Disrupted Sarcomere Reorganization of Cardiomyopathy-Prone Human iPSC-Derived Cardiomyocytes on a Dynamic Mechanical Substrate

Figures

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  Fig. 1. The SMP fabrication and mechanical characterization. a Schematic of the fabrication of tBA-BA co-polymer SMP substrate. b The SMP was manually stretched to 40% strain (Scale bar: 1 cm). c Recovery of the SMP at 37 °C in DPBS over a period of 5 hours is depicted (Scale bar: 1 mm). d The recovery rate of the SMP at 37 °C in DPBS after 5 hours is presented. The Tg of the SMPs were approximately 43 °C in e dry conditions and 37 °C in f wet conditions, as measured by differential scanning calorimetry (DSC). g A 3-D plot of the dynamic mechanical analysis (DMA) cycles revealed the SMP can deform and retain its shapes in high-temperature and low-temperature segments with applied stress, respectively

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  Fig. 2. Sarcomere organization on both static and dynamic SMP substrate. a hiPSC-CMs were seeded onto static and dynamic SMP substrate with 40% strain. b Schematic illustration of cardiac myofibril structures. Fluorescent images of hiPSC-CMs on static and dynamic substrates 5 h after shape change triggered by the temperature stimuli, staining with ACTN2 and MYH10 on c dynamic and e static substrate. Fluorescent images of hiPSC-CMs staining with MYOM1and F-actin on d dynamic and f static substrate (scale bar: 10 μm)

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  Fig. 3. Myofibril remodeling of WT and BAG3-/- hiPSC-CMs was analyzed on both dynamic and static substrates at 5 h. a Fluorescent images were used to measure the lengths and distance of the sarcomere Z-lines and M-lines (Scale bar: 10 µm). b The cell area of BAG3-/- hiPSC-CMs was reduced on the dynamic substrate compared to the static substrate. c The elongation of WT hiPSC-CMs was less than that of BAG3-/- hiPSC-CMs on both substrates. WT and BAG3-/- hiPSC-CMs showed a significant difference in d Z-lines distances, e but not M-lines. f The Z-line length in BAG3-/- hiPSC-CMs decreased on the dynamic substrate. g M-line length showed a difference between WT and BAG3-/- hiPSC-CMs on static substrate. Sarcomere density only showed a difference on M-lines between WT and BAG3-/- hiPSC-CMs on static substrate. j Dynamic mechanical cues improved the Z-line alignment in WT hiPSC-CMs, but not seen from BAG3-/- hiPSC-CMs. k No difference was observed on M-line alignment. Statistics: two-way ANOVA with multiple t-test with p-value < 0.05 (*), < 0.01 (**), < 0.001 (***), and < 0.0001 (****), n ≥ 15 represents the number of cells collected from multiple SMP substrates

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  Fig. 4. Myofibril remodeling in WT and BAG3-/- hiPSC-CMs on static and dynamic substrates after 24 h. Fluorescence images of WT (a, c) and BAG3-/- hiPSC-CMs (b, d) on static and dynamic substrates 24 h after shape, stained for Z-lines (ACTN2) and M-lines (MYOM1) (Scale bar: 10 μm) e BAG3-/- CM area on static substrates was significantly larger than both static WT CMs and dynamic BAG3-/- CMs. f BAG3-/- CMs on dynamic substrates were significantly more elongated compared with WT CMs on dynamic substrates and BAG3-/- CMs on static substrates. g Z-line and k M-line distance were greater in static BAG3-/- CMs compared with static WT CMs and dynamic BAG3-/- CMs. h Z-line length in static BAG3-/- CMs on was slightly greater than dynamic BAG3-/- CMs. j WT CMs displayed longer M-line length than BAG3-/- CMs on static substrates. Both WT and BAG3-/- CMs exhibited longer M-lines on static compared with dynamic substrates. i Z-line density and j Z-line orientation order parameter (OOP) showed no significant differences between WT and BAG3-/- hiPSC-CMs. m WT CMs exhibited higher M-line density than BAG3-/- CMs on both substrates. n M-line alignment was higher in BAG3-/- hiPSC-CMs cultured on dynamic…

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  Fig. 5. Contractile function of single hiPSC-CMs on static and dynamic substrates after 24 h. Beating activity was visualized using a motion velocity heatmaps (Scale bar: 25 µm). BAG3-/- CMs on dynamic substrates exhibited significantly higher b beat rate and d contraction velocity compared with WT CMs on dynamic substrates and BAG3-/- CMs on static substrates. WT CMs on static substrates demonstrated no significance on c relaxation velocity relative to dynamic substrates. BAG3-/- iPSC-CMs on static substrates showed longer e corrected contraction–relaxation (C–R) time interval compared with BAG3-/- CMs on dynamic substrates and WT CMs on static substrates. Statistics: two-way ANOVA with multiple t-test with p-value < 0.05 (*), < 0.01 (**), < 0.001 (***), and < 0.0001 (****), n ≥ 6 represents the number of cells collected from multiple SMP substrates