3D skeletal muscle model recapitulating the myostatin knockout phenotypic and mitochondrial metabolic features

Figures

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  FIGURE 1. Establishment of 3D C2C12 self‐organized skeletal muscle microtissues. (a) A diagram illustrating the 3D microtissue culture process: Ring‐shaped tissues are constructed and cultured on two micropillars, which serve as anchors during differentiation. The morphological, metabolic, and functional characteristics of the tissues are subsequently analyzed. (b) eNUVIO OMEGAMP devices are assembled in a standard 12‐well plate, allowing for the observation of the resulting 3D skeletal muscle microtissue with a binocular loupe. (c) Brightfield and immunofluorescence images of Troponin T (green) are captured before and after clearing using a spinning‐disk Andor W1 microscope at a magnification of ×10 (scale bar = 200 μm). (d) Confocal immunofluorescence images of C2C12 myotubes on day 14 are stained with anti‐troponin T antibodies (green). Tile scan images at 4× magnification are shown (scale bar = 1000 μm), along with images at 20× magnification (scale bar = 100 μm).

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  FIGURE 2. Morphological characterization of 3D C2C12‐type skeletal muscle microtissues on days 7, 14, and 21 (a) Confocal immunofluorescence images of C2C12 myotubes stained with anti‐sarcomeric α‐actinin and anti‐troponin T antibodies (green). Nuclei were visualized by Hoescht staining (blue). Images at 20X magnification (scale bar = 100 μm; independent preparations, n = 3). (b) Analysis of the diameters of C2C12 muscle myotubes during differentiation. The histograms represented means ± SEM. At least 50 myotubes at each time of differentiation were analyzed. A Kruskall‐Wallis non‐parametric test followed by Dunns multiple comparison test was used to determine the effect of differentiation time. (c) Analysis of gene expression of 3D myotube markers. Gene expression was calculated as relative abundance (2−DDCt) for each condition (n = 3). The histograms represented means ± SEM of 3 independent cultures, statistical analysis were performed using a Mann–Whitney non‐parametric test, p < 0.05 vs Day 0. Markers: Mki67, Antigen identified by monoclonal antibody Ki‐67; Myh2, myosin heavy chain 2; Myh4, myosin heavy chain 4; Mgn, myogenin; Myh7, myosin heavy chain 7. (d) Western blot analysis…

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  FIGURE 3. Mitochondrial respiration of 3D C2C12‐type skeletal muscle microtissues on day 14. (a) Substrate‐uncoupler‐inhibitor titration protocol (SUIT‐008_O2_ce‐pce_D025). Sequential titrations and respiratory states. 1ce1: Routine (basal) cell respiration R in living cells. 1Dig: Plasma membrane permeabilisation with digitonin. 1 pm: NADH‐pathway (N‐pathway) in the presence of 5 mM pyruvate and 2 mM malate in the N‐LEAK state. 2D: Addition of saturating ADP (5 mM) (N‐OXPHOS state). 2c: 10 μM cytochrome c for evaluating the integrity of the outer mitochondrial membrane (N‐OXPHOS‐c state). 3G: 10 mM glutamate as an additional NADH‐linked substrate (N‐OXPHOS‐G state). 4S: NS‐pathway evaluation with 10 mM succinate, (NS‐OXPHOS state). 5 U: Uncoupler titrations to evaluate the electron transfer system (ETS) capacity (NS‐ETS state). 6Rot: Inhibition of CI by 0.5 μM rotenone and evaluation of succinate pathway (S‐ETS state). 7Ama: Inhibition of CIII by 5 μM antimycin A (residual oxygen consumption, ROX). (b) Representative respiration trace with 3D C2C12 muscle following SUIT‐008_O2_ce‐pce_D025. (c) Mitochondrial respiration is expressed as oxygen flux per microtissue [pmol·s−1·amount] and as…

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  FIGURE 4. Morphological characterization and mitochondrial respiration of 3D skeletal muscle microtissues derived from primary mouse myotubes on day 14. (a) Confocal immunofluorescence images of 3D primary muscle myotubes, stained with anti‐troponin T and anti‐sarcomeric α‐actinin antibodies, at 20× magnification (scale bar = 50 μm for the zoomed images; n = 3 independent culture). Nuclei were revealed by Hoescht staining (blue). The arrow indicates the organized sarcomere structures in this myotube. (b) Analysis of myotube diameters of WT and Mstn −/− 3D primary muscle. Histograms are means ± SEM for three independent cultures. At least 100 myotubes stained with anti‐sarcomeric α‐actinin antibody were analyzed for WT and primary muscles. A paired Student t‐test was used to determine genotype effect. (c) Representative respiration trace with WT (red) and Mstn −/− (green) 3D primary muscle following SUIT‐008_O2_ce‐pce_D025. (d) Analysis of mitochondrial respiration of WT (red) and Mstn −/− (green) 3D primary muscles. Mitochondrial respiration is expressed as oxygen flux per microtissue [pmol·s−1·amount]. The respiratory control ratio (RCR) was set as the ratio of N‐OXPHOS respiration over…