Exercise enhances skeletal muscle regeneration by promoting senescence in fibro-adipogenic progenitors

Idiopathic inflammatory myopathies (IIMs) are rare autoimmune disorders characterized by muscle weakness, inflammation, and fibrosis. While immunosuppressive therapy (e.g., prednisolone, methotrexate) is standard, physical exercise is increasingly recognized as a potential adjunct therapy. However, exercise can yield mixed outcomes: beneficial regeneration in some contexts but exacerbation of inflammation and fibrosis in chronic inflammatory myopathy (CIM). Fibro-adipogenic progenitors (FAPs), PDGFRα+ mesenchymal cells distinct from satellite cells, support regeneration acutely but promote fibrosis when persistently activated. Prior work indicates that FAPs can adopt either a pro-regenerative, pro-apoptotic phenotype or a pro-fibrotic, apoptosis-resistant phenotype. The study investigates how exercise influences FAP phenotype and tests the hypothesis that FAP senescence in response to damage is required to establish a regenerative inflammatory milieu, whereas failure to induce FAP senescence in chronic myopathy leads to apoptosis resistance, accumulation of FAPs, and fibrosis.

Background work shows: (1) Acute muscle injury triggers transient FAP proliferation supportive of satellite cell differentiation, followed by apoptosis and clearance; chronic inflammation sustains FAPs and impairs their apoptotic clearance, resulting in fibrosis. (2) Exercise can activate FAPs and enhance regeneration in healthy or certain transgenic contexts, yet excessive or chronic settings can worsen fibrosis. (3) Senescence, via factors like p53 and p16INK4A, can promote apoptosis, SASP-mediated immune recruitment, and resolution of fibrosis; lack of senescence associates with cell accumulation and fibrotic progression in other tissues. (4) AMPK activation is linked to senescence induction and is triggered by metabolic stressors including exercise, suggesting a mechanistic route to modulate FAP fate. These findings frame the rationale to examine FAP senescence as a determinant of exercise outcomes in myopathy.

• Animal models: Female BALB/c and C57BL/6 mice (>6 weeks) housed pathogen-free. CIM (experimental autoimmune myositis) induced by immunization with partially purified skeletal muscle myosin (with myosin-binding protein C) emulsified in complete Freund’s adjuvant, given at three sites over three weeks; pertussis toxin administered after first immunization. AMI (acute muscle injury) induced by intramuscular injection of 50 μL 1.2% BaCl2 into triceps surae. Trp53(+/+) and Trp53(-/-) mice used for senescence/apoptosis mechanistic studies and FAP transplantation.
• Exercise protocols: Downhill treadmill (-20°, 17 m/min). Acute: single 30-min bout with tissue harvest 24 h later. Long-term: daily 30-min sessions for 2 weeks. CIM mice assigned to: sedentary, exercise-only, AICAR-only (250 mg/kg/day i.p.), or exercise+AICAR; tissues collected 24 h after final session.
• Pharmacology: AICAR (AMPK activator) 250 mg/kg/day i.p. in vivo; 0.5 mM in vitro where specified.
• FAP isolation: Muscles digested with collagenase; FAPs enriched via MACS or EasySep negative selection for Lin-/CD31-/α7-integrin- cells followed by PDGFRα positivity. Cultured adherent PDGFRα+/Sca-1+ cells defined as FAPs. Purity and gating validated by flow cytometry.
• Molecular assays: qRT-PCR for senescence (Cdkn2a/p16INK4A, Trp53, P21, p19Arf), cytokines/regenerative (Tnfaip6, Il33), fibrotic markers (Tgfb1, Acta1), immune evasion (Cd274/PD-L1, Pdcd1lg2/PD-L2, Cd47), others (Fst, Klb). PrimerArray profiling of cytokine–receptor interactions; hierarchical clustering and PCA.
• Senescence and DNA damage markers: SPIDER-β-gal staining in tissue; γH2AX by flow cytometry; immunofluorescence for p16INK4A and p53.
• Apoptosis/viability: LDH release assay; Annexin V/ethidium homodimer staining after TNF-α (1–100 ng/mL) or H2O2 (50–1000 μM) exposure.
• Histology/immunofluorescence: H&E; collagen I; PDGFRα; laminin; active caspase-3; confocal imaging. Quantification of fibrotic area and FAP counts.
• Co-culture assays: (1) Transwell co-culture of freshly isolated FAPs (AMI vs CIM) with satellite cells; assessment of myogenic differentiation and fusion indices via MyHC staining. (2) Co-culture of FAPs (Trp53 genotypes; ±H2O2) with RAW264.7 macrophages for phagocytosis assessment by dual labeling. (3) Transwell co-culture of FAPs (±H2O2) with C2C12 myoblasts to assess differentiation.
• FAP transplantation: Injection of PKH26-labeled Trp53(+/+) or Trp53(-/-) FAPs (8×10^4 cells) into C57BL/6 triceps surae, followed by BaCl2 injury 7 days later; outcomes at 10 and 20 dpi include muscle wet weight, cross-sectional area, histology, and persistence of transplanted FAPs.
• Functional tests: Exhaustion treadmill test and hindlimb grip strength pre- and post-intervention.
• Statistics: Shapiro–Wilk for normality; t tests for pairwise comparisons; one-way ANOVA with Holm correction for multiple comparisons; data presented as dot plots and box–whisker plots. Investigators unblinded except during behavioral assessments; no predefined power calculations.

• FAP dynamics in injury vs chronic inflammation: PDGFRα+ FAPs increased in both AMI and CIM vs control, but returned to baseline by day 7 in AMI and continued accumulating to day 14 in CIM. Fibrotic collagen-positive area was greater in CIM than control and AMI.
• Apoptosis sensitivity: AMI-FAPs exhibited TNF-α–induced cytotoxicity and increased Annexin V+ apoptosis in a dose-dependent manner; CIM-FAPs were resistant. Active caspase-3+ FAPs increased in AMI muscle but not in CIM.
• Transcriptional profiles: AMI-FAPs upregulated TNF receptor superfamily members (Fas, Cd27, Tnfrsf14, Tnfrsf19) and CC-chemokines, consistent with a pro-inflammatory/pro-apoptotic phenotype. CIM-FAPs upregulated TGF-β/BMP-related genes (Amhr2, Csfl) consistent with survival and pro-fibrotic signaling.
• Senescence markers: AMI-FAPs showed higher Cdkn2a (p16INK4A) and Trp53 mRNA, more SPIDER-β-gal+ FAPs, and higher γH2AX positivity than control and CIM; CIM-FAPs had reduced Cdkn2a/Trp53 and senescence readouts.
• Immune evasion correlates: CIM-FAPs had elevated Cd274 (PD-L1), Pdcd1lg2 (PD-L2), and Cd47 mRNA, correlating positively with Cdkn2a expression levels across samples; CIM-FAPs expressed higher Bcl-2 and lower p53 protein.
• Regenerative cytokines: Tnfaip6 (TSG-6) and Il33 were higher in AMI-FAPs and reduced in CIM-FAPs. IL-33 co-localized with p16INK4A+ cells. Senescence gene expression (Cdkn2a, Trp53, P21, p19Arf) correlated with Il33/Tnfaip6 expression and muscle function.
• Myogenesis support: FAPs from AMI, but not CIM, enhanced satellite cell differentiation and fusion in transwell assays (greater MyHC+ myotubes, higher differentiation/fusion indices).
• mdx model parallels: FAPs in mdx and CIM lacked p16INK4A expression seen in AMI; β-Klotho (Klb), a negative regulator of p16INK4A, was higher in CIM-FAPs and inversely correlated with Cdkn2a.
• p53 dependence: Transplantation of Trp53(-/-) FAPs impaired regeneration post-AMI, yielding lower muscle weight and fiber CSA, increased fibrosis, and persistence of transplanted FAPs vs Trp53(+/+) FAPs. In vitro, Trp53(-/-) FAPs were resistant to H2O2- and TNF-α–induced cytotoxicity; failed to upregulate P21/Cdkn2a; showed higher Cd274 and Cd47 (further increased by H2O2); and reduced follistatin induction. H2O2-treated Trp53(+/+) FAPs were more readily phagocytosed by RAW264.7 and promoted C2C12 differentiation; Trp53(-/-) FAPs showed the opposite.
• Exercise effects: In normal mice, acute downhill exercise increased FAP number and upregulated Cdkn2a, P21, Tnfaip6, and Il33 in FAPs; p16INK4A/p53 protein and phospho-p38 MAPK and NF-κB p65 increased. In CIM, exercise did not increase FAP number; Cdkn2a and P21 decreased; fibrotic genes (Tgfb1, Acta1) increased; p16INK4A/p53 decreased; NF-κB p65 increased while p38 MAPK activation decreased.
• Therapeutic intervention: Two-week exercise improved endurance, grip strength, muscle fiber CSA, and regenerating fibers in controls but had poor effects in CIM. AICAR alone improved endurance and reduced FAP number in CIM. Combined exercise + AICAR in CIM markedly improved function and histology (CSA and regenerating fibers) and increased the proportion of p16INK4A+ and β-gal+ FAPs; cytokine–receptor expression profiles by clustering and PCA shifted CIM FAPs toward AMI/healthy-exercise-like pro-inflammatory/pro-apoptotic phenotypes.

The study addresses why exercise can be beneficial or detrimental in chronic myopathies by identifying FAP senescence as a pivotal switch. In acute injury, FAPs enter a senescent, SASP-expressing state with elevated p16INK4A/p53, DNA damage signaling (γH2AX), and death receptor expression, enabling apoptosis and immune clearance (via recruitment of CD11b+ phagocytes and expression of IL-33/TSG-6), which supports regenerative inflammation and restoration of tissue architecture. In chronic inflammatory contexts (CIM and mdx), FAPs fail to senesce, instead upregulating survival and immune-evasion pathways (Bcl-2, PD-L1/PD-L2, CD47) and TGF-β/BMP-related signaling, leading to apoptosis resistance, impaired clearance, accumulation, and fibrosis. p53 is required for FAPs to adopt the pro-regenerative senescent phenotype: loss of Trp53 confers apoptosis resistance, persistent accumulation, impaired macrophage phagocytosis, and a reduced capacity to support myogenesis. Exercise in healthy muscle promotes FAP senescence and pro-regenerative signaling (p38 MAPK, NF-κB), but in CIM it paradoxically suppresses senescence markers, elevates NF-κB with reduced p38 MAPK, and increases fibrotic gene expression, explaining poor outcomes. AMPK activation (AICAR) restores a pro-senescent, pro-apoptotic FAP phenotype in CIM, especially when combined with exercise, likely by enhancing metabolic stress that balances mechanotransductive stress, shifting signaling toward p16INK4A/p53 activation and improving macrophage-mediated clearance. Thus, modulating FAP senescence reconciles the dual effects of exercise and provides a therapeutic entry point in chronic inflammatory myopathies.

FAP acquisition of senescent features after muscle damage is necessary to establish regenerative inflammation that supports muscle repair. In CIM, failure to induce FAP senescence results in apoptosis resistance, immune evasion, FAP accumulation, and fibrosis, worsening muscle degeneration. Exercise induces beneficial FAP senescence in healthy muscle but not in CIM; however, combining exercise with AMPK activation (AICAR) reprograms CIM-FAPs toward a pro-inflammatory, pro-apoptotic phenotype, restoring muscle function and regeneration in mice. These findings highlight FAP-targeted pro-senescent strategies, in concert with tailored exercise, as potential therapies for chronic inflammatory myopathies. Future work should optimize dosing and timing of exercise and AMPK activation, delineate mechanistic crosstalk between metabolic and mechanical stress, assess effects on satellite cells and broader immune compartments, and evaluate translatability and safety in more severe dystrophic models and ultimately in humans.

• Disease models: The CIM model is autoimmune and milder than severe dystrophic models; while mdx data support parallels, efficacy in advanced dystrophy remains to be established.
• Experimental design: No a priori power calculations; limited sample sizes in several assays; allocation not randomized; investigators unblinded for most procedures (except behavioral assessment), which may introduce bias.
• Mechanistic scope: Direct demonstration of SASP components and in vivo phagocyte dynamics is inferential; effects on satellite cell senescence after AICAR/exercise are not fully resolved.
• Generalizability: Findings are in mice; human FAP responses to exercise and AMPK activation require validation; optimal exercise intensity and AICAR dosing regimens may vary by disease severity and tissue mechanics.
• Biomarker specificity: Senescence markers (p16INK4A, γH2AX, SA-β-gal) are supportive but not exclusive to senescence; additional lineage-specific and temporal analyses could refine interpretations.