Development of the excitation-contraction coupling machinery and its relation to myofibrillogenesis in human iPSC-derived skeletal myocytes

Figures

• 

  Fig. 1. Myofibrillogenesis and SR-TT junctions in differentiating human iPSC-derived skeletal myocytes. A1–A5 Day 4 post-differentiation. Immunofluorescence labeling of α-actinin (A1) and electron micrographs of ultrathin longitudinal cell sections (A2–A5). Nascent myofibrils with Z-bodies (A2–A3); the boxed area is enlarged in A3 for further details. A4–A5 SR-TT junctions from different cells. Whenever possible, the SR is highlighted by artificial post-coloring. Note that T-tubules (T) are surrounded and/or associated with numerous caveolae. B1–B6 Day 5 post-differentiation. Transition from Z-bodies to Z-bands as revealed by α-actinin labeling (B1) and confirmed by electron microscopy (B2–B4). Upper and lower boxed areas in B2 are enlarged in B3 and B4, as indicated. B5–B6 SR-TT junctions from different cells; feet-like structures are visible and caveolae surround the T-tubules. C1–C5 Day 7 post-differentiation. A significant increase in the number of α-actinin+ cells with prevalent Z-bands (C1). C2–C3 Myofibrils with well-defined A-I banding pattern; the boxed area is enlarged in C3. C4–C5 Large and multiple SR-TT junctions from different cells with noticeable rows of feet-like…

• 

  Fig. 2. Alpha-actinin immunofluorescence and ultrastructure of human iPSC-derived skeletal myocytes on day 14 post-differentiation in culture. The left panel shows immunofluorescence labeling of α-actinin photographed at low (A) or high (B) magnification. The right panel depicts the ultrastructure of 14-day-old skeletal myocytes at increasing magnifications from C to E; the boxes delineate regions that are subsequently enlarged. A general view of the sarcomeric banding pattern is presented in C, followed by a detailed image of the different bands, as indicated. Myofibrils full maturity is attested by the presence of H-bands and M-lines. Multiple junctions between SR elements and T-tubules are illustrated in E; note the granular aspect of the SR lumen and the presence of regularly spaced (∼ 25 nm) electron-opaque feet-like structures between SR and T-tubules apposed membranes

• 

  Fig. 3. Immunofluorescence detection of RyR1 and Cav1.1 channels in differentiating human iPSC-derived skeletal myocytes. A1–A4 Immunofluorescence labeling of RyR1 (red) and nuclei (blue) after 4 to 22 days of cell differentiation as indicated. B1–B4 Immunofluorescence labeling of Cav1.1 (green) and nuclei (blue); same time sequence after differentiation as in A1–A4, scale: 10 μm. C Frequency distribution histograms of RyR1 immunofluorescent particle area at early (7 days) or mature (22 days) stages of differentiation. Insets represent enlarged areas of RyRs immunolabeled cells to highlight the difference in size of the fluorescent particles between 7 and 22 days post-differentiation (scale: 10 µm). Data were obtained from three independent experiments, and immunofluorescent particles were counted from 13 and 8 cells in 7- and 22-day-old myocytes, respectively

• 

  Fig. 4. Percentage of α-actinin, RyR1, and Cav1.1 positive cells during differentiation. Symbols represent mean ± SEM of the percentage of α-actinin+/RyR1+/Cav1.1+ cells as counted from immunofluorescent microphotographs of iPSC-derived skeletal myocytes at different stages of their differentiation in culture. The total number of cells was determined by counting all Hoechst-stained nuclei on the microphotograph. Immunofluorescent cells were counted from four to ten frames per independent experiment (×60 magnification); three independent experiments were performed at each time point for each type of protein (labeled separately). Note that the percentage of immunolabeled cells for the three proteins reaches a stable level after the second week of differentiation

• 

  Fig. 5. Depolarization-induced Ca2+ transients in differentiating human iPSC-derived skeletal myocytes. A Fura-2 ratiometric (340/380) fluorescence reflecting changes in intracellular Ca2+ level during 180 s of membrane depolarization by 50 mM extracellular [K+]. Symbols represent mean data (± SEM) obtained at different times post-differentiation as indicated (M: mature stages > 3 weeks post-differentiation). B–D Mean values (± SEM) of the three different characteristics of the Ca2+ signal at different times post-differentiation. Note that the value of time to peak was not included for day 4 (NA) as the signal has not reached a single peak profile necessary for this measurement