Using induced pluripotent stem cells to investigate cardiac phenotypes in Timothy syndrome

Abstract

Individuals with congenital or acquired prolongation of the QT interval, or long QT syndrome (LQTS), are at risk of life-threatening ventricular arrhythmia. LQTS is commonly genetic in origin but can also be caused or exacerbated by environmental factors. A missense mutation in the L-type calcium channel Ca(V)1.2 leads to LQTS in patients with Timothy syndrome. To explore the effect of the Timothy syndrome mutation on the electrical activity and contraction of human cardiomyocytes, we reprogrammed human skin cells from Timothy syndrome patients to generate induced pluripotent stem cells, and differentiated these cells into cardiomyocytes. Electrophysiological recording and calcium (Ca(2+)) imaging studies of these cells revealed irregular contraction, excess Ca(2+) influx, prolonged action potentials, irregular electrical activity and abnormal calcium transients in ventricular-like cells. We found that roscovitine, a compound that increases the voltage-dependent inactivation of Ca(V)1.2 (refs 6-8), restored the electrical and Ca(2+) signalling properties of cardiomyocytes from Timothy syndrome patients. This study provides new opportunities for studying the molecular and cellular mechanisms of cardiac arrhythmias in humans, and provides a robust assay for developing new drugs to treat these diseases.

Figure 1. Generation of cardiomyocytes from control and TS iPSCs

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).

Figure 2. Electrophysiological features of TS cardiomyocytes

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).

Figure 3. Ca²⁺ signaling in TS and control cardiomyocytes

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).

Figure 4. Roscovitine rescues the cellular phenotypes of TS cardiomyocytes

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.).