS, n=5 lines, 113 EBs, mean ± s.e.m.). h) Histogram of inter-contraction intervals for control EBs (black column, n=3,241 contractions in 5 lines) and TS EBs (red column, n=3,998 in 5 lines, mean ± s.d.). Statistical analyses were conducted using Student’s t-test (*P<0.05, **P<0.01).

Representative line-scan images (a, c, top) and spontaneous Ca²⁺ transients (a, c, bottom) in control (left) and TS CMs (right). TS CMs showed more irregular timing (b) and amplitude (d) of spontaneous Ca²⁺ transients compared to control cells (see Supplementary Fig. 6 and Methods for details about the analysis, control, n=102 cells in 4 lines; TS, n=149 in 4 lines, mean ± s.e.m.; *P<0.05, **P<0.01., students T-test).