Activation of the ubiquitin proteasome pathway in a mouse model of inflammatory myopathy: a potential therapeutic target

Abstract

Objective: Myositis is characterized by severe muscle weakness. We and others have previously shown that endoplasmic reticulum (ER) stress plays a role in the pathogenesis of myositis. The present study was undertaken to identify perturbed pathways and assess their contribution to muscle disease in a mouse myositis model.

Methods: Stable isotope labeling with amino acids in cell culture (SILAC) was used to identify alterations in the skeletal muscle proteome of myositic mice in vivo. Differentially altered protein levels identified in the initial comparisons were validated using a liquid chromatography tandem mass spectrometry spike-in strategy and further confirmed by immunoblotting. In addition, we evaluated the effect of a proteasome inhibitor, bortezomib, on the disease phenotype, using well-standardized functional, histologic, and biochemical assessments.

Results: With the SILAC technique we identified significant alterations in levels of proteins belonging to the ER stress response, ubiquitin proteasome pathway (UPP), oxidative phosphorylation, glycolysis, cytoskeleton, and muscle contractile apparatus categories. We validated the myositis-related changes in the UPP and demonstrated a significant increase in the ubiquitination of muscle proteins as well as a specific increase in ubiquitin carboxyl-terminal hydrolase isozyme L1 (UCHL-1) in myositis, but not in muscle affected by other dystrophies or normal muscle. Inhibition of the UPP with bortezomib significantly improved muscle function and also significantly reduced tumor necrosis factor α expression in the skeletal muscle of mice with myositis.

Conclusion: Our findings indicate that ER stress activates downstream UPPs and contributes to muscle degeneration and that UCHL-1 is a potential biomarker for disease progression. UPP inhibition offers a potential therapeutic strategy for myositis.

Figure 1

Differentially modulated proteins and pathways involved in experimental myositis in mice, identified by proteomic profiling using stable isotope labeling with amino acids in cell culture (SILAC). Quadriceps muscle was harvested from SILAC-treated (labeled [L]) C57BL/6 (BL6) mice, from age-matched major histocompatibility complex–overexpressing double-transgenic (HT) myositic mice that had not undergone SILAC (unlabeled [U]), and from age-matched single-transgenic (H or T) control mice that had not undergone SILAC (n = 3 per group). A, Log-transformed protein ratio distribution in muscle from unlabeled normal mice versus normal SILAC mice and in unlabeled mice with myositis versus normal SILAC mice, showing a broader distribution of up-regulated and down-regulated proteins in the myositic muscle. The ratios of unlabeled-to-labeled peptide pairs were obtained using IP2 software. B, Proteins with significant modulations (≥1.5-fold) annotated to specific pathways based on function, using the UniProt knowledge database. The proportion of proteins annotated to each pathway (as a percentage of the total number of proteins) is shown. ER = endoplasmic reticulum. Color figure can be viewed in the online issue, which is available at http://onlinelibrary.wiley.com/doi/10.1002/art.38180/abstract.

Figure 2

Up-regulation of the ubiquitin proteasome pathway (UPP) in myositic muscle. Quadriceps muscle was obtained from SILAC-treated mice, HT mice that had not undergone SILAC, and H or T mice that had not undergone SILAC (n = 3 per group), and mass spectrometry analysis was performed. A, Mean ± SEM ratios of UPP proteins in muscle from unlabeled versus labeled myositic mice and from unlabeled versus labeled control mice. B, Homogenates of quadriceps muscle lysates from 4 H or T mice and from 4 HT mice, immunoblotted with anti–ubiquitin carboxyl-terminal hydrolase isozyme L1 (anti–UCHL-1) antibody or with β-actin as a loading control. C, Mean ± SEM ratio of UCHL-1 to β-actin, calculated by densitometric analysis using Quantity One software. *** = P < 0.001 versus H or T mice, by Student's t-test. UBC = ubiquitin C; RS27A = ubiquitin 40S ribosomal protein S27A; RL40 = ubiquitin 60S ribosomal protein L40; TERA = transitional endoplasmic reticulum ATPase (see Figure 1 for other definitions).

Figure 3

Expression of ubiquitin carboxyl-terminal hydrolase isozyme L1 (UCHL-1) is specific to myositis muscle and does not occur in other types of dystrophic muscle. A, Quadriceps muscle lysates from 4 H or T mice and from 4 HT mice were obtained, and homogenates were immunoblotted for ubiquitin. B, Another set of quadriceps muscle lysates was obtained from 16-week-old H or T mice, HT mice, and age- and sex-matched SJL/J and mdx mice (n = 3 per group). The homogenates were immunoblotted with UCHL-1 or with β-actin as a loading control. C and D, The ratios of the respective proteins to β-actin from the experiments described in A and B, respectively, were calculated. Values are the mean ± SEM. *** = P < 0.001 versus other groups, by Student's t-test (C) or one-way analysis of variance (D). See Figure 1 for other definitions.

Figure 4

Bortezomib (Bort) treatment improves muscle function in the mouse model of myositis. Major histocompatibility complex–overexpressing double-transgenic (HT) myositic mice and single-transgenic (H or T) control mice were divided into 3 groups: H or T (n = 5), untreated (Unt) HT (n = 4), and bortezomib-treated HT (n = 4). Bortezomib (0.75 mg/kg body weight) was injected intraperitoneally twice a week for 4 weeks. A, Proteasomal activity in quadriceps muscle lysates, shown as relative fluorescence units (RFU) normalized to protein units. B, Body weight. C, Gastrocnemius muscle weight. D, Maximal muscle force, determined by in vitro testing of extensor digitorum longus (EDL) muscles. E, Percent force recovery for extensor digitorum longus muscle. Values are the mean ± SEM. * = P < 0.05; ** = P < 0.01; *** = P < 0.001 versus H or T control mice (a) or versus untreated HT mice (b), by one-way analysis of variance.

Figure 5

Bortezomib treatment decreases muscle inflammation in the mouse model of myositis. Myositic (HT) mice and control (H or T) mice were divided into 3 groups: H or T (n = 5), untreated HT (n = 4), and bortezomib-treated HT (n = 4). Bortezomib (0.75 mg/kg body weight) was injected intraperitoneally twice a week for 4 weeks. Extensor digitorum longus muscles were collected in formalin, and tissue sections were stained with hematoxylin and eosin. A–C, Representative photomicrographs of muscle from an H or T mouse (A), an untreated HT mouse (B), and a bortezomib-treated HT mouse (C). Original magnification × 20. D, Inflammation scores of stained sections (on a scale of 0–5 [0 = no inflammation, 5 = highest inflammation]), determined under blinded conditions. E and F, Levels of tumor necrosis factor α (TNFα) transcript (E) and intercellular adhesion molecule 1 (ICAM-1) transcript (F) in quadriceps muscle lysates, determined by quantitative polymerase chain reaction and expressed as the fold change in relation to hypoxanthine guanine phosphoribosyltransferase. Values in D–F are the mean ± SEM. * = P < 0.05 versus H or T control mice (a) or versus untreated HT mice (b), by Wilcoxon rank sum test. See Figure 4 for other definitions.

Figure 6

Bortezomib treatment decreases glucose regulatory protein 78 (Grp78) levels and increases regeneration in myositic mouse muscle. A, Immunoblotting for Grp78 in quadriceps muscle lysates from 4 control (H or T) mice, 4 untreated myositic (HT) mice, and 4 bortezomib-treated HT mice. B, Quantification of Grp78 levels in relation to β-actin levels. C, Number of embryonic myosin heavy chain (eMHC)–positive fibers per section. Values in B and C are the mean ± SEM. * = P < 0.05; *** = P < 0.001 versus H or T control mice (a) or versus untreated HT mice (b), by one-way analysis of variance (B) or Wilcoxon rank sum test (C). D–F, Representative photomicrographs showing staining for embryonic myosin heavy chain (arrows) in muscle from an H or T mouse (D), an untreated HT mouse (E), and a bortezomib-treated HT mouse (F). Bars = 100 μM. See Figure 4 for other definitions.