Calpain inhibition attenuated morphological and molecular changes in skeletal muscle of experimental allergic encephalomyelitis rats

Abstract

Muscle weakness and atrophy are important manifestations of multiple sclerosis (MS). To investigate the pathophysiological mechanisms of skeletal muscle change in MS, we induced experimental autoimmune encephalomyelitis (EAE) in Lewis male rats and examined morphological and molecular changes in skeletal muscle. We also treated EAE rats with calpepetin, a calpain inhibitor, to examine its beneficial effects on skeletal muscle damage. Morphological changes in muscle tissue of EAE rats included smaller and irregularly shaped muscle fibers and fibrosis. Western blot analysis demonstrated increased calpain:calpastatin ratio, inflammation-related transcription factors (nuclear factor-κB:inhibitor of κB α ratio), and proinflammatory enzymes (cyclooxygenase-2). TUNEL-positive myonuclei in skeletal muscle cells of EAE rats indicated cell death. In addition, markers of apoptotic cell death (Bax:Bcl-2 ratio and caspase-12 protein levels) were elevated. Expression of muscle-specific ubiquitin ligases (muscle atrophy F-box and muscle ring finger protein 1), was upregulated in muscle tissue of EAE-vehicle animals. Both prophylactic and therapeutic treatment with calpeptin partially attenuated muscle changes noted in EAE animals. These results indicate that morphological and molecular changes including apoptotic cell death and protein breakdown develop in skeletal muscle of EAE animals and that these changes can be reversed by calpain inhibition.

Fig. 1.

Morphological alterations in skeletal muscle of EAE rats. A: H&E staining demonstrated various changes, including smaller, irregularly shaped muscle fibers and fibrosis in thin section slices (10 μm) obtained from EAE rats treated with vehicle prophylactically or therapeutically compared with control rats. B: Assessment of muscle atrophy in EAE animals by measurement of muscle fiber diameter. A significant reduction in fiber diameter was noted in EAE-vehicle animals. These changes were attenuated in EAE animals treated with calpeptin prophylactically or therapeutically. *P < 0.05 vs. control, **P < 0.01 vs. control; ^#P < 0.05 vs. EAE-vehicle, ^##P < 0.01 vs. EAE-vehicle. CP, calpeptin. ×200.

Fig. 2.

Changes in calpain and inflammation-related protein expression in skeletal muscle of EAE rats. Calpain:calpastatin ratio, NF-κB:I-κB α ratio, STAT3, COX-2, and IL-2 expression were increased in EAE rats, whereas calpeptin treatment reduced these proinflammatory changes. Representative Western blot and determination of 80-kDa calpain:110-kDa calpastatin ratio (A), 65-kDa NF-κB:37-kDa I-κB α ratio (B), percentage change of 86-kDa STAT3 expression (C), 72-kDa COX-2 expression (D), and 15-kDa IL-2 expression (E). *P < 0.05 vs. control, **P < 0.01 vs. control; ^#P < 0.05 vs. EAE-vehicle, ^##P < 0.01 vs. EAE-vehicle. CP, calpeptin.

Fig. 3.

Inhibition of calpain reduced apoptotic changes in skeletal muscle of EAE rats. A: TUNEL-positive myonuclei were detected in muscle of EAE rats treated with vehicle prophylactically or therapeutically. Arrows indicate nicked DNA of apoptotic cells. Posttreatment with calpeptin attenuated these changes. Sections (10 μm) of muscle tissue were costained with TUNEL (green), and DAPI (blue; ×200). Representative Western blot and determination of 23-kDa Bax:26-kDa Bcl-2 ratio (B) and percentage change of 50-kDa caspase-12 (C) and 20-kDa active caspase-3 (D). *P < 0.05 vs. control, **P < 0.01 vs. control; ^#P < 0.05 vs. EAE-vehicle, ^##P < 0.01 vs. EAE-vehicle. CP, calpeptin.

Fig. 4.

Alteration of protein degradation-related proteins resulting from EAE induction. Posttreatment with calpeptin significantly attenuated muscle-specific E3 ligase expression in EAE rat skeletal muscles. Representative Western blot and determination of percentage changes in 42-kDa MAFbx (A) and 44-kDa MuRF1 (B). *P < 0.05 vs. control, **P < 0.01 vs. control; ^#P < 0.05 vs. EAE-vehicle, ^##P < 0.01 vs. EAE-vehicle. CP, calpeptin.

Fig. 5.

Changes in degradation-related protein expression by inflammatory stimulation (IFN-γ 500 U/ml) and their attenuation by calpeptin treatment (1 and 5 μM) in L6 rat skeletal myoblast cells. Representative Western blot and determination of percentage changes in 42-kDa MAFbx (A) and 44-kDa MuRF1 (B; n = 3 per group). *P < 0.05 vs. control, **P < 0.01 vs. control; ^#P < 0.05 vs. IFN-γ 500 U/ml, ^##P < 0.01 vs. IFN-γ 500 U/ml. CP, calpeptin.