We previously generated and characterized a strain of DMD rat [22, 33]. In this strain of DMD rat, a deletion of 329 bp around the splice site in intron2 leads to exon3 skipping, and an insertion of 1 bp in exon16 causes the generation of a stop codon, resulting in a loss of dystrophin protein [22, 33]. Adult XDmdX female rats were mated with wild-type (WT) male rats to generate male WT (XY) and DMD (XDmdY) rats. They were maintained under controlled environmental conditions, at 23 °C with a light/dark cycle (lights on 0800–2000). Laboratory chow (Labo MR Standard, Nihon Nousan Co., Yokohama, Japan) and water were given ad libitum. All animal experiments performed in this study were in accordance with the Guide for the Care and Use of Laboratory Animals of the University of Tokyo and were approved by the Institutional Animal Care and Use Committee of the University of Tokyo (P18-125).

At 3 or 7 months old, the rats were transferred individually to a plastic cage and their amount of food intake was measured for two consecutive days. The measured values were divided by 2 and expressed as food intake per day.

At indicated age, the rats were killed by inhalation of carbon dioxide gas. After removal of facial skin, the area corresponding to the masseter muscle was measured. The masseter muscle was considered as an ellipse (Fig. 1B), and its long and short diameters were measured with calipers to calculate the area. Then, the lower half part of the masseter muscle was removed. The tongue was cut out at the root. Collected tissues were fixed in 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS) for paraffin-embedded sections or snap-frozen in liquid nitrogen-cooled isopentane for cryosections.

Food intake and morphometrical analyses of masseter and tongue muscles of WT and DMD rats. A The amount of food intake per day was measured at 3 and 7 months old. The data are expressed as mean ± SE. **, p < 0.01 by unpaired Student’s t-test. B The masseter muscle was considered as an ellipse (dotted white circle) and its long and short diameters were measured with calipers to calculate the area. C Changes in the area of the masseter muscle at 1, 3, 6, and 8 months old. The data are expressed as mean ± SE. **, p < 0.01 by unpaired Student’s t-test. D The tongue was cut at the root (dotted black line) (left) and the width (dotted black line) and area (dotted red circle) were measured. Scale bar = 1 cm. E Changes in the width and cross-sectional area of the tongue at 1, 3, and 8 months old. The data are expressed as mean ± SE. **, p < 0.01 by unpaired Student’s t-test

The paraffin-embedded sections were used for hematoxylin–eosin (HE) or Masson’s trichrome staining. The sections were observed and photographed using a microscope (BX51, Olympus, Tokyo, Japan) equipped with a digital camera (DP73, Olympus). The HE-stained tongue sections were used for measurement of the width and area of the tongue (Fig. 1D). Masson’s trichrome-stained sections were used for quantitative analysis of the fibrotic area. Two fields were randomly selected in the section using a 4 × objective and the area occupied by fibrotic tissues stained blue and the total area of sections were calculated using ImageJ software (v1.47; National Institutes of Health, Bethesda, MD, USA).

The cryosections (7 μm) were used for immunohistochemistry. The sections were fixed with 4% PFA in PBS for 15 min, followed by blocking with 5% normal donkey serum (NDS) in PBS. The sections were incubated with primary antibodies overnight at 4 °C, then secondary antibodies for 1 h.

For identification of satellite cells, vascular endothelial cells, and mesenchymal progenitor cells, anti-Pax7 (1:200 with 5% NDS in PBS, mouse, clone P3U1; Developmental Studies Hybridoma Bank (DSHB), Iowa City, IA, USA), anti-CD31 (1:400 with 5% NDS in PBS, rabbit, NB100-2284, Novus Biological, Centennial, CO, USA), and anti-chondroitin sulfate proteoglycan 4 (CSPG4) (1:50 with 5% NDS in PBS, mouse, clone 5C12 [34]) were used as primary antibodies, respectively, followed by AlexaFluor-labeled donkey anti-mouse IgG (1:500 with 5% NDS in PBS, Jackson ImmunoResearch, West Grove, PA, USA).

For identification of necrotic myofibers, anti-laminin (1:400 with 5% NDS in PBS, rabbit, L9393 Sigma, St Louis, MO, USA) was used as the primary antibody, and AlexaFluor-labeled goat anti-rat IgG and donkey anti-rabbit IgG (1:500 with 2.5% NDS/2.5% normal goat serum (NGS) in PBS, Jackson ImmunoResearch, West Grove) were used as secondary antibodies.

For identification of newly formed regenerated myofibers, anti-embryonic myosin heavy chain (eMHC) (1:400 with 5% NDS in PBS, mouse, clone F1.652, DSHB) and anti-laminin were used as primary antibodies, and AlexaFluor-labeled donkey anti-mouse IgG and donkey anti-rabbit IgG (1:500 with 5% NDS in PBS, Jackson ImmunoResearch) were used as secondary antibodies. After the reaction with secondary antibodies, nuclei were counterstained with Hoechst 33258. The sections were observed and photographed using a microscope equipped with a digital camera.

For quantification of Pax7-positive cells, IgG-positive necrotic myofibers, and embryonic myosin heavy chain (eMHC)-positive myofibers, several numbers of the field were photographed using × 4 or × 10 objectives, and the number of positive cells or myofibers were counted. Myofibers were identified by double-staining with an anti-laminin antibody. Data were expressed as percent positive myofibers or number of positive cells per area.

For quantification of myofiber size and the number of nuclei per myofiber, the sections were immunostained with anti-laminin, followed by labeling with AlexaFluor-labeled secondary antibody. The nuclei were counterstained with Hoechst 33258. The sections were photographed as described above. The myofibers were automatically identified by Cellpose Google Colab script (https://colab.research.google.com/drive/1958UQIH-XAYogKvbxnaUHALYvR73KLj2) [38], and their diameters (minimum Feret diameters) and numbers were measured using ImageJ software. The number of nuclei per myofiber was calculated by dividing the total number of nuclei by the number of myofibers.

The area occupied by CD31-positive cells and CSPG4-positive cells was calculated by ImageJ.

Mononuclear cells were isolated from skeletal muscle as described previously [39]. In brief, the rats were killed by inhalation of carbon dioxide gas, and their tongue and masseter muscles were extirpated. As described above, the lower half part of the masseter muscle and tongue cut out at the root were used for cell isolation. They were minced with scissors and digested with 1.25 mg/mL protease (from Streptomyces griseus, type XIV; Sigma) at 37 °C for 1 h. Cells were separated from myofiber fragments through differential centrifugation and plated onto poly-l-lysine- and fibronectin-coated 48-well plates. Cells from each muscle piece were divided into two equal portions (one for immunocytochemistry of Pax7 and another for MyoD) and plated. Cells were cultured in Dulbecco’s modified Eagle medium (Gibco, Life Technologies, Palo Alto, CA, USA) containing 10% fetal bovine serum, 100 U/mL penicillin, 100 μg/mL streptomycin, and 50 μg/mL gentamicin for 4 days.

The cells were fixed with 4% PFA/PBS for 15 min and then blocked with 5% NGS/PBS containing 0.1% Triton X-100 for 10 min. The cells were incubated with anti-Pax7 (1:200 with 5% NGS/PBS) or anti-MyoD (1:200, with 5% NGS/PBS, mouse, clone 5.8A, Novocastra, Newcastle upon Tyne, UK) overnight at 4 °C. After washing, they were incubated with AlexaFluor-labeled secondary antibody for 1 h. Nuclei were counterstained with Hoechst 33,258. The Pax7- and MyoD-positive cells were counted in randomly selected five fields at 20 × objective of a fluorescence microscope (BX50, Olympus). The total numbers of nuclei were also counted. The data were expressed as percent positive cells.

Total RNA was extracted from 100 cryosections (7 μm) with FastGene RNA Basic kit (Nippon Genetics Co., Ltd., Tokyo, Japan) and reverse transcribed to cDNA using Super Script II kit (Invitrogen). Quantitative real-time PCR was performed on a Light Cycler 2.0 (Roche Diagnostics, Roche, Basel, Switzerland) with the Thunderbird SYBR qPCR Mix (TOYOBO, Osaka, Japan). The following primer sets were used: p16: forward, 5′-TTC ACC AAA CGC CCC GAA CA-3′; reverse, 5′-CAG GAG AGC TGC CAC TTT GAC-3′; p19: forward, 5ʹ-GTG TTG AGG CCA GAG AGG AT-3ʹ; reverse, 5ʹ-TTG CCC ATC ATC ATC ACC T-3ʹ; p21: forward, 5ʹ-GAC ATC TCA GGG CCG AAA-3ʹ; reverse, 5ʹ-GGC GCT TGG AGT GAT AGA AA-3ʹ; p53: forward, 5ʹ-AGA GAG CAC TGC CCA CCA-3ʹ; reverse, 5ʹ-AAC ATC TCG AAG CGC TCA C-3ʹ; Utrn: forward, 5ʹ‐TAG AGC AAT ACG CCA CAC GA‐3ʹ; reverse, 5ʹ‐ACG CTC TTC CTT CTC CAC AG‐3ʹ; MuRF1: forward, 5ʹ‐AGG ACT CCT GCC GAG TGA C-3′; reverse, 5′-TTG TGG CTC AGT TCC TCC TT-3′; Atrogin1: forward, 5′-GAA GAC CGG CTA CTG TGG AA-3′; Atrogin1: reverse, 5′-ATC AAT CGC TTG CGG ATC T-3′, and HPRT: forward, 5ʹ‐GAC CGG TTC TGT CAT GTC G‐3ʹ; reverse, 5ʹ‐ACC TGG TTC ATC ATC ACT AAT CAC‐3ʹ. The expression of each gene was analyzed using the crossing‐point method and expressed after normalization with that of HPRT.

Two hundred cryosections (7 μm) of the tongue muscle were homogenized using radioimmunoprecipitation assay (RIPA) buffer [10 mM NaH2PO4, 150 mM NaCl, 2 mM ethylenediaminetetraacetic acid (EDTA), 0.1% sodium deoxycholate, 1% Nonidet P-40, 10 μg/mL leupeptin, 5 μg/mL pepstatin, 1.84 g/L Na3VO4, 10 mg/mL p-nitrophenylphosphate (PNPP), 100 KIU/mL aprotinin, 20 μg/mL phenylmethanesulfonylfluoride (PMSF)], and protein concentration of the lysates was determined using Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). Lysates were, then, diluted with RIPA buffer to the same concentrations of protein. The samples were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting as described in the previous study [1]. Antibodies used were as follows: anti-phospho-Akt antibody (Ser473, Cell Signaling Technology, Danvers, MA, USA, #9271), anti-Akt antibody (Cell Signaling Technology, #9272), anti-phospho-p70 S6K antibody (Thr389, Cell Signaling Technology, #9234), anti-p70 S6K antibody (Santa Cruz Biotechnology, C-18, sc-230), anti-phospho-S6 ribosomal protein antibody (Ser240/244, Cell Signaling Technology, #2215), anti-S6 ribosomal protein antibody (Cell Signaling Technology, 54D2, #2317), anti-4EBP1 antibody (Cell Signaling Technology, #9452), anti-α-tubulin antibody (Sigma Aldrich, T9026), anti-rabbit IgG, horseradish peroxidase (HRP)-linked whole antibody from donkey (GE healthcare, NA934V), and anti-mouse IgG, HRP-linked whole antibody from sheep (GE healthcare, NA931V). Band intensities of each blot were quantified using ImageJ.

Except for an analysis of myofiber diameters, graphed data are expressed as means ± SE. Unpaired Student’s t-test (between two groups) and one-way analysis of variance (ANOVA) followed by a Tukey–Kramer test (for multiple group comparison) were used to evaluate statistical differences. For the experiment to assess the difference of myofiber diameters, the p-value was determined using the Wilcoxon rank sum test. p-values less than 0.05 were considered statistically significant.