Cardiomyocyte apoptosis contributes to contractile dysfunction in stem cell model of MYH7 E848G hypertrophic cardiomyopathy

Figures

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  Figure 1.. MYH7 E848G increases cardiomyocyte size and reduces engineered heart tissue contractility in human stem cell model. (a) Family pedigree for MYH7 E848G. (+), WT/E848G; (−), WT/WT; black, HCM; white, no HCM. (b) Schematic of CRISPR/Cas9 gene editing strategy to generate isogenic hiPSC-CMs expressing MYH7-EGFP fusion protein under control of endogenous MYH7 locus. (c) (L) Sanger sequencing chromatograms of MYH7 Exon 22 for patient-derived hiPSCs. (R) Schematic of relationship between isogenic hiPSC-CM lines, with Sanger sequencing chromatograms of MYH7 Exon 22 and representative confocal microscopy images of sarcomeric striations at differentiation day 35. Scale bar 10 μm. (d) Representative confocal microscopy images for Hoechst-stained MYH7WT/WT-EGFP and MYH7WT/E848G-EGFP hiPSC-CMs, differentiation day 33. Scale bar 100 µm. (e) 2D area of EGFP+ MYH7WT/WT-EGFP and MYH7WT/E848G-EGFP hiPSC-CMs, differentiation day 33, from confocal images. Mean and cell replicates shown, n = 150 cells. (f) Representative histogram of forward scatter area (FSC-A) as measured by flow cytometry of isogenic hiPSC-CMs, differentiation day 40. n = 10,000 cells. (g) Normalized FSC-A as measured by flow…

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  Figure 2.. MYH7 E848G reduces hiPSC-CM survival in monolayer and EHT culture. (a) Schematic of iPSC-CM differentiation and seeding protocol for monolayer culture. (b) Total EGFP+ cell count for isogenic hiPSC-CMs and cTnT+ cell count for patient-derived hiPSC-CMs in DMEM-based monolayer culture, normalized to day 2 post-seed. Mean ± SEM, n = 4–6 biological replicates. (c) Representative confocal microscopy images for Hoechst- and TUNEL-stained MYH7WT/WT-EGFP and MYH7WT/E848G-EGFP hiPSC-CMs, differentiation day 33. Scale bar 50 μm. (d) Quantification of percentage of TUNEL+ nuclei in EGFP+ hiPSC-CMs from (I), n = 2 biological replicates shown, n = 150 cells per replicate. * indicates p < 0.05 significance calculated by Student’s t-test. (e) Schematic for casting protocol for engineered heart tissues (EHTs) and papain-based digestion for FACS-based analysis. (f) Representative histogram of EGFP intensity as measured by flow cytometry in MYH7WT/WT, MYH7WT/WT-EGFP, MYH7WT/E848G-EGFP EHTs, cast week 1. n = 10,000 cells. (g) Total EGFP+ cells as percentage of initial EHT cast input for MYH7WT/WT-EGFP and MYH7WT/E848G-EGFP EHTs, cast week 1 and 3, as measured by FACS. Mean and cast replicates…

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  Figure 3.. MYH7 E848G induces p53-associated intrinsic apoptosis in monolayer and EHT culture. (a) Human apoptosis antibody array for MYH7WT/WT-EGFP and MYH7WT/E848G-EGFP hiPSC-CMs, differentiation day 35, with select targets highlighted. (b) Quantification of protein expression from (A) normalized to loading control and MYH7WT/WT-EGFP. Mean and biological replicates shown, n = 2 technical replicates. (c) Representative western blot for p53 and α-sarcomeric actinin protein expression in MYH7WT/WT-EGFP and MYH7WT/E848G-EGFP hiPSC-CMs, differentiation day 40. (d) Quantification of p53 protein expression from (c) normalized to α-sarcomeric actinin and MYH7WT/WT-EGFP. Mean and biological replicates shown. (e) TP53 mRNA expression for MYH7WT/WT-EGFP and MYH7WT/E848G-EGFP hiPSC-CMs normalized to HPRT and MYH7WT/WT-EGFP, differentiation day 40, as measured by RT-qPCR. Mean and biological replicates shown. (f) TP53 mRNA expression for MYH7WT/WT-EGFP and MYH7WT/E848G-EGFP EGFP+ hiPSC-CMs sorted from cast week 1 EHTs, normalized to HPRT and MYH7WT/WT-EGFP, as measured by RT-qPCR. Mean and biological replicates shown. * in (b, d-f) indicates p < 0.05 significance calculated by Student’s t-test.

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  Figure 4.. TP53 ablation does not rescue contractile function, hypertrophy, or cardiomyocyte survival in MYH7 E848G expressing hiPSC-CMs. (a) Schematic of CRISPR/Cas9 gene editing strategy to generate MYH7WT/E848G-EGFP TP53−/− hiPSC-CMs. (b) Schematic of deletions in MYH7WT/E848G-EGFP TP53−/− clones. (c) Maximum twitch force of EHTs at cast week 3 for isogenic hiPSC-CMs. Mean and tissue replicates shown. (d) Representative histogram of EGFP intensity as measured by flow cytometry in MYH7WT/WT, MYH7WT/WT-EGFP, MYH7WT/E848G-EGFP, and MYH7WT/E848G-EGFP TP53−/− EHTs, cast week 1. n = 10,000 cells. (e) Total EGFP+ cells as percentage of initial EHT cast input for MYH7WT/WT-EGFP, MYH7WT/E848G-EGFP, and MYH7WT/E848G-EGFP TP53−/− EHTs, cast week 1 and 3, as measured by FACS. Mean and cast replicates shown, 3–6 tissues per cast. (f) Normalized FSC-A of EGFP+ cells in MYH7WT/WT-EGFP, MYH7WT/E848G-EGFP, and MYH7WT/E848G-EGFP TP53−/− EHTs, cast week 1 and 3, as measured by FACS. Mean and cast replicates shown, 3–6 tissues per cast. * in (c, e-f) indicates p < 0.05 significance calculated by Student’s t-test.