Evaluation of functional erythropoietin receptor status in skeletal muscle in vivo: acute and prolonged studies in healthy human subjects

Abstract

Background: Erythropoietin receptors have been identified in human skeletal muscle tissue, but downstream signal transduction has not been investigated. We therefore studied in vivo effects of systemic erythropoietin exposure in human skeletal muscle.

Methodology/principal findings: The protocols involved 1) acute effects of a single bolus injection of erythropoietin followed by consecutive muscle biopsies for 1-10 hours, and 2) a separate study with prolonged administration for 16 days with biopsies obtained before and after. The presence of erythropoietin receptors in muscle tissue as well as activation of Epo signalling pathways (STAT5, MAPK, Akt, IKK) were analysed by western blotting. Changes in muscle protein profiles after prolonged erythropoietin treatment were evaluated by 2D gel-electrophoresis and mass spectrometry. The presence of the erythropoietin receptor in skeletal muscle was confirmed, by the M20 but not the C20 antibody. However, no significant changes in phosphorylation of the Epo-R, STAT5, MAPK, Akt, Lyn, IKK, and p70S6K after erythropoietin administration were detected. The level of 8 protein spots were significantly altered after 16 days of rHuEpo treatment; one isoform of myosin light chain 3 and one of desmin/actin were decreased, while three isoforms of creatine kinase and two of glyceraldehyd-3-phosphate dehydrogenase were increased.

Conclusions/significance: Acute exposure to recombinant human erythropoietin is not associated by detectable activation of the Epo-R or downstream signalling targets in human skeletal muscle in the resting situation, whereas more prolonged exposure induces significant changes in the skeletal muscle proteome. The absence of functional Epo receptor activity in human skeletal muscle indicates that the long-term effects are indirect and probably related to an increased oxidative capacity in this tissue.

Figure 1. Study B: Western blot and PCR results.

A+B: Identification of the Epo-R in skeletal muscle tissue in the non-stimulated state in all 10 subjects with the C20 (A) and M20 (B) antibodies. Positive control is the k-562 cells. M is the molecular marker. C: Activation of the Epo-R by phosphorylation evaluated by western blotting. The samples from the same subject in the stimulated and un-stimulated situation are loaded next to each other D: Phosphorylation of Epo-R, STAT5, p38-MAPK, Akt, p70S6 kinase, Lyn, and IKK, all normalized to the levels of the given protein. The mRNA levels of SOCS3 and IGF-I are normalized to beta-actin mRNA content. Black bars: placebo, white bars: rHuEpo. All results are from biopsies taken 1 h post treatment with either placebo or rHuEpo.

Figure 2. Study A: Western blot and PCR results.

Phosphorylation of STAT5, p38-MAPK, Akt, p70S6 kinase, Lyn, and IKK, all normalized to the total levels of the given protein. Black bars: placebo, white bars: rHuEpo. The levels of mRNA are measured at 10 h post rHuEpo administration, and are normalized to beta-actin mRNA levels. Level of significance p<0.05, * compared to baseline, # compared to control (same timepoint), § compared to rHuEpo 2 h. The interactions were as follows; pEpo-R p = 0.318, p38-MAPK p = 0.058 (treatment effect p = 0.030), pSTAT5 p = 0.562, p-Akt(ser) p = 0.001, pAkt(thr) p = 0.007, p-IKK p = 0.742 (time effect p = 0.017), p-LYN p = 0.427, p-p70S6kinase p = 0.164 (time effect p = 0.033).

Figure 3. Representative 2D-gel.

Representative 2D-gel of human skeletal muscle tissue. Protein spots that changed significantly (p<0.05) after 16 days of treatment with rHuEpo are shown and their identity given.

Figure 4. Changes in muscle proteins; Desmin/Actin (A), Myosin light chain 1 V/sB (B), Creatin kinase M-type (C, D, E), Glyceraldehyd-3-phosphat-dehydrogenase (F, G), and unidentified spot (H).

I. Representative 3D image of spots showing intensity changes between baseline and day 16. Images were obtained using the 3D viewer tool from PDQuest, which converts intensity to topographical peaks. All spot images belong to the same subject. II. Mean changes in intensity for each spot. The difference between baseline and day 16 are significant (p<0.05).