Proteomic analysis of altered protein expression in skeletal muscle of rats in a hypermetabolic state induced by burn sepsis

Abstract

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 HSPbeta6 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.

Figure 1. Effect of systemic injury on rat EDL muscle wet weight

Sham-burn or burn injury was administrated, followed by a sham-CLP or CLP operation 2 days later. Muscles were harvested on post-burn day 4. *P<0.05; **P<0.001 versus Sham-Sham by Student's t test.

Figure 2. Two-dimensional profiles of EDL muscles from control (Sham-Sham) and Burn-CLP rats

Protein extracts (50 μg) were separated with two overlapping two-dimensional gels and visualized with Sypro Ruby dye. Arrows point to the 17 protein spots that were found to be significantly altered. The altered spots that overlap on the pI 3–6 and pI 5–8 images are marked with the same identifying number and count as one regulated spot. Results shown are for one representative sample in each group.

Figure 3. Identification of transferrin by peptide mass ‘fingerprinting’ and peptide sequencing

(A) A total of 16 peptide mass peaks (grey line) matched the theoretical mass of the tryptic peptides of transferrin with a mass tolerance <0.1 Da. (B) The sequence of one representative peptide DQYELLCLDNTR (corresponding to the peak mass 1539.68 in (A) was generated by the MS/MS spectrum obtained using a ThermoFinnigan ion-trap mass spectrometer. All the matched Y and B ions were singly charged, with the exception of the m/z 761.4 value, which matched doubly charged B-12 ion.

Figure 4. Western blotting detection of transferrin (A) and HSP27 (B) to confirm their changes observed by two-dimensional analysis

A 40 μg portion of EDL muscle protein from each animal was loaded for immunoblotting. The quantified band volume was used to represent the relative amount of the protein, and is shown in the lower panel. Results are the means±S.D. for three animals in each group. The upper panel shows one representative blot (lane 1, Sham-Sham; lane 2, Burn-CLP). *P<0.05 versus Sham-Sham by Student's t test.

Figure 5. Effect of burn-CLP on HSP70 expression

Western-blot analysis of HSP70 was carried out on 40 μg of EDL muscle protein. The quantified band volume is used to represent the relative amount of the protein. Results shown represent the means±S.D. for three animals in each group. The upper-right panel shows a representative blot (lane 1, Sham-Sham; lane 2, Burn-CLP). *P<0.05 versus Sham-Sham by Student's t test. kD, kDa.

Figure 6. Effect of Burn-CLP on protein ubiquitination

Western-blot analysis of ubiquitin-conjugated proteins was carried out on 40 μg of EDL muscles, and the quantified volume for all the bands of >43 kDa was used to represent the relative content of ubiquitin-conjugated proteins. Results shown represent the means±S.D. for three animals per group. The upright corner shows one representative blot (lane 1, Sham-Sham; lane 2, Burn-CLP). *P<0.05 versus Sham-Sham by Student's t test.

Figure 7. Effect of Burn-CLP on oxidative damage

(A) Protein carbonyl-group levels expressed as the means±S.D. for data from EDL muscles from four animals in each group. (B) SOD-1 levels measured by Western blotting in 40 μg of EDL muscle protein extracts. The quantified band volume was used to represent the relative amount of the protein. Results shown are the means±S.D. for three animals in each group. The upper-right panel shows one representative blot (lane 1, Sham-Sham; lane 2, Burn-CLP). *P<0.05 versus Sham-Sham by Student's t test.

Figure 8. Effect of H₂O₂ and heat shock on protein degradation in C2C12 myotubes

Cells were labelled with [³H]tyrosine for 48 h, subjected to heat shock (42 °C for 2 h, followed by 4 h recovery at 37 °C) or incubated at 37 °C (control), and then treatment with H₂O₂ for 1 h. The protein degradation was then measured as described in the Experimental section. Results shown are means±S.D. normalized to the values for the 0 mM H₂O₂ in the heat-shock and control groups. The baseline values for protein degradation (in the absence of H₂O₂) were 37.6±0.7% in the control group and 38.9±0.4% in the heat-shock group. n=4 in each group.

Scheme 1. Summary of the potential roles of the differentially regulated proteins identified in the present study within the proteolytic and protein synthetic pathways

Mediators (e.g. TNF) released following severe injury induce oxidative stress, which leads to increased oxidized proteins (more susceptible to degradation) and up-regulation of the ubiquitin–proteasome pathway. These mediators also down-regulate HSPs, which are normally protective against oxidative stress and participate in repair mechanisms for oxidized proteins, further exacerbating oxidative stress-induced proteolysis. A decrease in HSPs and metabolic enzymes, both of which are essential for protein synthesis, may explain, at least partially, decreased protein synthesis after severe injury. −, negative effect; +, positive effect; ↓, protein or cellular event down-regulated; ↑, protein or cellular event up-regulated.