Spatiotemporal cell junction assembly in human iPSC-CM models of arrhythmogenic cardiomyopathy

Figures

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  Figure 1. Spatiotemporal cell-cell junction assembly of WT hiPSC-CMs (A–L) See also Figures S1–S3. Immunofluorescence images, averaged heatmaps, and quantified protein localization of N-cadherin (A–C), plakoglobin (D–F), PKP2 (G–I), and CX43 (J–L). Representative images of WT hiPSC-CM cell pairs stained for nuclei (blue), α-actinin (red), and cell junction proteins (white). (B, E, H, K) Overlaid heatmaps represent the distribution of individual proteins averaged over all imaged cells for each time point. Yellow arrows indicate junctional localization. Quantified center and distribution of N-cadherin (C), plakoglobin (F), PKP2 (I), and CX43 (L). Mean ± SD. One-way ANOVA followed by Tukey’s honest significant difference (HSD) test: ∗p < 0.05. Cell pairs were aggregated from three to four independent differentiations.

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  Figure 2. Compromised spatiotemporal assembly of cell-cell junctions in ACM hiPSC-CMs (A–H) See also Figure S4. Representative immunostained images and averaged heatmaps of ACM hiPSC-CM cell pairs stained for nuclei (blue), α-actinin (red), and cell junction proteins (white). Quantified centers and distributions of intercalated disc protein localization in isogenic WT control and ACM hiPSC-CM cell pairs for cell junction proteins are shown on the right. Mean ± SD. two-way ANOVA followed by Tukey’s multiple comparisons test: ∗p < 0.05, ∗∗p < 0.01. Cell pairs were aggregated from three to five independent differentiations.

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  Figure 3. Compromised spatiotemporal assembly of the cytoskeletal filaments in ACM hiPSC-CMs (A and B) Representative images of isogenic WT (A) and ACM (B) hiPSC-CM cell pairs stained for F-actin (green), sarcomeric α-actinin (red), and DAPI (blue). Day 1 skeletonized F-actin images, pseudo-colored based on fibril angular orientation, are shown below with boxed region magnified in the inset. (C and D) Representative images of day 9 WT (C) and ACM (D) hiPSC-CM cell pairs with skeletonized α-actinin images below. Boxed regions are magnified in the inset. (E–G) Quantified values of cytoskeletal organization over 9 days in culture. ACM cell pairs showed slower F-actin alignment (E), comparable α-actinin alignment (F), and reduced Z-disc presence (G). Mean ± SD. Two-way ANOVA followed by Tukey’s multiple comparison test: ∗p < 0.05. Cell pairs were aggregated from four to five independent differentiations.

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  Figure 4. SB restores cytoskeletal integrity in day 6 and 9 ACM hiPSC-CMs (A–F) Day 6 WT and ACM hiPSC-CM cell pairs treated with DMSO (veh) or SB. Representative images of cell pairs stained for F-actin (green) and α-actinin (red), with corresponding skeletonized fibrils pseudo-colored based on angular orientation. (E) Quantified values for cytoskeletal organization (F-actin and sarcomere OOP) were unaffected, but modest improvement to sarcomere formation (z-disc presence) was observed in ACM + SB (F). (G–L) Day 9 WT and ACM hiPSC-CM cell pairs treated with veh or SB. Similar trends are observed with more pronounced significant improvements to sarcomere formation in ACM + SB were observed (L). Whereas SB improved cytoskeletal integrity in ACM cell pairs, it reduced these metrics (E, F, K, L) in WT cell pairs. Mean ± SD. Two-way ANOVA followed by Tukey’s multiple comparisons test: ∗p < 0.05. Cell pairs were aggregated from three to five independent differentiations.

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  Figure 5. SB normalizes cell junction protein distribution in day 9 ACM hiPSC-CMs See also Figures S5 and S6. WT or ACM hiPSC-CM cell pairs treated with DMSO (veh) or SB and stained at day 9 for nuclei, α-actinin, and cell junction proteins. (A, B, D, E, G, H, J, and K). Representative images and corresponding averaged heatmaps. Yellow arrows indicate junctional localization. (C, F, I, and L) Quantitative analysis of cell junction protein localization center (left) and distribution (right) in WT or ACM hiPSC-CMs at day 9. Significantly impaired plakoglobin and CX43 localization in ACM + veh were restored by SB. N-cadherin and PKP2 were unaffected by SB in ACM hiPSC-CMs. Mean ± SD. Two-way ANOVA followed by Tukey’s multiple comparisons test: ∗p < 0.05.

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  Figure 6. Abundance of proteins in cytoplasmic, nuclear, and membrane fractions of day 9 hiPSC-CMs (A and B) See also Figure S7. Capillary western analysis of PKP2 and GAPDH. Relative changes to PKP2 protein and transcript levels with SB treatment in WT and ACM hiPSC-CMs. Transcript levels were measured by qRT-PCR and normalized to GAPDH. (C–F) Capillary western analysis of cell junction proteins in cytosolic or membrane fractions. Relative levels of cell junction proteins were normalized to GAPDH (cytosolic fraction) or SERCA2 (membrane fraction) and expressed relative to WT + veh. (G and H) β-Catenin in cytosolic, nuclear, and membrane fractions was measured by capillary western. Relative β-catenin was normalized LMNB1 (nuclear fraction), GAPDH (cytosolic fraction), or SERCA2 (membrane fraction) and expressed relative to WT + veh. Mean ± SD. Two-way ANOVA followed by Tukey’s multiple comparisons test: ∗p < 0.05. Data represent three technical replicates from two to three independent differentiations.

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  Figure 7. Ca2+ wave propagation velocity is reduced in ACM tissues and restored by SB (A) Schematic illustration of engineered tissue fabrication. (B) Design of tissue constructs for Ca2+ optical mapping experiments. Representative images of anisotropic tissues stained for sarcomeric α-actinin and nuclei demonstrate iPSC-CM alignment by micro-molded gelatin substrate. (C) Calcium wavefront isochrone maps for day 9 WT + veh, WT + SB, ACM + veh, and ACM + SB tissues at two representative time points (35 and 80 ms). (D) Ca2+ wave propagation velocities of WT and ACM tissues at day 6 and day 9, with and without SB treatment. Mean ± SD. Two-way ANOVA followed by Tukey’s multiple comparisons test: ∗p < 0.05.