Dystrophin myonuclear domain restoration governs treatment efficacy in dystrophic muscle.

Dystrophin is essential for muscle health: its sarcolemmal absence causes the fatal, X-linked condition, Duchenne muscular dystrophy (DMD). However, its normal, spatial organization remains poorly understood, which hinders the interpretation of efficacy of its therapeutic restoration. Using female reporter mice heterozygous for fluorescently tagged dystrophin (DmdEGFP), we here reveal that dystrophin distribution is unexpectedly compartmentalized, being restricted to myonuclear-defined sarcolemmal territories extending ~80 µm, which we called "basal sarcolemmal dystrophin units (BSDUs)." These territories were further specialized at myotendinous junctions, where both Dmd transcripts and dystrophin protein were enriched. Genome-level correction in X-linked muscular dystrophy mice via CRISPR/Cas9 gene editing restored a mosaic of separated dystrophin domains, whereas transcript-level Dmd correction, following treatment with tricyclo-DNA antisense oligonucleotides, restored dystrophin initially at junctions before extending along the entire fiber-with levels ~2% sufficient to moderate the dystrophic process. We conclude that widespread restoration of fiber dystrophin is likely critical for therapeutic success in DMD, perhaps most importantly, at muscle-tendon junctions.

Trilobatin contributes to the improvement of myopathy in a mouse model of Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) occurs due to genetic mutations that lead to a deficiency in dystrophin production and consequent progressive degeneration of skeletal muscle fibres, through oxidative stress and an exacerbated inflammatory process. The flavonoid trilobatin (TLB) demonstrates antioxidant and anti-inflammatory potential. Its high safety profile and effective action make it a potent therapy for the process of dystrophic muscle myonecrosis. Thus, we sought to investigate the action of TLB on damage in a DMD model, the mdx mouse. Eight-week-old male animals were treated with 160 mg/kg/day of trilobatin for 8 weeks. Control animals were treated with saline. Following treatment, muscle strength, serum creatine kinase (CK) levels, histopathology (necrotic myofibres, regenerated fibres/central nuclei, Feret's diameter and inflammatory area) and the levels of catalase and NF-κB (western blotting) of the quadriceps (QUA), diaphragm (DIA) and tibialis anterior (TA) muscles were measured. TLB was able to significantly increase muscle strength and reduce serum CK levels in dystrophic animals. The QUA of mdx mice showed a reduction in catalase and the number of fibres with a centralized nucleus after treatment with TLB. In the DIA of dystrophic animals, TLB reduced the necrotic myofibres, inflammatory area and NF-κB and increased the number of regenerated fibres and the total fibre diameter. In TA, TLB increased the number of regenerated fibres and reduced catalase levels in these animals. It is concluded that in the mdx experimental model, treatment with TLB was beneficial in the treatment of DMD.

Sarcoplasmic reticulum calcium handling in unbranched, immediately post-necrotic fast-twitch mdx fibres is similar to wild-type littermates.

NEW FINDINGS: What is the central question of this study? What are the early effects of dystrophin deficiency on sarcoplasmic reticulum Ca2+ handling in the mdx mouse? What is the main finding and its importance? In the mdx mouse, Ca2+ handling by the sarcoplasmic reticulum is little affected by the absence of dystrophin when looking at fibres without branches that have recently regenerated after massive myonecrosis. This has important implications for our understanding of Ca2+ pathology in the mdx mouse. ABSTRACT: There is a variety of results in the literature regarding the effects of dystrophin deficiency on the Ca2+ handling properties of the sarcoplasmic reticulum (SR) in the mdx mouse, an animal model of Duchenne muscular dystrophy. One possible source of variation is the presence of branched fibres. Fibre branching, a consequence of degenerative-regenerative processes such as muscular dystrophy, has in itself a significant influence on the function of the SR. In this study, we attempted to detect early effects of dystrophin deficiency on SR Ca2+ handling by using unbranched fibres from the immediate post-necrotic stage in mdx mice (recently regenerated after massive necrosis). Using kinetically corrected fura-2 fluorescence signals measured during twitch and tetanus, we analysed the amplitude, rise time and decay time of Δ[Ca2+ ]i in unfatigued and fatigued fibres. Decay was also resolved into SR pump and SR leak components. Fibres from mdx mice were similar in all respects to fibres from wild-type littermates apart from: (1) a smaller amplitude of the initial spike of Δ[Ca2+ ]i during a tetanus; and (2) a mitigation of the fall in Δ[Ca2+ ]i amplitude during the course of fatigue. Our findings suggest that the early effects of a loss of dystrophin on SR Ca2+ handling in mdx mice are subtle, and we emphasize the importance of distinguishing between Ca2+ pathology that is attributable to lack of dystrophin and Ca2+ pathology that is attributable to muscle degeneration.

Fiber optic Raman spectroscopy for the evaluation of disease state in Duchenne muscular dystrophy: An assessment using the mdx model and human muscle.

INTRODUCTION/AIMS: Raman spectroscopy is an emerging technique for the evaluation of muscle disease. In this study we evaluate the ability of in vivo intramuscular Raman spectroscopy to detect the effects of voluntary running in the mdx model of Duchenne muscular dystrophy (DMD). We also compare mdx data with muscle spectra from human DMD patients. METHODS: Thirty 90-day-old mdx mice were randomly allocated to an exercised group (48-hour access to a running wheel) and an unexercised group (n = 15 per group). In vivo Raman spectra were collected from both gastrocnemius muscles and histopathological assessment subsequently performed. Raman data were analyzed using principal component analysis-fed linear discriminant analysis (PCA-LDA). Exercised and unexercised mdx muscle spectra were compared with human DMD samples using cosine similarity. RESULTS: Exercised mice ran an average of 6.5 km over 48 hours, which induced a significant increase in muscle necrosis (P = .03). PCA-LDA scores were significantly different between the exercised and unexercised groups (P < .0001) and correlated significantly with distance run (P = .01). Raman spectra from exercised mice more closely resembled human spectra than those from unexercised mice. DISCUSSION: Raman spectroscopy provides a readout of the biochemical alterations in muscle in both the mdx mouse and human DMD muscle.

A New Method of Myostatin Inhibition in Mice via Oral Administration of Lactobacillus casei Expressing Modified Myostatin Protein, BLS-M22.

Myostatin is a member of the transforming growth factor-beta superfamily and is an endogenous negative regulator of muscle growth. This study aimed to determine whether an oral administration of Lactobacillus casei expressing modified human myostatin (BLS-M22) could elicit sufficient levels of myostatin-specific antibody and improve the dystrophic features of an animal model of Duchenne muscular dystrophy (DMD; mdx mouse). BLS-M22 is a recombinant L. casei engineered to harbor the pKV vector and poly-gamma-glutamic acid gene linked to a modified human myostatin gene. Serological analysis showed that anti-myostatin IgG titers were significantly increased, and serum creatine kinase was significantly reduced in the BLS-M22-treated mdx mice compared to the control mice. In addition, treatment of BLS-M22 resulted in a significant increase in body weight and motor function (Rotarod behavior test). Histological analysis showed an improvement in the dystrophic features (fibrosis and muscle hypertrophy) of the mdx mice with the administration of BLS-M22. The circulating antibodies generated after BLS-M22 oral administration successfully lowered serum myostatin concentration. Myostatin blockade resulted in serological, histological, and functional improvements in mdx mice. Overall, the findings suggest the potential of BLS-M22 to treat DMD; however, further clinical trials are essential to ascertain its efficacy and safety in humans.

Striated muscle activator of Rho signalling (STARS) overexpression in the mdx mouse enhances muscle functional capacity and regulates the actin cytoskeleton and oxidative phosphorylation pathways.

NEW FINDINGS: What is the central question of this study? Striated muscle activator of rho signalling (STARS) is an actin-binding protein that regulates transcriptional pathways controlling muscle function, growth and myogenesis, processes that are impaired in dystrophic muscle: what is the regulation of the STARS pathway in Duchenne muscular dystrophy (DMD)? What is the main finding and its importance? Members of the STARS signalling pathway are reduced in the quadriceps of patients with DMD and in mouse models of muscular dystrophy. Overexpression of STARS in the dystrophic deficient mdx mouse model increased maximal isometric specific force and upregulated members of the actin cytoskeleton and oxidative phosphorylation pathways. Regulating STARS may be a therapeutic approach to enhance muscle health. ABSTRACT: Duchenne muscular dystrophy (DMD) is characterised by impaired cytoskeleton organisation, cytosolic calcium handling, oxidative stress and mitochondrial dysfunction. This results in progressive muscle damage, wasting and weakness and premature death. The striated muscle activator of rho signalling (STARS) is an actin-binding protein that activates the myocardin-related transcription factor-A (MRTFA)/serum response factor (SRF) transcriptional pathway, a pathway regulating cytoskeletal structure and muscle function, growth and repair. We investigated the regulation of the STARS pathway in the quadriceps muscle from patients with DMD and in the tibialis anterior (TA) muscle from the dystrophin-deficient mdx and dko (utrophin and dystrophin null) mice. Protein levels of STARS, SRF and RHOA were reduced in patients with DMD. STARS, SRF and MRTFA mRNA levels were also decreased in DMD muscle, while Stars mRNA levels were decreased in the mdx mice and Srf and Mrtfa mRNAs decreased in the dko mice. Overexpressing human STARS (hSTARS) in the TA muscles of mdx mice increased maximal isometric specific force by 13% (P < 0.05). This was not associated with changes in muscle mass, fibre cross-sectional area, fibre type, centralised nuclei or collagen deposition. Proteomics screening followed by pathway enrichment analysis identified that hSTARS overexpression resulted in 31 upregulated and 22 downregulated proteins belonging to the actin cytoskeleton and oxidative phosphorylation pathways. These pathways are impaired in dystrophic muscle and regulate processes that are vital for muscle function. Increasing the STARS protein in dystrophic muscle improves muscle force production, potentially via synergistic regulation of cytoskeletal structure and energy production.

Dystrophin Deficiency Causes Progressive Depletion of Cardiovascular Progenitor Cells in the Heart.

Duchenne muscular dystrophy (DMD) is a devastating condition shortening the lifespan of young men. DMD patients suffer from age-related dilated cardiomyopathy (DCM) that leads to heart failure. Several molecular mechanisms leading to cardiomyocyte death in DMD have been described. However, the pathological progression of DMD-associated DCM remains unclear. In skeletal muscle, a dramatic decrease in stem cells, so-called satellite cells, has been shown in DMD patients. Whether similar dysfunction occurs with cardiac muscle cardiovascular progenitor cells (CVPCs) in DMD remains to be explored. We hypothesized that the number of CVPCs decreases in the dystrophin-deficient heart with age and disease state, contributing to DCM progression. We used the dystrophin-deficient mouse model (mdx) to investigate age-dependent CVPC properties. Using quantitative PCR, flow cytometry, speckle tracking echocardiography, and immunofluorescence, we revealed that young mdx mice exhibit elevated CVPCs. We observed a rapid age-related CVPC depletion, coinciding with the progressive onset of cardiac dysfunction. Moreover, mdx CVPCs displayed increased DNA damage, suggesting impaired cardiac muscle homeostasis. Overall, our results identify the early recruitment of CVPCs in dystrophic hearts and their fast depletion with ageing. This latter depletion may participate in the fibrosis development and the acceleration onset of the cardiomyopathy.

Reduction of circulating sphingosine-1-phosphate worsens mdx soleus muscle dystrophic phenotype.

NEW FINDINGS: What is the central question of the study? What are the consequences of reducing circulating sphingosine-1-phosphate (S1P) for muscle physiology in the murine model of Duchenne muscular dystrophy (DMD)? What is the main result and its importance? Reduction of the circulating S1P level in mdx mice aggravates the dystrophic phenotype, as seen by an increase in fibre atrophy, fibrosis and loss of specific force, suggesting that S1P signalling is a potential therapeutic target in DMD. Although further studies are needed, plasma S1P levels have the intriguing possibility of being used as a biomarker for disease severity, an important issue in DMD. ABSTRACT: Sphingosine-1-phosphate (S1P) is an important regulator of skeletal muscle properties. The dystrophin-deficient mdx mouse possesses low levels of S1P (∼50%) compared with wild type. Increased S1P availability was demonstrated to ameliorate the dystrophic phenotype in Drosophila and in mdx mice. Here, we analysed the effects produced by further reduction of S1P availability on the mass, force and regenerative capacity of dystrophic mdx soleus. Circulating S1P was neutralized by a specific anti-S1P antibody (S1P-Ab) known to lower the extracellular concentration of this signalling lipid. The S1P-Ab was administered intraperitoneally in adult mdx mice every 2 days for the duration of experiments. Soleus muscle properties were analysed 7 or 14 days after the first injection. The decreased availability of circulating S1P after the 14 day treatment reduced mdx soleus fibre cross-sectional area (-16%, P < 0.05), an effect that was associated with an increase in markers of proteolytic (MuRF1 and atrogin-1) and autophagic (p62 and LC3-II/LC3-I ratio) pathways. Moreover, an increase of fibrosis was also observed (+26%, P < 0.05). Notably, the treatment also caused a reduction of specific tetanic tension (-29%, P < 0.05). The mdx soleus regenerative capacity was only slightly influenced by reduced S1P. In conclusion, neutralization of circulating S1P reduces the mass and specific force and increases fibrosis of mdx soleus muscle, thus worsening the dystrophic phenotype. The results confirm that active, functional S1P signalling might counteract the progression of soleus mdx pathology and validate the pathway as a potential therapeutic target for muscular dystrophies.

What is the level of dystrophin expression required for effective therapy of Duchenne muscular dystrophy?

Duchenne muscular dystrophy (DMD) is a fatal X-linked muscle wasting disease. The disease is due to mutations in the DMD gene that encodes for a large intracellular protein called dystrophin. Dystrophin plays a critical role in linking the internal cytoskeleton of the striated muscle cell with the extracellular matrix as well as having cell signalling functions. In its absence muscle contraction is associated with cycles of damage, repair, inflammation and fibrosis with eventual loss of muscle and replacement with fat. Experiments in animal models of DMD have generated a number of different approaches to the induction of dystrophin including viral vector mediated delivery of a recombinant dystrophin gene, antisense oligonucleotide mediated exon-skipping to restore the open reading frame in the dystrophin mRNA, read-through of premature stop mutations, genome modification using CRISPR-Cas9 or cell based transfer of a functional dystrophin gene. In all cases, it will be important to understand how much dystrophin expression is required for a clinically effective therapy and this review examines the data from humans and animal models to estimate the percentage of endogenous dystrophin that is likely to have significant clinical benefit. While there are a number of important caveats to consider, including the appropriate outcome measures, this review suggests that approximately 20% of endogenous levels uniformly distributed within the skeletal muscles and the heart may be sufficient to largely prevent disease progression.

Inhibition of microRNA-92a increases blood vessels and satellite cells in skeletal muscle but does not improve duchenne muscular dystrophy-related phenotype in mdx mice.

INTRODUCTION: The vasculature and blood flow in muscle are perturbed in Duchenne muscular dystrophy (DMD) and its mdx mouse model. MicroRNA-92a (miR-92a) is enriched in endothelial cells, especially during ischemic injury. METHODS: Because antagonizing miR-92a was shown to result in increased proliferation and migration of endothelial cells and recovery from ischemia, we assessed the effects of Antagomir-92a in vitro in muscle stem cell culture and in vivo in mdx mice. RESULTS: miR-92a was found to be highly expressed in muscle endothelial cells and satellite cells. Treatment with Antagomir-92a increased capillary density and tissue perfusion, which was accompanied by an increase in satellite cells. However, Antagomir-92a-treated mdx mice showed no histological improvement and had worse muscle function. Antagomir-92a suppressed myogenic differentiation in satellite cell culture. DISCUSSION: AntagomiR-92a improves the vasculature but not the muscle in mdx mice, possibly due to its side effects on satellite cell differentiation. Muscle Nerve 59:594-594, 2019.

Proof-of-concept validation of the mechanism of action of Src tyrosine kinase inhibitors in dystrophic mdx mouse muscle: in vivo and in vitro studies.

Src tyrosine kinase (TK), a redox-sensitive protein overexpressed in dystrophin-deficient muscles, can contribute to damaging signaling by phosphorylation and degradation of ß-dystroglycan (ß-DG). We performed a proof-of-concept preclinical study to validate this hypothesis and the benefit-safety ratio of a pharmacological inhibition of Src-TK in Duchenne muscular dystrophy (DMD). Src-TK inhibitors PP2 and dasatinib were administered for 5 weeks to treadmill-exercised mdx mice. The outcome was evaluated in vivo and ex vivo on functional, histological and biochemical disease-related parameters. Considering the importance to maintain a proper myogenic program, the potential cytotoxic effects of both compounds, as well as their cytoprotection against oxidative stress-induced damage, was also assessed in C2C12 cells. In line with the hypothesis, both compounds restored the level of ß-DG and reduced its phosphorylated form without changing basal expression of genes of interest, corroborating a mechanism at post-translational level. The histological profile of gastrocnemius muscle was slightly improved as well as the level of plasma biomarkers. However, amelioration of in vivo and ex vivo functional parameters was modest, with PP2 being more effective than dasatinib. Both compounds reached appreciable levels in skeletal muscle and liver, supporting proper animal exposure. Dasatinib exerted a greater concentration-dependent cytotoxic effect on C2C12 cells than the more selective PP2, while being less protective against H2O2 cytotoxicity, even though at concentrations higher than those experienced during in vivo treatments. Our results support the interest of Src-TK as drug target in dystrophinopathies, although further studies are necessary to assess the therapeutic potential of inhibitors in DMD.

Mitochondrial content is preserved throughout disease progression in the mdx mouse model of Duchenne muscular dystrophy, regardless of taurine supplementation.

Mitochondrial dysfunction is a pathological feature of Duchenne muscular dystrophy (DMD), a debilitating and fatal neuromuscular disorder characterized by progressive muscle wasting and weakness. Mitochondria are a source of cellular ATP involved in Ca2+ regulation and apoptotic signaling. Ameliorating aberrant mitochondrial function has therapeutic potential for reducing DMD disease severity. The dystrophic mdx mouse exhibits peak muscle damage at 21-28 days, which stabilizes after 8 wk. The amino acid taurine is implicated in mitochondrial health and function, with endogenous concentrations low when measured during the cycle of peak muscle damage in mdx mice. Using whole soleus and extensor digitorum longus (EDL) muscle homogenates from 28- and 70-day mdx mice, we found that there was no change in native state mitochondrial complexes using blue native-PAGE. NADH:ubiquinone oxidotreductase subunit-A9 (NDUFA9) protein abundance was lower in soleus muscle of 28- and 70-day mdx mice and EDL muscle of 70-day mdx mice compared with same muscles in WT (C57/BL10ScSn) animals. There were age-dependent increases in both NDUFA9 protein abundance and citrate synthase activity in soleus muscles of mdx and wild-type mice. There was no change in abundances of mitochondrial dynamics proteins mitofusin 2 (Mfn2) and mitochondrial dynamics protein 49 (MiD49). Taurine administration essentially did not affect any measurements of mitochondria. Collectively, these findings suggest mitochondrial content and dynamics are not reduced in the mdx mouse regardless of disease severity. We also elucidate that taurine affords no significant benefit to mitochondrial content or dynamics in the mdx mouse at either 28 or 70 days.

Divergent impact of Toll-like receptor 2 deficiency on repair mechanisms in healthy muscle versus Duchenne muscular dystrophy.

Injury to skeletal muscle, whether acute or chronic, triggers macrophage-mediated innate immunity in a manner which can be either beneficial or harmful for subsequent repair. Endogenous ligands for Toll-like receptor 2 (TLR2) are released by damaged tissues and might play an important role in activating the innate immune system following muscle injury. To test this hypothesis, we compared macrophage behaviour and muscle repair mechanisms in mice lacking TLR2 under conditions of either acute (cardiotoxin-induced) or chronic (mdx mouse genetic model of Duchenne muscular dystrophy; DMD) muscle damage. In previously healthy muscle subjected to acute damage, TLR2 deficiency reduced macrophage numbers in the muscle post-injury but did not alter the expression pattern of the prototypical macrophage polarization markers iNOS and CD206. In addition, there was abnormal persistence of necrotic fibres and impaired regeneration in TLR2-/- muscles after acute injury. In contrast, TLR2 ablation in chronically diseased muscles of mdx mice not only resulted in significantly reduced macrophage numbers but additionally modified their phenotype by shifting from inflammatory (iNOS(pos) CD206(neg) ) to more anti-inflammatory (iNOS(neg) CD206(pos) ) characteristics. This decrease in macrophage-mediated inflammation was associated with ameliorated muscle histopathology and improved force-generating capacity of the dystrophic muscle. Our results suggest that the role of TLR2 in macrophage function and skeletal muscle repair depends greatly upon the muscle injury context, and raise the possibility that inhibition of TLR2 could serve as a useful therapeutic measure in DMD.

Glucocorticoids Improve Myogenic Differentiation In Vitro by Suppressing the Synthesis of Versican, a Transitional Matrix Protein Overexpressed in Dystrophic Skeletal Muscles.

In Duchenne muscular dystrophy (DMD), a dysregulated extracellular matrix (ECM) directly exacerbates pathology. Glucocorticoids are beneficial therapeutics in DMD, and have pleiotropic effects on the composition and processing of ECM proteins in other biological contexts. The synthesis and remodelling of a transitional versican-rich matrix is necessary for myogenesis; whether glucocorticoids modulate this transitional matrix is not known. Here, versican expression and processing were examined in hindlimb and diaphragm muscles from mdx dystrophin-deficient mice and C57BL/10 wild type mice. V0/V1 versican (Vcan) mRNA transcripts and protein levels were upregulated in dystrophic compared to wild type muscles, especially in the more severely affected mdx diaphragm. Processed versican (versikine) was detected in wild type and dystrophic muscles, and immunoreactivity was highly associated with newly regenerated myofibres. Glucocorticoids enhanced C2C12 myoblast fusion by modulating the expression of genes regulating transitional matrix synthesis and processing. Specifically, Tgfß1, Vcan and hyaluronan synthase-2 (Has2) mRNA transcripts were decreased by 50% and Adamts1 mRNA transcripts were increased three-fold by glucocorticoid treatment. The addition of exogenous versican impaired myoblast fusion, whilst glucocorticoids alleviated this inhibition in fusion. In dystrophic mdx muscles, versican upregulation correlated with pathology. We propose that versican is a novel and relevant target gene in DMD, given its suppression by glucocorticoids and that in excess it impairs myoblast fusion, a process key for muscle regeneration.

Opposing roles of miR-21 and miR-29 in the progression of fibrosis in Duchenne muscular dystrophy.

Excessive extracellular matrix deposition progressively replacing muscle fibres is the endpoint of most severe muscle diseases. Recent data indicate major involvement of microRNAs in regulating pro- and anti-fibrotic genes. To investigate the roles of miR-21 and miR-29 in muscle fibrosis in Duchenne muscle dystrophy, we evaluated their expression in muscle biopsies from 14 patients, and in muscle-derived fibroblasts and myoblasts. In Duchenne muscle biopsies, miR-21 expression was significantly increased, and correlated directly with COL1A1 and COL6A1 transcript levels. MiR-21 expression was also significantly increased in Duchenne fibroblasts, more so after TGF-ß1 treatment. In Duchenne fibroblasts the expression of miR-21 target transcripts PTEN (phosphatase and tensin homolog deleted on chromosome 10) and SPRY-1 (Sprouty homolog 1) was significantly reduced; while collagen I and VI transcript levels and soluble collagen production were significantly increased. MiR-29a and miR-29c were significantly reduced in Duchenne muscle and myoblasts, and miR-29 target transcripts, COL3A1, FBN1 and YY1, significantly increased. MiR-21 silencing in mdx mice reduced fibrosis in the diaphragm muscle and in both Duchenne fibroblasts and mdx mice restored PTEN and SPRY-1 expression, and significantly reduced collagen I and VI expression; while miR-29 mimicking in Duchenne myoblasts significantly decreased miR-29 target transcripts. These findings indicate that miR-21 and miR-29 play opposing roles in Duchenne muscle fibrosis and suggest that pharmacological modulation of their expression has therapeutic potential for reducing fibrosis in this condition.

Treatment with human immunoglobulin G improves the early disease course in a mouse model of Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a severe hereditary myopathy. Standard treatment by glucocorticosteroids is limited because of numerous side effects. The aim of this study was to test immunomodulation by human immunoglobulin G (IgG) as treatment in the experimental mouse model (mdx) of DMD. 2 g/kg human IgG compared to human albumin was injected intraperitoneally in mdx mice at the age of 3 and 7 weeks. Advanced voluntary wheel running parameters were recorded continuously. At the age of 11 weeks, animals were killed so that blood, diaphragm, and lower limb muscles could be removed for quantitative PCR, histological analysis and ex vivo muscle contraction tests. IgG compared to albumin significantly improved the voluntary running performance and reduced muscle fatigability in an ex vivo muscle contraction test. Upon IgG treatment, serum creatine kinase values were diminished and mRNA expression levels of relevant inflammatory markers were reduced in the diaphragm and limb muscles. Macrophage infiltration and myopathic damage were significantly ameliorated in the quadriceps muscle. Collectively, this study demonstrates that, in the early disease course of mdx mice, human IgG improves the running performance and diminishes myopathic damage and inflammation in the muscle. Therefore, IgG may be a promising approach for treatment of DMD. Two monthly intraperitoneal injections of human immunoglobulin G (IgG) improved the early 11-week disease phase of mdx mice. Voluntary running was improved and serum levels of creatine kinase were diminished. In the skeletal muscle, myopathic damage was ameliorated and key inflammatory markers such as mRNA expression of SPP1 and infiltration by macrophages were reduced. The study suggests that IgG could be explored as a potential treatment option for Duchenne muscular dystrophy and that pre-clinical long-term studies should be helpful.

The high correlation between counts and area fractions of lipofuscin granules, a biomarker of oxidative stress in muscular dystrophies.

Images of cryostat unstained sections of two skeletal muscles, diaphragm and extensor digitorum longus (EDL), from wild-type normal and dystrophic mdx mice were captured with a fluorescence microscope, binarised and analysed by an automated procedure using ImageJ free software. The numbers, Feret diameters and areas of autofluorescent lipofuscin (LF)-like granules in the sections were determined from the binary images. The mean numbers of counted LF granules per mm3 muscle tissue correlated highly (r ≥ 0.9) with the area fractions of the granules in sections of both normal and mdx muscles. The similar distribution patterns of granule sizes in sections of diaphragm and EDL muscles are consistent with the high correlations.

Degenerative and regenerative features of myofibers differ among skeletal muscles in a murine model of muscular dystrophy.

Skeletal muscle myofibers constantly undergo degeneration and regeneration. Histopathological features of 6 skeletal muscles (cranial tibial [CT], gastrocnemius, quadriceps femoris, triceps brachii [TB], lumbar longissimus muscles, and costal part of the diaphragm [CPD]) were compared using C57BL/10ScSn-Dmd mdx (mdx) mice, a model for muscular dystrophy versus control, C57BL/10 mice. Body weight and skeletal muscle mass were lower in mdx mice than the control at 4 weeks of age; these results were similar at 6-30 weeks. Additionally, muscular lesions were observed in all examined skeletal muscles in mdx mice after 4 weeks, but none were noted in the controls. Immunohistochemical staining revealed numerous paired box 7-positive satellite cells surrounding the embryonic myosin heavy chain-positive regenerating myofibers, while the number of the former and staining intensity of the latter decreased as myofiber regeneration progressed. Persistent muscular lesions were observed in skeletal muscles of mdx mice between 4 and 14 weeks of age, and normal myofibers decreased with age. Number of muscular lesions was lowest in CPD at all ages examined, while the ratio of normal myofibers was lowest in TB at 6 weeks. In CT, TB, and CPD, Iba1-positive macrophages, the main inflammatory cells in skeletal muscle lesions, showed a significant positive correlation with the appearance of regenerating myofibers. Additionally, B220-positive B-cells showed positive and negative correlation with regenerating and regenerated myofibers, respectively. Our data suggest that degenerative and regenerative features of myofibers differ among skeletal muscles and that inflammatory cells are strongly associated with regenerative features of myofibers in mdx mice.

Age-related T2 changes in hindlimb muscles of mdx mice.

INTRODUCTION: Magnetic resonance imaging (MRI) was used to monitor changes in the transverse relaxation time constant (T2) in lower hindlimb muscles of mdx mice at different ages. METHODS: Young (5 weeks), adult (44 weeks), and old mdx (96 weeks), and age-matched control mice were studied. Young mdx mice were imaged longitudinally, whereas adult and old mdx mice were imaged at a single time-point. RESULTS: Mean muscle T2 and percent of pixels with elevated T2 were significantly different between mdx and control mice at all ages. In young mdx mice, mean muscle T2 peaked at 7-8 weeks and declined at 9-11 weeks. In old mdx mice, mean muscle T2 was decreased compared with young and adult mice, which could be attributed to fibrosis. CONCLUSIONS: MRI captured longitudinal changes in skeletal muscle integrity of mdx mice. This information will be valuable for pre-clinical testing of potential therapeutic interventions for muscular dystrophy.

In vivo treatment with the NF-κB inhibitor ursodeoxycholic acid (UDCA) improves tension development in the isolated mdx costal diaphragm.

INTRODUCTION: Previous experiments have indicated that in vivo administration of ursodeoxycholic acid (UDCA) inhibits nuclear NF-κB activation and has beneficial effects on the structure and function of dystrophic (mdx) muscle. We examined the effect of UDCA on tension development in dystrophic muscle. METHODS: Isometric tension development was examined in costal diaphragms that were freshly isolated from vehicle and UDCA treated mdx mice. Percent recovery scores were obtained by directly comparing these measurements to those obtained from age-matched nondystrophic mice. RESULTS: Vehicle treated mdx mice exhibited significantly reduced optimal muscle lengths (lo ) and specific twitch and tetanic tensions compared with age-matched nondystrophic mice. UDCA treated preparations exhibited significantly improved tension development with a 33% recovery score. CONCLUSIONS: Because UDCA is used in treating certain clinical disorders, these results provide a rationale for human clinical trials using this and related drugs for treatment of Duchenne and related muscular dystrophies.