ACTN2 Mutant Causes Proteopathy in Human iPSC-Derived Cardiomyocytes

Figures

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  Figure 1. Disease modeling in 30-day-old, 2D-cultured hiPSC-CMs. (A) Representative immunofluorescence images of hiPSC-CMs (scale bar = 50 µm), including a higher magnification on the right (scale bar = 10 µm). After 30 days, hiPSC-CMs were fixed and stained with antibodies against ACTN2 and TNNT2, and with Hoechst for nuclei. (B) Blinded analysis of myofibrillar disarray using high-resolution pictures (ACTN2wt and ACTN2mut: N/d = 16/3). (C) Quantification of ACTN2 aggregates analyzed with Fiji software (ACTN2wt: N/d = 14/3, ACTN2mut: N/d = 15/3). (D) Quantification of cell area was analyzed with Fiji software (ACTN2wt: N/n/d = 548/3/3, ACTN2mut: N/n/d 319/3/3). (E) Quantification of cell volume analyzed with Imaris software (ACTN2wt: N/n/d = 54/3/3, ACTN2mut: N/n/d = 29/3/3). (F) Quantification of nucleus number per hiPSC-CM analyzed with Fiji software and expressed as percentage (ACTN2wt: N/n/d = 548/3/3, ACTN2mut: N/n/d = 319/3/3; Chi-square = 280.1, p < 0.0001). Data are expressed as mean ± SEM (panels B–E) or percentages (panel F), with p-values obtained with the unpaired Student’s t-test (panels B–E) or two-tailed Chi-square test (panel F). Abbreviations: TNNT2, cardiac troponin T;…

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  Figure 2. Live cell imaging and immunofluorescence in 2D-cultured hiPSC-CMs after 7 or 30 days in culture. (A) Representative images of ACTN2wt and ACTN2mut hiPSC-CMs transduced with an AAV6 carrying either the ACTN2-MUT-HaloTag® or ACTN2-WT-HaloTag® construct and seeded in 96-well plates (2500–5000 cells/well). After 7 days, live cell imaging was performed by adding TMR-ligand to stain ACTN2-HaloTag® protein and Hoechst for nuclei staining (Scale bars = 50 µm; zoom = 20 µm). (B) Blinded quantification of sarcomere integration or aggregation of exogenous MUT-ACTN2 in ACTN2wt hiPSC-CMs (N/n/d = 292/3/1) or WT-ACTN2 in ACTN2mut hiPSC-CMs (N/n/d = 234/3/1). (C) Quantification of ACTN2 aggregates in hiPSC-CM lines. Analysis was performed with Fiji software (ACTN2wt+MUT-ACTN2: N/d = 26/1, ACTN2mut+WT-ACTN2: N/d = 9/1). (D,E) Western blot stained for ACTN2 and TNNT2 and quantification of non-transduced (NT) ACTN2wt or ACTN2wt transduced with ACTN2-MUT-HaloTag® (n/d = 3/1) after 30 days. (F,G) Western blot stained for ACTN2 and TNNT2 and quantification of non-transduced (NT) ACTN2mut or ACTN2mut transduced with ACTN2-WT-HaloTag® (n/d = 3/1) after 30 days. (H) Representative images of fixed…

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  Figure 3. MS and RNA-seq analysis of 30-day-old, 2D-cultured hiPSC-CMs. (A) Alterations in protein levels between ACTN2mut vs. ACTN2wt (250 up, 231 down) hiPSC-CMs based on MS are displayed in a volcano plot that shows the -Log10 of p-value vs. magnitude of change (log2 ratio) whereby light grey dots indicate p > 0.05 and dark grey dots p < 0.05. (B) Selected hits of significantly dysregulated canonical pathways and biological functions in 2D-cultured ACTN2mut vs. ACTN2wt hiPSC-CMs based on MS analysis using Ingenuity Pathway Analysis (IPA). Unsupervised IPA was performed for significantly altered proteins (Fisher’s exact test; p < 0.05). (C) Alterations in mRNA levels in ACTN2mut vs. ACTN2wt (164 up, 180 down) hiPSC-CMs based on RNA-seq analysis are displayed in a volcano plot that shows the -Log10 of Padj-value vs. magnitude of change (log2 ratio) whereby light grey dots indicate FDR > 0.1 and dark grey dots FDR < 0.1. (D) Selected hits of significantly dysregulated canonical pathways and biological functions in 2D-cultured ACTN2mut vs. ACTN2wt hiPSC-CMs based on RNA-seq analysis using IPA. Unsupervised IPA was performed for significantly altered genes (Fisher’s exact test; FDR < 0.1).

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  Figure 4. Evaluation of the impact of the ACTN2 gene variant on the proteolytic systems in 30-day-old, 2D-cultured hiPSC-CMs. Representative Western blots and Ponceau (A), and quantification, normalized to Ponceau, of the levels of (B) (poly)ubiquitinated proteins, (C) SQSTM1, (D) ACTN2 in hiPSC-CMs treated with DMSO (0.05%) or epoxomicin (Epoxo; 250 nM) for 16.5 h at 37 °C (ACTN2wt: n/d = 7–8/3, ACTN2mut: n/d = 7–8/3). (E) Chymotrypsin-like activity of the proteasome in hiPSC-CMs (ACTN2wt: n/d = 8/3, ACTN2mut: n/d = 8/3). (F) Representative Western blots, Ponceau, and quantification of protein levels of (G) LC3-II and (H) ACTN2 of hiPSC-CMs treated with DMSO (0.05%) or bafilomycin A1 (Bafilo; 50 nM) for 3 h at 37 °C (ACTN2wt: n/d = 6–7/3, ACTN2mut: n/d = 6–7/3). (I) The ALP activity was indirectly measured by determining the number of autophagosomes (green puncta) and autolysosomes (red minus green puncta) whereby the number of puncta is related to the CM number per well (ACTN2wt and ACTN2mut: n = 9). Data are expressed as mean ± SEM, with p-values obtained with two-way ANOVA and Tukey’s post-test (panels B–D, G–I) or with unpaired Student’s t-test (panels E). Abbreviations: n/d, number…

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  Figure 5. Force measurements in 3D-cultured hiPSC-engineered heart tissues. Representative images of (A) ACTN2wt and (B) ACTN2mut EHTs cultured for 30 days (scale bar = 1 mm). (C) Force and (D) beats per minute were measured under unpaced conditions in EHT culture medium or in 1.8 mM Ca2+ Tyrode’s solution at 37 °C (ACTN2wt: N/d = 27–44/3, ACTN2mut: N/d = 32–44/3). (E) Average force, (F) force, (G) normalized force, (H) time to peak −80% (TTP−80%), and (I) relaxation time to baseline 80% (RT80%) were measured under paced conditions at 1 Hz in 1.8 mM Ca2+ Tyrode’s solution at 37 °C (ACTN2wt: N/d = 37/3, ACTN2mut: N/d = 37/3); data are expressed as mean ± SEM, with p-values obtained with a mixed-effects analysis plus Sidak’s multiple comparison tests performed on pooled batches (Panels C,D) or with the nested t-test vs. ACTN2wt (Panels F,H,I). Abbreviations: EHTs, engineered heart tissues; N/d, number of EHTs/differentiations.