Allopurinol partially prevents disuse muscle atrophy in mice and humans

Abstract

Disuse muscle wasting will likely affect everyone in his or her lifetime in response to pathologies such as joint immobilization, inactivity or bed rest. There are no good therapies to treat it. We previously found that allopurinol, a drug widely used to treat gout, protects muscle damage after exhaustive exercise and results in functional gains in old individuals. Thus, we decided to test its effect in the prevention of soleus muscle atrophy after two weeks of hindlimb unloading in mice, and lower leg immobilization following ankle sprain in humans (EudraCT: 2011-003541-17). Our results show that allopurinol partially protects against muscle atrophy in both mice and humans. The protective effect of allopurinol is similar to that of resistance exercise which is the best-known way to prevent muscle mass loss in disuse human models. We report that allopurinol protects against the loss of muscle mass by inhibiting the expression of ubiquitin ligases. Our results suggest that the ubiquitin-proteasome pathway is an appropriate therapeutic target to inhibit muscle wasting and emphasizes the role of allopurinol as a non-hormonal intervention to treat disuse muscle atrophy.

Figure 1

Allopurinol-treated mice are partially protected against hindlimb unloading-induced skeletal muscle atrophy. (A) Hematoxylin and eosin staining (Original magnification, 4×. Scale bar, 100.00 µm). (B) Myosin staining specific of MHC (type I fibers were deeply stained, while type II were lightly stained. Original magnification 20×. Scale bar, 100.00 µm). (C) Shows quantitative analysis of the cross-sectional area of the soleus muscle. (D) Representative western blotting and densitometric analysis of soleus MHC I (full-length blots are included in supplementary information. See Supplementary Figure 1). Actin was used as a loading control. Data are shown as mean ± SD. C = Control (n = 5–7); U = Unloading (n = 5–7); UA = Unloading treated with allopurinol (n = 5–7).

Figure 2

Allopurinol-treated mice are partially protected against hindlimb unloading-induced inflammation and oxidative stress. (A) Representative western blotting of soleus proteins (full-length blots are included in supplementary information. See Supplementary Figure 2). (B) Shows plasma MDA levels measured by HPLC. (C and D) show the densitometric analysis quantified by using relative expression in arbitrary units of MnSOD (C), and p-p38 (D). (E and F) Show NF-κB p65 binding activity (E), and plasma 6-keto prostaglandin F1α (F) Determined by ELISA. Actin was used as a loading control. Data are shown as mean ± SD. C (n = 4–7), U (n = 4–6), and UA (n = 4–6).

Figure 3

Allopurinol up-regulates the IGF-1/Akt pathway and down-regulates muscle atrophy-related E3 ubiquitin ligases in skeletal muscle. (A) Representative western blotting of soleus proteins (full-length blots are included in supplementary information. See Supplementary Figure 3). (B) Shows mRNA levels of Cbl-b in soleus muscle. (C to E) Show the densitometric analysis quantified using relative expression in arbitrary units of IRS-1 (C), p-Akt (D), p-FoxO3 (E). (F and G) Show the soleus muscle mRNA levels of MuRF-1 (F) and MAFbx (G). (H to J) Show the densitometric analysis quantified using relative expression in arbitrary units of Beclin-1 (H), p62 (I), and LC3-II (J). Actin and GAPDH were used as a loading control in the western blots, as indicated. Data are shown as mean ± SD, C (n = 4–6), U (n = 4–5), and UA (n = 4–5).

Figure 4

Allopurinol-treated ankle sprain patients are partially protected against unloading-induced skeletal muscle atrophy. (A) Quantification of the changes in soleus muscle volume during lower leg immobilization with a posterior ankle splint. (B) Quantification of the changes in soleus muscle CSA during lower leg immobilization with a posterior ankle splint. (C) Representative volume images after analysis and segmentation of the soleus muscle in control and allopurinol-treated subjects. R = right, L = left, A = anterior, P = posterior, T = top, B = bottom. (D) Representative magnetic resonance images of control and allopurinol-treated subjects. The soleus muscle is shown in yellow.

Figure 5

Proposed signaling pathways activated during muscle atrophy and the effect of allopurinol administration.