Tenascin-C from the tissue microenvironment promotes muscle stem cell maintenance and function through Annexin A2

Abstract

Skeletal muscle regeneration occurs through the finely timed activation of resident muscle stem cells (MuSC). Following injury, MuSC exit quiescence, undergo myogenic commitment, and regenerate the muscle. This process is coordinated by tissue microenvironment cues, however the underlying mechanisms regulating MuSC function are still poorly understood. Here, we demonstrate that the extracellular matrix protein Tenascin-C (TnC) promotes MuSC self-renewal and function. Mice lacking TnC exhibit reduced number of MuSC, and defects in MuSC self-renewal, myogenic commitment, and repair. We show that fibro-adipogenic progenitors are the primary cellular source of TnC during regeneration, and that MuSC respond through the surface receptor Annexin A2. We further demonstrate that TnC declines during aging, leading to impaired MuSC function. Aged MuSC exposed to soluble TnC show a rescued ability to both migrate and self-renew in vitro. Overall, our results highlight the pivotal role of TnC during muscle repair in healthy and aging muscle.

Fig. 1. TnC regulates MuSC quiescence and myogenic commitment.

a Whole tibialis anterior (TA) muscles from wild-type (WT) and TnC knockout (TnC-KO) adult mice and quantification of their wet weight (mg) (WT n = 3; KO n = 4). Scale bar = 1 mm. b Representative immunofluorescence (IF) images of uninjured TA muscle cross-sections from WT and TnC-KO adult mice (Pax7, green; laminin, gray; DAPI, blue). Pax7⁺ are indicated with yellow arrows. Scale bar = 50 µm. Quantification of the myofiber cross-sectional area (CSA) in µm² (n = 3). c Quantification of the number of Pax7⁺ nuclei (yellow arrows in b) per mm² (WT n = 6; KO n = 5). d Representative IF images of cross-sections from P14 TA muscles of WT and TnC-KO mice (MyoD, red; laminin, gray; DAPI, blue). Scale bar = 50 µm. Quantification of the number of MyoD⁺ nuclei (yellow arrows) per mm² (n = 4). e Representative IF images of cross-sections from uninjured TA muscles of WT and TnC-KO P14 mice for quantification of Pax7⁺ cell localization (Pax7, red; laminin, gray; DAPI, blue). Scale bar = 10 µm. Quantification of Pax7⁺ cell localization under the basal lamina (white arrows) or in the interstitial space (yellow arrows) in WT versus TnC-KO muscles (WT n = 6; KO n = 4). f Representative IF images of WT and TnC-KO freshly isolated myofibers from adult mice (F-actin, green; Pax7, red; α-tubulin, magenta; DAPI, blue). Scale bar = 20 µm. Quantification of the distribution of quiescence projection length, and the number of projections per cell (n = 3). g Representative IF images of WT and TnC-KO differentiated cells from adult mice (Myogenin, red; myosin heavy chain, MHC, gray; DAPI, blue). Scale bar = 50 µm. Quantification of the percentage of myogenin⁺ cells, differentiation index, and fusion index (n = 3). Data are represented as mean ± SEM; *p < 0.05, ***p < 0.001, ****p < 0.0001, t-test (a, b, c, d, e, f); *p < 0.05, Two-way ANOVA (e, f, g). Data are represented as the median with quartiles; **p < 0.01, ****p < 0.0001, t-test (f, g).

Fig. 2. TnC is required for MuSC self-renewal and skeletal muscle regeneration.

a Representative Western blot of TnC expression kinetics in uninjured and regenerating whole muscle protein lysates from adult mice and quantification (n = 3). b Representative immunofluorescence (IF) images of cross-sections from uninjured and injured (5 DPI and 14 DPI) TA muscles of WT adult mice (TnC, red; DAPI, blue). Scale bar = 50 µm. c Representative IF images for embryonic myosin heavy chain (eMyHC) of injured TA muscles (5 DPI) in adult WT and TnC-KO mice (eMyHC, green; laminin, gray; DAPI, blue). Scale bar = 50 µm. Quantification of the cross-sectional area (CSA) (n = 4). d Representative IF images of Pax7⁺ cells (yellow arrows) in injured (5 DPI) TA muscles in adult WT and TnC-KO mice (Pax7, green; laminin, gray; DAPI, blue). Scale bar = 50 µm. Quantification of the number of Pax7⁺ nuclei per mm² (n = 4). e Representative IF images of MyoD⁺ cells in injured (5 DPI) TA muscles in adult WT and TnC-KO mice (MyoD, red; laminin, gray; DAPI, blue). Scale bar = 50 µm. Quantification of MyoD⁺ cells per mm² (n = 3). f Representative IF images of Pax7⁺ and MyoD⁺ cells in injured (5 DPI) TA muscles in adult WT and TnC-KO mice (Pax7, red; MyoD, green; laminin, gray; DAPI, blue). Scale bar = 50 µm. Quantification of percentage double positive Pax7⁺ MyoD⁺ (yellow arrows) and Pax7⁺ MyoD^- (white arrows) cells (n = 3). g Representative IF images of Pax7⁺ cells (yellow arrows) in injured (30 DPI post 1st injury) or double-injured (30 DPI post 2nd injury) TA muscles in adult WT and TnC-KO mice (Pax7, green; laminin, red; DAPI, blue) (Scale bar = 50 µm) and quantification per mm² (n = 3). Data are represented as mean ± SEM; *p < 0.05, one-way ANOVA (a); *p < 0.05, **p < 0.01, t-test (c, d, e); *p < 0.05, ***p < 0.001, Two-way ANOVA (f, g).

Fig. 3. TnC from the tissue microenvironment is required for MuSC function.

a scRNAseq analysis of TnC expression at 5 DPI TA muscles of adult WT mice. Data originally from Oprescu et al. (n = 3). b, c TnC mRNA expression dynamics in MuSC and FAPs from uninjured and regenerating TA muscles at different DPIs (0.5–21). Data originally from Oprescu et al. (n = 3). d, e TnC mRNA expression dynamics in MuSC and FAPs in uninjured and at 3 and 7 DPIs by qPCR (n = 3). f, g Chord diagrams representing the number of inter-cluster interactions within the TA muscle in uninjured and 5 DPI. Data originally from Oprescu et al. (n = 3). h, i Sender–Receiver Probability Heatmap of the Tenascin signaling network in uninjured and 5 DPI TA muscles. Data originally from Oprescu et al. (n = 3). j Representative immunofluorescence (IF) images representing the contribution of transplanted RFP-labeled WT MuSC from adult 3-month-old donor mice to regenerating TA muscles of WT or TnC-KO age-matched recipient mice (donor-derived fibers – RFP, red; DAPI blue) (Scale bar = 50 µm) and quantification of RFP⁺ myofibers per section (WT n = 4; KO n = 3). k Representative IF images of TnC expression in WT MuSC:WT FAP co-cultures from adult mice (TnC, red; Pax7, green; DAPI, blue). Yellow arrows indicate FAPs. Scale bar = 50 µm. Quantification of TnC fluorescent signal intensity in WT MuSC and WT FAPs normalized on TnC-KO FAPs (n = 3). l Representative IF images of MuSC and FAPs co-cultures from WT and TnC-KO adult mice (Pax7, red; PDGFRα, green; DAPI, blue) (Scale bar = 50 µm). Quantification of Pax7⁺ cells normalized on WT MuSC monoculture (n = 5). Data are represented as mean ± SEM; **p < 0.01, ***p < 0.001, one-way ANOVA (d, e), *p < 0.05, ****p < 0.0001, t-test (j, k), *p < 0.05, **p < 0.01, one-way ANOVA (l).

Fig. 4. TnC signals MuSC through Annexin A2.

a Violin plots depicting the different expression dynamics Anxa2, Pax7, and Pdgfra at different DPIs (0, 3.5, 5, 21) in MuSC and FAPs. Data originally from Oprescu et al. (n = 3). b Validation by immunofluorescence (IF) of Annexin A2 expression in WT MuSC in vitro (Annexin A2, red; Pax7, green; DAPI, blue) (Scale bar = 50 µm). Quantification of the percentage of Pax7⁺ cells Annexin A2⁺ or Annexin A2^- (n = 3). c Western blot analysis for Annexin A2 on TnC-immunoprecipitated 5 DPI TA muscle protein lysates. d Dot plot representing the changes in the relative expression to the threshold of TnC and the percentages of TnC expressing cell populations within Pax7⁺Anxa2⁺ and Pax7⁺Anxa2^- MuSC and Pdgfra⁺Anxa2⁺ and Pdgfra⁺Anxa2^- FAPs in uninjured TA muscles and across different timepoints post injury (3.5, 5, 21 DPI). Data originally from Oprescu et al. (n = 3). e Representative IF images of GFP control and Annexin A2 (AnxA2) knockdown (KD) being treated or not with recombinant TnC (48 h treatment) (Pax7, red; GFP, green; DAPI, blue) (scale bar = 50 µm) and quantification of Pax7⁺ cells normalized on non-treated GFP-control MuSC (n = 3). Data are represented as mean ± SEM; ****p < 0.0001, one-way ANOVA (e).

Fig. 5. TnC promotes MuSC migration in vitro.

a Representative images of time-lapse microscopy to track cultured MuSC from adult WT and TnC-KO mice. Scale bar = 50 µm. b Quantification of total distance covered (n = 3, N_cell ≥ 75). c Quantification of average velocity of cells (n = 3, N_cell ≥ 75). d Quantification of migrated TnC-KO cells through transwell matrix normalized to WT (n = 6). e Quantification of the percentage of proliferating cells by EdU staining (timepoint = 48 h) (n = 3). f Representative immunofluorescence (IF) images of migrated WT and TnC-KO MuSC from adult mice with or without recombinant TnC treatment (EdU, gray; DAPI, blue) (scale bar = 50 µm). Quantification of the number of migrated WT and TnC-KO cells through transwell matrix after vehicle or TnC treatment (per mm²) (timepoint = 48 h) and quantification of the percentage of proliferating cells through EdU staining (timepoint = 48 h) (n = 3). Data are represented as mean ± SEM; *p < 0.05, t-test (b, c, d), *p < 0.05, **p < 0.01, one-way ANOVA (f).

Fig. 6. TnC declines with aging and is required for muscle maintenance and regeneration of aged skeletal muscles.

a Western blot for TnC on young and old WT mice and quantification of TnC protein relative expression (n = 4). b Representative immunofluorescence (IF) images of 5 DPI TA muscles of young and old mice (TnC, red; laminin, gray; DAPI, blue) (n = 3). Scale bar = 50 µm. c Representative IF images of cross-sections of uninjured TA muscles from young and old WT and TnC-KO mice (laminin, green; DAPI, blue) (scale bar = 50 µm) and quantification of the cross-sectional area (CSA) in µm² (n = 3). d Representative IF images of cross-sections of uninjured TA muscles from old WT and TnC-KO mice (Pax7, green; laminin, red; DAPI, blue) (scale bar = 50 µm) for Pax7⁺ cell quantification. Pax7⁺ cells are indicated with yellow arrows (n = 3). e Representative IF images of cross-sections of injured (5 DPI) TA muscles from young and old WT and TnC-KO mice (laminin, green; eMyHC, red; DAPI, blue) (scale bar = 50 µm) and quantification of the percentage of regenerating area (eMyHC⁺) (n = 3). f Quantification of migrated aged WT MuSC through transwell matrix (48 h culture) normalized to WT young mice (WT young n = 6; WT old n = 4). g Representative IF images of migrated aged WT MuSC with or without recombinant TnC treatment (timepoint= 48 h) (DAPI, gray) (scale bar = 50 µm) and quantification of migrated aged WT MuSC through transwell matrix after TnC treatment normalized to control non-treated cells (n = 4). h Representative IF images of Pax7⁺ young and old WT MuSC cells (yellow arrows) treated with or without recombinant TnC (timepoint = 48 h) (Pax7, red; DAPI, blue) (scale bar = 50 µm) and quantification of Pax7⁺ cells after TnC treatment normalized on control non-treated WT young cells (n = 3). Data are represented as mean ± SEM; *p < 0.05, **p < 0.01, t-test (a, d, f, g), *p < 0.05, **p < 0.005, ***p < 0.001, ****p < 0.0001, one-way ANOVA (c, e, h).