AMP-activated protein kinase signaling regulated expression of urea cycle enzymes in response to changes in dietary protein intake.

Abundance of urea cycle enzymes in the liver is regulated by dietary protein intake. Although urea cycle enzyme levels rise in response to a high-protein (HP) diet, signaling networks that sense dietary protein intake and trigger changes in expression of urea cycle genes have not been identified. The aim of this study was to identify signaling pathway(s) that respond to changes in protein intake and regulate expression of urea cycle genes in mice and human hepatocytes. Mice were adapted to either HP or low-protein diets followed by isolation of liver protein and mRNA and integrated analysis of the proteomic and transcriptomic data. HP diet led to increased expression of mRNA and enzymes in amino acid degradation pathways and decreased expression of mRNA and enzymes in carbohydrate and fat metabolism, which implicated adenosine monophosphate-activated protein kinase (AMPK) as a possible regulator. Primary human hepatocytes, treated with 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) an activator of AMPK, were used to test whether AMPK regulates expression of urea cycle genes. The abundance of carbamoylphosphate synthetase 1 and ornithine transcarbamylase mRNA increased in hepatocytes treated with AICAR, which supports a role for AMPK signaling in regulation of the urea cycle. Because AMPK is either a target of drugs used to treat type-2 diabetes, these drugs might increase the expression of urea cycle enzymes in patients with partial urea cycle disorders, which could be the basis of a new therapeutic approach.

Mitochondria mediate cell membrane repair and contribute to Duchenne muscular dystrophy.

Dystrophin deficiency is the genetic basis for Duchenne muscular dystrophy (DMD), but the cellular basis of progressive myofiber death in DMD is not fully understood. Using two dystrophin-deficient mdx mouse models, we find that the mitochondrial dysfunction is among the earliest cellular deficits of mdx muscles. Mitochondria in dystrophic myofibers also respond poorly to sarcolemmal injury. These mitochondrial deficits reduce the ability of dystrophic muscle cell membranes to repair and are associated with a compensatory increase in dysferlin-mediated membrane repair proteins. Dysferlin deficit in mdx mice further compromises myofiber cell membrane repair and enhances the muscle pathology at an asymptomatic age for dysferlin-deficient mice. Restoring partial dystrophin expression by exon skipping improves mitochondrial function and offers potential to improve myofiber repair. These findings identify that mitochondrial deficit in muscular dystrophy compromises the repair of injured myofibers and show that this repair mechanism is distinct from and complimentary to the dysferlin-mediated repair of injured myofibers.

Effect of the IL-1 Receptor Antagonist Kineret® on Disease Phenotype in mdx Mice.

Duchenne muscular dystrophy (DMD) is an X-linked muscle disease caused by mutations in the dystrophin gene. The pathology of DMD manifests in patients with progressive muscle weakness, loss of ambulation and ultimately death. One of the characteristics of DMD is muscle inflammation, and dystrophin-deficient skeletal muscles produce higher levels of the pro-inflammatory cytokine interleukin 1ß (IL-1ß) in response to toll like receptor (TLR) stimulation compared to controls; therefore, blocking the IL-1ß pathway could improve the disease phenotype in mdx mice, a mouse model of DMD. Kineret® or IL-1Ra is a recombinant IL-1 receptor antagonist approved by the FDA for treating rheumatoid arthritis. To determine the efficacy of IL-1Ra in a DMD model, we administered subcutaneous injections of saline control or IL-1Ra (25 mg/kg/day) to mdx mice daily for 45 days beginning at 5 weeks of age. Functional and histological parameters were measured at the conclusion of the study. IL-1Ra only partially inhibited this signaling pathway in this study; however, there were still interesting observations to be noted. For example, although not significantly changed, splenocytes from the IL-1Ra-treated group secreted less IL-1ß after LPS stimulation compared to control mice indicating a blunted response and incomplete inhibition of the pathway (37% decrease). In addition, normalized forelimb grip strength was significantly increased in IL-1Ra-treated mice. There were no changes in EDL muscle-specific force measurements, histological parameters, or motor coordination assessments in the dystrophic mice after IL-1Ra treatment. There was a significant 27% decrease in the movement time and total distance traveled by the IL-1Ra treated mice, correlating with previous studies examining effects of IL-1 on behavior. Our studies indicate partial blocking of IL-1ß with IL-1Ra significantly altered only a few behavioral and strength related disease parameters; however, treatment with inhibitors that completely block IL-1ß, pathways upstream of IL-1ß production or combining various inhibitors may produce more favorable outcomes.

Muscle myeloid type I interferon gene expression may predict therapeutic responses to rituximab in myositis patients.

OBJECTIVE: To identify muscle gene expression patterns that predict rituximab responses and assess the effects of rituximab on muscle gene expression in PM and DM. METHODS: In an attempt to understand the molecular mechanism of response and non-response to rituximab therapy, we performed Affymetrix gene expression array analyses on muscle biopsy specimens taken before and after rituximab therapy from eight PM and two DM patients in the Rituximab in Myositis study. We also analysed selected muscle-infiltrating cell phenotypes in these biopsies by immunohistochemical staining. Partek and Ingenuity pathway analyses assessed the gene pathways and networks. RESULTS: Myeloid type I IFN signature genes were expressed at higher levels at baseline in the skeletal muscle of rituximab responders than in non-responders, whereas classic non-myeloid IFN signature genes were expressed at higher levels in non-responders at baseline. Also, rituximab responders have a greater reduction of the myeloid and non-myeloid type I IFN signatures than non-responders. The decrease in the type I IFN signature following administration of rituximab may be associated with the decreases in muscle-infiltrating CD19(+) B cells and CD68(+) macrophages in responders. CONCLUSION: Our findings suggest that high levels of myeloid type I IFN gene expression in skeletal muscle predict responses to rituximab in PM/DM and that rituximab responders also have a greater decrease in the expression of these genes. These data add further evidence to recent studies defining the type I IFN signature as both a predictor of therapeutic responses and a biomarker of myositis disease activity.

The use of proteomic analysis to study trafficking defects in axons.

Mutations in microtubule subunits and microtubule-associated proteins are the causes of many neurological disorders. These human conditions are usually associated with axonal tract defects or degeneration. The molecular mechanisms of these axonal dysfunction are still largely unknown. Conventional methods may not yield a complete analysis of downstream molecules related to axonal dysfunctions. Therefore, we devised a simple unbiased method to screen molecular motors and axonal molecules, which might be involved in axonal defects. We performed our analysis in the mouse with a targeted deletion in the doublecortin (Dcx) gene. Dcx is a microtubule-associated protein with direct effects on microtubule motors. Furthermore, the knockout of Dcx and its functionally redundant structurally similar paralog, doublecortin-like kinase 1 (Dclk1), in mouse results in thinner or absent axon tracts, including the corpus callosum and anterior commissures. We compared protein profiles of corpus callosum from Dcx knockout and wild-type mouse of P0-P2 using mass spectrometry. This strategy allowed us to identify novel candidates downstream of Dcx involved in axon transport.

Effect of genetic background on the dystrophic phenotype in mdx mice.

Genetic background significantly affects phenotype in multiple mouse models of human diseases, including muscular dystrophy. This phenotypic variability is partly attributed to genetic modifiers that regulate the disease process. Studies have demonstrated that introduction of the γ-sarcoglycan-null allele onto the DBA/2J background confers a more severe muscular dystrophy phenotype than the original strain, demonstrating the presence of genetic modifier loci in the DBA/2J background. To characterize the phenotype of dystrophin deficiency on the DBA/2J background, we created and phenotyped DBA/2J-congenic Dmdmdx mice (D2-mdx) and compared them with the original, C57BL/10ScSn-Dmdmdx (B10-mdx) model. These strains were compared with their respective control strains at multiple time points between 6 and 52 weeks of age. Skeletal and cardiac muscle function, inflammation, regeneration, histology and biochemistry were characterized. We found that D2-mdx mice showed significantly reduced skeletal muscle function as early as 7 weeks and reduced cardiac function by 28 weeks, suggesting that the disease phenotype is more severe than in B10-mdx mice. In addition, D2-mdx mice showed fewer central myonuclei and increased calcifications in the skeletal muscle, heart and diaphragm at 7 weeks, suggesting that their pathology is different from the B10-mdx mice. The new D2-mdx model with an earlier onset and more pronounced dystrophy phenotype may be useful for evaluating therapies that target cardiac and skeletal muscle function in dystrophin-deficient mice. Our data align the D2-mdx with Duchenne muscular dystrophy patients with the LTBP4 genetic modifier, making it one of the few instances of cross-species genetic modifiers of monogenic traits.

Elusive sources of variability of dystrophin rescue by exon skipping.

BACKGROUND: Systemic delivery of anti-sense oligonucleotides to Duchenne muscular dystrophy (DMD) patients to induce de novo dystrophin protein expression in muscle (exon skipping) is a promising therapy. Treatment with Phosphorodiamidate morpholino oligomers (PMO) lead to shorter de novo dystrophin protein in both animal models and DMD boys who otherwise lack dystrophin; however, restoration of dystrophin has been observed to be highly variable. Understanding the factors causing highly variable induction of dystrophin expression in pre-clinical models would likely lead to more effective means of exon skipping in both pre-clinical studies and human clinical trials. METHODS: In the present study, we investigated possible factors that might lead to the variable success of exon skipping using morpholino drugs in the mdx mouse model. We tested whether specific muscle groups or fiber types showed better success than others and also correlated residual PMO concentration in muscle with the amount of de novo dystrophin protein 1 month after a single high-dose morpholino injection (800 mg/kg). We compared the results from six muscle groups using three different methods of dystrophin quantification: immunostaining, immunoblotting, and mass spectrometry assays. RESULTS: The triceps muscle showed the greatest degree of rescue (average 38±28 % by immunostaining). All three dystrophin detection methods were generally concordant for all muscles. We show that dystrophin rescue occurs in a sporadic patchy pattern with high geographic variability across muscle sections. We did not find a correlation between residual morpholino drug in muscle tissue and the degree of dystrophin expression. CONCLUSIONS: While we found some evidence of muscle group enhancement and successful rescue, our data also suggest that other yet-undefined factors may underlie the observed variability in the success of exon skipping. Our study highlights the challenges associated with quantifying dystrophin in clinical trials where a single small muscle biopsy is taken from a DMD patient.

The use of urinary and kidney SILAM proteomics to monitor kidney response to high dose morpholino oligonucleotides in the mdx mouse.

Phosphorodiamidate morpholino oligonucleotides (PMO) are used as a promising exon-skipping gene therapy for Duchenne Muscular Dystrophy (DMD). One potential complication of high dose PMO therapy is its transient accumulation in the kidneys. Therefore new urinary biomarkers are needed to monitor this treatment. Here, we carried out a pilot proteomic profiling study using stable isotope labeling in mammals (SILAM) strategy to identify new biomarkers to monitor the effect of PMO on the kidneys of the dystrophin deficient mouse model for DMD (mdx-23). We first assessed the baseline renal status of the mdx-23 mouse compared to the wild type (C57BL10) mouse, and then followed the renal outcome of mdx-23 mouse treated with a single high dose intravenous PMO injection (800 mg/kg). Surprisingly, untreated mdx-23 mice showed evidence of renal injury at baseline, which was manifested by albuminuria, increased urine output, and changes in established urinary biomarker of acute kidney injury (AKI). The PMO treatment induced further transient renal injury, which peaked at 7 days, and returned to almost the baseline status at 30 days post-treatment. In the kidney, the SILAM approach followed by western blot validation identified changes in Meprin A subunit alpha at day 2, then returned to normal levels at day 7 and 30 after PMO injection. In the urine, SILAM approach identified an increase in Clusterin and γ-glutamyl transpeptidase 1 as potential candidates to monitor the transient renal accumulation of PMO. These results, which were confirmed by Western blots or ELISA, demonstrate the value of the SILAM approach to identify new candidate biomarkers of renal injury in mdx-23 mice treated with high dose PMO. Chemical compounds studied in this article: Phosphorodiamidate morpholino (PubChem CID: 22140692); isoflurane (PubChem CID: 3763); formic acid (PubChem CID: 284); acetonitrile (PubChem CID: 6342); acetone (PubChem CID: 180); methanol (PubChem CID: 887).

Comparative Risk Assessment of Formulation Changes in Generic Drug Products: A Pharmacology/Toxicology Perspective.

This review highlights general toxicology concerns caused by formulation differences between generic and innovator drugs. It underscores the importance of a scientific, clinically oriented, evidence-based comparative safety evaluation procedure for generic drugs and discusses representative case studies from a pharmacology-toxicology perspective. For consideration by generic drug industry stakeholders, this article provides an overview of comparative risk assessments for generic drug products.

Inhibition of inflammation with celastrol fails to improve muscle function in dysferlin-deficient A/J mice.

The dysferlin-deficient A/J mouse strain represents a homologous model for limb-girdle muscular dystrophy 2B. We evaluated the disease phenotype in 10 month old A/J mice compared to two dysferlin-sufficient, C57BL/6 and A/JOlaHsd, mouse lines to determine which functional end-points are sufficiently sensitive to define the disease phenotype for use in preclinical studies in the A/J strain. A/J mice had significantly lower open field behavioral activity (horizontal activity, total distance, movement time and vertical activity) when compared to C57BL/6 and A/JoIaHsd mice. Both A/J and A/JOIaHsd mice showed decreases in latency to fall with rotarod compared to C57BL/6. No changes were detected in grip strength, force measurements or motor coordination between these three groups. Furthermore, we have found that A/J muscle shows significantly increased levels of the pro-inflammatory cytokine TNF-α when compared to C57BL/6 mice, indicating an activation of NF-κB signaling as part of the inflammatory response in dysferlin-deficient muscle. Therefore, we assessed the effect of celastrol (a potent NF-κB inhibitor) on the disease phenotype in female A/J mice. Celastrol treatment for four months significantly reduced the inflammation in A/J muscle; however, it had no beneficial effect in improving muscle function, as assessed by grip strength, open field activity, and in vitro force contraction. In fact, celastrol treated mice showed a decrease in body mass, hindlimb grip strength and maximal EDL force. These findings suggest that inhibition of inflammation alone may not be sufficient to improve the muscle disease phenotype in dysferlin-deficient mice and may require combination therapies that target membrane stability to achieve a functional improvement in skeletal muscle.

Discovery of serum protein biomarkers in the mdx mouse model and cross-species comparison to Duchenne muscular dystrophy patients.

It is expected that serum protein biomarkers in Duchenne muscular dystrophy (DMD) will reflect disease pathogenesis, progression and aid future therapy developments. Here, we describe use of quantitative in vivo stable isotope labeling in mammals to accurately compare serum proteomes of wild-type and dystrophin-deficient mdx mice. Biomarkers identified in serum from two independent dystrophin-deficient mouse models (mdx-Δ52 and mdx-23) were concordant with those identified in sera samples of DMD patients. Of the 355 mouse sera proteins, 23 were significantly elevated and 4 significantly lower in mdx relative to wild-type mice (P-value < 0.001). Elevated proteins were mostly of muscle origin: including myofibrillar proteins (titin, myosin light chain 1/3, myomesin 3 and filamin-C), glycolytic enzymes (aldolase, phosphoglycerate mutase 2, beta enolase and glycogen phosphorylase), transport proteins (fatty acid-binding protein, myoglobin and somatic cytochrome-C) and others (creatine kinase M, malate dehydrogenase cytosolic, fibrinogen and parvalbumin). Decreased proteins, mostly of extracellular origin, included adiponectin, lumican, plasminogen and leukemia inhibitory factor receptor. Analysis of sera from 1 week to 7 months old mdx mice revealed age-dependent changes in the level of these biomarkers with most biomarkers acutely elevated at 3 weeks of age. Serum analysis of DMD patients, with ages ranging from 4 to 15 years old, confirmed elevation of 20 of the murine biomarkers in DMD, with similar age-related changes. This study provides a panel of biomarkers that reflect muscle activity and pathogenesis and should prove valuable tool to complement natural history studies and to monitor treatment efficacy in future clinical trials.

Role of Toll-like receptors in the pathogenesis of dystrophin-deficient skeletal and heart muscle.

Although the cause of Duchenne muscular dystrophy (DMD) is known, the specific factors that initiate and perpetuate disease progression are not well understood. We hypothesized that leaky dystrophin-deficient skeletal muscle releases endogenous danger signals (TLR ligands), which bind to Toll-like receptors (TLRs) on muscle and immune cells and activate downstream processes that facilitate degeneration and regeneration in dystrophic skeletal muscle. Here, we demonstrate that dystrophin-deficient mouse muscle cells show increased expression of several cell-surface and endosomal TLRs. In vitro screening identified ssRNA as a relevant endogenous TLR7 ligand. TLR7 activation led to myd88-dependent production of pro-inflammatory cytokines in dystrophin-deficient muscle cells, and cause significant degeneration/regeneration in vivo in mdx mouse muscle. Also, knockout of the central TLR adaptor protein, myd88 in mdx mice significantly improved skeletal and cardiac muscle function. Likewise, proof-of-concept experiments showed that treating young mdx mice with a TLR7/9 antagonist significantly reduced skeletal muscle inflammation and increased muscle force, suggesting that blocking this pathway may have therapeutic potential for DMD.

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

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.

The effects of MyD88 deficiency on disease phenotype in dysferlin-deficient A/J mice: role of endogenous TLR ligands.

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.

Idiopathic inflammatory myopathies: pathogenic mechanisms of muscle weakness.

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.

Daily supplementation of D-ribose shows no therapeutic benefits in the MHC-I transgenic mouse model of inflammatory myositis.

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.

Omigapil treatment decreases fibrosis and improves respiratory rate in dy(2J) mouse model of congenital muscular dystrophy.

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: dy(2J) 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: dy(2J) mice treated with omigapil showed improved respiratory rates compared to vehicle treated dy(2J) 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 dy(2J) and controls at baseline or after 17.5 weeks and no significant differences seen among the dy(2J) treatment groups. At 30-33 weeks of age, dy(2J) mice treated with 0.1 mg/kg omigapil showed significantly more movement time and less rest time compared to vehicle treated. dy(2J) mice showed normal cardiac systolic function throughout the trial. dy(2J) 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. dy(2J) 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 dy(2J) mice demonstrated decreased apoptosis. CONCLUSION: Omigapil therapy (0.1 mg/kg) improved respiratory rate and decreased skeletal and respiratory muscle fibrosis in dy(2J) mice. These results support a putative role for the use of omigapil in laminin deficient congenital muscular dystrophy patients.

Identification of disease specific pathways using in vivo SILAC proteomics in dystrophin deficient mdx mouse.

Duchenne muscular dystrophy (DMD) is an X-linked neuromuscular disorder caused by a mutation in the dystrophin gene. DMD is characterized by progressive weakness of skeletal, cardiac, and respiratory muscles. The molecular mechanisms underlying dystrophy-associated muscle weakness and damage are not well understood. Quantitative proteomics techniques could help to identify disease-specific pathways. Recent advances in the in vivo labeling strategies such as stable isotope labeling in mouse (SILAC mouse) with (13)C6-lysine or stable isotope labeling in mammals (SILAM) with (15)N have enabled accurate quantitative analysis of the proteomes of whole organs and tissues as a function of disease. Here we describe the use of the SILAC mouse strategy to define the underlying pathological mechanisms in dystrophin-deficient skeletal muscle. Differential SILAC proteome profiling was performed on the gastrocnemius muscles of 3-week-old (early stage) dystrophin-deficient mdx mice and wild-type (normal) mice. The generated data were further confirmed in an independent set of mdx and normal mice using a SILAC spike-in strategy. A total of 789 proteins were quantified; of these, 73 were found to be significantly altered between mdx and normal mice (p < 0.05). Bioinformatics analyses using Ingenuity Pathway software established that the integrin-linked kinase pathway, actin cytoskeleton signaling, mitochondrial energy metabolism, and calcium homeostasis are the pathways initially affected in dystrophin-deficient muscle at early stages of pathogenesis. The key proteins involved in these pathways were validated by means of immunoblotting and immunohistochemistry in independent sets of mdx mice and in human DMD muscle biopsies. The specific involvement of these molecular networks early in dystrophic pathology makes them potential therapeutic targets. In sum, our findings indicate that SILAC mouse strategy has uncovered previously unidentified pathological pathways in mouse models of human skeletal muscle disease.

Use of quantitative membrane proteomics identifies a novel role of mitochondria in healing injured muscles.

Skeletal muscles are proficient at healing from a variety of injuries. Healing occurs in two phases, early and late phase. Early phase involves healing the injured sarcolemma and restricting the spread of damage to the injured myofiber. Late phase of healing occurs a few days postinjury and involves interaction of injured myofibers with regenerative and inflammatory cells. Of the two phases, cellular and molecular processes involved in the early phase of healing are poorly understood. We have implemented an improved sarcolemmal proteomics approach together with in vivo labeling of proteins with modified amino acids in mice to study acute changes in the sarcolemmal proteome in early phase of myofiber injury. We find that a notable early phase response to muscle injury is an increased association of mitochondria with the injured sarcolemma. Real-time imaging of live myofibers during injury demonstrated that the increased association of mitochondria with the injured sarcolemma involves translocation of mitochondria to the site of injury, a response that is lacking in cultured myoblasts. Inhibiting mitochondrial function at the time of injury inhibited healing of the injured myofibers. This identifies a novel role of mitochondria in the early phase of healing injured myofibers.

The molecular basis of skeletal muscle weakness in a mouse model of inflammatory myopathy.

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 these patients. This study was undertaken to examine whether nonimmune mechanisms also contribute to muscle weakness. In particular, it has been suggested that an acquired deficiency of AMP deaminase 1 (AMPD1) may be responsible for muscle weakness in myositis. METHODS: We performed comprehensive functional, behavioral, histologic, molecular, enzymatic, and metabolic assessments before and after the onset of inflammation in a class I major histocompatibility complex (MHC)-transgenic mouse model of autoimmune inflammatory myositis. RESULTS: 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 AMPD1 expression and was myositis specific. Decreasing AMPD1 expression resulted in decreased muscle strength in healthy mice. Fiber typing suggested that fast-twitch muscles were converted to slow-twitch muscles as myositis progressed, and microarray results indicated that AMPD1 and other purine nucleotide pathway genes were 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.