Calcium Signaling Consequences of RyR2-S4938F Mutation Expressed in Human iPSC-Derived Cardiomyocytes

Figures

• 

  Figure 1. Introduction of S4938F mutation into the ryanodine receptor 2 (RYR2) gene of hiPSC-CMs. (A) Schematic of gene editing showing RYR2 gene was digested by Cas9 exonuclease at the target sequence. (B) The targeted locus of the genome was amplified by polymerase chain reaction (PCR) with primers F6 and R4 (see Section 4.2), followed by restriction digestion by HpyCH4IV. Total size of unrestricted PCR was 527 bp. WT PCR product was cut into two fragments by the enzyme indicated with red triangle (119 + 408 bp). The larger fragment was cut into two fragments in the PCR product of homozygous S4938F mutant, resulting in total of three digested fragments (119 + 161 + 247 bp). (C) Sequencing of PCR products show S4938F (TCT to TTT) mutation (*) and a silent mutation creating a new HpyCHIV4 restriction enzyme site (#).

• 

  Figure 2. Spontaneous cytoplasmic and SR Ca2+ oscillations in intact WT and S4938F-RyR2 mutant hiPSC-CMs. (A) Representative traces of spontaneous Ca2+ transients measured in WT and S4938F mutant hiPSC-CMs incubated with Fluo-4 AM showing increased spontaneous frequency and decreased Ca2+ transients in S4938F. (B) Quantified data show that S4938F-RyR2 mutant hiPSC-CMs exhibited decreased spontaneous Ca2+ transients as compared with WT hiPSC-CMs (0.92 ± 0.09 ΔF/F0 in S4938F vs. 1.23 ± 0.11 ΔF/F0 in WT, n = 39–36, p = 0.027). (C) Representative traces of spontaneous SR Ca2+ releases and accompanying Ca2+ transients in WT and S4938F hiPSC-CMs. SR Ca2+ release was detected by R-CEPIA1-ER (red) and the cytosolic Ca2+ levels were monitored using Fluo-4 AM (green). (D) Quantification of spontaneous SR Ca2+ release and cytosolic Ca2+ changes show greatly reduced SR Ca2+ release in the mutant cells (0.02 ± 0.002 ΔF/F0 in S4938F vs. 0.05 ± 0.006 ΔF/F0 in WT, n = 19–19, p = 1.39 × 10−5) with suppressed cytosolic Ca2+ rise compared with those of WT hiPSC-CMS (0.95 ± 0.10 ΔF/F0 in S4938F vs. 1.48 ± 0.23 ΔF/F0 in WT, n = 19–19, p = 0.04). (E) Spontaneous Ca2+ transients of Fluo-4 AM loaded mutant cells…

• 

  Figure 3. Sarcoplasmic reticulum Ca2+ content measured by 5 mM caffeine. (A) Representative traces and quantified data of 5 mM caffeine-triggered Ca2+ transients measured by Fluo-4 AM in intact WT and S4938F mutant hiPSC-CMs. Caffeine-induced Ca2+ transients were significantly higher in S4938F cells (1.73 ± 0.15 ΔF/F0 vs. 1.12 ± 0.1 ΔF/F0, n = 38–44, p = 0.0007). (B) Simultaneously measured caffeine-triggered cytosolic Ca2+ transient (green) and SR Ca2+ release (red) also showed higher Ca2+ release (0.25 ± 0.019 ΔF/F0 vs. 0.18 ± 0.016 ΔF/F0, n = 31–22, p = 0.009) and cytosolic Ca2+ rise in S4938F cells compared with those of WT cells (2.74 ± 0.16 ΔF/F0 vs. 1.88 ± 0.24 ΔF/F0, n = 31–22, p = 0.003). (C) Quantified data and representative traces show that fractional release of Ca2+ as the ratio of spontaneous Ca2+ release to caffeine-induced Ca2+ release was significantly smaller in S4938F mutant cells (R-CEPIA1er: 0.08 ± 0.01 vs. 0.31 ± 0.08, n = 14–11, p = 0.003; Fluo-4 AM: 0.52 ± 0.04 vs. 0.98 ± 0.09, n = 14–11, p = 4.6 × 10−5). Data are shown as scatter plots with mean ± SEM, ** <0.01.

• 

  Figure 4. INCX and caffeine-induced Ca2+ release from patch-clamped and TIRF-imaged WT and S4938F mutant hiPSC-CMs. (A) Representative traces of INCX from WT and S4938F mutant cells. (B) Quantified INCX densities from WT and mutant cells. Scatter plots show that both peak INCX (5.42 ± 1.03 pA/pF vs. 2.45 ± 0.2 pA/pF, n = 10–20, p = 0.0005) and the carried Ca2+ by INCX (14.5 ± 4.4 pC/pF vs. 6.8 ± 0.8 pC/pF, n = 8–17, p = 0.02) were significantly higher in S4938F mutant hiPS-CMs compared with those of WT cells in 5 mM extracellular calcium. (C) Representative traces of caffeine-induced SR Ca2+ release (ER-GCaMP6, green) and cytosolic Ca2+ rise (Fura-2, blue) in WT and S4938F mutant CMs. (D) Scatter plots depict no significant difference in the caffeine-induced Ca2+ release or Ca2+ transients in patched conditions compared with WT (0.34 ± 0.06 ΔF/F0 in S4938F vs. 0.39 ± 0.03 ΔF/F0 in WT, n = 10–20, p = 0.4, and 0.27 ± 0.05 ΔF/F0 in S4938F vs. 0.26 ± 0.02 ΔF/F0 in WT, n = 10–20, p = 0.7, respectively). Data are shown as scatter plots with mean ± SEM. * p < 0.05, ** p < 0.01.

• 

  Figure 5. L-type Ca2+ current (ICa) and Ca2+-induced Ca2+ release (CICR). (A) Representative traces of ICa recorded on depolarizations to zero mV from holding potential of –50 mV in WT and S4938F mutant hiPSC-CMs. (B) Quantified ICa density (bottom panel) and ICa inactivation (top panel) from WT and S4938F-RyR2 mutant hiPSC-CMs. ICa density was significantly smaller in S4938F hiPSC-CMs in 2 mM extracellular Ca2+ solutions (12.35 ± 1.1 pA/pF vs. 16.62 ± 1.05 pA/pF, n = 22–17, p = 0.009), and the current inactivated significantly slower (inactivation tau: 18.74 ± 1.45 ms in S4938F vs. 10.41 ± 0.82 ms in WT, n = 20–17, p = 2.3 × 10−5). Increasing the extracellular Ca2+ concentration from 2 mM to 5 mM, increased the ICa density in both cell types (21.0 ± 2.05 pA/pF in S4938F vs. 19.6 ± 2.4 pA/pF in WT, n = 23–24, p = 0.66). (C) Representative traces of ICa-activated Ca2+ transients and SR Ca2+ release in WT and S4938F hiPSC-CMs measured by Fura2 pentasodium salt (blue) and SR targeted probe (ER-GCaMP6, green). (D) CICR was suppressed in S4938F hiPSC-CMs compared with levels in WT hiPSC-CMs (0 ΔF/F0 in S4938F vs. 0.07 ± 0.02 ΔF/F0 in WT, n = 22–17, p = 0.0001). Increasing the extracellular Ca2+…

• 

  Figure 6. Ca2+ sparks recorded from WT and S4938F-RyR2 mutant hiPSC-CMs. (A) Representative time courses of normalized Ca2+ sparks from different color-coded regions in WT and S4938F mutant cells. (B) Scatter plot shows significantly higher spark frequency of S4938F mutant hiPSC-CMs compared with WT cells (29.0 spark/s in S4938F vs. 15.7 spark/s in WT, n = 55–24, p = 2.9 × 10−5). (C) Histogram and distribution curves depict the spark duration measured at half-maximum amplitude (63.08 ± 1.17 ms in S4938F vs. 74.30 ± 2.86 ms in WT, n = 212–662, p = 2.17 × 10−5). (D) Total internal reflection fluorescence images show the evolution of the # labeled sparks from WT and S4938F mutant hiPSC-CMs. Data are shown as scatter plots with mean ± SEM, ** p < 0.01.

• 

  Figure 7. Ca2+ leak and load levels in WT and S4938F mutant hiPSC-CMs. (A) Representative time courses of normalized Ca2+ fluorescence changes in spontaneously beating WT and S4938F mutant hiPSC-CMs after rapid exposure to zero Ca2+/Na+ solution containing 1 mM tetracaine followed by 5 mM caffeine. (B) Scatter plots show decreased raw sarcoplasmic reticulum (SR) Ca2+ leak levels (0.09 ± 0.01 ΔF/F0 vs. 0.18 ± 0.02 ΔF/F0, n = 26–31, p = 0.0007) and decreased SR Ca2+ levels normalized to the SR Ca2+ store size (0.04 ± 0.005 ΔF/F0 vs. 0.14 ± 0.012 ΔF/F0, n = 26–31, p = 2.7 × 10−9). (C) Along with the reduced SR Ca2+ leak, S4938F cells showed higher SR Ca2+ load triggered by caffeine (2.27 ± 0.15 ΔF/F0 vs. 1.35 ± 0.13 ΔF/F0, n = 26–31, p = 1.94 × 10−5). Data are shown as scatter plots with mean ± SEM, ** p < 0.01.