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.

Δ-9,11 modification of glucocorticoids dissociates nuclear factor-κB inhibitory efficacy from glucocorticoid response element-associated side effects.

Glucocorticoids are standard of care for many inflammatory conditions, but chronic use is associated with a broad array of side effects. This has led to a search for dissociative glucocorticoids--drugs able to retain or improve efficacy associated with transrepression [nuclear factor-κB (NF-κB) inhibition] but with the loss of side effects associated with transactivation (receptor-mediated transcriptional activation through glucocorticoid response element gene promoter elements). We investigated a glucocorticoid derivative with a Δ-9,11 modification as a dissociative steroid. The Δ-9,11 analog showed potent inhibition of tumor necrosis factor-α-induced NF-κB signaling in cell reporter assays, and this transrepression activity was blocked by 17ß-hydroxy-11ß-[4-dimethylamino phenyl]-17α-[1-propynyl]estra-4,9-dien-3-one (RU-486), showing the requirement for the glucocorticoid receptor (GR). The Δ-9,11 analog induced the nuclear translocation of GR but showed the loss of transactivation as assayed by GR-luciferase constructs as well as mRNA profiles of treated cells. The Δ-9,11 analog was tested for efficacy and side effects in two mouse models of muscular dystrophy: mdx (dystrophin deficiency), and SJL (dysferlin deficiency). Daily oral delivery of the Δ-9,11 analog showed a reduction of muscle inflammation and improvements in multiple muscle function assays yet no reductions in body weight or spleen size, suggesting the loss of key side effects. Our data demonstrate that a Δ-9,11 analog dissociates the GR-mediated transcriptional activities from anti-inflammatory activities. Accordingly, Δ-9,11 analogs may hold promise as a source of safer therapeutic agents for chronic inflammatory disorders.

One-year treatment of morpholino antisense oligomer improves skeletal and cardiac muscle functions in dystrophic mdx mice.

Antisense therapy has been successful to skip targeted dystrophin exon with correction of frameshift and nonsense mutations of Duchenne muscular dystrophy (DMD). Systemic production of truncated but functional dystrophin proteins has been achieved in animal models. Furthermore, phase I/II clinical trials in United Kingdom and the Netherlands have demonstrated dystrophin induction by local and systemic administrations of antisense oligomers. However, long-term efficacy and potential toxicity remain to be determined. The present study examined 1-year systemic effect of phosphorodiamidate morpholino oligomers (PMO) treatment targeting mutated dystrophin exon 23 in mdx mice. PMO induced dystrophin expression dose-dependently and significantly improved skeletal muscle pathology and function with reduced creatine kinase (CK) levels by a regimen of 60 mg/kg biweekly administration. This regimen induced <2% dystrophin expression in the heart, but improved cardiac functions demonstrated by hemodynamics analysis. The results suggest that low levels of dystrophin induction may be able to provide detectable benefit to cardiac muscle with limited myopathy. Body weight, serum enzyme tests, and histology analysis showed no sign of toxicity in the mice treated with up to 1.5 g/kg PMO for 6 months. These results indicate that PMO could be used safely as effective drugs for long-term systemic treatment of DMD.

IL-6 signaling blockade increases inflammation but does not affect muscle function in the mdx mouse.

BACKGROUND: IL-6 is a pleiotropic cytokine that modulates inflammatory responses and plays critical roles in muscle maintenance and remodeling. In the mouse model (mdx) of Duchenne Muscular Dystrophy, IL-6 and muscle inflammation are elevated, which is believed to contribute to the chronic inflammation and failure of muscle regeneration in DMD. The purpose of the current study was to examine the effect of blocking IL-6 signaling on the muscle phenotype including muscle weakness and pathology in the mdx mouse. METHODS: A monoclonal antibody against the IL-6 receptor (IL-6r mAb) that blocks local and systemic IL-6 signaling was administered to mdx and BL-10 mice for 5 weeks and muscle function, histology, and inflammation were examined. RESULTS: IL-6r mAb treatment increased mdx muscle inflammation including total inflammation score and ICAM-1 positive lumens in muscles. There was no significant improvement in muscle strength nor muscle pathology due to IL-6r mAb treatment in mdx mice. CONCLUSIONS: These results showed that instead of reducing inflammation, IL-6 signaling blockade for 5 weeks caused an increase in muscle inflammation, with no significant change in indices related to muscle regeneration and muscle function. The results suggest a potential anti-inflammatory instead of the original hypothesized pro-inflammatory role of IL-6 signaling in the mdx mice.

Membrane sealant Poloxamer P188 protects against isoproterenol induced cardiomyopathy in dystrophin deficient mice.

BACKGROUND: Cardiomyopathy in Duchenne muscular dystrophy (DMD) is an increasing cause of death in patients. The absence of dystrophin leads to loss of membrane integrity, cell death and fibrosis in cardiac muscle. Treatment of cardiomyocyte membrane instability could help prevent cardiomyopathy. METHODS: Three month old female mdx mice were exposed to the ß(1) receptor agonist isoproterenol subcutaneously and treated with the non-ionic tri-block copolymer Poloxamer P188 (P188) (460 mg/kg/dose i.p. daily). Cardiac function was assessed using high frequency echocardiography. Tissue was evaluated with Evans Blue Dye (EBD) and picrosirius red staining. RESULTS: BL10 control mice tolerated 30 mg/kg/day of isoproterenol for 4 weeks while death occurred in mdx mice at 30, 15, 10, 5 and 1 mg/kg/day within 24 hours. Mdx mice tolerated a low dose of 0.5 mg/kg/day. Isoproterenol exposed mdx mice showed significantly increased heart rates (p < 0.02) and cardiac fibrosis (p < 0.01) over 4 weeks compared to unexposed controls. P188 treatment of mdx mice significantly increased heart rate (median 593 vs. 667 bpm; p < 0.001) after 2 weeks and prevented a decrease in cardiac function in isoproterenol exposed mice (Shortening Fraction = 46 ± 6% vs. 35 ± 6%; p = 0.007) after 4 weeks. P188 treated mdx mice did not show significant differences in cardiac fibrosis, but demonstrated significantly increased EBD positive fibers. CONCLUSIONS: This model suggests that chronic intermittent intraperitoneal P188 treatment can prevent isoproterenol induced cardiomyopathy in dystrophin deficient mdx mice.

Effective rescue of dystrophin improves cardiac function in dystrophin-deficient mice by a modified morpholino oligomer.

Antisense oligonucleotide-mediated exon skipping is able to correct out-of-frame mutations in Duchenne muscular dystrophy and restore truncated yet functional dystrophins. However, its application is limited by low potency and inefficiency in systemic delivery, especially failure to restore dystrophin in heart. Here, we conjugate a phosphorodiamidate morpholino oligomer with a designed cell-penetrating peptide (PPMO) targeting a mutated dystrophin exon. Systemic delivery of the novel PPMO restores dystrophin to almost normal levels in the cardiac and skeletal muscles in dystrophic mdx mouse. This leads to increase in muscle strength and prevents cardiac pump failure induced by dobutamine stress in vivo. Muscle pathology and function continue to improve during the 12-week course of biweekly treatment, with significant reduction in levels of serum creatine kinase. The high degree of potency of the oligomer in targeting all muscles and the lack of detectable toxicity and immune response support the feasibility of testing the novel oligomer in treating Duchenne muscular dystrophy patients.

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.

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.

The proton pump inhibitor lansoprazole improves the skeletal phenotype in dystrophin deficient mdx mice.

BACKGROUND: In Duchenne muscular dystrophy (DMD), loss of the membrane stabilizing protein dystrophin results in myofiber damage. Microinjury to dystrophic myofibers also causes secondary imbalances in sarcolemmic ion permeability and resting membrane potential, which modifies excitation-contraction coupling and increases proinflammatory/apoptotic signaling cascades. Although glucocorticoids remain the standard of care for the treatment of DMD, there is a need to investigate the efficacy of other pharmacological agents targeting the involvement of imbalances in ion flux on dystrophic pathology. METHODOLOGY/PRINCIPAL FINDINGS: We designed a preclinical trial to investigate the effects of lansoprazole (LANZO) administration, a proton pump inhibitor, on the dystrophic muscle phenotype in dystrophin deficient (mdx) mice. Eight to ten week-old female mice were assigned to one of four treatment groups (n = 12 per group): (1) vehicle control; (2) 5 mg/kg/day LANZO; (3) 5 mg/kg/day prednisolone; and (4) combined treatment of 5 mg/kg/day prednisolone (PRED) and 5 mg/kg/day LANZO. Treatment was administered orally 5 d/wk for 3 months. At the end of the study, behavioral (Digiscan) and functional outcomes (grip strength and Rotarod) were assessed prior to sacrifice. After sacrifice, body, tissue and organ masses, muscle histology, in vitro muscle force, and creatine kinase levels were measured. Mice in the combined treatment groups displayed significant reductions in the number of degenerating muscle fibers and number of inflammatory foci per muscle field relative to vehicle control. Additionally, mice in the combined treatment group displayed less of a decline in normalized forelimb and hindlimb grip strength and declines in in vitro EDL force after repeated eccentric contractions. CONCLUSIONS/SIGNIFICANCE: Together our findings suggest that combined treatment of LANZO and prednisolone attenuates some components of dystrophic pathology in mdx mice. Our findings warrant future investigation of the clinical efficacy of LANZO and prednisolone combined treatment regimens in dystrophic pathology.

VBP15, a novel anti-inflammatory and membrane-stabilizer, improves muscular dystrophy without side effects.

Absence of dystrophin makes skeletal muscle more susceptible to injury, resulting in breaches of the plasma membrane and chronic inflammation in Duchenne muscular dystrophy (DMD). Current management by glucocorticoids has unclear molecular benefits and harsh side effects. It is uncertain whether therapies that avoid hormonal stunting of growth and development, and/or immunosuppression, would be more or less beneficial. Here, we discover an oral drug with mechanisms that provide efficacy through anti-inflammatory signaling and membrane-stabilizing pathways, independent of hormonal or immunosuppressive effects. We find VBP15 protects and promotes efficient repair of skeletal muscle cells upon laser injury, in opposition to prednisolone. Potent inhibition of NF-κB is mediated through protein interactions of the glucocorticoid receptor, however VBP15 shows significantly reduced hormonal receptor transcriptional activity. The translation of these drug mechanisms into DMD model mice improves muscle strength, live-imaging and pathology through both preventive and post-onset intervention regimens. These data demonstrate successful improvement of dystrophy independent of hormonal, growth, or immunosuppressive effects, indicating VBP15 merits clinical investigation for DMD and would benefit other chronic inflammatory diseases.

Glucocorticoid-treated mice are an inappropriate positive control for long-term preclinical studies in the mdx mouse.

BACKGROUND: Dmd(mdx) (mdx) mice are used as a genetic and biochemical model of dystrophin deficiency. The long-term consequences of glucocorticoid (GC) treatment on dystrophin-deficient skeletal and heart muscle are not yet known. Here we used systematic phenotyping to assess the long-term consequences of GC treatment in mdx mice. Our investigation addressed not only the effects of GC on the disease phenotype but also the question of whether GCs can be used as a positive control for preclinical drug evaluations. METHODS AND FINDINGS: We performed nine pre-clinical efficacy trials (treated N = 129, untreated N = 106) of different durations in 9-to-50-week-old dystrophic mdx mice over a 3-year time period using standardized methods. In all these trials, we used either 1 mg/kg body weight of prednisone or 5 mg/kg body weight of prednisolone as positive controls to compare the efficacy of various test drugs. Data from untreated controls and GC-treated mice in the various trials have been pooled and analyzed to assess the effects of GCs on dystrophin-deficient skeletal and cardiac muscles of mdx mice. Our results indicate that continuous GC treatment results in early (e.g., at 50 days) improvements in normalized parameters such as grip strength, motor coordination and maximal in vitro force contractions on isolated EDL muscle, but these initial benefits are followed by a progressive loss of muscle strength after 100 days. We also found a significant increase in heart fibrosis that is reflected in a significant deterioration in cardiac systolic function after 100 days of treatment. CONCLUSION: Continuous administration of prednisone to mdx mice initially improves skeletal muscle strength, but further therapy result in deterioration of muscle strength and cardiac function associated with enhanced cardiac fibrosis. These results suggest that GCs may not serve as an appropriate positive control for long-term mdx mouse preclinical trials.

Non-invasive optical imaging of muscle pathology in mdx mice using cathepsin caged near-infrared imaging.

PURPOSE: To develop a reliable live-animal imaging method for monitoring muscle pathology in mouse models of myopathy. PROCEDURES: A caged near-infrared Cathepsin B (CTSB) substrate, ProSense 680, is evaluated in the dystrophin deficient mdx mice, a genetic homologue of Duchenne muscular dystrophy via optical imaging. RESULTS: We show high levels of infrared signal in dystrophic muscle relative to healthy muscle at 24 h post-injection. Imaging for CTSB presence revealed localization to inflammatory infiltrates and regenerating muscle fibers. A time series myotoxin-induced muscle injury experiment showed that CTSB activity and its mRNA levels peaked at the interface between inflammation and myoblast fusion stage of recovery. Prednisone treatment in mdx mice resulted in decreased CTSB activity and increased grip strength in forelimbs and hindlimbs. CONCLUSIONS: Optical imaging of CTSB activity is an ideal method to sensitively monitor inflammation, regeneration, and response to therapy in myopathic skeletal muscle.

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-Dmd(mdx)/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.

Evaluation of skeletal and cardiac muscle function after chronic administration of thymosin beta-4 in the dystrophin deficient mouse.

Thymosin beta-4 (Tbeta4) is a ubiquitous protein with many properties relating to cell proliferation and differentiation that promotes wound healing and modulates inflammatory mediators. We studied the effects of chronic administration of Tbeta4 on the skeletal and cardiac muscle of dystrophin deficient mdx mice, the mouse model of Duchenne muscular dystrophy. Female wild type (C57BL10/ScSnJ) and mdx mice, 8-10 weeks old, were treated with 150 microg of Tbeta4 twice a week for 6 months. To promote muscle pathology, mice were exercised for 30 minutes twice a week. Skeletal and cardiac muscle function were assessed via grip strength and high frequency echocardiography. Localization of Tbeta4 and amount of fibrosis were quantified using immunohistochemistry and Gomori's tri-chrome staining, respectively. Mdx mice treated with Tbeta4 showed a significant increase in skeletal muscle regenerating fibers compared to untreated mdx mice. Tbeta4 stained exclusively in the regenerating fibers of mdx mice. Although untreated mdx mice had significantly decreased skeletal muscle strength compared to untreated wild type, there were no significant improvements in mdx mice after treatment. Systolic cardiac function, measured as percent shortening fraction, was decreased in untreated mdx mice compared to untreated wild type and there was no significant difference after treatment in mdx mice. Skeletal and cardiac muscle fibrosis were also significantly increased in untreated mdx mice compared to wild type, but there was no significant improvement in treated mdx mice. In exercised dystrophin deficient mice, chronic administration of Tbeta4 increased the number of regenerating fibers in skeletal muscle and could have a potential role in treatment of skeletal muscle disease in Duchenne muscular dystrophy.

Functional and molecular effects of arginine butyrate and prednisone on muscle and heart in the mdx mouse model of Duchenne Muscular Dystrophy.

BACKGROUND: The number of promising therapeutic interventions for Duchenne Muscular Dystrophy (DMD) is increasing rapidly. One of the proposed strategies is to use drugs that are known to act by multiple different mechanisms including inducing of homologous fetal form of adult genes, for example utrophin in place of dystrophin. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we have treated mdx mice with arginine butyrate, prednisone, or a combination of arginine butyrate and prednisone for 6 months, beginning at 3 months of age, and have comprehensively evaluated the functional, biochemical, histological, and molecular effects of the treatments in this DMD model. Arginine butyrate treatment improved grip strength and decreased fibrosis in the gastrocnemius muscle, but did not produce significant improvement in muscle and cardiac histology, heart function, behavioral measurements, or serum creatine kinase levels. In contrast, 6 months of chronic continuous prednisone treatment resulted in deterioration in functional, histological, and biochemical measures. Arginine butyrate-treated mice gene expression profiling experiments revealed that several genes that control cell proliferation, growth and differentiation are differentially expressed consistent with its histone deacetylase inhibitory activity when compared to control (saline-treated) mdx mice. Prednisone and combination treated groups showed alterations in the expression of genes that control fibrosis, inflammation, myogenesis and atrophy. CONCLUSIONS/SIGNIFICANCE: These data indicate that 6 months treatment with arginine butyrate can produce modest beneficial effects on dystrophic pathology in mdx mice by reducing fibrosis and promoting muscle function while chronic continuous treatment with prednisone showed deleterious effects to skeletal and cardiac muscle. Our results clearly indicate the usefulness of multiple assays systems to monitor both beneficial and toxic effects of drugs with broad range of in vivo activity.

Preclinical drug trials in the mdx mouse: assessment of reliable and sensitive outcome measures.

The availability of animal models for Duchenne muscular dystrophy has led to extensive preclinical research on potential therapeutics. Few studies have focused on reliability and sensitivity of endpoints for mdx mouse drug trials. Therefore, we sought to compare a wide variety of reported and novel endpoint measures in exercised mdx and normal control mice at 10, 20, and 40 weeks of age. Statistical analysis as well as power calculations for expected effect sizes in mdx preclinical drug trials across different ages showed that body weight, normalized grip strength, horizontal activity, rest time, cardiac function measurements, blood pressure, total central/peripheral nuclei per fiber, and serum creatine kinase are the most effective measurements for detecting drug-induced changes. These data provide an experimental basis upon which standardization of preclinical drug testing can be developed. Muscle Nerve, 2008.