Senescence dynamics define therapeutic windows for Duchenne muscular dystrophy in DBA/2-mdx mice.

Figures

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  Fig. 1. Senescent cells accumulate in the dystrophic skeletal muscle of young DBA/2-mdx mice and DMD patients. A Quantification of collagen deposition in the tibialis anterior (TA) muscle of wild-type (WT) and DBA/2-mdx mice at the indicated ages (left), and representative images of Sirius Red staining (right). B Four-limb grip strength of wild-type (WT) and DBA/2-mdx mice. C Maximum specific force of the extensor digitorum longus (EDL) muscle. D Serum creatine kinase (CK) levels in WT and DBA/2-mdx mice across age groups. E Quantification and representative images of SA-β-gal⁺ cells in the TA muscle of WT and DBA/2-mdx mice across age groups. F Quantification and representative images of DNA damage by γH2AX intensity in extra-myofiber nuclei of TA from WT and DBA/2-mdx mice at young ages. G Quantification and representative images of percentage of cells with telomeric DNA damage response (TeloDDR, upper graph) and average number of TeloDDR foci (bottom graph) in extra-myofiber nuclei of TA from WT and DBA/2-mdx mice at young ages. H Relative RNA levels of senescent markers in TA of WT and DBA/2-mdx mice at young ages assessed by RT-qPCR. I Quantification and representative images of…

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  Fig. 2. Pharmacological reduction of senescent cells improves skeletal muscle function in young dystrophic mice. A Three-month-old DBA/2-mdx mice were treated with vehicle or D + Q twice weekly for 8 weeks. B–E Histology from TA; Histology from diaphragm is from I-L. B Quantification and representative images of SA-β-gal⁺ cells in the TA muscle of DBA/2-mdx mice. C Mean cross-sectional area (CSA) of fibers and frequency distribution of muscle fiber size in the TA of DBA/2-mdx mice based on H&E-stained sections. D Percentage of centrally nucleated fibers in the TA based on H&E-stained sections. E Quantification of collagen deposition and representative Sirius Red staining images. F Maximum specific force and force–frequency curves of the EDL muscle in vehicle and D + Q–treated mice. G Four-limb grip strength. H Serum creatine kinase (CK) levels in DBA/2-mdx mice treated with vehicle or D + Q. I Quantification and representative images of SA-β-gal⁺ cells in the diaphragm of DBA/2-mdx mice. J Mean cross-sectional area (CSA) of fibers and frequency distribution of muscle fiber size in the diaphragm of DBA/2-mdx mice based on H&E-stained sections. K Percentage of centrally nucleated fibers in…

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  Fig. 3. Genetic ablation of senescent cells improves skeletal muscle function in DBA/2-mdxp16−3MR dystrophic mice. A Luciferase activity in the TA muscles of young (3–5 months) p16-3MR and DBA/2-mdxp16−3MR mice. B Three-month-old DBA/2-mdxp16−3MR mice were treated with PBS or GCV twice weekly for 8 weeks. C–F Histology from TA; (I–L), histology from diaphragm. C Quantification and representative images of SA-β-gal⁺ cells in the TA. D Mean cross-sectional area (CSA) of fibers and frequency distribution of muscle fiber size based on H&E-stained sections. E Percentage of centrally nucleated fibers in the TA based on H&E-stained sections. F Quantification of collagen deposition and representative Sirius Red staining images in the TA. G Maximum specific force and force–frequency curves of the EDL muscle in PBS and GCV–treated mice. H Four-limb grip strength. I Quantification and representative images of SA-β-gal⁺ cells in the diaphragm of DBA/2-mdxp16−3MR mice. J Percentage of centrally nucleated fibers in the diaphragm based on H&E-stained sections. K Mean cross-sectional area (CSA) of fibers and frequency distribution of muscle fiber size in the diaphragm of DBA/2-mdxp16−3MR mice based on…

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  Fig. 4. Transcriptomic changes in TAs of DBA/2-mdx mice after D + Q treatment for 2 months in young mice. A Principal component analysis (PCA) plot of gene expression data of TAs from vehicle- and D + Q–treated mice. B Volcano plot of differentially expressed genes (DEGs; adj p < 0.05 and log2FoldChange > 0.5) comparing D + Q– vs. vehicle-treated TAs. C qPCR levels of selected genes upregulated or downregulated in the RNA-seq. D Heatmap showing selected up- and downregulated DEGs in the D + Q group related to muscle-relevant functions. E Dot plot representing selected enriched GO pathways in D + Q vs. vehicle TAs. In (C), data are presented as mean ± SEM. The Mann–Whitney test was used for all comparisons. Statistical significance is indicated by *p < 0.05

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  Fig. 5. Senolytic treatment improves heart pathology in dystrophic mice at advanced disease stages. A Collagen quantification by Sirius Red staining in the hearts of wild-type (WT) and DBA/2-mdx mice at the indicated ages. Representative images of aged hearts from both genotypes are shown. B SA-β-gal⁺ cell number and representative pictures of heart sections from wild-type (WT) or DBA/2-mdx mice at each age group. C Relative RNA levels of senescent markers in the hearts of WT and DBA/2-mdx mice at adult ages (12 months) assessed by RT-qPCR. D 12-month-old DBA/2-mdx mice were treated twice weekly for 4 months with D + Q or vehicle. E Quantification of SA-β-gal⁺ cells and representative images of the hearts after treatment with vehicle or D + Q. F Collagen deposition in hearts from mice treated with vehicle or D + Q. G Fractional shortening (FS) and ejection fraction (EF) of hearts assessed by echocardiography. Data are presented as mean ± SEM. The Mann–Whitney test was used for all comparisons (in A and B, individual comparisons were performed between age-matched WT and DBA/2-mdx mice). The unpaired Student’s t-test was used in (F). Statistical significance is indicated by *p < 0.05; **p…

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  Fig. 6. D + Q treatment in dystrophic mice activates a similar transcriptomic signature in skeletal muscle and heart. A Heatmaps showing the expression of shared up- and downregulated DEGs in TA and heart from vehicle- or D + Q–treated DBA/2-mdx mice. Vehicle, gray; D + Q, yellow. B, C Dot plots showing selected enriched pathways from the SysMyo gene sets in the TA (B) and heart (C) after D + Q treatment of DBA/2-mdx mice