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. 2018 Nov 30;9(1):5104.
doi: 10.1038/s41467-018-07639-3.

Loss of peroxiredoxin-2 Exacerbates Eccentric Contraction-Induced Force Loss in Dystrophin-Deficient Muscle

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Free PMC article

Loss of peroxiredoxin-2 Exacerbates Eccentric Contraction-Induced Force Loss in Dystrophin-Deficient Muscle

John T Olthoff et al. Nat Commun. .
Free PMC article

Abstract

Force loss in skeletal muscle exposed to eccentric contraction is often attributed to injury. We show that EDL muscles from dystrophin-deficient mdx mice recover 65% of lost force within 120 min of eccentric contraction and exhibit minimal force loss when the interval between contractions is increased from 3 to 30 min. A proteomic screen of mdx muscle identified an 80% reduction in the antioxidant peroxiredoxin-2, likely due to proteolytic degradation following hyperoxidation by NADPH Oxidase 2. Eccentric contraction-induced force loss in mdx muscle was exacerbated by peroxiredoxin-2 ablation, and improved by peroxiredoxin-2 overexpression or myoglobin knockout. Finally, overexpression of γcyto- or βcyto-actin protects mdx muscle from eccentric contraction-induced force loss by blocking NADPH Oxidase 2 through a mechanism dependent on cysteine 272 unique to cytoplasmic actins. Our data suggest that eccentric contraction-induced force loss may function as an adaptive circuit breaker that protects mdx muscle from injurious contractions.

Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Eccentric contraction-induced force loss in mdx muscle recovers rapidly and is partially protected by overexpression of γcyto-actin, but not αcardiac-actin. a Recovery of isometric force production in isolated extensor digitorum longus (EDL) muscles from mdx mice subjected to 10 maximal isometric or eccentric contractions. Values are expressed as a percentage of the isometric force measured before the 10 contractions (Pre) for each timepoint listed; n = 4 for both conditions. *P < 0.05, ***P < 0.001 compared with Post 0’; two-way ANOVA. b Increasing the time interval between eccentric contractions from 3 to 30 min significantly diminishes the measured force loss in EDL muscles from mdx mice; n = 4 for both conditions. *P < 0.05, **P < 0.01, ***P < 0.001 compared to 3 min Rest; two-way ANOVA. c Immunoblot analysis of γcyto-actin in tibialis anterior (TA), extensor digitorum longus (EDL), gastrocnemius (Gastroc), and soleus muscles from mdx/Actg1-TG mice versus non-transgenic mdx littermates. d Immunofluorescence analysis of γcyto-actin (green), laminin (red), and DAPI (blue) in 10 µm cryosections of quadriceps muscle from mdx/Actg1-TG mice versus non-transgenic mdx littermates. e EDL muscles isolated from mdx/Actg1-TG mice and non-transgenic mdx littermates were subjected to 10 eccentric contractions and the force measured at each contraction expressed as a percentage of the force produced during the first contraction; n = 4 for both genotypes. *P < 0.05, **P < 0.01, ***P < 0.001 compared to mdx; two-way ANOVA. f Immunoblot analysis of αca-actin in tibialis anterior (TA), extensor digitorum longus (EDL), gastrocnemius (Gastroc), and soleus muscles from mdx/Coco mice versus non-transgenic mdx littermates. g Immunofluorescence analysis of αca-actin (green), laminin (red), and DAPI (blue) in 10 µm cryosections of quadriceps muscle from mdx/Coco mice versus non-transgenic mdx littermates. h EDL muscles isolated from mdx/Coco mice and non-transgenic mdx littermates were subjected to 10 eccentric contractions and the force measured at each contraction expressed as a percentage of the force produced during the first contraction; n = 4 for both genotypes. Throughout, error bars represent means ± SEM
Fig. 2
Fig. 2
Peroxiredoxin-2 is significantly decreased in mdx skeletal muscle and restored by γcyto-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 γcyto-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
Fig. 3
Genetic ablation of myoglobin partially protects mdx muscle from eccentric contraction-induced force loss. a Immunoblot analysis demonstrated the absence of myoglobin in mdx/mb–/– muscle, that myoglobin levels are decreased in mdx, and that myoglobin levels are partially restored in mdx/p47–/– gastrocnemius muscle; n = 4 for each genotype. *P < 0.05, **P < 0.01, ***P < 0.001; one-way ANOVA. b EDL muscles isolated from mdx and mdx/mb–/– mice, or mdx muscles treated with 20 mM N-acetylcysteine (NAC) 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+NAC; n = 9 for mdx/mb–/–; *mdx+NAC significantly different from mdx (P ≤ 0.05); #mdx/mb–/– significantly different from mdx (P ≤ 0.05); two-way ANOVA. Throughout, error bars represent means ± SEM
Fig. 4
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
Fig. 5
Fig. 5
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. 6
Fig. 6
Cysteine 272 of γcyto-actin is necessary for protection of mdx muscle from eccentric contraction-induced force loss. a Immunoblot comparison of γcyto (Actg1-TG), γcytoC272A (C272A-TG), and βcyto (Actb-TG) overexpression in mdx gastrocnemius muscle. b Immunofluorescence analysis demonstrates similar distributions of γcyto, γcytoC272A, and βcyto in 10 µm quadriceps cryosections. Scale bar = 50 µm. c EDL muscles isolated from WT, mdx, mdx/Actg1-TG, mdx/C272A-TG, and mdx/Actb-TG 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 = 8 for WT and mdx/C272A-TG; n = 5 for mdx; n = 6 for mdx/Actg1-TG and mdx/Actb-TG. *The mdx/Actg1-TG significantly different from mdx (P ≤ 0.05), #mdx/Actb-TG significantly different from mdx (P ≤ 0.05); two-way ANOVA. d Rate of DCF fluorescence in single flexor digitorum brevis (FDB) muscles from WT, mdx, mdx/Actg1-TG, mdx/C272A-TG, and mdx/Actb-TG mice exposed to cyclic stretch in the presence of DMSO (vehicle) or the NOX2 inhibitor gp91ds-tat; n = 9 for WT, mdx/Actg1-TG, and mdx/Actb-TG; n = 12 for mdx; n = 7 for mdx/C272A-TG. ***P < 0.001, ns no significance compared to WT; one-way ANOVA. Throughout, error bars represent means ± SEM
Fig. 7
Fig. 7
Muscle-specific peroxiredoxin-2 overexpression leads to increased sarcolemmal damage following long-term injurious eccentric contractions in mdx mice. a Fluorescent microscopy of WT, mdx, and mdx/PrxII-TG (58×) for Evans blue dye (EBD; red) and laminin (green). No ECC contralateral TA not subjected to eccentric contractions, ECC TA subjected to 70 eccentric contractions performed in vivo. Scale bar = 50 µm. b Quantification of the percentage of EBD-positive myofibers in WT, mdx, and mdx/PrxII-TG TA muscle either subjected to 70 eccentric contractions (ECCs) or not (no ECC); n = 3 for WT and mdx; n = 4 for mdx/PrxII-TG. *P < 0.05 compared to WT-ECC, #P < 0.05 compared to mdx-ECC; one-way ANOVA

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