Longitudinal assessment of skeletal muscle functional mechanics in the DE50-MD dog model of Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD), caused by mutations in the dystrophin (DMD) gene, is associated with fatal muscle degeneration and atrophy. Patients with DMD have progressive reductions in skeletal muscle strength and resistance to eccentric muscle stretch. Using the DE50-MD dog model of DMD, we assessed tibiotarsal joint (TTJ) flexor and extensor force dynamics, and the resistance of dystrophic muscle to eccentric stretch. Male DE50-MD and wild-type (WT) dogs were analysed every 3 months until 18 months of age. There was an age-associated decline in eccentric contraction resistance in DE50-MD TTJ flexors that discriminated, with high statistical power, WT from DE50-MD individuals. For isometric contraction, at the majority of timepoints, DE50-MD dogs had lower maximum absolute and relative TTJ flexor force, reduced TTJ muscle contraction times and prolonged relaxation compared to those in WT dogs. Cranial tibial muscles, the primary TTJ flexor, of 18-month-old DE50-MD dogs had significant numbers of regenerating fibres as expected, but also fewer type I fibres and more hybrid fibres than those in WT dogs. We conclude that these parameters, in particular, the eccentric contraction decrement, could be used as objective outcome measures for pre-clinical assessment in DE50-MD dogs.

Serum inflammatory cytokines as disease biomarkers in the DE50-MD dog model of Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a fatal muscle-wasting disease, caused by mutations in the dystrophin gene, characterised by cycles of muscle degeneration, inflammation and regeneration. Recently, there has been renewed interest specifically in drugs that ameliorate muscle inflammation in DMD patients. The DE50-MD dog is a model of DMD that closely mimics the human DMD phenotype. We quantified inflammatory proteins in serum from wild-type (WT) and DE50-MD dogs aged 3-18 months to identify biomarkers for future pre-clinical trials. Significantly higher concentrations of C-C motif chemokine ligand 2 (CCL2), granulocyte-macrophage colony-stimulating factor (GM-CSF or CSF2), keratinocyte chemotactic-like (KC-like, homologous to mouse CXCL1), TNFα (or TNF), and interleukins IL2, IL6, IL7, IL8 (CXCL8), IL10, IL15 and IL18 were detected in DE50-MD serum compared to WT serum. Of these, CCL2 best differentiated the two genotypes. The relative level of CCL2 mRNA was greater in the vastus lateralis muscle of DE50-MD dogs than in that of WT dogs, and CCL2 was expressed both within and at the periphery of damaged myofibres. Serum CCL2 concentration was significantly associated with acid phosphatase staining in vastus lateralis biopsy samples in DE50-MD dogs. In conclusion, the serum cytokine profile suggests that inflammation is a feature of the DE50-MD phenotype. Quantification of serum CCL2 in particular is a useful non-invasive biomarker of the DE50-MD phenotype.

The skeletal muscle phenotype of the DE50-MD dog model of Duchenne muscular dystrophy.

Background: Animal models of Duchenne muscular dystrophy (DMD) are essential to study disease progression and assess efficacy of therapeutic intervention, however dystrophic mice fail to display a clinically relevant phenotype, limiting translational utility. Dystrophin-deficient dogs exhibit disease similar to humans, making them increasingly important for late-stage preclinical evaluation of candidate therapeutics. The DE50-MD canine model of DMD carries a mutation within a human 'hotspot' region of the dystrophin gene, amenable to exon-skipping and gene editing strategies. As part of a large natural history study of disease progression, we have characterised the DE50-MD skeletal muscle phenotype to identify parameters that could serve as efficacy biomarkers in future preclinical trials. Methods: Vastus lateralis muscles were biopsied from a large cohort of DE50-MD dogs and healthy male littermates at 3-monthly intervals (3-18 months) for longitudinal analysis, with multiple muscles collected post-mortem to evaluate body-wide changes. Pathology was characterised quantitatively using histology and measurement of gene expression to determine statistical power and sample sizes appropriate for future work. Results: DE50-MD skeletal muscle exhibits widespread degeneration/regeneration, fibrosis, atrophy and inflammation. Degenerative/inflammatory changes peak during the first year of life, while fibrotic remodelling appears more gradual. Pathology is similar in most skeletal muscles, but in the diaphragm, fibrosis is more prominent, associated with fibre splitting and pathological hypertrophy. Picrosirius red and acid phosphatase staining represent useful quantitative histological biomarkers for fibrosis and inflammation respectively, while qPCR can be used to measure regeneration ( MYH3, MYH8), fibrosis ( COL1A1), inflammation ( SPP1), and stability of DE50-MD dp427 transcripts. Conclusion: The DE50-MD dog is a valuable model of DMD, with pathological features similar to young, ambulant human patients. Sample size and power calculations show that our panel of muscle biomarkers are of strong pre-clinical value, able to detect therapeutic improvements of even 25%, using trials with only six animals per group.

A highly prevalent SINE mutation in the myostatin (MSTN) gene promoter is associated with low circulating myostatin concentration in Thoroughbred racehorses.

Horse racing is a popular and financially important industry worldwide and researchers and horse owners are interested in genetic and training influences that maximise athletic performance. An association has been found between the presence of a short interspersed nuclear element (SINE) mutation in the myostatin (MSTN) gene promoter and optimal race distance in Thoroughbred horses. There is previous laboratory evidence that this mutation reduces MSTN expression in a cell culture model and influences skeletal muscle fibre type proportions in horses. Manipulating MSTN expression has been proposed for illicit gene doping in human and equine athletes and already, researchers have generated homozygous and heterozygous MSTN-null horse embryos following CRISPR/Cas9 editing at the equine MSTN locus and nuclear transfer, aiming artificially to enhance performance. To date however, the role of the naturally-occurring equine MSTN SINE mutation in vivo has remained unclear; here we hypothesised that it reduces, but does not ablate circulating myostatin expression. Following validation of an ELISA for detection of myostatin in equine serum and using residual whole blood and serum samples from 176 Thoroughbred racehorses under identical management, horses were genotyped for the SINE mutation by PCR and their serum myostatin concentrations measured. In our population, the proportions of SINE homozygotes, heterozygotes and normal horses were 27%, 46% and 27% respectively. Results indicated that horses that are homozygous for the SINE mutation have detectable, but significantly lower (p < 0.0001) serum myostatin concentrations (226.8 pg/ml; 69.3-895.4 pg/ml; median; minimum-maximum) than heterozygous (766 pg/ml; 64.6-1182 pg/ml) and normal horses (1099 pg/ml; 187.8-1743 pg/ml). Heterozygotes have significantly lower (p < 0.0001) myostatin concentrations than normal horses. Variation in serum myostatin concentrations across horses was not influenced by age or sex. This is the first study to reveal the direct functional effect of a highly prevalent mutation in the equine MSTN gene associated with exercise performance. Determining the reason for variation in expression of myostatin within SINE-genotyped groups might identify additional performance-associated environmental or genetic influences in Thoroughbreds. Understanding the mechanism by which altered myostatin expression influences skeletal muscle fibre type remains to be determined.

Longitudinal assessment of blood-borne musculoskeletal disease biomarkers in the DE50-MD dog model of Duchenne muscular dystrophy.

Background: Duchenne muscular dystrophy (DMD) is a fatal muscle wasting disease caused by mutations in the dystrophin gene. Due to their phenotypic similarity to human patients, large animal models are invaluable tools for pre-clinical trials. The DE50-MD dog is a relatively new model of DMD, and carries a therapeutically-tractable mutation lying within the hotspot for human patients, making it especially valuable. Prior to conducting therapeutic trials using this novel animal model, it is essential to establish a panel of viable biomarkers. Methods: We evaluated a panel of blood-borne biomarkers of musculoskeletal disease in the DE50-MD dog. Venous blood samples were obtained monthly throughout an 18-month study period in DE50-MD (N=18) and wild-type (WT) control (N=14) dogs. A panel of potential plasma/serum biomarkers of DMD was measured and their theoretical utility in future clinical trials determined using sample size calculations. Results: Compared to WT dogs, DE50-MD dogs had substantially higher circulating creatine kinase (CK) activities, myomesin-3 (MYOM3), and the dystromiRs miR-1, miR-133a and miR-206, but significantly lower serum myostatin concentrations. An age-associated pattern, similar to that observed in DMD patients, was seen for CK and MYOM3. Sample size calculations suggested that low cohort sizes (N≤3) could be used to detect up to a 50% improvement in DE50-MD results towards WT levels for each biomarker or a combination thereof (via principal component analysis); as few as N=3 animals should enable detection of a 25% improvement using a combined biomarker approach (alpha 0.05, power 0.8). Conclusions: We have established a panel of blood-borne biomarkers that could be used to monitor musculoskeletal disease or response to a therapeutic intervention in the DE50-MD dog using low numbers of animals. The blood biomarker profile closely mimics that of DMD patients, supporting the hypothesis that this DMD model would be suitable for use in pre-clinical trials.

Single-transcript multiplex in situ hybridisation reveals unique patterns of dystrophin isoform expression in the developing mammalian embryo.

Background: The dystrophin gene has multiple isoforms: full-length dystrophin (dp427) is principally known for its expression in skeletal and cardiac muscle, but is also expressed in the brain, and several internal promoters give rise to shorter, N-terminally truncated isoforms with wider tissue expression patterns (dp260 in the retina, dp140 in the brain and dp71 in many tissues). These isoforms are believed to play unique cellular roles both during embryogenesis and in adulthood, but their shared sequence identity at both mRNA and protein levels makes study of distinct isoforms challenging by conventional methods. Methods: RNAscope is a novel in-situ hybridisation technique that offers single-transcript resolution and the ability to multiplex, with different target sequences assigned to distinct fluorophores. Using probes designed to different regions of the dystrophin transcript (targeting 5', central and 3' sequences of the long dp427 mRNA), we can simultaneously detect and distinguish multiple dystrophin mRNA isoforms at sub-cellular histological levels. We have used these probes in healthy and dystrophic canine embryos to gain unique insights into isoform expression and distribution in the developing mammal. Results: Dp427 is found in developing muscle as expected, apparently enriched at nascent myotendinous junctions. Endothelial and epithelial surfaces express dp71 only. Within the brain and spinal cord, all three isoforms are expressed in spatially distinct regions: dp71 predominates within proliferating germinal layer cells, dp140 within maturing, migrating cells and dp427 appears within more established cell populations. Dystrophin is also found within developing bones and teeth, something previously unreported, and our data suggests orchestrated involvement of multiple isoforms in formation of these tissues. Conclusions: Overall, shorter isoforms appear associated with proliferation and migration, and longer isoforms with terminal lineage commitment: we discuss the distinct structural contributions and transcriptional demands suggested by these findings.