Fibroblast growth factor-inducible 14 regulates satellite cell self-renewal and expansion during skeletal muscle repair

Abstract

Skeletal muscle regeneration in adults is predominantly driven by satellite cells. Loss of satellite cell pool and function leads to skeletal muscle wasting in many conditions and disease states. Here, we demonstrate that the levels of fibroblast growth factor-inducible 14 (Fn14) are increased in satellite cells after muscle injury. Conditional ablation of Fn14 in Pax7-expressing satellite cells drastically reduces their expansion and skeletal muscle regeneration following injury. Fn14 is required for satellite cell self-renewal and proliferation as well as to prevent precocious differentiation. Targeted deletion of Fn14 inhibits Notch signaling but leads to the spurious activation of STAT3 signaling in regenerating skeletal muscle and in cultured muscle progenitor cells. Silencing of STAT3 improves proliferation and inhibits premature differentiation of Fn14-deficient satellite cells. Furthermore, conditional ablation of Fn14 in satellite cells exacerbates myopathy in the mdx mouse model of Duchenne muscular dystrophy (DMD) whereas its overexpression improves the engraftment of exogenous muscle progenitor cells into the dystrophic muscle of mdx mice. Altogether, our study highlights the crucial role of Fn14 in the regulation of satellite cell fate and function and suggests that Fn14 can be a potential molecular target to improve muscle regeneration in muscular disorders.

Keywords: Muscle regeneration; Muscular dystrophy; Notch; Satellite cells; Stat3.

FIGURE 1.. Expression of Fn14 in satellite cells.

(A) Violin plots showing gene expression of Fn14 (gene name: Tnfrsf12a) and TWEAK (gene name: Tnfsf12) in satellite cells at different time points after muscle injury in mice analyzed from a publicly available scRNA-seq dataset (GSE143435). (B) Single cells isolated from uninjured and injured TA muscle of WT mice were subjected to FACS analysis for the expression of α7-integrin and Fn14 protein. Representative scatter plots of FACS-based analysis demonstrate presence of Fn14⁺ cells amongst α7-integrin⁺ population (lower panel). (C) Satellite cells isolated from TA muscle of WT mice at day 0, 5, or 21 post-injury were analyzed for mRNA levels of Fn14 by performing qPCR. n= 3 mice in each group. Data are presented as mean ± SEM and analyzed by one-way ANOVA followed by Tukey’s multiple comparison test. #p ≤ 0.05, values significantly different from uninjured muscle (day 0). *p ≤ 0.05, values significantly different from 5d-injured TA muscle. (D) Representative photomicrographs showing expression of Fn14 in Pax7⁺ cells in cultured mouse primary myoblasts. Scale bar: 50 μm.

FIGURE 2.. Satellite cell-specific ablation of Fn14 inhibits muscle regeneration.

(A) Schematic representation of the experimental design indicating tamoxifen regimen for Fn14 deletion, muscle injury, and collection. (B) Body weight of Fn14^fl/fl and Fn14^scKO mice. (C) Uninjured and 5d-injured TA muscle wet weight normalized by body weight (BW) of Fn14^fl/fl and Fn14^scKO mice. (D) Uninjured and 14d-injured TA muscle wet weight normalized by BW of Fn14^fl/fl and Fn14^scKO mice. (E) Representative photomicrographs of H&E-stained transverse sections of TA muscle of Fn14^fl/fl and Fn14^scKO mice at indicated time points after injury. Scale bar: 50 μm. Quantitative analysis of average myofiber cross-sectional area (CSA) in TA muscle of Fn14^fl/fl and Fn14^scKO mice on (F) day 0, (G) day 5 and (H) day 14 post-injury. (I) Representative photomicrographs of transverse sections of 5d-injured TA muscle of Fn14^fl/fl and Fn14^scKO mice after immunostaining for embryonic isoform of myosin heavy chain (eMyHC) and laminin protein. Nuclei were identified by staining with DAPI. Scale bar: 50 μm. Quantification of (J) average CSA of eMyHC⁺ laminin⁺ myofibers, (K) percentage of eMyHC⁺ laminin⁺ myofibers containing 2 or more centrally located nuclei in 5d-injured TA muscle of Fn14^fl/fl and Fn14^scKO mice. n= 3–6 mice in each group. All data are presented as mean ± SEM. *p ≤ 0.05, values significantly different from contralateral uninjured muscle of Fn14^fl/fl or Fn14^scKO mice; #p ≤ 0.05, values significantly different from corresponding 5d- or 14d-injured TA muscle of Fn14^fl/fl mice analyzed by two-way ANOVA followed by Tukey’s multiple comparison test. ^@p ≤ 0.05, values significantly different from corresponding Fn14^fl/fl mice analyzed by unpaired Student t test.

FIGURE 3.. Fn14 regulates satellite cell number in regenerating skeletal muscle.

(A) Representative photomicrographs of 5d-injured TA muscle sections of Fn14^fl/fl and Fn14^scKO mice after immunostaining for Pax7 (red) and laminin (green) protein. Nuclei were identified by staining with DAPI. Scale bar: 50 μm. White arrows point to Pax7⁺ satellite cells. (D) Average number of Pax7⁺ cells per unit area in (B) 5d-injured and (C) 14d-injured TA muscle of Fn14^fl/fl and Fn14^scKO mice. (D) Single cell suspensions were isolated from TA muscle of Fn14^fl/fl and Fn14^scKO mice were subjected to FACS analysis for satellite cells. Representative FACS dot plots demonstrating the percentage of α7-integrin⁺ cells in 5d-injured TA muscle of Fn14^fl/fl and Fn14^scKO mice. (E) Quantification of α7-integrin⁺ satellite cells in 5d-injured TA muscle of Fn14^fl/fl and Fn14^scKO mice assayed by FACS. Relative mRNA levels of (E) Pax7, (F) Myod1, Myh3 and Myog in 5d-injured TA muscle of Fn14^fl/fl and Fn14^scKO mice assayed by performing qPCR. n= 3–4 mice in each group. All data are presented as mean ± SEM. #p ≤ 0.05, values significantly different from corresponding muscle of Fn14^fl/fl mice analyzed by unpaired Student t test.

FIGURE 4.. Fn14 mediates activation and proliferation of satellite cells.

(A) Gene ontology (GO) biological processes associated with downregulated and upregulated genes. (B) Heatmap representing selected genes involved in the regulation of cell proliferation in cultured WT and Fn14-KO myogenic cells generated after analysis of RNA-seq dataset. (C) WT and Fn14-KO myogenic cultures were pulse-labeled with EdU for 60 min. Representative images of the cultures after detection of EdU and Hoechst staining (nuclei detection). Scale bar: 50 μm. (D) Quantification of percentage of EdU⁺ cells in WT and Fn14-KO cultures. n= 3 biological replicates in each group. (E) TA muscle of Fn14^fl/fl and Fn14^scKO mice was injured by intramuscular injection of 1.2% BaCl₂ solution. After 3 days, the mice were given an intraperitoneal injection of EdU and 11 days later TA muscles were collected, transverse muscle sections were generated and stained to detect EdU, anti-laminin, and nuclei. Representative photomicrographs after EdU, laminin, and DAPI staining are presented here. Scale bar: 50 μm. (F) Quantification of the EdU⁺ nuclei per unit area. n= 4 mice in each group. (G) Single myofibers were isolated from EDL muscle of Fn14^fl/fl and Fn14^scKO mice. After 48 h of culturing, the myofibers were pulse-labeled with EdU for 60 min. Representative merged images of EdU⁺ and DAPI-stained myofibers are presented here. Scale bar: 100 μm. (H) Quantification of number of EdU⁺ cells per myofiber. n= 3 mice in each group. Analysis was done using 15–20 myofibers for each mouse. n= 3 mice in each group. All data are presented as mean ± SEM. #p ≤ 0.05, values significantly different from WT myoblast, or corresponding muscle of Fn14^fl/fl mice, analyzed by unpaired Student t test.

FIGURE 5.. Fn14 promotes satellite cell self-renewal.

(A) TA muscle of Fn14^fl/fl and Fn14^scKO mice was injured by intramuscular injection of 1.2% BaCl₂ solution. After 21 days, the same TA muscle was injured again, and the muscle was isolated 5 days later. Representative photomicrograph of H&E- and Sirius red-stained TA muscle sections. Quantification of (B) percentage of myofibers containing two or more centrally located nuclei and (C) percentage of total area stained with Sirius red. Scale bar: 50 μm. (D) Representative photomicrograph and (E) quantification of Pax7⁺ cells per unit area in double injured TA muscle sections of Fn14^fl/fl and Fn14^scKO mice after immunostaining for Pax7 (red) and laminin (green) protein. Nuclei were identified by staining with DAPI. Scale bar: 50 μm. n= 3–4 mice in each group. (F) Heatmap of selected genes associated with stem cell population maintenance in WT and Fn14-KO cultures generated after analysis of RNA-seq dataset. (G) Immunoblots and (H) densitometry analysis showing levels of cleaved Notch1 and total Notch1 protein in WT and Fn14-KO cultures. n= 3 biological replicates in each group. (I) Relative mRNA levels of Notch receptors (Notch1, Notch2, Notch3), Notch ligands (Jagged1, Jagged2, Dll1, and Dll4), and Notch targets (Hes1, Hes6, Heyl, and Hey1) in 5d-injured TA muscle of Fn14^fl/fl and Fn14^scKO mice. n= 4 mice in each group. All data are presented as mean ± SEM. #p ≤ 0.05, values significantly different from corresponding muscle of Fn14^fl/fl mice analyzed by unpaired Student t test.

FIGURE 6.. Deletion of Fn14 leads to precocious differentiation of satellite cells.

(A) Heatmap representing selected genes associated with muscle cell differentiation in WT and Fn14-KO myogenic cultures generated after analysis of RNA-seq dataset. (B) Immunoblots and (C) densitometry analysis of protein levels of Pax7, myogenin, Fn14, and unrelated protein GAPDH in WT and Fn14-KO cultures. n= 3 biological replicates in each group. (D) Single myofibers were isolated from EDL muscle of Fn14^fl/fl and Fn14^scKO mice and fixed at 0 h or at 72 h of culturing and stained for Pax7 and MyoD protein. Nuclei were counterstained with DAPI. Representative individual-stained and merged images of 0 h and 72 h cultured myofibers from Fn14^fl/fl and Fn14^scKO mice. Scale bars: 100 μm. Quantification of (E) number of Pax7⁺ cells per myofiber, and (F) number of MyoD⁺ cells per myofiber at 0 h. Quantification of (G) number of clusters per myofiber, percentage of (H) Pax7⁺/MyoD⁻ (self-renewing), (I) Pax7⁺/MyoD⁺ (activated/proliferating), and (J) Pax7⁻/MyoD⁺ (differentiating) cells per myofiber following 72 h of culturing. Analysis was done using 15–20 myofibers for each mouse at each time point. n= 3 mice in each group. All data are presented as mean ± SEM. #p ≤ 0.05, values significantly different from WT myoblast, or corresponding muscle of Fn14^fl/fl mice analyzed by unpaired Student t test.

FIGURE 7.. Fn14 regulates STAT signaling in myogenic cells.

(A) Heatmap of selected genes associated with positive regulation of Jak-Stat pathway, target genes, and inhibitors of Jak-Stat signaling in WT and Fn14-KO cultures generated after analysis of RNA-seq dataset. (B) Immunoblots showing protein levels of phosphorylated and total levels of Stat2, Stat3 and Stat5 protein in uninjured and 5d-injured TA muscle of Fn14^fl/fl and Fn14^scKO mice. n= 3–4 mice in each group. (C) Immunoblots showing protein levels of phosphorylated and total Stat3 and Fn14 protein in WT and Fn14-KO cultures. (D) WT and Fn14-KO myoblasts were transfected with control and Stat3 siRNA. After 24 h, the cells were pulse-labeled with EdU for 60 min and analyzed for EdU incorporation. Representative photomicrographs are presented here. Scale bar: 100 μm. (E) Quantification of percentage of EdU⁺/Hoechst33258⁺ cells in WT and Fn14-KO cultures transfected with control or Stat3 siRNA. n= 3 biological replicates in each group. (F) Representative immunoblots and (G) densitometry analysis showing protein levels of Pax7, myogenin, Stat3 and Fn14 in WT and Fn14-KO myogenic cultures transfected with control or Stat3 siRNA. n= 6 biological replicates in each group. All data are presented as mean ± SEM. *p ≤ 0.05, values significantly different from corresponding cultures transfected with control siRNA; #p ≤ 0.05, values significantly different from corresponding WT cultures analyzed by two-way ANOVA followed by Tukey’s multiple comparison test.

FIGURE 8.. Satellite cell-specific ablation of Fn14 exacerbate dystrophic phenotype in mdx mice.

(A) Heatmaps showing expression of Fn14 (gene name: Tnfrsf12a), TWEAK (gene name: Tnfsf12), Nfkb1, Nfkb2, Rela, Relb, and Nfkbia in muscle stem cells of WT and mdx, and WT and D2.mdx mice analyzed from a publicly available single cell dataset (GSE213925). (B) Representative pictures of 7-week-old mdx;Fn14^fl/fl and mdx;Fn14^scKO mice. Quantitative analysis of (C) body weight, and (D) wet weight of tibialis anterior (TA), gastrocnemius (GA), and quadriceps (QUAD) muscle normalized by BW of corresponding mice. (E) Representative photomicrographs of GA muscle sections of 7-week-old mdx;Fn14^fl/fl and mdx;Fn14^scKO mice after H&E staining, anti-eMyHC staining, or anti-Pax7/anti-laminin/DAPI staining. White arrows point to satellite cells in muscle sections. Scale bar: 50 μm. Quantification of (F) number of centronucleated fibers (CNF) per field, (G) average CSA of eMyHC⁺ myofibers, and (H) average number of Pax7⁺ cells per unit area. (I) Western blot demonstrating levels of Fn14 protein in myoblasts transduced with EGFP (control) or Fn14 overexpressing (Fn14OE) retrovirus. (J) TA muscle of 8-week-old mdx mice was injured by intramuscular injection of 1.2% BaCl₂ solution. After 24 h, the muscle was injected with 1 × 10⁶ primary myoblasts stably transduced with control or Fn14OE retrovirus. After 28 days, the TA muscle was isolated and weighed. (K) Representative anti-dystrophin-stained images of TA muscle sections of mdx mice transplanted with control or Fn14OE myoblasts. (L) Quantitative analysis of average CSA of dystrophin⁺ myofibers in TA muscle of mdx mice transplanted with control or Fn14OE myoblasts. n= 3–6 mice in each group. All data are presented as mean ± SEM. #p ≤ 0.05, values significantly different from mdx;Fn14^fl/fl mice analyzed by unpaired Student t test. *p ≤ 0.05, values significantly different from TA muscle injected with control myoblasts by unpaired Student t test.