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1.  Losartan Decreases Cardiac Muscle Fibrosis and Improves Cardiac Function in Dystrophin-Deficient Mdx Mice 
Recent studies showed that chronic administration of losartan, an angiotensin II type I receptor antagonist, improved skeletal muscle function in dystrophin-deficient mdx mice. In this study, C57BL/10ScSn-Dmdmdx/J female mice were either untreated or treated with losartan (n = 15) in the drinking water at a dose of 600 mg/L over a 6-month period. Cardiac function was assessed via in vivo high frequency echocardiography and skeletal muscle function was assessed using grip strength testing, Digiscan monitoring, Rotarod timing, and in vitro force testing. Fibrosis was assessed using picrosirius red staining and Image J analysis. Gene expression was evaluated using real-time polymerized chain reaction (RT-PCR). Percentage shortening fraction was significantly decreased in untreated (26.9% ± 3.5%) mice compared to losartan-treated (32.2% ± 4.2%; P < .01) mice. Systolic blood pressure was significantly reduced in losartan-treated mice (56 ± 6 vs 69 ± 7 mm Hg; P < .0005). Percentage cardiac fibrosis was significantly reduced in losartan-treated hearts (P < .05) along with diaphragm (P < .01), extensor digitorum longus (P < .05), and gastrocnemius (P < .05) muscles compared to untreated mdx mice. There were no significant differences in skeletal muscle function between treated and untreated groups. Chronic treatment with losartan decreases cardiac and skeletal muscle fibrosis and improves cardiac systolic function in dystrophin-deficient mdx mice.
doi:10.1177/1074248410381757
PMCID: PMC4147941  PMID: 21304057
Duchenne muscular dystrophy; dystrophin; mice; cardiomyopathy; angiotensin; echocardiography
2.  Activation of the Ubiquitin Proteasome Pathway in a Mouse Model of Inflammatory Myopathy 
Arthritis and rheumatism  2013;65(12):3248-3258.
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.
doi:10.1002/art.38180
PMCID: PMC4080828  PMID: 24022788
3.  The effects of MyD88 deficiency on disease phenotype in dysferlin-deficient A/J mice: role of endogenous TLR ligands 
The Journal of pathology  2013;231(2):199-209.
An absence of dysferlin leads to activation of innate immune receptors such as Toll-like receptors (TLRs) and skeletal muscle inflammation. Myeloid differentiation primary response gene 88 (MyD88) is a key mediator of TLR-dependent innate immune signalling. We hypothesized that endogenous TLR ligands released from the leaking dysferlin-deficient muscle fibres engage TLRs on muscle and immune cells and contribute to disease progression. To test this hypothesis, we generated and characterized dysferlin and MyD88 double-deficient mice. Double-deficient mice exhibited improved body weight, grip strength, and maximum muscle contractile force at 6–8 months of age when compared to MyD88-sufficient, dysferlin-deficient A/J mice. Double-deficient mice also showed a decrease in total fibre number, which contributed to the observed increase in the number of central nuclei/fibres. These results indicate that there was less regeneration in the double-deficient mice. We next tested the hypothesis that endogenous ligands, such as single-stranded ribonucleic acids (ssRNAs), released from damaged muscle cells bind to TLR-7/8 and perpetuate the disease progression. We found that injection of ssRNA into the skeletal muscle of pre-symptomatic mice (2 months old) resulted in a significant increase in degenerative fibres, inflammation, and regenerating fibres in A/J mice. In contrast, characteristic histological features were significantly decreased in double-deficient mice. These data point to a clear role for the TLR pathway in the pathogenesis of dysferlin deficiency and suggest that TLR-7/8 antagonists may have therapeutic value in this disease.
doi:10.1002/path.4207
PMCID: PMC4071457  PMID: 23857504
Toll-like receptor; dysferlin; single-stranded RNA; regeneration; degeneration; inflammation; osteopontin
4.  The molecular basis of skeletal muscle weakness in a mouse model of inflammatory myopathy 
Arthritis and rheumatism  2012;64(11):3750-3759.
OBJECTIVE
It is generally believed that muscle weakness in patients with polymyositis and dermatomyositis is due to autoimmune and inflammatory processes. However, it has been observed that there is a poor correlation between the suppression of inflammation and a recovery of muscle function in patients. We have therefore hypothesized that non-immune mechanisms also contribute to muscle weakness. In particular, it has been suggested that an acquired deficiency of AMP deaminase (AMPD1) may be responsible for muscle weakness in myositis.
METHODS
We have used comprehensive functional, behavioral, histological, molecular, enzymatic and metabolic assessments before and after the onset of inflammation in MHC class I mouse model of autoimmune inflammatory myositis.
RESULTS
We found that muscle weakness and metabolic disturbances were detectable in the mice prior to the appearance of infiltrating mononuclear cells. Force contraction analysis of muscle function revealed that weakness was correlated with AMDP1 expression and was myositis-specific. We also demonstrated that decreasing AMPD1 expression results in decreased muscle strength in healthy mice. Fiber typing suggested that fast-twitch muscles are converted to slow-twitch muscles as myositis progresses, and microarray results indicated that AMPD1 and other purine nucleotide pathway genes are suppressed, along with genes essential to glycolysis.
CONCLUSION
These data suggest that an AMPD1 deficiency is acquired prior to overt muscle inflammation and is responsible, at least in part, for the muscle weakness that occurs in the mouse model of myositis. AMPD1 is therefore a potential therapeutic target in myositis.
doi:10.1002/art.34625
PMCID: PMC3485437  PMID: 22806328
5.  Daily Supplementation of D-ribose Shows No Therapeutic Benefits in the MHC-I Transgenic Mouse Model of Inflammatory Myositis 
PLoS ONE  2013;8(6):e65970.
Background
Current treatments for idiopathic inflammatory myopathies (collectively called myositis) focus on the suppression of an autoimmune inflammatory response within the skeletal muscle. However, it has been observed that there is a poor correlation between the successful suppression of muscle inflammation and an improvement in muscle function. Some evidence in the literature suggests that metabolic abnormalities in the skeletal muscle underlie the weakness that continues despite successful immunosuppression. We have previously shown that decreased expression of a purine nucleotide cycle enzyme, adenosine monophosphate deaminase (AMPD1), leads to muscle weakness in a mouse model of myositis and may provide a mechanistic basis for muscle weakness. One of the downstream metabolites of this pathway, D-ribose, has been reported to alleviate symptoms of myalgia in patients with a congenital loss of AMPD1. Therefore, we hypothesized that supplementing exogenous D-ribose would improve muscle function in the mouse model of myositis. We treated normal and myositis mice with daily doses of D-ribose (4 mg/kg) over a 6-week time period and assessed its effects using a battery of behavioral, functional, histological and molecular measures.
Results
Treatment with D-ribose was found to have no statistically significant effects on body weight, grip strength, open field behavioral activity, maximal and specific forces of EDL, soleus muscles, or histological features. Histological and gene expression analysis indicated that muscle tissues remained inflamed despite treatment. Gene expression analysis also suggested that low levels of the ribokinase enzyme in the skeletal muscle might prevent skeletal muscle tissue from effectively utilizing D-ribose.
Conclusions
Treatment with daily oral doses of D-ribose showed no significant effect on either disease progression or muscle function in the mouse model of myositis.
doi:10.1371/journal.pone.0065970
PMCID: PMC3681851  PMID: 23785461
6.  Idiopathic inflammatory myopathies: pathogenic mechanisms of muscle weakness 
Skeletal Muscle  2013;3:13.
Idiopathic inflammatory myopathies (IIMs) are a heterogenous group of complex muscle diseases of unknown etiology. These diseases are characterized by progressive muscle weakness and damage, together with involvement of other organ systems. It is generally believed that the autoimmune response (autoreactive lymphocytes and autoantibodies) to skeletal muscle-derived antigens is responsible for the muscle fiber damage and muscle weakness in this group of disorders. Therefore, most of the current therapeutic strategies are directed at either suppressing or modifying immune cell activity. Recent studies have indicated that the underlying mechanisms that mediate muscle damage and dysfunction are multiple and complex. Emerging evidence indicates that not only autoimmune responses but also innate immune and non-immune metabolic pathways contribute to disease pathogenesis. However, the relative contributions of each of these mechanisms to disease pathogenesis are currently unknown. Here we discuss some of these complex pathways, their inter-relationships and their relation to muscle damage in myositis. Understanding the relative contributions of each of these pathways to disease pathogenesis would help us to identify suitable drug targets to alleviate muscle damage and also improve muscle weakness and quality of life for patients suffering from these debilitating muscle diseases.
doi:10.1186/2044-5040-3-13
PMCID: PMC3681571  PMID: 23758833
Adaptive immune; Autophagy; Cytokines; Endoplasmic reticulum stress; Innate immune; Myositis; Skeletal muscle; TLRs
7.  Omigapil Treatment Decreases Fibrosis and Improves Respiratory Rate in dy2J Mouse Model of Congenital Muscular Dystrophy 
PLoS ONE  2013;8(6):e65468.
Introduction
Congenital muscular dystrophy is a distinct group of diseases presenting with weakness in infancy or childhood and no current therapy. One form, MDC1A, is the result of laminin alpha-2 deficiency and results in significant weakness, respiratory insufficiency and early death. Modification of apoptosis is one potential pathway for therapy in these patients.
Methods
dy2J mice were treated with vehicle, 0.1 mg/kg or 1 mg/kg of omigapil daily via oral gavage over 17.5 weeks. Untreated age matched BL6 mice were used as controls. Functional, behavioral and histological measurements were collected.
Results
dy2J mice treated with omigapil showed improved respiratory rates compared to vehicle treated dy2J mice (396 to 402 vs. 371 breaths per minute, p<0.03) and similar to control mice. There were no statistical differences in normalized forelimb grip strength between dy2J and controls at baseline or after 17.5 weeks and no significant differences seen among the dy2J treatment groups. At 30–33 weeks of age, dy2J mice treated with 0.1 mg/kg omigapil showed significantly more movement time and less rest time compared to vehicle treated. dy2J mice showed normal cardiac systolic function throughout the trial. dy2J mice had significantly lower hindlimb maximal (p<0.001) and specific force (p<0.002) compared to the control group at the end of the trial. There were no statistically significant differences in maximal or specific force among treatments. dy2J mice treated with 0.1 mg/kg/day omigapil showed decreased percent fibrosis in both gastrocnemius (p<0.03) and diaphragm (p<0.001) compared to vehicle, and in diaphragm (p<0.013) when compared to 1 mg/kg/day omigapil treated mice. Omigapil treated dy2J mice demonstrated decreased apoptosis.
Conclusion
Omigapil therapy (0.1 mg/kg) improved respiratory rate and decreased skeletal and respiratory muscle fibrosis in dy2J mice. These results support a putative role for the use of omigapil in laminin deficient congenital muscular dystrophy patients.
doi:10.1371/journal.pone.0065468
PMCID: PMC3675144  PMID: 23762378
8.  Endoplasmic Reticulum Stress in Skeletal Muscle Homeostasis and Disease 
Current rheumatology reports  2012;14(3):238-243.
Our appreciation of the role of endoplasmic reticulum(ER) stress pathways in both skeletal muscle homeostasis and the progression of muscle diseases is gaining momentum. This review provides insight into ER stress mechanisms during physiologic and pathological disturbances in skeletal muscle. The role of ER stress in the response to dietary alterations and acute stressors, including its role in autoimmune and genetic muscle disorders, has been described. Recent studies identifying ER stress markers in diseased skeletal muscle are noted. The emerging evidence for ER–mitochondrial interplay in skeletal muscle and its importance during chronic ER stress in activation of both inflammatory and cell death pathways (autophagy, necrosis, and apoptosis) have been discussed. Thus, understanding the ER stress–related molecular pathways underlying physiologic and pathological phenotypes in healthy and diseased skeletal muscle should lead to novel therapeutic targets for muscle disease.
doi:10.1007/s11926-012-0247-5
PMCID: PMC3587844  PMID: 22410828
Skeletal muscle; Endoplasmic reticulum; Sarcoplasmic reticulum; Mitochondria; Myositis; ER stress; Autophagy; Necrosis; Apoptosis; Muscle disease
9.  Role of non-immune mechanisms of muscle damage in idiopathic inflammatory myopathies 
Idiopathic inflammatory myopathies (IIMs) comprise a group of autoimmune diseases that are characterized by symmetrical skeletal muscle weakness and muscle inflammation with no known cause. Like other autoimmune diseases, IIMs are treated with either glucocorticoids or immunosuppressive drugs. However, many patients with an IIM are frequently resistant to immunosuppressive treatments, and there is compelling evidence to indicate that not only adaptive immune but also several non-immune mechanisms play a role in the pathogenesis of these disorders. Here, we focus on some of the evidence related to pathologic mechanisms, such as the innate immune response, endoplasmic reticulum stress, non-immune consequences of MHC class I overexpression, metabolic disturbances, and hypoxia. These mechanisms may explain how IIM-related pathologic processes can continue even in the face of immunosuppressive therapies. These data indicate that therapeutic strategies in IIMs should be directed at both immune and non-immune mechanisms of muscle damage.
doi:10.1186/ar3791
PMCID: PMC3446443  PMID: 22546362
10.  Characterization of Dysferlin Deficient SJL/J Mice to Assess Preclinical Drug Efficacy: Fasudil Exacerbates Muscle Disease Phenotype 
PLoS ONE  2010;5(9):e12981.
The dysferlin deficient SJL/J mouse strain is commonly used to study dysferlin deficient myopathies. Therefore, we systematically evaluated behavior in relatively young (9–25 weeks) SJL/J mice and compared them to C57BL6 mice to determine which functional end points may be the most effective to use for preclinical studies in the SJL/J strain. SJL/J mice had reduced body weight, lower open field scores, higher creatine kinase levels, and less muscle force than did C57BL6 mice. Power calculations for expected effect sizes indicated that grip strength normalized to body weight and open field activity were the most sensitive indicators of functional status in SJL/J mice. Weight and open field scores of SJL/J mice deteriorated over the course of the study, indicating that progressive myopathy was ongoing even in relatively young (<6 months old) SJL/J mice. To further characterize SJL/J mice within the context of treatment, we assessed the effect of fasudil, a rho-kinase inhibitor, on disease phenotype. Fasudil was evaluated based on previous observations that Rho signaling may be overly activated as part of the inflammatory cascade in SJL/J mice. Fasudil treated SJL/J mice showed increased body weight, but decreased grip strength, horizontal activity, and soleus muscle force, compared to untreated SJL/J controls. Fasudil either improved or had no effect on these outcomes in C57BL6 mice. Fasudil also reduced the number of infiltrating macrophages/monocytes in SJL/J muscle tissue, but had no effect on muscle fiber degeneration/regeneration. These studies provide a basis for standardization of preclinical drug testing trials in the dysferlin deficient SJL/J mice, and identify measures of functional status that are potentially translatable to clinical trial outcomes. In addition, the data provide pharmacological evidence suggesting that activation of rho-kinase, at least in part, may represent a beneficial compensatory response in dysferlin deficient myopathies.
doi:10.1371/journal.pone.0012981
PMCID: PMC2945315  PMID: 20886045

Results 1-10 (10)