Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2018 Feb 1;28(4):275-295.
doi: 10.1089/ars.2017.7249. Epub 2017 Dec 12.

Comparison of Whole Body SOD1 Knockout with Muscle-Specific SOD1 Knockout Mice Reveals a Role for Nerve Redox Signaling in Regulation of Degenerative Pathways in Skeletal Muscle

Giorgos K Sakellariou  1 , Brian McDonagh  1 , Helen Porter  1 , Ifigeneia I Giakoumaki  1 , Kate E Earl  1 , Gareth A Nye  1 , Aphrodite Vasilaki  1 , Susan V Brooks  2 , Arlan Richardson  3   4 , Holly Van Remmen  4   5 , Anne McArdle  1 , Malcolm J Jackson  1
Affiliations

Comparison of Whole Body SOD1 Knockout with Muscle-Specific SOD1 Knockout Mice Reveals a Role for Nerve Redox Signaling in Regulation of Degenerative Pathways in Skeletal Muscle

Giorgos K Sakellariou et al. Antioxid Redox Signal. .

Abstract

Aims: Lack of Cu,Zn-superoxide dismutase (CuZnSOD) in homozygous knockout mice (Sod1-/-) leads to accelerated age-related muscle loss and weakness, but specific deletion of CuZnSOD in skeletal muscle (mSod1KO mice) or neurons (nSod1KO mice) resulted in only mild muscle functional deficits and failed to recapitulate the loss of mass and function observed in Sod1-/- mice. To dissect any underlying cross-talk between motor neurons and skeletal muscle in the degeneration in Sod1-/- mice, we characterized neuromuscular changes in the Sod1-/- model compared with mSod1KO mice and examined degenerative molecular mechanisms and pathways in peripheral nerve and skeletal muscle.

Results: In contrast to mSod1KO mice, myofiber atrophy in Sod1-/- mice was associated with increased muscle oxidative damage, neuromuscular junction degeneration, denervation, nerve demyelination, and upregulation of proteins involved in maintenance of myelin sheaths. Proteomic analyses confirmed increased proteasomal activity and adaptive stress responses in muscle of Sod1-/- mice that were absent in mSod1KO mice. Peripheral nerve from neither Sod1-/- nor mSod1KO mice showed increased oxidative damage or molecular responses to increased oxidation compared with wild type mice. Differential cysteine (Cys) labeling revealed a specific redox shift in the catalytic Cys residue of peroxiredoxin 6 (Cys47) in the peripheral nerve from Sod1-/- mice. Innovation and Conclusion: These findings demonstrate that neuromuscular integrity, redox mechanisms, and pathways are differentially altered in nerve and muscle of Sod1-/- and mSod1KO mice. Results support the concept that impaired redox signaling, rather than oxidative damage, in peripheral nerve plays a key role in muscle loss in Sod1-/- mice and potentially sarcopenia during aging. Antioxid. Redox Signal. 28, 275-295.

Keywords: 20S proteasome; mitochondria; myelin; peroxiredoxins 5 and 6; superoxide.

PubMed Disclaimer

Conflict of interest statement

No competing financial interests exist.

Figures

<b>FIG. 1.</b>
FIG. 1.
Characterization of muscle morphology and redox homeostasis in skeletal muscle of Sod1−/− and mSod1KO mice. (A) Representative Western blots showing CuZnSOD protein content in various tissues of Sod1−/− and the respective WT mice. (B) Representative Western blots depicting CuZnSOD protein expression in various tissues of mSod1KO and the respective WT mice. (C) Confocal image of a single isolated fiber from the FDB muscle of a WT (Sod1+/+) mouse under bright field. 20 × original magnification. Scale bar, 60 μm (top panel); protein expression of CuZnSOD and MnSOD in lysates from single isolated FDB fibers of mSod1KO and the respective WT mice (middle panel); native gel stained for SOD1 enzyme activity in lysates from single isolated FDB fibers of mSod1KO and the respective WT mice (bottom panel). (D, E) Example of hematoxylin and eosin stained transverse sections of the AT muscle from Sod1−/− (D), mSod1KO (E), and the respective WT littermate controls. White asterisks depict myofibers with central nuclei. 20 × original magnification. Scale bar, 70 μm. (F) Representative triple immunofluorescent staining of MHC isoforms; MHC IIA (green), MHC IIX (blue), and MHC IIB (red) performed on an AT cryosection obtained from a mSod1KO mouse. Right panel shows enlarged area marked by red box in the left panel to show the selective MHC isoform immunolabeling. Scale bar, 500 μm. (G) Histograms showing the fiber-type distribution of AT muscle from Sod1−/− (top panel), mSod1KO (bottom panel), and the respective WT mice. n = 3–4 mice/strain. (H, I) Western blot analysis (top panel) and quantification (bottom panel) of protein carbonyls in AT lysates of Sod1−/− (H), mSod1KO (I), and the respective WT mice. *p < 0.05 compared with values from the respective WT mice. (J) Transverse section of an AT muscle from an Sod1−/− mouse immune-labeled for 4-hydroxynonenal (4-HNE, green) protein adducts, WGA (5 μg/ml, red), to observe extracellular matrix, and a merged image as indicated and analyzed by confocal microscopy. Scale bar, 40 μm; 4-HNE content in AT skeletal muscle of Sod1−/−, mSod1KO, and the respective WT mice (bottom right panel). *p < 0.05 compared with values from the respective WT mice, n = 3 mice/strain. (K) Oxoguanine DNA glycosylase protein levels in AT skeletal muscle of Sod1−/− (top left panel), mSod1KO (bottom left panel), and the respective WT mice, and densitometric quantification of the blots (right panel). *p < 0.05 compared with values from the respective WT mice. (L) Transverse section of an AT muscle from an Sod1−/− mouse immune-labeled for 3-NT (purple) content, WGA (5 μg/ml, green), to observe extracellular matrix, and a merged image as indicated and analyzed by confocal microscopy. Scale bar, 40 μm; 3-NT content in AT skeletal muscle of Sod1−/−, mSod1KO, and the respective WT mice (bottom right panel). *p < 0.05 compared with values from the respective WT mice, n = 3 mice/strain. 3-NT, 3-nitrotyrosine; AT, anterior tibialis; EDL, extensor digitorum longus; FDB, flexor digitorum brevis; GTN, gastrocnemius; MHC, myosin heavy chain; SOL, soleus muscle; WGA, wheat germ agglutinin; WT, wild type.
<b>FIG. 2.</b>
FIG. 2.
Peripheral nerve redox homeostasis and NMJ structure in Sod1−/− and mSod1KO mice. (A, B) Confocal immunofluorescence imaging of NMJs of an AT muscle from an Sod1−/− mouse (A) and mSod1KO (B) mouse. Presynaptic motor neurons immunolabeled with neuronal class III β-tubulin monoclonal antibody, a neuronal marker (green), and postsynaptic motor endplate AChRs stained with AlexaFluor 594-conjugated α-bungarotoxin (red). Small panels show enlarged area marked by a box in the larger panel. 10 × original magnification (larger panel). Scale bar, 150 μm. (C, D) Western blots of myelin-associated proteins including MBP, MAG, PMP22, MPZ, PGP9.5, and CX32 in SN lysates of Sod1−/− (C), mSod1KO (D), and the respective WT mice. (E) Densitometric analysis of the Western blots shown in (C, D). *p < 0.05 compared with values from the respective WT mice. (F, G) Western blot analysis (top panel) and quantification (bottom panel) of protein carbonyls in SN lysates of Sod1−/− (F), mSod1KO (G), and the respective WT mice. (H, I) Western blot analysis (top panel) and quantification (bottom panel) of 4-HNE protein adducts in SN lysates of Sod1−/− (H), mSod1KO (I), and the respective WT mice. (J, K) Protein expression (top panel) of the main cytosolic and mitochondrial superoxide reducing enzymes, including SOD1 and SOD2 in SN lysates of Sod1−/− (J), mSod1KO (K), and the respective WT mice; densitometric analysis of the blots (bottom panel). AChRs, acetylcholine receptors; CX32, connexin 32/GJB1; MAG, myelin-associated glycoprotein; MBP, myelin basic protein; MPZ, myelin protein zero; NMJ, neuromuscular junction; PGP9.5, protein gene product 9.5; PMP22, peripheral myelin protein 22; SN, sciatic nerve; SOD, superoxide dismutase.
<b>FIG. 3.</b>
FIG. 3.
Global label-free proteomics of skeletal muscle from Sod1−/− and mSod1KO mice. (A, B) Heatmaps of significantly up- and downregulated proteins in skeletal muscle of Sod1−/− (A), mSod1KO (B), and the respective WT mice detected by PEAKS label-free quantification software (Significance −10log p > 20 or p < 0.01 and fold change ≥2) (top panel); volcano plots of label-free proteomic data showing changes in protein content between skeletal muscle of Sod1−/− (A), mSod1KO (B), compared with the respective WT mice (bottom panel). Proteins involved in redox regulation are highlighted in red, proteins involved in protein folding and stress response are highlighted in blue, and proteins involved in metabolism are in yellow. Proteins highlighted are alpha-1-antitrypsin 1–5 (A1AT5), acylphosphatase-2 (ACYP2), mitochondrial aldehyde dehydrogenase (ALDH2), ATP synthase subunits alpha and beta (ATPA and ATPB), carbonic anhydrase 2 and 3 (CAH2 and CAH3), catalase (CATA), heat shock protein 10 mitochondrial (CH10), cytochrome c oxidase subunit 2 (COX2), alpha crystallin B chain (CRYAB), delta (3 5)–delta (2 4)–dieonyl–CoA isomerase (ECH1), protein EMSY (EMSY), gamma enolase (ENOG), glutathione peroxidase 1 (GPX1), pyruvate kinase isozymes M1/M2 (KPYM), NADH dehydrogenase iron–sulfur protein 3 (NDUS3), protein disulfide isomerase (PDIA4), peroxiredoxins 2, 3, 5, and 6 (PRDX2, PRDX3, PRDX5, and PRDX6), phosphoglucomutase 1 (PGM1), CuZnSOD (SODC), and thioredoxin (THIO).
<b>FIG. 4.</b>
FIG. 4.
Pathway analysis of antioxidant mechanisms in skeletal muscle of Sod1−/− and mSod1KO mice. (A, B) Regulation of antioxidant proteins (as represented by Pathvisio and WikiPathways) in skeletal muscle of Sod1−/− (A) and mSod1KO mice (B). (C, D) Western blots of protein content of the PRX isoforms (PRX I–VI) in AT lysates of Sod1−/− (C), mSod1KO (D), and the respective WT mice. (E) Densitometric analysis of the Western blots shown in (C, D). *p < 0.05 compared with values from the respective WT mice. (F, G) Western blots for GPX1 and CAT protein levels (top panel) in AT skeletal muscle of Sod1−/− (F), mSod1KO (G), and the respective WT mice, and densitometric quantification of the blots (bottom panel). *p < 0.05 compared with values from the respective WT mice. CAT, catalase; GPX1, glutathione peroxidase 1; PRX, peroxiredoxin.
<b>FIG. 5.</b>
FIG. 5.
Pathway analysis reveal proteasomal activation in skeletal muscle of Sod1−/− but not in mSod1KO mice. (A, B) Pathways analysis of proteasomal regulation (as represented by Pathvisio and WikiPathways) in skeletal muscle of Sod1−/− (A) and mSod1KO mice (B). (C, D) Western blots (top panel) and quantification (bottom panel) of ubiquitin-bound proteins in AT skeletal muscle of Sod1−/− (C), mSod1KO (D), and the respective WT mice. *p < 0.05 compared with values from the respective WT mice. (E, F) Western blot analysis (top panel) and quantification (bottom panel) of α, β1, β2, and β5 proteasomal subunits in AT lysates of Sod1−/− (E), mSod1KO (F), and the respective WT mice. *p < 0.05 compared with values from the respective WT mice. (G) Proteasomal activity assessed by ATP-independent (20S proteasome, top panel) and the difference between ATP-independent and ATP-stimulated (26S proteasome, bottom panel) lactacystine-sensitive degradation of the fluorogenic peptide suc-LLVY-AMC in AT lysates of Sod1−/−, mSod1KO, and the respective WT littermate controls. *p < 0.05 compared with values from the respective WT mice, n = 4 mice/strain. (H, I) Proteolytic calpain I and calpastatin protein levels (left panel) in AT skeletal muscle of Sod1−/− (H), mSod1KO (I), and the respective WT mice, and densitometric quantification of the blots (right panel). *p < 0.05 compared with values from the respective WT mice. Suc-LLVY-AMC, succinyl-Leu-Leu-Val-Tyr-7-amido-4-methylcoumarin.
<b>FIG. 6.</b>
FIG. 6.
Global label-free proteomics and differential cysteine labeling in peripheral nerve of Sod1−/− and mSod1KO mice. (A, B) Heatmaps of significantly up- and downregulated proteins in SN of Sod1−/− (A), mSod1KO (B), and the respective WT mice detected by PEAKS label-free quantification software (p < 0.01 and at least one unique peptide) (top panel); volcano plots of label-free proteomic data showing changes in protein content between peripheral nerve of Sod1−/− (A), mSod1KO (B), compared with the respective WT mice (bottom panel). Proteins involved in redox regulation are highlighted in red, myelin-associated proteins are highlighted in blue, and metabolic proteins are highlighted in yellow. Proteins highlighted are alpha-1 antitrypsin (A1AT), apolipoprotein A-I and A-II (APOA1 and APOA2), carbonic anhydrase 3 (CAH3), catalase (CATA), heat shock protein 10 mitochondrial (CH10), electron transport flavoprotein subunit alpha (ETFA), gelsolin (GELS), glutathione peroxidase 3 (GPX3), isocitrate dehydrogenase [NAD] alpha mitochondrial (IDH3A), myelin basic protein (MBP), myelin proteolipid protein (MYPR), myelin protein P0 (MPZ), protein DJ-1 (PARK7) peroxiredoxins 3 and 5 (PRDX3 and PRDX5), CuZnSOD (SODC), extracellular superoxide dismutase (SODE), sphingosine 1-phosphate receptor 4 (S1PR4), thioredoxin reductase 1 (TRXR1), and transketolase (TKT). (C, D) Redox state of Cys47 from PRDX 6 (C) and Cys95 from PRDX 5 (D) in peripheral nerve of Sod1−/− (left panel), mSod1KO (right panel), and their respective controls. Blue bars represent the reduced state of the Cys residue and red bars the reversibe oxidation of the Cys residue. * Indicates a significant shift (p < 0.05) in the redox state toward reversible oxidation compared with values from the respective WT mice. Cys, cysteine
<b>FIG. 7.</b>
FIG. 7.
Pathway analysis of antioxidant mechanisms in peripheral nerve of Sod1−/− and mSod1KO mice. (A, B) Regulation of antioxidant proteins in pathways in peripheral nerve of Sod1−/− (A) and mSod1KO mice (B). (C, D) Western blots (top panel) of the main cytosolic and mitochondrial H2O2 reducing enzymes, including CAT and PRXIII in SN lysates of Sod1−/− (C), mSod1KO (D), and the respective WT mice; densitometric analysis of the blots (bottom panel). (E, F) Protein expression (top panel) of endothelial nitric oxide synthase (eNOS) isoform in SN lysates of Sod1−/− (E), mSod1KO (F), and the respective WT mice; densitometric analysis of the blots (bottom panel). (G, H) Protein expression of HSPs, including mitochondrial HSP10, HSP25, HSP60, and HSC70 in SN lysates of Sod1−/− (G), mSod1KO (H), and the respective WT mice. (I) Densitometric analysis of the Western blots shown in (G, H). (J, K) Western blots for total and Phospho IκB-α and P65 content (total and phosphorylated), in SN lysates of Sod1−/− (J), mSod1KO (K), and the respective WT mice. (L) Densitometric analysis of the Western blots shown in (J, K). *p < 0.05 compared with values from the respective WT mice. H2O2, hydrogen peroxide; HSPs, heat shock proteins; Phospho IκB-α, phosphorylated IκB-α; PRXIII, peroxiredoxin III.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Bhattacharya A, Hamilton R, Jernigan A, Zhang Y, Sabia M, Rahman MM, Li Y, Wei R, Chaudhuri A, and Van Remmen H. Genetic ablation of 12/15-lipoxygenase but not 5-lipoxygenase protects against denervation-induced muscle atrophy. Free Radic Biol Med 67: 30–40, 2014 - PubMed
    1. Bhattacharya A, Muller FL, Liu Y, Sabia M, Liang H, Song W, Jang YC, Ran Q, and Van Remmen H. Denervation induces cytosolic phospholipase A2-mediated fatty acid hydroperoxide generation by muscle mitochondria. J Biol Chem 284: 46–55, 2009 - PMC - PubMed
    1. Bonaldo P. and Sandri M. Cellular and molecular mechanisms of muscle atrophy. Dis Model Mech 6: 25–39, 2013 - PMC - PubMed
    1. Bronstein JM. Function of tetraspan proteins in the myelin sheath. Curr Opin Neurobiol 10: 552–557, 2000 - PubMed
    1. Broome CS, Kayani AC, Palomero J, Dillmann WH, Mestril R, Jackson MJ, and McArdle A. Effect of lifelong overexpression of HSP70 in skeletal muscle on age-related oxidative stress and adaptation after nondamaging contractile activity. FASEB J 20: 1549–1551, 2006 - PubMed

Publication types

MeSH terms