Biochem. J. (2006) 397
(149–158) (Printed in Great Britain)
Proteomic analysis of altered protein expression in skeletal muscle of rats in a hypermetabolic state induced by burn sepsis
Xunbao DUAN, François BERTHIAUME, David YARMUSH and Martin L. YARMUSH1
The Surgical Services/Center for Engineering in Medicine, Massachusetts General Hospital, Harvard Medical School and Shriners Hospitals for Children, Boston, MA 02114, U.S.A.
mRNA profiling has been extensively used to study muscle wasting. mRNA level changes may not reflect that of proteins, especially in catabolic muscle where there is decreased synthesis and increased degradation. As sepsis is often associated with burn injury, and burn superimposed by sepsis has been shown to result in significant loss of lean tissues, we characterized changes in the skeletal-muscle proteome of rats subjected to a cutaneous burn covering 20% of the total body surface area, followed 2 days later by sepsis induced by CLP (caecal ligation and puncture). EDL (extensor digitorum longus) muscles were dissected from Burn-CLP animals (n=4) and controls (sham-burned and sham-CLP-treated, n=4). Burn-CLP injury resulted in a rapid loss of EDL weight, increased ubiquitin-conjugated proteins and increased protein carbonyl groups. EDL protein profiles were obtained by two-dimensional gel electrophoresis using two immobilized pH gradient strips with overlapping pH range covering a pH 3–8 range. Seventeen spots were significantly altered in the Burn-CLP compared with the control group, representing 15 different proteins identified by peptide mass fingerprinting. The identities of three proteins including transferrin were further confirmed by liquid chromatography–tandem MS. The significant changes in transferrin and HSP27 (heat-shock protein 27) were verified by Western-blot analysis. HSP60, HSP27 and HSPb6 were down-regulated, along with HSP70, as detected by Western blotting. Six metabolic enzymes related to energy production were also down-regulated. A simultaneous decrease in chaperone proteins and metabolic enzymes could decrease protein synthesis. Furthermore, decreased HSPs could increase oxidative damage, thus accelerating protein degradation. Using cultured C2C12 myotubes, we showed that H2O2-induced protein degradation in vitro could be partially attenuated by prior heat-shock treatment, consistent with a protective role of HSP70 and/or other HSPs against proteolysis.
Key words: burn sepsis, heat-shock protein, metabolic enzyme, muscle atrophy, proteomic analysis, skeletal muscle.
Abbreviations used: CLP, caecal ligation and puncture; DMEM, Dulbecco's modified Eagle's medium; DNPH, dinitrophenylhydrazine; DTT, dithiothreitol; EDL muscle, extensor digitorum longus muscle; HS, horse serum; HSP, heat-shock protein; IPG, immobilized pH gradient; MAFbx, muscle atrophy F-box; MALDI–TOF, matrix-assisted laser-desorption ionization–time-of-flight; MS/MS, tandem MS; mt-GrpE#1, mitochondrial GrpE protein homologue 1; MuRF1, muscle RING finger 1; MyBP-H, myosin-binding protein H; nano-LC–ESI, nano-liquid chromatography–electrospray ionization; NF-kB, nuclear factor kB; SAGE, serial analysis of gene expression; SOD-1, superoxide dismutase-1; TNFa, tumour necrosis factor a.
1To whom correspondence should be addressed, at Shriners Hospitals for Children, 51 Blossom Street, Boston, MA 02114, U.S.A. (email ireis@sbi.org).
Received 21 October 2005/16 January 2006; accepted 16 February 2006
Published as BJ Immediate Publication 16 February 2006, doi:10.1042/BJ20051710
The Biochemical Society, London ©2006
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