Benfotiamine improves dystrophic pathology and exercise capacity in mdx mice by reducing inflammation and fibrosis.

Duchenne Muscular Dystrophy (DMD) is a progressive and fatal neuromuscular disease. Cycles of myofibre degeneration and regeneration are hallmarks of the disease where immune cells infiltrate to repair damaged skeletal muscle. Benfotiamine is a lipid soluble precursor to thiamine, shown clinically to reduce inflammation in diabetic related complications. We assessed whether benfotiamine administration could reduce inflammation related dystrophic pathology. Benfotiamine (10 mg/kg/day) was fed to male mdx mice (n = 7) for 15 weeks from 4 weeks of age. Treated mice had an increased growth weight (5-7 weeks) and myofibre size at treatment completion. Markers of dystrophic pathology (area of damaged necrotic tissue, central nuclei) were reduced in benfotiamine mdx quadriceps. Grip strength was increased and improved exercise capacity was found in mdx treated with benfotiamine for 12 weeks, before being placed into individual cages and allowed access to an exercise wheel for 3 weeks. Global gene expression profiling (RNAseq) in the gastrocnemius revealed benfotiamine regulated signalling pathways relevant to dystrophic pathology (Inflammatory Response, Myogenesis) and fibrotic gene markers (Col1a1, Col1a2, Col4a5, Col5a2, Col6a2, Col6a2, Col6a3, Lum) towards wildtype levels. In addition, we observed a reduction in gene expression of inflammatory gene markers in the quadriceps (Emr1, Cd163, Cd4, Cd8, Ifng). Overall, these data suggest that benfotiamine reduces dystrophic pathology by acting on inflammatory and fibrotic gene markers and signalling pathways. Given benfotiamine's excellent safety profile and current clinical use, it could be used in combination with glucocorticoids to treat DMD patients.

Expression profiling in exercised mdx suggests a role for extracellular proteins in the dystrophic muscle immune response.

Duchenne muscular dystrophy (DMD) is a lethal muscle wasting disorder caused by mutations in the DMD gene that leads to the absence or severe reduction of dystrophin protein in muscle. The mdx mouse, also dystrophin deficient, is the model most widely used to study the pathology and test potential therapies, but the phenotype is milder than human DMD. This limits the magnitude and range of histological damage parameters and molecular changes that can be measured in pre-clinical drug testing. We used 3 weeks of voluntary wheel running to exacerbate the mdx phenotype. In mdx mice, voluntary exercise increased the amount of damaged necrotic tissue and macrophage infiltration. Global gene expression profiling revealed that exercise induced additional and larger gene expression changes in mdx mice and the pathways most impacted by exercise were all related to immune function or cell-extracellular matrix (ECM) interactions. When we compared the matrisome and inflammation genes that were dysregulated in mdx with those commonly differentially expressed in DMD, we found the exercised mdx molecular signature more closely resembled that of DMD. These gene expression changes in the exercised mdx model thus provide more scope to assess the effects of pre-clinical treatments. Our gene profiling comparisons also highlighted upregulation of ECM proteins involved in innate immunity pathways, proteases that can release them, downstream receptors and signaling molecules in exercised mdx and DMD, suggesting that the ECM could be a major source of pro-inflammatory molecules that trigger and maintain the immune response in dystrophic muscle.

UV-adVISor: Attention-Based Recurrent Neural Networks to Predict UV-Vis Spectra.

Ultraviolet-visible (UV-Vis) absorption spectra are routinely collected as part of high-performance liquid chromatography (HPLC) analysis systems and can be used to identify chemical reaction products by comparison to the reference spectra. Here, we present UV-adVISor as a new computational tool for predicting the UV-Vis spectra from a molecule's structure alone. UV-Vis prediction was approached as a sequence-to-sequence problem. We utilized Long-Short Term Memory and attention-based neural networks with Extended Connectivity Fingerprint Diameter 6 or molecule SMILES to generate predictive models for the UV spectra. We have produced two spectrum datasets (dataset I, N = 949, and dataset II, N = 2222) using different compound collections and spectrum acquisition methods to train, validate, and test our models. We evaluated the prediction accuracy of the complete spectra by the correspondence of wavelengths of absorbance maxima and with a series of statistical measures (the best test set median model parameters are in parentheses for model II), including RMSE (0.064), R2 (0.71), and dynamic time warping (DTW, 0.194) of the entire spectrum curve. Scrambling molecule structures with the experimental spectra during training resulted in a degraded R2, confirming the utility of the approaches for prediction. UV-adVISor is able to provide fast and accurate predictions for libraries of compounds.

Resveratrol Promotes Hypertrophy in Wildtype Skeletal Muscle and Reduces Muscle Necrosis and Gene Expression of Inflammatory Markers in Mdx Mice.

Duchenne muscular dystrophy (DMD) is a progressive fatal neuromuscular disorder with no cure. Therapies to restore dystrophin deficiency have been approved in some jurisdictions but long-term effectiveness is yet to be established. There is a need to develop alternative strategies to treat DMD. Resveratrol is a nutraceutical with anti-inflammatory properties. Previous studies have shown high doses (100-400 mg/kg bodyweight/day) benefit mdx mice. We treated 4-week-old mdx and wildtype mice with a lower dose of resveratrol (5 mg/kg bodyweight/day) for 15 weeks. Voluntary exercise was used to test if a lower dosage than previously tested could reduce exercise-induced damage where a greater inflammatory infiltrate is present. We found resveratrol promoted skeletal muscle hypertrophy in wildtype mice. In dystrophic muscle, resveratrol reduced exercise-induced muscle necrosis. Gene expression of immune cell markers, CD86 and CD163 were reduced; however, signalling targets associated with resveratrol's mechanism of action including Sirt1 and NF-κB were unchanged. In conclusion, a lower dose of resveratrol compared to the dosage used by other studies reduced necrosis and gene expression of inflammatory cell markers in dystrophic muscle suggesting it as a therapeutic candidate for treating DMD.

Hyaluronan synthesis and myogenesis: a requirement for hyaluronan synthesis during myogenic differentiation independent of pericellular matrix formation.

Exogenous hyaluronan is known to alter muscle precursor cell proliferation, migration, and differentiation, ultimately inhibiting myogenesis in vitro. The aim of the current study was to investigate the role of endogenous hyaluronan synthesis during myogenesis. In quantitative PCR studies, the genes responsible for synthesizing hyaluronan were found to be differentially regulated during muscle growth, repair, and pathology. Although all Has genes (Has1, Has2, and Has3) were differentially regulated in these models, only Has2 gene expression consistently associated with myogenic differentiation. During myogenic differentiation in vitro, Has2 was the most highly expressed of the synthases and increased after induction of differentiation. To test whether this association between Has2 expression and myogenesis relates to a role for Has2 in myoblast differentiation and fusion, C2C12 myoblasts were depleted of Has2 by siRNA and induced to differentiate. Depletion of Has2 inhibited differentiation and caused a loss of cell-associated hyaluronan and the hyaluronan-dependent pericellular matrix. The inhibition of differentiation caused by loss of hyaluronan was confirmed with the hyaluronan synthesis inhibitor 4-methylumbelliferone. In hyaluronan synthesis-blocked cultures, restoration of the pericellular matrix could be achieved through the addition of exogenous hyaluronan and the proteoglycan versican, but this was not sufficient to restore differentiation to control levels. These data indicate that intrinsic hyaluronan synthesis is necessary for myoblasts to differentiate and form syncytial muscle cells, but the hyaluronan-dependent pericellular matrix is not sufficient to support differentiation alone; additional hyaluronan-dependent cell functions that are yet unknown may be required for myogenic differentiation.

Versican processing by a disintegrin-like and metalloproteinase domain with thrombospondin-1 repeats proteinases-5 and -15 facilitates myoblast fusion.

Skeletal muscle development and regeneration requires the fusion of myoblasts into multinucleated myotubes. Because the enzymatic proteolysis of a hyaluronan and versican-rich matrix by ADAMTS versicanases is required for developmental morphogenesis, we hypothesized that the clearance of versican may facilitate the fusion of myoblasts during myogenesis. Here, we used transgenic mice and an in vitro model of myoblast fusion, C2C12 cells, to determine a potential role for ADAMTS versicanases. Versican processing was observed during in vivo myogenesis at the time when myoblasts were fusing to form multinucleated myotubes. Relevant ADAMTS genes, chief among them Adamts5 and Adamts15, were expressed both in developing embryonic muscle and differentiating C2C12 cells. Reducing the levels of Adamts5 mRNA in vitro impaired myoblast fusion, which could be rescued with catalytically active but not the inactive forms of ADAMTS5 or ADAMTS15. The addition of inactive ADAMTS5, ADAMTS15, or full-length V1 versican effectively impaired myoblast fusion. Finally, the expansion of a hyaluronan and versican-rich matrix was observed upon reducing the levels of Adamts5 mRNA in myoblasts. These data indicate that these ADAMTS proteinases contribute to the formation of multinucleated myotubes such as is necessary for both skeletal muscle development and during regeneration, by remodeling a versican-rich pericellular matrix of myoblasts. Our study identifies a possible pathway to target for the improvement of myogenesis in a plethora of diseases including cancer cachexia, sarcopenia, and muscular dystrophy.

An anti-inflammatory role for leukemia inhibitory factor receptor signaling in regenerating skeletal muscle.

Skeletal muscle regeneration in pathology and following injury requires the coordinated actions of inflammatory cells and myogenic cells to remove damaged tissue and rebuild syncytial muscle cells, respectively. Following contusion injury to muscle, the cytokine leukemia inhibitor factor (LIF) is up-regulated and knockout of Lif negatively impacts on morphometric parameters of muscle regeneration. Although it was speculated that LIF regulates muscle regeneration through direct effects on myogenic cells, the inflammatory effects of LIF have not been examined in regenerating skeletal muscle. Therefore, the expression and function of LIF was examined using the antagonist MH35-BD during specific inflammatory and myogenic stages of notexin-induced muscle regeneration in mice. LIF protein and mRNA were up-regulated in two distinct phases following intramuscular injection of notexin into tibialis anterior muscles. The first phase of LIF up-regulation coincided with the increased expression of pro-inflammatory cytokines; the second phase coincided with myogenic differentiation and formation of new myotubes. Administration of the LIF receptor antagonist MH35-BD during the second phase of LIF up-regulation had no significant effects on transcript expression of genes required for myogenic differentiation or associated with inflammation; there were no significant differences in morphometric parameters of the regenerating muscle. Conversely, when MH35-BD was administered during the acute inflammatory phase, increased gene transcripts for the pro-inflammatory cytokines Tnf (Tumor necrosis factor), Il1b (Interleukin-1ß) and Il6 (Interleukin-6) alongside an increase in the number of Ly6G positive neutrophils infiltrating the muscle were observed. This was followed by a reduction in Myog (Myogenin) mRNA, which is required for myogenic differentiation, and the subsequent number of myotubes formed was significantly decreased in MH35-BD-treated groups compared to sham. Thus, antagonism of the LIF receptor during the inflammatory phase of skeletal muscle regeneration appeared to induce an inflammatory response that inhibited subsequent myotube formation. We propose that the predominant role of LIF in skeletal muscle regeneration appears to be in regulating the inflammatory response rather than directly effecting myogenic cells.

Effect of pH, salt and chemical rinses on bacterial attachment to extracellular matrix proteins.

Microbial contamination of carcass surfaces occurs during slaughter and post-slaughter processing steps, therefore interventions are needed to enhance meat safety and quality. Although many studies have been done at the macro-level, little is known about specific processes that influence bacterial attachment to carcass surfaces, particularly the role of extracellular matrix (ECM) proteins. In the present study, the effect of pH and salt (NaCl, KCl and CaCl2) on attachment of Escherichia coli and Salmonella isolates to dominant ECM proteins: collagen I, fibronectin, collagen IV and laminin were assessed. Also, the effects of three chemical rinses commonly used in abattoirs (2% acetic acid, 2% lactic acid and 10% trisodium phosphate (TSP)) were tested. Within a pH range of 5-9, there was no significant effect on attachment to ECM proteins, whereas the effect of salt type and concentration varied depending on combination of strain and ECM protein. A concentration-dependant effect was observed with NaCl and KCl (0.1-0.85%) on attachment of E. coli M23Sr, but only to collagen I. One-tenth percent CaCl2 produced the highest level of attachment to ECM proteins for E. coli M23Sr and EC614. In contrast, higher concentrations of CaCl2 increased attachment of E. coli EC473 to collagen IV. Rinses containing TSP produced >95% reduction in attachment to all ECM proteins. These observations will assist in the design of targeted interventions to prevent or disrupt contamination of meat surfaces, thus improving meat safety and quality.

Increased Yearling Weight Gain Is Associated with a Distinct Faecal Microbial Profile.

Microbial communities inhabiting the gut have the ability to influence physiological processes contributing to livestock production and performance. Livestock enterprises rely on animal production traits such as growth performance for profit. Previous studies have shown that gut microbiota are correlated to growth performance and could even influence it. The aim of this study was to characterise the faecal microbial profiles of Angus steers with high and low ADG at both weaning and yearling stages by profiling 16S rRNA gene sequences from rectal faecal samples. When microbial profiles were compared in terms of relative abundances, LEfSe analysis, alpha diversity metrics, and beta diversity, at the weaning stage, few significant differences were found between the high and low ADG groups. However, at yearling stage, microbial profiles significantly differed between the high and low ADG groups. The relative abundances of eight phyla and six genera significantly differed between the two groups. Alpha diversity metrics showed a significant decrease (p = 0.001) in species richness in the high ADG group. Similarly, beta diversity analysis showed that samples clustered clearly according to high and low ADG groups at yearling stage, indicating that phylogenetic similarity between the two ADG groups was significantly reduced (p = 0.005).

Preserved Left Ventricular Function despite Myocardial Fibrosis and Myopathy in the Dystrophin-Deficient D2.B10-Dmd mdx /J Mouse.

Duchenne muscular dystrophy involves an absence of dystrophin, a cytoskeletal protein which supports cell structural integrity and scaffolding for signalling molecules in myocytes. Affected individuals experience progressive muscle degeneration that leads to irreversible loss of ambulation and respiratory diaphragm function. Although clinical management has greatly advanced, heart failure due to myocardial cell loss and fibrosis remains the major cause of death. We examined cardiac morphology and function in D2.B10-Dmd mdx /J (D2-mdx) mice, a relatively new mouse model of muscular dystrophy, which we compared to their wild-type background DBA/2J mice (DBA/2). We also tested whether drug treatment with a specific blocker of mitochondrial permeability transition pore opening (Debio-025), or ACE inhibition (Perindopril), had any effect on dystrophy-related cardiomyopathy. D2-mdx mice were treated for six weeks with Vehicle control, Debio-025 (20 mg/kg/day), Perindopril (2 mg/kg/day), or a combination (n = 8/group). At 18 weeks, compared to DBA/2, D2-mdx hearts displayed greater ventricular collagen, lower cell density, greater cell diameter, and greater protein expression levels of IL-6, TLR4, BAX/Bcl2, caspase-3, PGC-1α, and notably monoamine oxidases A and B. Remarkably, these adaptations in D2-mdx mice were associated with preserved resting left ventricular function similar to DBA/2 mice. Compared to vehicle, although Perindopril partly attenuated the increase in heart weight and collagen at 18 weeks, the drug treatments had no marked impact on dystrophic cardiomyopathy.

Temporal Changes in the Faecal Microbiota of Beef Cattle on Feedlot Placement.

The microbial communities that inhabit the intestinal tract play an important role in modulating health and productivity. Environmental stressors can impact microbial communities, which can significantly influence host physiology. Cattle are subjected to several environmental stressors when placed on feedlots, such as transportation stress, exposure to feedlot environments and change in diet and management. Exposure to these stressors could influence host gut microbiota, which in turn, could potentially influence host health and performance. The aim of the current study was to characterise the temporal changes that occur in intestinal microbiota as a consequence of feedlot placement by profiling 16s rRNA sequences in rectal faecal samples. When faecal microbiome profiles were compared in terms of relative abundances and alpha diversity metrics, the results showed significant, observable changes in profiles 2 days post-feedlot induction. Furthermore, beta-diversity analysis indicated that the phylogenetic similarity between samples significantly decreased on day 2 (PERMANOVA, p < 0.001). These trends were suggestive of a short-term reduction in microbial diversity coupled with decreased similarity between animals. These changes warrant further investigation and could provide opportunities for improved performance, health and even welfare of feedlot cattle in future.

Conversion of large-amplitude vibration to electron excitation at a metal surface.

Gaining insight into the nature and dynamics of the transition state is the essence of mechanistic investigations of chemical reactions, yet the fleeting configuration when existing chemical bonds dissociate while new ones form is extremely difficult to examine directly. Adiabatic potential-energy surfaces--usually derived using quantum chemical methods that assume mutually independent nuclear and electronic motion--quantify the fundamental forces between atoms involved in reaction and thus provide accurate descriptions of a reacting system as it moves through its transition state. This approach, widely tested for gas-phase reactions, is now also commonly applied to chemical reactions at metal surfaces. There is, however, some evidence calling into question the correctness of this theoretical approach for surface reactions: electronic excitation upon highly exothermic chemisorption has been observed, and indirect evidence suggests that large-amplitude vibrations of reactant molecules can excite electrons at metal surfaces. Here we report the detection of 'hot' electrons leaving a metal surface as vibrationally highly excited NO molecules collide with it. Electron emission only occurs once the vibrational energy exceeds the surface work function, and is at least 10,000 times more efficient than the emissions seen in similar systems where large-amplitude vibrations were not involved. These observations unambiguously demonstrate the direct conversion of vibrational to electronic excitation, thus questioning one of the basic assumptions currently used in theoretical approaches to describing bond-dissociation at metal surfaces.

Production of the NO photofragment in the desorption of RDX and HMX from surfaces.

A promising scheme for the remote detection of nitrate-based explosives, which have low vapor pressure, involves two lasers: the first to desorb, vaporize, and photofragment the explosive molecule and the second to create laser-induced fluorescence in the NO fragment. It is desirable to use for the first a powerful 532 nm frequency-doubled Nd:YAG laser. In this study, we investigate the degree of photofragmentation into NO resulting from the irradiation of the explosives RDX and HMX coated on a variety of surfaces. The desorption step is followed by femtosecond laser ionization and time-of-flight mass spectrometry to reveal the fragments produced in the first step. We find that modest laser power of 532 nm desorbs the explosive and produces adequate amounts of NO.

Thermal denaturation of proteins in the muscle fibre and connective tissue from bovine muscles composed of type I (masseter) or type II (cutaneous trunci) fibres: DSC and FTIR microspectroscopy study.

The changes in secondary structure of proteins with heating were characterised and compared for bovine masseter (fibre type I) and cutaneous trunci (fibre type II) muscles by Differential Scanning Calorimetry (DSC) and Fourier Transform InfraRed (FTIR) microspectroscopy. Heating led to a decrease in α- helices, and an increase in aggregated strands, random coils and aromatic side chains in the muscle fibres of both muscles. In the intramuscular connective tissue (IMCT) of both muscles, a decrease in α- helix, turn and unordered structures was complemented with an increase in aggregated strands. At temperatures < 60 °C, the greater thermal denaturation of proteins in cutaneous trunci than in masseter (FTIR), supported by a myosin associated peak at 55.8 °C for cutaneous trunci and no peak for masseter (DSC), indicates that myosin in type II fibres is more sensitive to thermal denaturation than myosin in type I fibres and this should be considered in thermal meat processing.

Immunohistochemical analysis of laryngeal muscle of horses clinically affected with recurrent laryngeal neuropathy.

BACKGROUND: As myosin heavy chain (MyHC) profile of muscle fibres is heavily influenced by neural input, changes in MyHC expression are expected in horses clinically affected with recurrent laryngeal neuropathy (RLN) yet, this has not been thoroughly investigated. OBJECTIVES: To describe the changes in MyHC and fibre diameter in left cricoarytenoideus dorsalis (L-CAD) muscle of horses with clinical signs of RLN. STUDY DESIGN: Observational cohort study. METHODS: Immunohistochemistry was used to assess the MyHC-based fibre-type proportion, size and grouping in the L-CAD of 10 Thoroughbred horses, five clinically affected with RLN and five unaffected controls based on resting endoscopic examination. The Mann-Whitney U test was used to compare the two groups. RESULTS: Compared to controls (of mean age 3.0 ± 1.7 years) which only expressed type I, IIA and IIX MyHC, the L-CAD of affected horses (of mean age 2.8 ± 0.8 years) had obvious fibre-type grouping, and despite apparent compensatory hypertrophy of a small number of fibres, a decrease in overall fibre diameter (median difference -35.2 µm, 95% CI -47.4 to -7.9, P = .02) and diameter of type IIA fibres (median difference -46.8 µm, 95% CI -52.1 to -5.0, P = .03) was observed. Anti-fast MyHC (MY32) cross-immunoreacted with embryonic-MyHC. Whereas MY32-positive fibres were identified as type IIX in controls, in affected horses these fibres were less than 50 µm diameter with internal nuclei and were MYH3-positive for embryonic myosin indicating depletion of type IIX fibres, yet active regeneration and fibre renewal. MAIN LIMITATIONS: Small sample size that did not include subclinical cases. Fibre size and appearance rather than staining colour were relied upon to differentiate embryonic from type IIX MyHC. CONCLUSIONS: Horses clinically affected with RLN have overall atrophy of fibres, loss of IIX fibres and expression of embryonic myosin indicating regenerative capacity. Despite hypertrophy of some remaining fibres, the overall decline in the bulk of fibres, including those most fatigue-resistant, may be the critical change that results in failure to maintain arytenoid abduction during exercise although direct comparison to subclinical cases is needed to confirm this.

Ageing and cathepsin inhibition affect the shrinkage of fibre fragments of bovine semitendinosus, biceps femoris and psoas major during heating.

This study examines the effects of ageing (1, 14 days), cathepsin inhibition (No or Yes) and temperature (25-90 °C) on the shrinkage of fibre fragments from three bovine muscles (semitendinosus, biceps femoris and psoas major) during heating. Shrinkage was quantified using light microscopy images. Muscle fibres (except in psoas major) had greater transverse shrinkage, and less longitudinal shrinkage in aged than in unaged muscles at temperatures ≥60-75 °C. In addition, cathepsin inhibition during heating at ≥65-90 °C caused greater transverse shrinkage in semitendinosus fibres, and reduced longitudinal shrinkage for all muscles. At temperatures ≥75 °C, the longitudinal and transverse shrinkage of the fibres was correlated for all muscles. Ageing of biceps femoris increases volume shrinkage on a fibre level, and hence potentially cooking loss, while cathepsin activity in the semitendinosus reduces volume shrinkage. In conclusion, cathepsin activity and ageing influence the shrinkage that occurs during heating and these factors should be explored further to enable optimisation of thermal meat processing.

A gene network switch enhances the oxidative capacity of ovine skeletal muscle during late fetal development.

BACKGROUND: The developmental transition between the late fetus and a newborn animal is associated with profound changes in skeletal muscle function as it adapts to the new physiological demands of locomotion and postural support against gravity. The mechanisms underpinning this adaption process are unclear but are likely to be initiated by changes in hormone levels. We tested the hypothesis that this developmental transition is associated with large coordinated changes in the transcription of skeletal muscle genes. RESULTS: Using an ovine model, transcriptional profiling was performed on Longissimus dorsi skeletal muscle taken at three fetal developmental time points (80, 100 and 120 d of fetal development) and two postnatal time points, one approximately 3 days postpartum and a second at 3 months of age. The developmental time course was dominated by large changes in expression of 2,471 genes during the interval between late fetal development (120 d fetal development) and 1-3 days postpartum. Analysis of the functions of genes that were uniquely up-regulated in this interval showed strong enrichment for oxidative metabolism and the tricarboxylic acid cycle indicating enhanced mitochondrial activity. Histological examination of tissues from these developmental time points directly confirmed a marked increase in mitochondrial activity between the late fetal and early postnatal samples. The promoters of genes that were up-regulated during this fetal to neonatal transition were enriched for estrogen receptor 1 and estrogen related receptor alpha cis-regulatory motifs. The genes down-regulated during this interval highlighted de-emphasis of an array of functions including Wnt signaling, cell adhesion and differentiation. There were also changes in gene expression prior to this late fetal--postnatal transition and between the two postnatal time points. The former genes were enriched for functions involving the extracellular matrix and immune response while the latter principally involved functions associated with transcriptional regulation of metabolic processes. CONCLUSIONS: It is concluded that during late skeletal muscle development there are substantial and coordinated changes in the transcription of a large number of genes many of which are probably triggered by increased estrogen levels. These changes probably underpin the adaption of muscle to new physiological demands in the postnatal environment.

Adenylosuccinic acid therapy ameliorates murine Duchenne Muscular Dystrophy.

Arising from the ablation of the cytoskeletal protein dystrophin, Duchenne Muscular Dystrophy (DMD) is a debilitating and fatal skeletal muscle wasting disease underpinned by metabolic insufficiency. The inability to facilitate adequate energy production may impede calcium (Ca2+) buffering within, and the regenerative capacity of, dystrophic muscle. Therefore, increasing the metabogenic potential could represent an effective treatment avenue. The aim of our study was to determine the efficacy of adenylosuccinic acid (ASA), a purine nucleotide cycle metabolite, to stimulate metabolism and buffer skeletal muscle damage in the mdx mouse model of DMD. Dystrophin-positive control (C57BL/10) and dystrophin-deficient mdx mice were treated with ASA (3000 µg.mL-1) in drinking water. Following the 8-week treatment period, metabolism, mitochondrial density, viability and superoxide (O2-) production, as well as skeletal muscle histopathology, were assessed. ASA treatment significantly improved the histopathological features of murine DMD by reducing damage area, the number of centronucleated fibres, lipid accumulation, connective tissue infiltration and Ca2+ content of mdx tibialis anterior. These effects were independent of upregulated utrophin expression in the tibialis anterior. ASA treatment also increased mitochondrial viability in mdx flexor digitorum brevis fibres and concomitantly reduced O2- production, an effect that was also observed in cultured immortalised human DMD myoblasts. Our data indicates that ASA has a protective effect on mdx skeletal muscles.

Analysis of the callipyge phenotype through skeletal muscle development; association of Dlk1 with muscle precursor cells.

The callipyge mutation in sheep in the form of the paternal heterozygote results in skeletal muscle hypertrophy, which is most pronounced in the hindquarters. Overexpression of one of the genes in the region of the causative single-nucleotide polymorphism, Dlk1, is postulated to be a primary cause of the muscle hypertrophy although the mechanism is not clear. This study examined the expression of Dlk1 mRNA and its encoded protein in skeletal muscles of callipyge and wild-type sheep. The muscles examined included those that demonstrate hypertrophy in callipyge sheep as well as an unaffected muscle. The expression pattern of Dlk1 protein in these muscles was also measured over a developmental time course ranging from 80 days of gestation to 12 weeks after birth. Quantitative reverse transcription-polymerase chain reaction demonstrated that Dlk1 mRNA was significantly increased in affected, but not unaffected, muscles from callipyge sheep at 120 days of gestation through to 12 weeks of age. Immuno-localization of Dlk1 was pronounced in the interstitial connective tissue of fetal muscle but was less intense at later ages. No clear difference in Dlk1 immuno-localization was noted between genotypes in the fetal samples. Strong myofiber-specific Dlk1 immuno-localization was observed in hypertrophied callipyge muscles at 12 weeks of age. This staining was exclusively associated with fast type II myofibers and these had a significantly larger mean cross-sectional area, compared with fast type II myofibers in control sheep that did not overexpress Dlk1. In addition, Dlk1 immuno-localization was associated with a sub-population of Pax7-positive mononucleated cells in all skeletal muscles examined during fetal development and at birth, but this was not apparent at 12 weeks. There were no genotype-dependent alterations in the mRNA expression patterns of a number of promyogenic transcription factors indicating that the callipyge mutation was not affecting muscle cell differentiation per se. We postulate that Dlk1 is implicated in the commitment and/or proliferation of fetal myoblasts as well as in the maintenance of hypertrophy in fully differentiated myofibers.

Myoblasts isolated from hypertrophy-responsive callipyge muscles show altered growth rates and increased resistance to serum deprivation-induced apoptosis.

Back and hind limb muscles of sheep paternally heterozygous for the callipyge single nucleotide polymorphism undergo extensive hypertrophy shortly after birth. We have established cell cultures from foetal semitendinosus and longissimus dorsi muscles of normal and callipyge animals. Cultures were assessed for rates of proliferation, cell death, myogenicity and DLK1 expression. Myoblasts from callipyge semitendinosus, but not longissimus dorsi muscles, proliferated faster than myoblasts isolated from normal semitendinosus muscle, and cells isolated from either callipyge muscle were more resistant to serum deprivation-induced apoptosis than equivalent cells isolated from normal individuals. These observations indicate that there are intrinsic differences in the behaviour of isolated myoblasts, which are associated with their muscle and genotype of origin. As myoblasts are the cells responsible for hypertrophy of muscle fibres, the observed differences in cell growth may play a role in the hypertrophy of certain muscles in callipyge animals.