Cooperation between myofibril growth and costamere maturation in human cardiomyocytes

Figures

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  FIGURE 1. Myofibrils and costameres in cardiomyocytes (A) Schematics showing the components of sarcomeres and costameres in cardiomyocytes. (B) Fluorescent images of Z-lines (α-actinin), M-lines (myomesin), and costameres (zyxin). Intensity profiles to demonstrate the measurement of Z/M-line distance, Z/M-costamere distance, and Z/M-costamere intensity. Scale bar: 10 μm.

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  FIGURE 2. Assembly of costameres in hiPSC-CMs during cell attachment. (A) Schematics showing the sampling of hiPSC-CMs in the initial stage of cell attachment. (B) Fluorescent images of Z-lines, M-lines and costameres. The region containing mature costameres and mature myofibrils is indicated by a red dashed line. Nascent myofibrils with striated-like Z-lines colocalized with pre-costameres are indicated by an orange dashed line. Pre-myofibrils with Z-bodies colocalized with pre-costameres in the cell peripheral region are indicated by a yellow dashed line. Scale bar: 5 μm. To characterize the assembly and growth of myofibrils within hiPSC-CMs during cell attachment, we measured the (C) Z-line density, (D) M-line density, (E) Z-line distance, (F) M-line distance, (G) Z-line length and (H) M-line length. To track the assembly of costameres, we quantified the (I) Z-costamere distance (J) M-costamere distance (K) Z-costamere intensity and (L) M-costamere intensity. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001.

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  FIGURE 3. (A) Schematics showing sampling of hiPSC-CMs in the late stage of cell spreading. (B) Zoom-in images showing mature Z/M-costameres together with mature myofibrils within the hiPSC-CMs. Scale bar: 5 μm. To track the myofibril growth within hiPSC-CMs during cell spreading, we measured the (C) Z-line density, (D) M-line density, (E) Z-line distance, (F) M-line distance, (G) Z-line length and (H) M-line length. To track the costamere growth during cell spreading, we quantified (I) Z-costamere distance (J) M-costamere distance (K) Z-costamere intensity and (L) M-costamere intensity. *p <0.05, **p <0.01, ***p <0.001 and ****p <0.0001.

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  FIGURE 4. Costameres of hiPSC-CMs on different substrates. (A) Schematics showing the experiment of hiPSC-CMs seeding on PA hydrogels with different stiffness. (B) hiPSC-CMs cultured on the PA hydrogels with 10 kPa and 40 kPa modulus were analyzed for their mechanical output based on traction force microscopy. Scale bar: 10 μm. To characterize the responsiveness of myofibrils to different substrate stiffness, we measured the (C) Z-line density, (D) M-line density, (E) Z-line distance, (F) M-line distance, (G) Z-line length, (H) M-line length. To investigate the behaviors of Z-costameres and M-costameres of hiPSC-CMs on different substrates, we quantified the (I) Z-costamere distance (J) M-costamere distance (K) Z-costamere intensity and (L) M-costamere intensity. *p <0.05, **p <0.01, ***p <0.001 and ****p <0.0001.