IP3R-Mediated Compensatory Mechanism for Calcium Handling in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes With Cardiac Ryanodine Receptor Deficiency

Figures

• 

  FIGURE 1. Generation of human homozygous RYR2 knockout iPSCs (RYR2–/–-iPSCs). (A) Illustration of the RYR2 locus and gRNA1 designed for CRISPR/Cas9-mediated gene editing in exon 90 of RYR2. (B,C) DNA sequencing of Ctrl- (B) and CRISPR/Cas9-edited A3 RYR2–/–-iPSC lines (C). The A3 RYR2–/–-iPSC line displayed a single homozygous cytidine deletion in the RYR2 gene, which resulted in a premature termination codon (PTC).

• 

  FIGURE 2. Growth analysis of Ctrl- and RYR2–/–-iPSC-CMs during long-term culture. (A,B) Bright-field images of Ctrl-iPSC-CMs (A) and A3 RYR2–/–-iPSC-CMs (B) after replating at day 20 of differentiation. Ctrl-iPSC-CMs showed an increase in cell density from week 2 to week 10 post replating (A), while RYR2–/–-iPSC-CMs exhibited reduced cell density (B). Scale bar, 100 μm. (C) Quantification of the cardiomyocyte numbers. Ctrl- and A3 RYR2–/–-iPSC-CMs showed distinct growth curves during long-term cell culture. Ctrl-CMs from 2 different iPSC lines and 11 differentiation experiments (iBM76.1: n = 6 and iWTD2.1: n = 5) were analyzed. A3 RYR2–/–-CMs from six differentiation experiments were used. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001, RYR2–/– vs. Ctrl by using the two-way ANOVA with the Sidak’s multiple comparison test. (D) Percentage of Ki67-positive CMs (n = 10 samples each for Ctrl, A3 and A5, n = 300–400 cells per sample counted). (E) Cell viability of Ctrl-, A3 and A5 RYR2–/–-iPSC-CMs (Ctrl: n = 4 from two differentiation experiments; A3 RYR2–/–: n = 4 from two differentiation experiments; A5 RYR2–/–: n = 8 from two differentiation experiments). ****P < 0.0001 by using the…

• 

  FIGURE 3. Expression of RYR2 in 3-month-old Ctrl- and RYR2–/–-iPSC-CMs. (A) Western blot analysis of the full-length RYR2 and cardiac troponin T (cTNT) in Ctrl- and A3 RYR2–/–-iPSC-CMs (Ctrl: n = 3 and A3 RYR2–/–: n = 3 independent differentiation experiments). (B) Western blot analysis showing protein expression of N-terminal RYR2 and cTNT in Ctrl- and A3 RYR2–/–-iPSC-CMs (Ctrl: n = 3 and A3 RYR2–/–: n = 3 independent differentiation experiments). (C) Representative western blot analysis of RYR2 in response to treatment with MG132 and/or Bafilomycin A (BafA1) in Ctrl- and RYR2–/–-iPSC-CMs (Ctrl: n = 3, A3 RYR2–/–: n = 3 independent differentiation experiments). No degraded RYR2 proteins were detected in RYR2–/–-iPSC-CMs by inhibiting protein degradation with the proteasome and calpain inhibitor MG132 and the autophagy inhibitor BafA1. (D) Representative immunostaining of Ctrl- and RYR2–/–-iPSC-CMs using antibodies against RYR2 (green) and α-actinin (red). The cells were counterstained with DAPI (blue) to show the nucleus. Scale bar, 100 μm.

• 

  FIGURE 4. Expression of markers involved in Ca2+ signaling in RYR2–/–-iPSC-CMs compared to Ctrl-iPSC-CMs. (A) Reverse transcription-PCR analyses showing mRNA expression of transcripts for Ca2+ handling-related genes (RYR2, IP3R1, IP3R2, and CACNA1C), genes encoding sarcomeric proteins (TNNT2 and ACTN2), and the housekeeping gene GAPDH in Ctrl- and A3 RYR2–/–-iPSC-CMs (Ctrl: n = 6 and RYR2–/–-A3: n = 6 different differentiation experiments). M: DNA molecular-weight size marker. (B) Western blot showing expression of Ca2+ handling-associated proteins (IP3R, SERCA2A, and NCX1) in both Ctrl- and A3 RYR2–/–-iPSC-CMs. (C–E) Scatter dot plot showing protein levels of IP3R (C), SERCA2A (D), and NCX1 (E) normalized to GAPDH between Ctrl- and A3 RYR2–/–-iPSC-CMs (Ctrl: n = 4 and A3 RYR2–/–: n = 5 different differentiation experiments). (F) Representative immunostaining of Ctrl- and RYR2–/–-iPSC-CMs for IP3R (green) and α-actinin (red). Cells were counterstained with DAPI (blue). Scale bar, 100 μm.

• 

  FIGURE 5. RYR2–/–-iPSC-CMs showed defective Ca2+ cycling. (A,B) Ca2+ sparks in Ctrl- and A3 RYR2–/–-iPSC-CMs during β-adrenergic stimulation. (A) Spontaneous Ca2+ spark images in Ctrl-iPSC-CMs (A; ncell = 358, nspark = 1521) and A3 RYR2–/–-iPSC-CMs (B; ncell = 21, nspark = 38) after treatment with 100 nM isoproterenol. Both Ctrl- and RYR2–/–-iPSC-CMs were paced at 0.25 Hz. (C) Bar graphs summarize the key parameters of Ca2+ sparks in mean ± SEM values for Ctrl- and RYR2–/–-iPSC-CMs: spark frequency (100 μm–1s–1), amplitude (ΔF/F0), FDHM (ms), FWHM (μm) and relative SR Ca2+ leak. For A5 RYR2–/–-iPSC-CMs, ncell = 12, nspark = 20 were analyzed. ***P < 0.001 by using the one-way ANOVA with the Dunnett’s multiple comparison test. (D) Representative Ca2+ transients in spontaneously beating Ctrl- and A3 RYR2–/–-iPSC-CMs. (E) Representative abnormal Ca2+ transients in A3 RYR2–/–-iPSC-CMs. (F) Fractions of irregularly beating Ctrl- and A3- and A5 RYR2–/–-iPSC-CMs (Ctrl: CMs from six differentiation experiments; A3 RYR2–/–: CMs from four differentiation experiments; A5 RYR2–/–: CMs from three differentiation experiments. n > 15 cells per experiment). *P < 0.05 by using the one-way ANOVA with the…

• 

  FIGURE 6. Sensitivity of Ctrl- and RYR2–/–-iPSC-CMs to extracellular calcium and caffeine. (A,B) Cytosolic Ca2+ dynamics in Ctrl-iPSC-CMs (A) and A3 RYR2–/–-iPSC-CMs (B) perfused with increasing concentrations of extracellular Ca2+ ([Ca2+ ]o). Cytosolic Ca2+ dynamics in A3 RYR2–/–-iPSC-CMs (B) were inconsistent from cell to cell. (C) Frequency of Ca2+ events detected in Ctrl- and RYR2–/–-iPSC-CMs under different [Ca2+ ]o. (D) Percentage of CMs displaying Ca2+ events under different [Ca2+ ]o concentrations among the three groups (Ctrl: n = 20 cells from four differentiation experiments; A3 RYR2–/–: n = 15 cells from three differentiation experiments; A5 RYR2–/–: n = 12 cells from two differentiation experiments). (E) Representative traces of caffeine-induced Ca2+ release in Ctrl- and RYR2–/–-iPSC-CMs. (F) Relationship between Ca2+ release and caffeine concentration in CMs from the two groups. The amplitude of each caffeine-induced Ca2+ release was normalized to the maximum peak for each experiment (Ctrl: n = 20 cells from four differentiation experiments; A3 RYR2–/–: n = 6 cells from two differentiation experiments; A5 RYR2–/–: n = 6 cells from two differentiation experiments). **P < 0.01,…

• 

  FIGURE 7. Contributions of IP3R-mediated Ca2+ release to spontaneous Ca2+ transients in Ctrl- and RYR2–/–-iPSC-CMs. (A,B) Representative cytosolic Ca2+ dynamics in Ctrl- (A) and RYR2–/–-iPSC-CMs (B) before and after the addition of IP3R antagonist 2-APB (20 μM), and after the washout of the drug. (C) Pie charts depict the percentage of cells maintaining Ca2+ transients after the treatment with 2-APB for 500 s. (D,E) Changes of Ca2+ transient amplitude (D) and frequency (E) after the treatment and the following removal of 2-APB, which were normalized to those from the same cell under the basal condition (Ctrl: n = 13 cells from three differentiation experiments; A3 RYR2–/–: n = 14 cells from three differentiation experiments). (F,G) Representative cytosolic Ca2+ dynamics in Ctrl- (F) and RYR2–/–-iPSC-CMs (G) before and after the addition of IP3R inhibitor Xestospongin C (XeC;1 μM), and after the washout of the drug. (H) Pie charts depict the percentage of cells maintaining Ca2+ transients after the treatment with XeC for 200 s. (I,J) Changes of Ca2+ transient amplitude (I) and frequency (J) after the treatment and following the washout of XeC, which were normalized to those from the same…

• 

  FIGURE 8. Contributions of SERCA-mediated SR Ca2+ uptake to spontaneous Ca2+ transients in Ctrl- and RYR2–/–-iPSC-CMs. (A,B) Representative cytosolic Ca2+ dynamics in Ctrl- (A) and RYR2–/–-iPSC-CMs (B) before and after the addition of SERCA inhibitor thapsigargin (5 μM). (C) Pie charts depict the percentage of cells maintaining Ca2+ transients after the treatment with thapsigargin for 500 s. (D,E) Changes of Ca2+ transient amplitude (D) and frequency (E) after the treatment with thapsigargin, which were normalized to those from the same cell under the basal condition. (Ctrl: n = 14 cells from three differentiation experiments; A3 RYR2–/–: n = 10 cells from three differentiation experiments; A5 RYR2–/–: n = 8 cells from two differentiation experiments). *P < 0.05; ***P < 0.001; ****P < 0.0001 by using the one-way ANOVA with the Dunnett’s multiple comparison test.

• 

  FIGURE 9. Electrophysiological analysis of ICaL in Ctrl- and RYR2–/–-iPSC-CMs. (A,B) Representative ICaL traces of 3-month-old Ctrl- (A) and A3 RYR2–/–-iPSC-CMs (B). (C) Average current-voltage (I–V) curves for peak ICaL in 1-month-old Ctrl- and RYR2–/–-iPSC-CMs (Ctrl: n = 12 cells from three differentiation experiments; RYR2–/–: n = 15 cells from two differentiation experiments). (D) Average current-voltage (I–V) curves for peak ICaL in 3-month-old Ctrl- and RYR2–/–-iPSC-CMs (Ctrl: n = 32 cells from four differentiation experiments; A3 RYR2–/–: n = 24 cells from three differentiation experiments; A5 RYR2–/–: n = 18 cells from two differentiation experiments). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 RYR2–/– vs. Ctrl by the two-way ANOVA with Sidak’s multiple comparison test. (E–G) Western blot analysis of the voltage-dependent calcium channel alpha 1C subunit (Cav1.2) in Ctrl- and A3 RYR2–/–-iPSC-CMs. Protein levels of Cav1.2 normalized to EEF2 (F) and cTNT (G) in Ctrl- and A3 RYR2–/–-iPSC-CMs. Ctrl: n = 4 and A3 RYR2–/–: n = 4 independent differentiation experiments.

• 

  FIGURE 10. Analysis of action potentials in Ctrl- and RYR2–/–-iPSC-CMs. (A,B) Representative traces of APs revealed normal beating rhythm in Ctrl-iPSC-CMs (A) and arrhythmic beating in A3 RYR2–/–-iPSC-CMs (B). (C–F) Scatter dot plot comparing RMP (C), APA (D), maximal upstroke velocity Vmax (E), and beating frequency (F) between Ctrl- and RYR2–/–-iPSC-CMs (Ctrl: n = 12 cells from three differentiation experiments; A3 RYR2–/–: n = 13 cells from two differentiation experiments; A5 RYR2–/–: n = 13 cells from two differentiation experiments). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 by the one-way ANOVA with the Dunnett’s multiple comparison test.