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

Abstract

Background: Human induced pluripotent stem cells-derived myogenic progenitors develop functional and ultrastructural features typical of skeletal muscle when differentiated in culture. Besides disease-modeling, such a system can be used to clarify basic aspects of human skeletal muscle development. In the present study, we focus on the development of the excitation-contraction (E-C) coupling, a process that is essential both in muscle physiology and as a tool to differentiate between the skeletal and cardiac muscle. The occurrence and maturation of E-C coupling structures (Sarcoplasmic Reticulum-Transverse Tubule (SR-TT) junctions), key molecular components, and Ca²⁺ signaling were examined, along with myofibrillogenesis.

Methods: Pax7⁺-myogenic progenitors were differentiated in culture, and developmental changes were examined from a few days up to several weeks. Ion channels directly involved in the skeletal muscle E-C coupling (RyR1 and Cav1.1 voltage-gated Ca²⁺ channels) were labeled using indirect immunofluorescence. Ultrastructural changes of differentiating cells were visualized by transmission electron microscopy. On the functional side, depolarization-induced intracellular Ca²⁺ transients mediating E-C coupling were recorded using Fura-2 ratiometric Ca²⁺ imaging, and myocyte contraction was captured by digital photomicrography.

Results: We show that the E-C coupling machinery occurs and operates within a few days post-differentiation, as soon as the myofilaments align. However, Ca²⁺ transients become effective in triggering myocyte contraction after 1 week of differentiation, when nascent myofibrils show alternate A-I bands. At later stages, myofibrils become fully organized into adult-like sarcomeres but SR-TT junctions do not reach their triadic structure and typical A-I location. This is mirrored by the absence of cross-striated distribution pattern of both RyR1 and Cav1.1 channels.

Conclusions: The E-C coupling machinery occurs and operates within the first week of muscle cells differentiation. However, while early development of SR-TT junctions is coordinated with that of nascent myofibrils, their respective maturation is not. Formation of typical triads requires other factors/conditions, and this should be taken into account when using in-vitro models to explore skeletal muscle diseases, especially those affecting E-C coupling.

Keywords: Ca2+ signaling; Cav1.1; Excitation-contraction coupling; Human-induced pluripotent stem cells; Myofibrillogenesis; RyR1; SR-TT junctions; iPS-derived skeletal muscle cells.

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 structures in the interspace. D1–D4 Mature stages (> 3 weeks post-differentiation). D1 Alpha-actinin labeling at day 26 post-differentiation showing large skeletal myocytes with clear-cut Z-lines. D2–D3 Ultrastructure of 29-day-old myocytes showing parallel bundles of myofibrils (D2) with fully mature and aligned banding pattern including H-bands and M-lines as can be seen from subsequent enlargement of the boxed areas in D3 and D4. SR-TT junctions (arrows in D4) are enlarged in boxes on the right. Notice the smaller size of these structures and their proximity to the myofibrils compared with earlier stages

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