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. Author manuscript; available in PMC: 2022 Aug 3.
Published in final edited form as: Circulation. 2021 Apr 30;144(5):382–392. doi: 10.1161/CIRCULATIONAHA.120.049844

Figure 4. Defining the role of the UPR pathway in R14del hiPSC-CMs.

Figure 4.

a, Schematics of UPR signaling including stress sensors IRE1, ATF6, and PERK at the ER membrane and the three corresponding downstream transcriptional effectors.

b-e, Functional analysis of UPR pathway perturbation in isogenic hiPSC-CMs. Cells were transfected with the indicated siRNAs and 2D monolayer contractility was measured after four days either under baseline conditions (b,c) or with 0.1 μM isoproterenol (d,e). Contractility represented as percent change from control siRNA (6 batches, n = 2-3 wells each). Circles(●), squares(■), triangles(▲), diamonds (♦) represent experimental batches; dark-colored symbols represent hiPSC-CMs from healthy donor background, light-colored symbols from the patient background. All data are presented as mean ± SEM. * p < 0.05, *** p < 0.0005 vs siRNA controls (d,e).

f-h, Assessment of contractility in hiPSC-CMs after pharmacological activation of the UPR with the small molecule BiX. Overview of BiX dose-response kinetics in hiPSC-CMs showing effects on UPR activation and contractility (f, see also Figure IV in the Supplement). Assessment of 2D contractility measurements after 72 hours BiX (or DMSO vehicle control) treatment with 0.1 μM isoproterenol in isogenic hiPSC-CMs (g,h; 4 batches, n = 12-16 wells each). Contractility is represented as percent change from the DMSO control per batch (g) or actual peak contraction amplitude (h). Circles(●), squares(■), triangles(▲), diamonds (♦) represent experimental batches; dark-colored symbols represent hiPSC-CMs from healthy donor background, light-colored symbols from the patient background. All data are presented as mean ± SEM. ** p < 0.005, **** p < 0.00005 vs DMSO vehicle controls (g,h).

i-j, Force generation of 3D-EHTs derived from isogenic hiPSC-CM at baseline and following treatment with BiX for 72h. Force generation is represented as percent change from baseline (i) or as total generated force (j) (4 batches, n=2-8 EHT each). ** p < 0.005, *** p < 0.0005, **** p < 0.00005 by paired student t-test.