Fig 2: Peroxiredoxin-2 is significantly decreased in mdx skeletal muscle and restored by ?cyto-actin overexpression and genetic ablation of NOX2 activity. a Immunoblot analysis of PrxII, dystrophin, utrophin, and GAPDH in WT, mdx, mdx/Actg1-TG, and mdx/Coco gastrocnemius muscles. b Immunoblot quantitation demonstrated that PrxII levels in mdx skeletal muscle were 16.5 ± 0.03% of WT and restored in mdx/Actg1-TG muscle to levels not different from WT, but not in mdx/Coco muscle; n = 5 for WT and mdx; n = 7 for mdxActg1-TG and mdx/Coco. ***P < 0.001, ns no significance; one-way ANOVA. c Immunoblot analysis of peroxiredoxins 1–6 in gastrocnemius muscles from WT, mdx, and mdx/Actg1-TG mice. PrxII was the only peroxiredoxin isoform that was both altered in mdx compared to WT, and also restored to its WT level by muscle-specific ?cyto-actin overexpression. d Immunoblot analysis of PrxII in WT, mdx, and mdx/p47–/– gastrocnemius muscles demonstrated a restoration of PrxII to WT levels in mdx/p47–/– muscle; n = 4 for each genotype. ***P < 0.001, ns no significance; one-way ANOVA. e Immunoblot analysis demonstrated significantly elevated hyperoxidized peroxiredoxin (PrxSO3) in mdx compared to WT, and restored to WT levels in mdx/p47–/– gastrocnemius muscles; n = 4 for each genotype. ***P < 0.001, ns no significance; one-way ANOVA. f EDL muscles isolated from WT, mdx, p47–/–, and mdx/p47–/– mice were subjected to 10 eccentric contractions and the forces measured expressed as a percentage of the force generated during the first eccentric contraction; n = 4 for WT and mdx; n = 3 for p47–/–; n = 7 for mdx/p47–/–. *P < 0.05, ***P < 0.001 compared to mdx; two-way ANOVA. Throughout, error bars represent means ± SEM

Fig 3: Muscle-specific peroxiredoxin-2 overexpression partially protects mdx muscle from eccentric contraction-induced force loss. a EDL muscles isolated from WT, mdx, and mdx/PrxII-TG lines expressing PrxII at 1-, 12-, 58-, and 112-fold relative to WT were subjected to 10 eccentric contractions and the forces measured expressed as a percentage of the force generated during the first eccentric contraction; n = 8 for WT; n = 4 for mdx; n = 5 for 1× and 58×; n = 6 for 12× and 112×. @1× Significantly different from mdx (P < 0.05), #12× significantly different from mdx (P < 0.05), *58× significantly different from mdx (P < 0.001), &112× significantly different from mdx (P < 0.05); two-way ANOVA. b The force produced at contraction 5 for each line was presented as a percentage of initial force; n = same as in (a). *P < 0.05, **P < 0.01, ***P < 0.001 compared to mdx; one-way ANOVA. c Representative images of 10 µm cryosections of TA from WT, mdx, PrxII-TG, and mdx/PrxII-TG (58×) stained with H&E. Scale bar: 50 µm. d The 58-fold PrxII overexpression caused a small but significant decrease in the percentage of centrally nucleated fibers (%CNFs) in mdx TA muscle; n = 3 for WT and PrxII-TG; n = 6 for mdx n; = 7 for mdx/PrxII-TG. **P < 0.01 for mdx/PrxII-TG compared to mdx; one-way ANOVA. Throughout, error bars represent means ± SEM

Fig 4: Genetic ablation of peroxiredoxin-2 further sensitizes mdx muscle to eccentric contraction-induced force loss. a Immunoblot analysis of PrxII in WT, mdx, PrxII–/–, and mdx/PrxII–/– gastrocnemius demonstrated the absence of PrxII in PrxII–/– and mdx/PrxII–/– muscle. b A small but significant increase in the percentage of centrally nucleated fibers (%CNFs) was seen in mdx/PrxII–/– versus mdx muscle quantified from 10 µm cryosections of TA stained with H&E. n = 3 for each genotype. **P < 0.01 for mdx/PrxII–/– compared to mdx; one-way ANOVA. c Representative images of 10 µm cryosections of TA from WT, mdx, PrxII–/–, and mdx/PrxII–/– stained with H&E. Scale bar: 50 µm. d EDL muscles isolated from mdx and mdx/PrxII–/– mice were subjected to 10 eccentric contractions with either a 5% or 10% length change, and the forces measured expressed as a percentage of the force generated during the first eccentric contraction. There was no significant difference between mdx and mdx/PrxII–/– with a 10% length change, but a 5% length change revealed a significant difference between mdx and mdx/PrxII–/– for contractions 6–10; n = 4 for each genotype/condition. *P < 0.05, **P < 0.01, ***P < 0.001 compared to mdx; two-way ANOVA. Throughout, error bars represent means ± SEM