Fibro-adipogenic progenitors prevent skeletal muscle degeneration at acute phase upon tendon rupture in a murine tibialis anterior tenotomy model

Figure 1 Change in fibro-adipogenic progenitors population after tendon rupture observed in fibro-adipogenic progenitor-tracing mice. A-H: Images of frozen sections of tibialis anterior muscle at 0 day, 3 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, and 6 weeks after tibialis anterior tenotomy in Pdgfra-H2B::eGFP mice, respectively; I: The statistical analysis of the number of EGFP⁺ cells per field counted under microscopy. ^aP < 0.001, 3 days compare to 0 day; ^bP < 0.001, 1 week compare to 0 day; ^cP < 0.001, 2 weeks compare to 0 day; ^dP < 0.01, 3 weeks compare to 0 day; ^eP < 0.01, 4 weeks compare to 0 day; ^fP < 0.01, 5 weeks compare to 0 day; ^gP > 0.05, 6 weeks compare to 0 day. Pdgfra⁺ cells were labeled with enhanced green fluorescence protein. Yellow arrowheads indicate EGFP⁺ cells (i.e., fibro-adipogenic progenitors). Scale bar = 50 μm. TAT: Tibialis anterior tenotomy; FAPs: Fibro-adipogenic progenitors.

Figure 2 Change in fibro-adipogenic progenitors population after tendon rupture observed in C57BL/6 mice. A: The gating strategy of fluorescence-activated cell sorting for fibro-adipogenic progenitors (FAPs) in the tibialis anterior muscle of C57BL/6 mice; B: The fluorescence-activated cell sorting results of FAPs in the muscle at 0 day, 3 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, and 6 weeks, respectively, after tibialis anterior tenotomy (TAT) or sham surgery (SS) in C57BL/6 mice; C: Subsequent analysis showing the proportion of FAPs among CD45^-CD31^- cells (^aP < 0.001, ^bP < 0.001, ^cP < 0.001, ^dP < 0.001, ^eP < 0.001, ^fP < 0.001, ^gP < 0.001, TAT compared to SS side at 3 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, and 6 weeks, respectively); D: The proportion of FAPs among live cells (^aP < 0.001, ^bP < 0.001, ^cP < 0.001, ^dP < 0.001, ^eP < 0.001, TAT compared to SS side at 3 days, 1 week, 2 weeks, 3 weeks, and 4 weeks, respectively); E: The number of FAPs per gram tibialis anterior muscle (^aP < 0.001, ^bP < 0.001, ^cP < 0.001, ^dP < 0.001, ^eP < 0.001, ^fP < 0.001, ^gP < 0.001, TAT compared to SS side at 3 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, and 6 weeks, respectively). TAT: Tibialis anterior tenotomy; SS: Sham surgery; FAPs: Fibro-adipogenic progenitors.

Figure 3 The progression of muscle atrophy after tendon rupture vs sham surgery in C57BL/6 mice. A: The hematoxylin and eosin staining results of the tibialis anterior muscle at 0 day, 1 week, 2 weeks, 4 weeks, and 6 weeks after tibialis anterior tenotomy (TAT) or sham surgery (SS) in C57BL/6 mice; B: The statistical analysis of average myofiber cross-sectional area which represents the severity of muscle atrophy (^aP < 0.05, ^bP < 0.05, TAT compared to SS side at 4 weeks and 6 weeks, respectively); C: The FBXO32 immunohistochemistry staining results of the muscle at 0 day, 1 week, 2 weeks, 4 weeks, and 6 weeks after TAT or SS in C57BL/6 mice; D: The statistical analysis of the area fraction of positive stained area which represents the tendency towards muscle atrophy (compared to SS side, ^aP < 0.001, ^bP < 0.001, ^cP < 0.001, ^dP < 0.001, TAT compared to SS side at 1 week, 2 weeks, 4 weeks, and 6 weeks, respectively). Scale bar = 100 μm. TAT: Tibialis anterior tenotomy; SS: Sham surgery.

Figure 4 The progression of muscle fibrotic degeneration after tendon rupture vs sham surgery in C57BL/6 mice. A: The modified Masson trichrome staining results of the tibialis anterior muscle at 0 day, 1 week, 2 weeks, 4 weeks, and 6 weeks after tibialis anterior tenotomy (TAT) or sham surgery (SS) in C57BL/6 mice; B: The statistical analysis of area fraction of collagen which represents the severity of fibrosis (^aP < 0.05, ^bP < 0.001, TAT compared to SS side at 4 weeks and 6 weeks, respectively); C: The alpha-smooth muscle actin immunohistochemistry staining results of the muscle at 0 day, 1 week, 2 weeks, 4 weeks, and 6 weeks after TAT or SS in C57BL/6 mice; D: The statistical analysis of the area fraction of positive stained area. Area fraction of collagen (%) = area of collagen staining of the entire sample area which represents the tendency of fibrosis (^aP < 0.001, ^bP < 0.001, ^cP < 0.001, ^dP < 0.001, TAT compared to SS side at 1 week, 2 weeks, 4 weeks, and 6 weeks, respectively). Scale bar = 100 μm. TAT: Tibialis anterior tenotomy; SS: Sham surgery.

Figure 5 The progression of muscle adipogenic degeneration after tendon rupture vs sham surgery in C57BL/6 mice. A: Images of perilipin 1 immunofluorescence staining of the tibialis anterior muscle at 0 day, 1 week, 2 weeks, 4 weeks, and 6 weeks after tibialis anterior tenotomy or sham surgery in C57BL/6 mice; B: The statistical analysis of the area fraction of positive stained area which represents the tendency of adipogenesis (^aP < 0.05, ^bP < 0.05, ^cP < 0.01, ^dP < 0.001, tibialis anterior tenotomy compared to sham surgery side at 1 week, 2 weeks, 4 weeks, and 6 weeks, respectively). Scale bar = 100 μm. TAT: Tibialis anterior tenotomy; SS: Sham surgery.

Figure 6 The progression of muscle atrophy after tendon rupture in fibro-adipogenic progenitor-depleted mice. A: The hematoxylin and eosin staining results of the tibialis anterior muscle at 0 day, 1 week, and 6 weeks after tibialis anterior tenotomy (TAT) in PdgfraCre^ERT::Rosa^DTA mice induced with Tamoxifen or corn oil; B: The statistical analysis of average myofiber cross-sectional area which represents the severity of muscle atrophy; C: The FBXO32 immunohistochemistry staining results of the tibialis anterior muscle at 0 day, 1 week, and 6 weeks after TAT in PdgfraCre^ERT::Rosa^DTA mice induced with Tamoxifen or corn oil; D: The statistical analysis of the area fraction of positive stained area which represents the tendency of muscle atrophy (^aP < 0.05, compared to control group). Scale bar = 100 μm. TAT: Tibialis anterior tenotomy.

Figure 7 The progression of muscle fibrotic degeneration after tendon rupture in fibro-adipogenic progenitor-depleted mice. A: The modified Masson trichrome staining results of the tibialis anterior muscle at 0 day, 1 week, and 6 weeks after tibialis anterior tenotomy (TAT) in PdgfraCre^ERT::Rosa^DTA mice induced with Tamoxifen or corn oil; B: The statistical analysis of area fraction of collagen which represents the severity of fibrosis (^aP < 0.05, ^bP < 0.001, compared to control group); C: The alpha-smooth muscle actin immunohistochemistry staining results of the tibialis anterior muscle at 0 day, 1 week, and 6 weeks after TAT in PdgfraCre^ERT::Rosa^DTA mice induced with Tamoxifen or corn oil; D: The statistical analysis of the area fraction of positive stained area which represents the tendency of fibrosis. Area fraction of collagen (%) = area of collagen staining of the entire sample area (^aP < 0.01, compared to control group). Scale bar = 100 μm. TAT: Tibialis anterior tenotomy.

Figure 8 The progression of muscle adipogenic degeneration after tendon rupture in fibro-adipogenic progenitor-depleted mice. A: The perilipin 1 immunofluorescence staining results of the tibialis anterior muscle at 0 day, 1 week, and 6 weeks after tibialis anterior tenotomy in PdgfraCre^ERT::Rosa^DTA mice induced with Tamoxifen or corn oil; B: The statistical analysis of the area fraction of positive stained area which represents the tendency of adipogenesis. ^aP < 0.05, compared to control group. Scale bar = 100 μm. TA: Tibialis anterior; TAT: Tibialis anterior tenotomy.

Figure 9 The main findings of the study. FAPs: Fibro-adipogenic progenitors.