Altered endoplasmic reticulum calcium loading in human PLN-R14del cardiomyopathy

FIGURE 1. CEPIAer immunofluorescence. Immunofluorescence staining of 2D hiPSC-CMs after transduction with the CEPIAer sensor (green), staining using antibodies against alpha-actinin, the ER-resident protein calnexin, and SERCA2 (red, as indicated), along with DAPI staining (blue); scale bar 50 µm.

FIGURE 2. Simultaneous recording of CEPIAer fluorescence intensity and force (A–C). (A) Average contraction (blue) and not baseline-corrected fluorescence intensity (cyan) of electrically stimulated EHTs from the control hiPSC line. (B) Same data plotted with baseline-corrected fluorescence intensity. (C) Average contraction (blue) and not baseline-corrected fluorescence intensity (cyan) of electrically stimulated control EHTs in the presence of myosin inhibitor butanedione monoxime (BDM, 10 mM). n = 7–8 EHTs per condition. Data are expressed as the mean. (D) Original baseline-corrected recording at baseline and (E) in the presence of BDM (10 mM). Vertical blue lines: electrical pacing signal; blue curve: non-filtered force curve; red line: Gaussian-filtered force curve; pink line: velocity of contraction/relaxation; cyan line: baseline-corrected CEPIAer fluorescence intensity.

FIGURE 3. Drug response to cyclopiazonic acid, thapsigargin, and ryanodine. Effects on CEPIAer fluorescence intensity (left), force (middle), and frequency (right) of spontaneously beating hiPSC-CM EHTs from control (O), PLNic (O), and PLN-R14del hiPSC line (O). Data are plotted relative to mean baseline and normalized to time/vehicle control (TVC) per EHT batch. (A) Cyclopiazonic acid (10.0 µM; control: n = 15 EHT; TVC = 14 EHT; two batches; PLNic: n = 23 EHT; TVC = 23 EHT; three batches; PLN-R14del: n = 32 EHT; TVC = 22 EHT; three batches). (B) Thapsigargin (1.0 µM; control: n = 16 EHT; TVC = 14 EHT; two batches; PLNic: n = 16 EHT; TVC = 11 EHT; two batches; PLN-R14del: n = 19 EHT; TVC = 20 EHT; three batches). (C) Ryanodine (10.0 µM; control: n = 14 EHT; TVC = 13 EHT; two batches; PLNic: n = 25 EHT; TVC = 20 EHT; three batches; PLN-R14del: n = 21 EHT; TVC = 19 EHT; three batches). (D) Ryanodine (200.0 µM; control: n = 8 EHT; TVC = 8 EHT; two batches; PLNic: n = 7 EHT; TVC = 8 EHT; two batches; PLN-R14del: n = 8 EHT; TVC = 8 EHT; one batch). One-way ANOVA versus baseline with Dunnett’s post-test, *p < 0.05. Mean ± SEM. Asterisk color code indicates the significance for each hiPSC line.…

FIGURE 4. Drug response to ATP, tetracaine, and istaroxime. (A) Effects of ATP on CEPIAer fluorescence intensity (left), force (middle), and frequency (right) of spontaneously beating hiPSC-CM EHTs from control (O), PLNic (O), and PLN-R14del hiPSC line (O). Data are plotted relative to mean baseline and normalized to TVC per EHT batch. ATP (10.0 mM; n = 35 EHT; TVC = 30 EHT; four batches; PLNic: n = 16 EHT; TVC = 15 EHT; two batches; PLN-R14del: n = 25 EHT; TVC = 23 EHT; three batches). (B) Effects of tetracaine (50 µM) on hiPSC-CM EHTs from control hiPSC line (O). Tetracaine (50.0 µM; n = 20 EHT; TVC = 17 EHT; two batches)—for the tetracaine experiment, extracellular calcium concentration was increased to 5 mM after the 20-min time point, as indicated in the graphs. (C, D) Effects of istaroxime on hiPSC-CM EHTs from control hiPSC line (O). (C) Istaroxime (1.0 µM; control: n = 18 EHT; TVC = 17 EHT; three batches). (D) Istaroxime (4.0 µM; n = 28 EHT; TVC = 23 EHT; four batches. One-way ANOVA versus baseline with Dunnett’s post-test, *p < 0.05. Mean ± SEM. Asterisk color code indicates the significance for each hiPSC line. The scatter plots of the same data for the different cell lines are…

FIGURE 5. Drug response to calcium (5 mM) and isoprenaline. Effects of calcium (5 mM) and isoprenaline (100 nM) on CEPIAer fluorescence intensity (left), force (middle), and frequency (right) of spontaneously beating hiPSC-CM EHTs from control (O), PLNic (O), and PLN-R14del hiPSC line (O). Data are plotted relative to mean baseline and normalized to TVC per EHT batch. (A) Ca2+ (5.0 mM; control: n = 25 EHT; TVC = 15 EHT; three batches; PLNic: n = 29 EHT; TVC = 24 EHT; three batches; PLN-R14del: n = 12 EHT; TVC = 9 EHT; one batch). (B) Isoprenaline (100.0 nM; control: n = 18 EHT; TVC = 15 EHT; three batches; PLNic: n = 34 EHT; TVC = 28 EHT; three batches; PLN-R14del: n = 28 EHT; TVC = 23 EHT; four batches). One-way ANOVA versus baseline with Dunnett’s post-test, *p < 0.05. Mean ± SEM. Asterisk color code indicates the significance for each hiPSC line. The scatter plots of the same data for the different cell lines are shown in Supplementary Figure S4.

FIGURE 6. (A) ER calcium loading assessment by flow cytometry and ER stress response. Flow cytometry analysis of CEPIAer fluorescence intensity of dissociated hiPSC-CM from PLNic and PLN-R14del EHTs, where each data point represents the average fluorescence intensity of 10,000 individual hiPSC-CM from one EHT. n = 11 PLN-R14del (three batches) and n = 12 PLNic EHTs (three batches). Unpaired Student’s t-test, *p < 0.05. Data are expressed as the mean ± SEM (B). (A) Quantitative PCR of ER/UPR stress marker genes in control hiPSC-CM EHT under time/vehicle control (ctrl) or experimental conditions (370 min incubation, cyclopiazonic acid (CPA) 10 μM, thapsigargin 1 μM, calcium 5.0 mM, and ryanodine 10 µM), as indicated. Gene expression was normalized to GUSB, and data were depicted as fold changes relative to control. n = 7–8 EHTs per condition. One-way ANOVA followed by Bonferroni’s post-test for multiple comparisons, *p < 0.05. Data are expressed as the mean ± SEM.