Patient-Specific Human Induced Pluripotent Stem Cell Model Assessed with Electrical Pacing Validates S107 as a Potential Therapeutic Agent for Catecholaminergic Polymorphic Ventricular Tachycardia

Figures

• 

  Fig 1. Characterization of CPVT-hiPSCs. (A) CPVT-hiPSC colonies derived from the dermal fibroblasts of a patient with CPVT expressed pluripotency markers, shown by immunostaining. Scale bars = 200 μm. (B) CPVT-hiPSCs maintained the normal karyotype. (C) Sequencing analysis of the RYR2 gene identified the I4587V heterozygous point mutation in the CPVT-hiPSCs. (D) Hematoxylin-eosin staining of teratomas formed from CPVT-hiPSCs showed differentiation of the cells into various tissues derived from all three germ layers: pigmented epithelium (ectoderm), gut-like structures (endoderm), and cartilage tissue (mesoderm). Scale bar = 100 μm.

• 

  Fig 2. Gene expression of calcium handling proteins in hiPSC-CMs. Quantitative real-time PCR of spontaneously contracting embryoid bodies differentiated from control- and CPVT-hiPSCs showed comparable expression levels of the studied calcium handling proteins. All values are relative to the adult human heart and were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH). PCR = polymerase chain reaction

• 

  Fig 3. Ca2+ imaging of spontaneously beating hiPSC-CMs. (A) Representative tracings of Ca2+ imaging in spontaneously contracting control- and CPVT-hiPSC-CMs. (B) Cycle length variability indices, defined as the standard deviation of the cycle length/mean cycle length of spontaneously beating hiPSC-CMs showed no significant difference between control and CPVT. (C) The percentage of spontaneously contracting control- and CPVT-hiPSC-CMs presenting diastolic Ca2+ waves at baseline and after isoproterenol administration.

• 

  Fig 4. Ca2+ imaging of electrically stimulated hiPSC-CMs. (A) Representative tracings of Ca2+ imaging in control- and CPVT-hiPSC-CMs with electrical stimulation at 0.5 Hz. Arrows indicate the diastolic Ca2+ waves and arrowheads indicate the triggered activity. Vertical bars indicate the time points that the hiPSC-CMs were stimulated at 0.5 Hz. (B) The percentage of control- and CPVT-hiPSC-CMs presenting diastolic Ca2+ waves at baseline and after isoproterenol administration (at 0.5 and 1 Hz pacing). *p < 0.05.

• 

  Fig 5. Ryanodine suppressed diastolic Ca2+ waves in CPVT-hiPSC-CMs. (A) Representative tracings of Ca2+ imaging in CPVT-hiPSC-CMs with (lower) and without (upper) ryanodine. Note diastolic Ca2+ waves (arrows) without ryanodine (upper, right), however, no diastolic Ca2+ waves with ryanodine (lower, right). Vertical bars indicate the time points that the CPVT-hiPSC-CMs were stimulated at 0.5 Hz. (B) Fraction (in %) of CPVT-hiPSC-CMs that showed diastolic Ca2+ waves with and without ryanodine. *p < 0.05.

• 

  Fig 6. AP recordings of hiPSC-CMs. (A) Representative tracings of AP recordings from spontaneously beating control- and CPVT-hiPSC-CMs. Both control- and CPVT-hiPSC-CMs showed DADs. (B) No significant difference was found in the percentage of spontaneously contracting hiPSC-CMs presenting DADs between control and CPVT. (C) Representative tracings of AP recordings during 1 Hz pacing from control- and CPVT-hiPSC-CMs. CPVT-hiPSC-CMs showed DADs, especially after isoproterenol administration. (D) The percentage of control- and CPVT-hiPSC-CMs that developed DADs at 1 and 1.5 Hz pacing. *p < 0.05.

• 

  Fig 7. S107 prevented DADs in CPVT-hiPSC-CMs. (A) Representative tracings of AP recordings from CPVT-hiPSC-CMs following a 2–3 h pre-incubation with 10 μM S107. DADs were not found after isoproterenol application. (B) Fraction (in %) of CPVT-hiPSC-CMs that showed DADs after isoproterenol administration with and without S107 pre-incubation. S107 suppressed DADs in a concentration-dependent manner. *p < 0.05 versus the absence of S107.