254 Chapter 9 Figure 4. Defining the role of the unfolded protein response (UPR) pathway in human induced pluripotent stem cell–derived cardiomyocytes (hiPSC-CMs) with loss of arginine at position 14 (R14del). A, Schematics of UPR signaling including stress sensors IRE1, ATF6, and PERK at the endoplasmic reticulum (ER) membrane and the 3 corresponding downstream transcriptional effectors. B through E, Functional analysis of UPR pathway perturbation in isogenic hiPSC-CMs. Cells were transfected with the indicated siRNAs, and 2-dimensional (2D) monolayer contractility was measured after 4 days either under baseline conditions (B and C) or with 0.1 µmol/L isoproterenol (D and E). Contractility represented as percent change from control siRNA (6 batches, n = 2–3 wells each). ●, ■, ▲, and ♦ represent experimental batches; dark symbols represent hiPSC-CMs from healthy donor background; light symbols represent hiPSC-CMs from the patient background. All data are presented as mean±SEM. **P<0.005; ****P<0.00005 vs DMSO vehicle controls (G and H). I and J, Force generation of 3-dimensional engineered heart tissues (EHTs) derived from isogenic hiPSC-CMs at baseline and after treatment with BiX for 72 hours. Force generation is represented as percent change from baseline (I) or as total generated force (J; 4 batches, n=2–8 EHTs each). WT indicates wild-type. **P<0.005; ***P<0.0005; ****P<0.00005 by paired Student t test. the contractile deficit without increasing the expression levels of myofilament proteins (Figure V and Table VI in the Data Supplement). At high concentrations, BiX decreased contractility in both the PLN R14del and isogenic controls (Emax=−25% at 10 µmol/L for both R14del and isogenic controls; Figure IVa in the Data Supplement). We considered whether the beneficial effect of BiX is mediated through the modulation of the calcium handling properties of PLN R14del hiPSC-CMs. Compared with a dimethyl sulfoxide control, there was no significant difference in the intracellular calcium handling kinetics in PLN R14del hiPSC-CMs
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