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# Metabolic reprogramming of fibro/adipogenic progenitors facilitates muscle regeneration.

^{1}, Rosina M

^{1}, Krahmer N

^{2}, Palma A

^{1}, Petrilli LL

^{1}, Maiolatesi G

^{1}, Massacci G

^{1}, Salvatori I

^{3}, Valle C

^{3,}

^{4}, Testa S

^{1}, Gargioli C

^{1}, Fuoco C

^{1}, Castagnoli L

^{5}, Cesareni G

^{6,}

^{3}, Sacco F

^{7}.

### Author information

- 1
- Department of Biology, University of Rome Tor Vergata, Rome, Italy.
- 2
- Department Proteomics and Signal Transduction, Max-Planck Institute of Biochemistry, Martinsried, Germany.
- 3
- Fondazione Santa Lucia Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS), Rome, Italy.
- 4
- Institute of Translational Pharmacology, Consiglio Nazionale delle Ricerche (CNR), Rome, Italy.
- 5
- Department of Biology, University of Rome Tor Vergata, Rome, Italy castagnoli@uniroma2.it.
- 6
- Department of Biology, University of Rome Tor Vergata, Rome, Italy cesareni@uniroma2.it.
- 7
- Department of Biology, University of Rome Tor Vergata, Rome, Italy francesca.sacco@uniroma2.it.

### Abstract

In Duchenne muscular dystrophy (DMD), the absence of the dystrophin protein causes a variety of poorly understood secondary effects. Notably, muscle fibers of dystrophic individuals are characterized by mitochondrial dysfunctions, as revealed by a reduced ATP production rate and by defective oxidative phosphorylation. Here, we show that in a mouse model of DMD (*mdx*), fibro/adipogenic progenitors (FAPs) are characterized by a dysfunctional mitochondrial metabolism which correlates with increased adipogenic potential. Using high-sensitivity mass spectrometry-based proteomics, we report that a short-term high-fat diet (HFD) reprograms dystrophic FAP metabolism in vivo. By combining our proteomic dataset with a literature-derived signaling network, we revealed that HFD modulates the β-catenin-follistatin axis. These changes are accompanied by significant amelioration of the histological phenotype in dystrophic mice. Transplantation of purified FAPs from HFD-fed mice into the muscles of dystrophic recipients demonstrates that modulation of FAP metabolism can be functional to ameliorate the dystrophic phenotype. Our study supports metabolic reprogramming of muscle interstitial progenitor cells as a novel approach to alleviate some of the adverse outcomes of DMD.

© 2020 Rosina et al.

*mdx*fibro/adipogenic progenitors (FAPs) reveals a significant alteration of key metabolic pathways.

**(A)**The metabolic pathway map derived from Wikipathways (http://www.wikipathways.org) of key metabolic enzymes significantly modulated in

*mdx*FAPs compared with

*wt*. For each detected enzyme, a corresponding square is color coded according to the log2 fold change of the protein expression level in

*mdx*compared with

*wt*FAPs.

**(B)**Boxplot representing the log2 fold change (

*mdx*/

*wt*) of the abundance of metabolic enzymes annotated with the GO terms

*glycolysis*,

*TCA cycle*,

*pentose phosphate*, and

*fatty acid metabolism*in FAPs.

**(C)**Western Blot of PKM2, CV-ATP5a, CIII-UQCRC2, and vinculin in FAPs isolated from the hind limbs of young (45-d old)

*wt*and

*mdx*mice (

*wt*FAPs

*n*= 3;

*mdx*FAPs

*n*= 5).

**(D)**Bar graphs representing the fold change of the enzymes PKM2, CV-ATP5a, and CIII-UQCRC2 in FAPs. Protein levels were normalized to vinculin.

**(E)**Western Blot of PKM2, CV-ATP5a, CIII-UQCRC2, and vinculin in muscle satellite cells (MuSCs) isolated from the hind limbs of

*wt*and

*mdx*mice (

*wt*MuSCs

*n*= 3;

*mdx*MuSCs

*n*= 5).

**(F)**Bar graphs representing the fold change of the enzymes PKM2, CV-ATP5a, and CIII-UQCRC2 in MuSCs. Protein levels were normalized to vinculin. Statistical significance was estimated by

*t*test. All data are represented as mean ± SEM and statistical significance is defined as *

*P*< 0.05; **

*P*< 0.01; ***

*P*< 0.001.

^{+}FAPs and ITGA7

^{+}MuSCs.

**(A)**Scatter plots of Sca1

^{+}FAPs (from 45-d old

*wt*mice) stained with antibodies raised against CD140a-APC (PDGFRα) (left panel). Scatter plots of ITGA7

^{+}MuSCs (from 45-d-old

*wt*mice) stained with antibodies raised against ITG7-APC and Pax7-FITC (right panel).

**(B)**Percentage of Sca1

^{+}FAPs expressing CD140a (PDGFRα) (left table). Percentage of ITGA7

^{+}MuSCs co-expressing ITGA7 and Pax7 (left table). Data from three independent cell preparations are shown.

**(C)**Representative immunofluorescence showing the expression of CD140a (PDGFRα) in FAPs and the expression of Pax7 and MyoD in MuSCs.

**(A)**Mitochondrial stress test profile of

*wt*and

*mdx*FAPs (from 45-d old

*wt*and mdx

*mice*) by Seahorse analysis. The oxygen consumption rate (OCR) (pmol/min/10

^{3}cells) was monitored for 80 min under basal conditions and upon sequential treatment with the mitochondrial inhibitors oligomycin, FCCP, and rotenone/antimycin (

*wt*FAPs

*n*= 4;

*mdx*FAPs

*n*= 6).

**(B, C, D, E, F, G, H, I, J)**Bar graphs representing basal OCR (B), ATP production (C), spare capacity (D), OCR/extracellular acidification rate ratio (E), coupling efficiency (% to the basal OCR) (F), spare capacity (% to the basal OCR) (G), proton leak (H), maximal respiration (I), and non-mitochondrial oxygen consumption (J) obtained by Seahorse Wave Desktop software. Statistical significance was estimated by the

*t*test.

**(K)**Bar graph representing the median fluorescence intensity (MFI) of MitoTracker RED (MT-Red) dye in flow cytometry in basal condition and under uncoupling with 10 μM FCCP, in

*wt*and

*mdx*FAPs (

*n*= 3). Statistical significance was estimated by the

*t*test.

**(L)**Bar graph representing median fluorescence intensity (MFI) of MitoTracker GREEN (MT Green) dye in flow cytometry in basal condition on

*wt*and

*mdx*FAPs (

*n*= 3). Statistical significance was estimated by the

*t*test.

**(M)**EdU labelling and growth curve profile of FAPs purified from 45-d old

*wt*and

*mdx*mice. FAPs were cultured for 20 d (

*wt n*= 3;

*mdx n*= 3). Statistical significance was estimated by two-way ANOVA.

**(N)**Representative EdU (green, 20× magnification; scale bar, 100 μm), Oil Red O (ORO) staining (red, 40× magnification; scale bar, 100 μm), and confocal micrographs of perilipin immunostaining (red, 20× magnification; scale bar, 70 μm) of FAP cells at 3, 7, and 20 d. Nuclei (blue) were revealed with Hoechst 33342.

**(N, O)**Bar graph resenting the adipogenic differentiation index of

*wt*and

*mdx*FAPs calculated as ORO-positive pixels/cell from the panel (N) (

*n*= 3).

**(N, P)**Bar graph representing the average lipid droplet area (μm

^{2}) of confocal images in panel (N) (

*wt n*= 4;

*mdx n*= 3).

**(N, Q)**Bar graph representing the frequency distribution of lipid droplet areas of confocal images in panel (N) (

*wt n*= 4;

*mdx n*= 3). Statistical significance was estimated by

*t*test.

**(R)**Representative ORO staining (10× magnification; scale bar, 100 μm) of FAPs from 45-d-old

*wt*and

*mdx*mice. Adipogenic differentiation was obtained by incubating FAPS in adipocyte differentiation medium (ADM) followed by the adipocyte maintenance medium (AMM) in the presence of 25 mM glucose (Glc) supplemented with DMSO (vehicle) or 250 μM 2-deoxyglucose (2-DG). Alternatively, FAPs were differentiated by incubating cells with the opportune differentiation media containing either 25 mM Glc, 2 mM Glc, or 10 mM galactose (Gal). Insets are enlarged views of the dashed areas (scale bar: 100 μm). Nuclei (blue) were revealed with Hoechst 33342.

**(S)**Bar plots reporting the adipogenic index (left) and the average number of nuclei per field (right) for FAPs differentiated in the presence of the vehicle or 250 μM 2-DG treatment.

**(T)**Bar plots reporting the adipogenic index (left) and the average number of nuclei per field (right) for FAPs differentiated in the presence of 25 mM Glc, 2 mM Glc, or 10 mM Gal treatment (

*wt n*= 3;

*mdx n*= 3). Statistical significance was estimated by two-way ANOVA. All data are represented as mean ± SEM and statistical significance is defined as *

*P*< 0.05; **

*P*< 0.01; ***

*P*< 0.001.

*mdx*fibro/adipogenic Progenitors. MuSC differentiation can be modulated by metabolic interventions. Referred to .

**(A)**. Mitochondrial stress test profile of

*wt*and

*mdx*MuSCs (from 45-d-old

*wt*and

*mdx*mice). Oxygen consumption rate (OCR) (pmol/min/10

^{3}cells) monitored in real time (for 80 min) under basal conditions and upon sequential treatment with mitochondrial inhibitors (

*wt n*= 4;

*mdx n*= 6).

**(B, C, D, E, F, G, H, I, J)**Bar graphs representing the basal OCR (B), ATP production (C), spare capacity (D), OCR/Extra-Cellular Acidification Rate ratio (E), percentage of coupling efficiency (F), percentage of spare respiratory capacity (G), proton leak (H), and maximal respiration (I) non-mitochondrial oxygen consumption (J). Statistical significance was estimated by

*t*test.

**(K)**Representative MyHC immunostaining (10× magnification; scale bar, 100 μm) of

*wt*and

*mdx*MuSC-derived myotubes (from 45-d old mice) upon incubation with DMSO (vehicle), 250 μm 2-DG, 25 mM Glc alone, 2 mM Glc, or 10 mM Gal. Nuclei (blue) were counterstained with Hoechst 33342.

**(L)**Bar plots showing the fusion index and the average number of nuclei per field of MuSCs differentiated in the presence of vehicle and 250 μM 2-DG (

*wt*MuSCs

*n*= 5;

*mdx*MuSCs

*n*= 7).

**(M)**Bar plots showing the fusion index and the average number of nuclei per field of MuSCs differentiated in the presence of 25 mM Glc, 2 mM Glc, or 10 mM Gal (

*wt*MuSCs

*n*= 5;

*mdx*MuSCs 25 mM Glc

*n*= 5;

*mdx*MuSCs 2 mM Glc

*n*= 6;

*mdx*MuSCs 10 mM Gal

*n*= 7). Statistical significance was estimated by two-way ANOVA.

**(N)**Representative TUNEL immunofluorescence showing pyknotic nuclear dots (red) in

*mdx*MuSCs (from 45-d-old mice) incubated in the presence of DMSO (vehicle), 250 μm 2-DG, 25 mM Glc alone, 2 mM Glc, or 10 mM Gal. Nuclei (blue) were counterstained with Hoechst 33342.

**(O)**Bar plot showing the percentage of pyknotic nuclear dots (TUNEL

^{+}dots) in

*mdx*MuSCs incubated in the presence of DMSO (vehicle) or 250 μm 2-DG (

*n*= 4). Statistical significance was estimated by

*t*test.

**(P)**Bar plot showing the percentage of pyknotic nuclear dots (TUNEL

^{+}dots) in

*mdx*MuSCs incubated in the presence of 25 mM Glc, 2 mM Glc, or 10 mM Gal (

*n*= 4). Statistical significance was estimated by one-way ANOVA.

**(Q)**Representative EdU labelling showing proliferating

*mdx*MuSCs (green) (from 45-d-old mice) incubated in the presence of DMSO (vehicle), 250 μm 2-DG, 25 mM Glc alone, 2 mM Glc, or 10 mM Gal. Nuclei (blue) were counterstained with Hoechst 33342.

**(R)**Bar plot showing the percentage of EdU

^{+}

*mdx*MuSCs upon incubation with DMSO (vehicle) or 250 μm 2-DG (

*n*= 4). Statistical significance was estimated by

*t*test.

**(S)**Bar plot showing the percentage of EdU

^{+}

*mdx*MuSCs upon incubation with 25 mM Glc, 2 mM Glc, or 10 mM Gal (

*n*= 4). Statistical significance was estimated by one-way ANOVA. All data are represented as mean ± SEM and statistical significance is defined as *

*P*< 0.05; **

*P*< 0.01; ***

*P*< 0.001.

**(A)**Bar plot showing the doubling time of proliferating

*wt*and

*mdx*FAPs (

*n*= 3). Statistical significance was estimated by

*t*test.

**(B)**Quantitation of the ATP in

*mdx*FAPs, purified from 45-d-old mice, treated with DMSO (vehicle), 250 μm 2-DG, 2 mM Glc, or 10 mM Gal (

*n*= 4). Statistical significance was estimated by one-way ANOVA.

**(C)**Representative Ki67 staining showing proliferating

*mdx*FAPs (red), from 45-d-old mice, incubated in the presence of DMSO (vehicle), 250 μm 2-DG, 25 mM Glc alone, 2 mM Glc, or 10 mM Gal. Nuclei (blue) were counterstained with Hoechst 33342. Scale bar 100 μm.

**(D)**Bar plot (left) showing the percentage of Ki67

^{+}FAPs incubated in the presence of DMSO (vehicle) or 250 μm 2-DG (

*n*= 4). Statistical significance was estimated by

*t*test. Bar plot (right) reporting the percentage of Ki67

^{+}FAPs incubated in the presence of 25 mM Glc (

*n*= 3), 2 mM Glc, or 10 mM Gal (

*n*= 4). Statistical significance was estimated by one-way ANOVA.

**(E)**Bar plot (left) showing the percentage of pyknotic nuclear dots (TUNEL

^{+}dots) in FAPs incubated in the presence of DMSO (vehicle) or 250 μm 2-DG (

*n*= 4). Statistical significance was estimated by

*t*test. Bar plot (right) reporting the percentage of pyknotic nuclear dots (TUNEL

^{+}dots) in FAPs incubated in the presence of 25 mM Glc, 2 mM Glc, or 10 mM Gal (

*n*= 4). Statistical significance was estimated by one-way ANOVA. All data are represented as mean ± SEM and statistical significance is defined as *

*P*< 0.05; **

*P*< 0.01; ***

*P*< 0.001.

*wt*and

*mdx*mice. Referred to .

**(A)**Weight (grams, g), food (g), and water (milliliters, ml) intake in 49-d-old

*wt*and

*mdx*mice were recorded every 2 d during the diet period.

**(B)**Bar plot (left) reporting the organ weight for the liver, spleen, heart, and brain of 49-d-old

*wt*and

*mdx*mice fed with low-fat diet (LFD) or HFD. Bar plot (right) reporting the weight of adipose depots BAT, brown adipose tissue; eWAT, epididymal adipose tissue; iWAT, inguinal adipose tissue explanted from

*wt*and

*mdx*mice fed with LFD or HFD (

*wt*LFD

*n*= 8 mice;

*wt*HFD

*n*= 7 mice;

*mdx*LFD

*n*= 6 mice;

*mdx*HFD

*n*= 8 mice).

**(C)**Box plot reporting the weight of the minced muscle tissue before the enzymatic digestion for each condition (

*wt*LFD

*n*= 8 mice;

*wt*HFD

*n*= 7 mice;

*mdx*LFD

*n*= 10 mice;

*mdx*HFD

*n*= 12 mice).

**(D)**Serum levels of triglycerides and cholesterol (milligrams per deciliter, mg/dl) from

*wt*and

*mdx*mice fed with LFD and HFD (

*wt*LFD

*n*= 5 mice;

*wt*HFD

*n*= 5 mice;

*mdx*LFD

*n*= 9 mice;

*mdx*HFD

*n*= 12 mice). Statistical significance was estimated by Two-way ANOVA. All data are represented as mean ± SEM and statistical significance is defined as *

*P*< 0.05; **

*P*< 0.01; ***

*P*< 0.001.

**(A)**Experimental workflow to analyze the proteome of FAPs and Muscle Satellite Cells purified from 49-d-old

*wt*and

*mdx*mice fed with low-fat diet (LFD) and HFD (

*wt*LFD

*n*= 3;

*wt*HFD

*n*= 2;

*mdx*LFD

*n*= 2;

*mdx*HFD

*n*= 3).

**(B)**Principal component analysis of the proteomic profiles of FAPs and muscle satellite cells from mice under LFD or HFD.

**(C)**The principal component analysis inset shows the sample separation of

*wt*/

*mdx*FAPs from mice fed with LFD and HFD.

**(D)**Scatterplot of the log2 fold change of protein expression level of 480 proteins significantly modulated in

*mdx*/

*wt*FAPs (y-axis) and HFD/LFD

*mdx*FAPs (x-axis).

**(E)**Two-dimensional annotation enrichment analysis of the significantly modulated proteins in

*mdx*/

*wt*FAPs (y-axis) and HFD/LFD

*mdx*FAPs (x-axis). Groups of related GO terms are labelled with the same color, as described in the inset.

**(F)**Quantitation of the ATP in FAPs purified from 49-d-old

*mdx*mice fed with LFD and HFD (

*n*= 3).

**(G)**Mitochondrial stress test profile of LFD and HFD

*mdx*FAPs. Oxygen consumption rate (pmol/min/10

^{3}cells) was monitored in real time (for 80 min) under basal condition and upon sequential treatment with mitochondrial inhibitors (

*mdx*LFD

*n*= 4;

*mdx*HFD

*n*= 4).

**(H, I)**Bar graphs representing basal oxygen consumption rate (H) and ATP production (I).

**(J, K)**Western blot (J) and relative densitometric analysis (K) of PKM2 and vinculin in FAPs from

*mdx*mice fed with LFD and HFD (

*mdx*LFD

*n*= 4;

*mdx*HFD

*n*= 4). Statistical significance was estimated by

*t*test. All data are represented as mean ± SEM and statistical significance is defined as *

*P*< 0.05; **

*P*< 0.01; ***

*P*< 0.001.

**(A)**Unsupervised hierarchical clustering of 4,500 proteins show that biological replicates cluster together.

**(B)**Heat map showing the Pearson correlation coefficients between the different biological replicates in the proteome.

**(C)**The loadings of the principal component analysis reveal that the proteins responsible for driving sample separations in component 1 and component 2 are significantly enriched for the reported GOBPs (FDR < 0.05).

**(D, E)**Statistically significant up- and down-regulated proteins in

*mdx*muscle satellite cells (MuSCs) (D) and fibro/adipogenic progenitors (FAPs) (E) upon high-fat diet (HFD) were identified by

*t*test (

*P*< 0.05) and represented as scatterplot.

**(F)**GOBPs and KEGG pathways significantly enriched (FDR < 0.08) in

*mdx*MuSCs and FAPs upon HFD were represented as bar graph.

**(G)**Scatterplot of the log2 fold change of expression level of 863 proteins significantly modulated in

*mdx*MuSCs and upon HFD.

**(H)**Two-dimensional annotation enrichment analysis of the significantly modulated proteins in

*mdx*MuSCs and upon HFD. Groups of related GO terms are labelled with the same color, as described in the inset.

**(I)**Boxplot of the HFD-dependent log2 fold change of the expression profile of metabolic enzymes involved in tricarboxylic acid cycle, OxPhos, and fatty acid oxidation in

*mdx*cells.

**(J)**Heat map of the HFD-dependent log2 fold change of the expression profile of metabolic enzymes involved in tricarboxylic acid cycle, OxPhos, and fatty acid oxidation in

*mdx*cells.

**(K)**LFQ intensity of PDK4 enzyme in

*mdx*FAPs and MuSCs.

**(A)**Cluster of proteins oppositely modulated by the disease state and high-fat diet treatment in fibro/adipogenic progenitors (right) and muscle satellite cells (left).

**(**Unsupervised hierarchical clustering of the 283 diet-cured proteins identified in fibro/adipogenic progenitors.

**)****B***mdx*fibro/adipogenic progenitors (FAPs). Referred to .

**(A, B)**The 283 HFD “cured” proteins were mapped onto the literature-derived network extracted from SIGNOR and Mentha database.

**(A, B)**Proteins were color coded according to their log2 fold change expression level comparing the

*mdx*versus

*wt*FAPs (A) and

*mdx*HFD versus LFD FAPs (B).

*mdx*muscle satellite cells (MuSCs). Referred to .

**(A, B)**The 220 HFD “cured” proteins were mapped onto the literature-derived network extracted from the SIGNOR and Mentha databases.

**(A, B)**Proteins were color coded according to their log2 fold change expression level comparing the

*mdx*versus

*wt*MuSCs (A) and

*mdx*HFD versus LFD MuSCs (B).

**(A)**Schematic representation of the main molecular events reverted by HFD treatment in

*mdx*FAPs.

**(B)**Representative confocal images of PDGFRα-positive FAPs (green) from 49-d-old

*wt*and

*mdx*mice fed with low-fat diet (LFD) and HFD (60× magnification; scale bar, 20 μm). Fibers (red) were stained using antibodies directed against the MyHC isoforms. Representative micrograph (20× magnification; scale bar, 100 μm) showing proliferating FAPs by coupling PDGFRα staining (green) with anti-Ki67 antibodies (red). Nuclei (blue) were revealed with Hoechst 33342.

**(C)**Bar plot reporting the number of PDGFRα-positive FAPs per cm

^{2}of muscle section (

*wt*LFD

*n*= 4;

*wt*HFD

*n*= 4;

*mdx*LFD

*n*= 3;

*mdx*HFD

*n*= 6).

**(D)**Bar plot reporting the fraction of Ki67-positive cells in PDGFRα-positive FAPs in TA cross-sections (

*wt*LFD

*n*= 4;

*wt*HFD

*n*= 4;

*mdx*LFD

*n*= 3;

*mdx*HFD

*n*= 6). Statistical significance was estimated by two-way ANOVA.

**(E)**Mass spectrometry–based quantitation of β-catenin and Mest in

*wt*and

*mdx*FAPs from mice fed with LFD and HFD.

**(F)**Quantitative PCR for β-catenin and Mest in

*mdx*FAPs from mice fed with HFD and LFD mice.

**(G)**Quantitative PCR of

*Follistatin*in

*wt*and

*mdx*FAPs from mice fed with LFD and HFD (

*wt*LFD n = 3 mice;

*mdx*LFD

*n*= 3 mice;

*mdx*HFD

*n*= 4 mice). Statistical significance was estimated by one-way ANOVA.

**(H)**Representative scheme summarizing the experimental procedure to treat, ex vivo, FAPs with BSA-coupled palmitate/oleate (50 μM/50 μM) and 100 μM carnitine.

**(H, I)**Quantitative PCR of Ctnnb1 and Fst transcripts in

*mdx*FAPs treated as shown in (H). Statistical significance was estimated by

*t*test.

**(J)**Representative scheme summarizing the experimental procedure to treat, ex vivo,

*mdx*FAPs with 20 nM LY2090314.

**(K)**Quantitative PCR of Ctnnb1 and Fst transcripts in

*mdx*FAPs treated with 20 nM LY2090314 for 48 and 72 h. Statistical significance was estimated by Two-way ANOVA (

*n*= 3).

**(L)**Bar plot reporting the concentrations of Follistatin in FAP-derived supernatants. Follistatin concentrations were analyzed via ELISA assay.

**(M)**Representative immunofluorescence (20× magnification; scale bar, 100 μm) of muscle satellite cell (MuSC)–derived myotubes (red) upon incubation with the control and LFD/HFD

*mdx*FAP-derived supernatants. Proliferating myoblasts (green) were detected using a Ki67 specific antibody.

**(N)**Bar plot reporting the fusion index (

*n*= 6) of differentiated MuSCs in each treatment condition.

**(O)**Bar plot reporting the fraction of Ki67-positive MuSCs in each treatment condition. Statistical significance was estimated by One-way ANOVA. All data are represented as mean ± SEM and Statistical significance is defined as *

*P*< 0.05; **

*P*< 0.01; ***

*P*< 0.001.

*mdx*phenotype.

**(A)**Serum creatine phosphokinase (units per liter, U/l) from 49-d-old

*wt*and

*mdx*mice fed with low-fat diet (LFD) or HFD (

*wt*LFD

*n*= 6;

*wt*HFD

*n*= 7;

*mdx*LFD

*n*= 6;

*mdx*HFD

*n*= 10).

**(B)**Representative hematoxylin and eosin staining of TA cross-sections from 49-d-old

*wt*and

*mdx*mice fed with LFD or HFD (20× magnification; scale bar, 100 µm).

**(C)**Percentage of centrally nucleated myofibers in TAs (

*wt*LFD

*n*= 8;

*wt*HFD

*n*= 6;

*mdx*LFD

*n*= 6;

*mdx*HFD

*n*= 8).

**(D)**Representative hematoxylin and eosin staining of diaphragm cross-sections from 49-d-old

*wt*and

*mdx*mice fed with LFD or HFD (20× magnification; scale bar, 100 μm).

**(E)**Percentage of centrally nucleated myofibers in diaphragms (

*wt*LFD

*n*= 3;

*wt*HFD

*n*= 3;

*mdx*LFD

*n*= 3;

*mdx*HFD

*n*= 3). Statistical significance was estimated by Two-way ANOVA.

**(F)**Representative picrosirius red staining of TA cross-sections (20× magnification; scale bar, 100 ìm).

**(G)**Bar plot showing the extent of picrosirius red area in TA cross-sections from 49-d-old

*wt*and

*mdx*mice fed with LFD or HFD (

*wt*LFD

*n*= 8;

*wt*HFD

*n*= 7;

*mdx*LFD

*n*= 6;

*mdx*HFD

*n*= 8).

**(H)**Representative picrosirius red staining of diaphragm cross-sections from 49-d-old

*wt*and

*mdx*mice fed with LFD or HFD (20× magnification; scale bar, 100 μm).

**(I)**Bar plot showing the extent of picrosirius red area in diaphragm cross-sections (

*wt*LFD

*n*= 3;

*wt*HFD

*n*= 3;

*mdx*LFD

*n*= 3;

*mdx*HFD

*n*= 3). Statistical significance was estimated by one-way ANOVA.

**(J)**Representative confocal micrographs of laminin-stained (green) TA cross-sections from 49-d-old

*wt*and

*mdx*mice fed with LFD or HFD (20× magnification; scale bar, 70 μm), left panel. Pseudo-color representation of the myofiber caliber, ranging from 0 to 5,000 μm

^{2}, right panel. Nuclei (blue) were revealed with Hoechst 33342.

**(K)**Bar plot reporting the average cross-sectional area in μm

^{2}(

*wt*LFD

*n*= 8;

*wt*HFD

*n*= 6;

*mdx*LFD

*n*= 6;

*mdx*HFD

*n*= 6). Statistical significance was estimated by two-way ANOVA.

**(L)**Immunoblot of TA lysates from

*wt*and

*mdx*mice fed with LFD and HFD. Samples were probed with anti-pmTOR (Ser2448), anti-pRPS6 (Ser240/244), anti-mTOR, anti-RPS6, and anti-vinculin antibodies.

**(M)**Bar plot showing the densitometric analysis of the phosphorylation level of mTOR (posho-mTOR).

**(N)**Bar plot showing the densitometric analysis of the phosphorylation level of RPS6 (posho-RPS6). Statistical significance was estimated by two-way ANOVA.

**(O)**Quantitative PCR of

*Igf1*in fibro/adipogenic progenitors from

*wt*and

*mdx*mice fed with LFD or HFD (

*wt*LFD

*n*= 3;

*wt*HFD

*n*= 4;

*mdx*LFD

*n*= 5;

*mdx*HFD

*n*= 7). Statistical significance was estimated by two-way ANOVA. All data are represented as mean ± SEM and statistical significance is defined as *

*P*< 0.05; **

*P*< 0.01; ***

*P*< 0.001.

*mdx*mice. Referred to .

**(A)**Representative immunofluorescent showing perilipin-positive adipocytes in diaphragm muscles from 49-d-old

*wt*and

*mdx*mice.

**(B)**Bar plot showing the average percentage of perilipin

^{+}area over the whole section area (

*n*= 3). Statistical significance was estimated by two-way ANOVA.

**(C)**Bar plot reporting the average percentage of the frequency distribution of the myofiber areas (μm

^{2}) in

*tibialis anterior*muscles for each experimental condition (

*wt*LFD

*n*= 8,

*wt*HFD

*n*= 6,

*mdx*LFD

*n*= 6,

*mdx*HFD

*n*= 6). Statistical significance was estimated by two-way ANOVA.

**(D)**Representative micrographs of laminin-stained (green) diaphragm cross-sections (20× magnification; scale bar, 100 μm).

**(E)**Bar plot reporting the average cross-sectional area (μm

^{2}). Statistical significance was estimated by two-way ANOVA.

**(F)**Bar plot reporting the average percentage of the frequency distribution of the myofiber areas for each experimental condition (

*wt*LFD

*n*= 3;

*wt*HFD

*n*= 3;

*mdx*LFD

*n*= 3;

*mdx*HFD

*n*= 3). Statistical significance was estimated by two-way ANOVA.

**(G)**Representative immunofluorescence of muscle satellite cells (MuSCs) purified from 49-d-old mdx mice treated for 28 d with LFD or HFD. MuSCs were immunostained with anti-Myod or anti-MyHC antibodies at the indicated time points. Nuclei (blue) were counterstained with Hoechst 33342.

**(H)**Bar plots reporting the fraction of MyoD

^{+}cells, EdU

^{+}cells, and average number of nuclei per field (

*mdx*MuSCs LFD

*n*= 3;

*mdx*MuSCs HFD

*n*= 4). At day 20, the fusion index and the average number of nuclei per field is reported for MuSC-derived myotubes (

*mdx*MuSCs LFD

*n*= 3;

*mdx*MuSCs HFD

*n*= 4). Statistical significance was estimated by

*t*test. All data are represented as mean ± SEM and statistical significance is defined as *

*P*< 0.05; **

*P*< 0.01; ***

*P*< 0.001.

**(A)**Workflow describing the FAP transduction and the transplantation strategies. FAPs were transplanted in 2-mo-old

*mdx*recipient mice. FAPs (1.0 × 10

^{5}cells) were resuspended in 100 μl of 1× PBS and injected in the TA of

*mdx*recipient mice.

**(B)**After 15 d from transplantation, GFP-positive cells (red) were found in the muscle interstitium (10× and 40× magnification; scale bar, 100 μm) (

*n*= 3). Fibers (grey) were probed using phalloidin-488.

**(C)**Representative immunofluorescence, showing that GFP-positive cells co-express the FAP distinctive marker PDGFRα (

*n*= 3). Nuclei (blue) were revealed with Hoechst 33342.

**(D)**Dystrophic (

*mdx*) TA muscles receiving FAPs from LFD- and HFD-treated

*mdx*mice were stained with anti-laminin antibodies to reveal the fiber outlines.

**(E)**Bar plot reporting the average percentage of the frequency distribution of the myofiber areas (μm

^{2}) in each experimental condition (

*n*= 3).

**(E, F)**Curves, inferred on the basis of the cross-sectional area distribution in (E), showing the shift toward higher fiber calibers upon transplantation of

*mdx*FAPs from mice fed with HFD (red line).

**(G)**Bar plot reporting the average cross-sectional area in μm

^{2}(

*n*= 3). Statistical significance was estimated by

*t*test. All data are represented as mean ± SEM and statistical significance is defined as *

*P*< 0.05; **

*P*< 0.01; ***

*P*< 0.001.