Erythropoietin Alleviates Burn-induced Muscle Wasting

Abstract

Background: Burn injury induces long-term skeletal muscle pathology. We hypothesized EPO could attenuate burn-induced muscle fiber atrophy. Methods: Rats were allocated into four groups: a sham burn group, an untreated burn group subjected to third degree hind paw burn, and two burn groups treated with weekly or daily EPO for four weeks. Gastrocnemius muscle was analyzed at four weeks post-burn. Results: EPO attenuated the reduction of mean myofiber cross-sectional area post-burn and the level of the protective effect was no significant difference between two EPO-treated groups (p=0.784). Furthermore, EPO decreased the expression of atrophy-related ubiquitin ligase, atrogin-1, which was up-regulated in response to burn. Compared to untreated burn rats, those receiving weekly or daily EPO groups had less cell apoptosis by TUNEL assay. EPO decreased the expression of cleaved caspase 3 (key factor in the caspase-dependent pathway) and apoptosis-inducing factor (implicated in the caspase-independent pathway) after burn. Furthermore, EPO alleviated connective tissue overproduction following burn via transforming growth factor beta 1-Smad2/3 pathway. Daily EPO group caused significant erythrocytosis compared with untreated burn group but not weekly EPO group. Conclusion: EPO therapy attenuated skeletal muscle apoptosis and fibrosis at four weeks post-burn. Weekly EPO may be a safe and effective option in muscle wasting post-burn.

Keywords: Apoptosis Inducing Factor; Burn injury; Erythropoietin; Muscle fiber atrophy; Transforming Growth Factor beta1.

Figure 1

EPO on burn-induced muscle fiber atrophy and atrophy-related ubiquitin ligase, atrogin-1 (A) Representative H&E staining of gastrocnemius muscle section (200x). Average myofiber cross-sectional area of 4 groups. (B) Representative western blot of atrogin-1. EPO decreased the elevation of atrogin-1 post-burn. All error bars represented the SEM. *p<0.05, **p<0.01.

Figure 2

EPO on cleaved caspase 3 mediated apoptosis. (A) Representative TUNEL stain (green) and immunofluorescence of cleaved caspase 3 (red). DAPI (blue) was used for nuclear counterstaining. Arrowheads indicated positive-staining cells. EPO-treated groups showed less TUNEL/cleaved caspase 3 positive cells. (B) The expression of cleaved caspase 3 by western blot. EPO decreased the elevation of cleaved caspase 3 post-burn. All error bars represented the SEM. *p<0.05, **p<0.01. Scale bar: 50 µm.

Figure 3

EPO on AIF mediated apoptosis. (A) Representative TUNEL stain (green) and immunofluorescence of AIF (red). DAPI (blue) was used for nuclear counterstaining. Arrowheads indicated positive-staining cells. TUNEL/AIF-positive cells were decreased in both EPO-treated groups. (B) Representative western blot of AIF. EPO decreased the elevation of AIF post-burn. AIF: Apoptosis-inducing factor. All error bars represented the SEM. *p<0.05, **p<0.01. Scale bar: 50 µm.

Figure 4

EPO on ECM overproduction following burn. (A) Immunofluorescence images of type I collagen, type III collagen and fibronectin. The expression of ECM proteins was significantly increased in untreated burn group but not in EPO-treated groups. (B) Western blot revealed EPO attenuated burn-induced ECM proteins elevation. ECM: extracellular matrix. All error bars represent the SEM. *p<0.05. Scare bars= 100 µm.

Figure 5

EPO alleviated burn-induced muscle fibrosis by suppressing TGF-β1/Smad pathway. (A) Representative western blot of CTGF and TGF-β1. EPO attenuated the overexpression of CTGF and TGF-β1 post-burn. (B) Immunofluorescence and western blot of pSmad2/3 in muscle sections. EPO attenuated the overexpression of pSmad2/3 after burn. TGF-β1: Transforming growth factor-β1, CTGF: Connective tissue growth factor, pSmad2/3: phosphorylated Smad2/3. *p<0.05.

Figure 6

Proposed mechanism of EPO on muscle fiber atrophy following burn. EPO attenuates burn-induced skeletal muscle apoptosis via decreasing the expression of cleaved caspase 3 and AIF at four weeks post-burn. Moreover, EPO modulates burn-induced overexpression of TGF-β1/Smad2/3 profibrotic pathway and decreases the elevation of CTGF. EPO is a potential therapeutic agent for burn-induced skeletal muscle wasting. Apoptosis-inducing factor (AIF), Transforming growth factor beta 1 (TGF-β1), phosphorylated Smad2/3 (pSmad2/3), Connective tissue growth factor (CTGF).