Journal: Skeletal Muscle

Article Title: Extensive striated muscle damage in a rat model of Duchenne muscular dystrophy with Dmd exons 10–17 duplication

doi: 10.1186/s13395-025-00386-2

Figure Lengend Snippet: Early death associated with severe muscle atrophy in dystrophin-deficient R-DMDdup10-17 (DMD) rats. (A) Scheme of CRISPR/Cas9-mediated duplication on the rat chromosome X of a ~ 125 kb genomic region encompassing exons 10 to 17 of the Dmd gene. The wild-type allele (X + ) is at the top, with the reading frame indicated by the blue colour of the exons. The allele containing the duplication (X DMD ) is at the bottom. Splicing between exon 17 and the duplicated exon 10 induces a premature stop codon in the duplicated exon 10 (white cross on red background) and the absence of translation of subsequent exons (grey). The position and orientation of the primers used for genotyping is indicated by arrows, as is the expected amplicon size for each of the two alleles. (B) Electrophoretic gel of amplicons obtained using a multiplex of the three primers indicated in (A) from genomic DNA of male rats from a litter born from a carrier female and a WT male. A band at 130 bp reveals the WT allele, while a band at 277 bp reveals a male hemizygous for the Dmd allele carrying the duplicated 10–17 region. (C) Body mass of WT and DMD littermates at 6 and 10 months of age. (D) Kaplan-Meier curve for the frequency of WT (black curve) and DMD (orange curve) rat survival. (E) Representative immunofluorescence for laminin (red) and dystrophin (orange) at 6 months on TA, diaphragm and heart sections of WT and DMD littermates, showing complete absence of a dystrophin signal in DMD muscles. Scale bars, 50 μm. (F) Plasma CK levels in 7-month-old WT and DMD littermates. One-tailed unpaired t test. (G) Upper panel: Capillary immunoelectrophoresis plasma protein analysis detecting the two fragments (100 and 130 kDa) of the sarcomeric myomesin-3 (MYOM3) protein in 7-month-old DMD rats, but not in their WT littermates; lower panel: total proteins for each lane. (H) Quantification of plasma MYOM3 levels assessed in ( G ), corresponding to the sum of both MYOM3-130 and MYOM3-100 signals normalised to total plasma proteins. One-tailed unpaired t test. (I) Plasma hs-cTNT levels in 10-month-old WT and DMD littermates. One-tailed unpaired t test

Article Snippet: MYOM3 plasma concentration was obtained with the SimpleProtein Jess analyzer (BioTechne) using the regular 12–230 kDa kit, the polyclonal anti-MYOM3 antibody (ProteinTech #17692-1-AP, 1:2000) and the anti-rabbit HRP (ProteinSimple).

Techniques: CRISPR, Amplification, Multiplex Assay, Immunofluorescence, Muscles, Clinical Proteomics, One-tailed Test, Immunoelectrophoresis

Journal: Wellcome Open Research

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

doi: 10.12688/wellcomeopenres.17398.2

Figure Lengend Snippet: Figure a ) representative MYOM3 western blot. This western blot shows a positive control mini-dilution series that was run alongside samples in every gel, followed by 5 samples of WT control dog serum, 5 samples of DE50-MD dog serum, and a negative control lane in which no protein was loaded. Figure b ) MYOM3 quantified by western blot in serum samples from dogs studied longitudinally between 1 and 18 months of age. DE50-MD: grey, total N=83 samples, from N=10 dogs, N=2-8 per age group; WT dogs: (not shown, see Underlying data ), total N=113 samples, from N=10 dogs, N=3-9 per age group. Bands corresponding to MYOM3 were not detected in any WT serum samples tested. Boxes extend from the 25 th to 75 th percentile, with a line within the box at the median value. Each point represents an individual sample, and whiskers show the minimum and maximum results for that age-group. Band intensity calculated by densitometry using ImageJ software. Figure c ) correlation between MYOM3 quantity and CK activity in blood samples from DE50-MD dogs studied longitudinally between 1 and 18 months, total of N=75 samples, from N=10 dogs. (MYOM3: myomesin-3; WT: wild type.)

Article Snippet: Membranes were blocked in 10% milk powder (Marvel) in phosphate buffered saline (PBS, Fisher Scientific, #BR0014G)/Tween 0.05% (Sigma, #P9416) for one hour at room temperature, before incubation with a polyclonal rabbit anti-human MYOM3 antibody (Proteintech, #17692-1-AP, 1:1000 dilution) and a polyclonal rabbit anti-canine albumin antibody (as internal loading control, Biorbyt Ltd, #orb242465; 1:1,000,000 dilution) overnight at 4 °C.

Techniques: Western Blot, Positive Control, Control, Negative Control, Software, Activity Assay

Journal: Wellcome Open Research

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

doi: 10.12688/wellcomeopenres.17398.2

Figure Lengend Snippet: Included in the analysis was plasma CK activity (log10 U/l), serum MYOM3 quantity (AU), serum MSTN concentration (ng/ml), serum miR-1, miR-133a and miR 206 levels (log10 relative quantity). Study population consisted of DE50-MD and wild-type (WT) dogs studied longitudinally between 3 and 18 months of age. DE50-MD: grey, N=10 dogs total, N=2-9 per age group; WT: white, N=7 dogs total, N=2-5 per age group. Where an individual was missing data for a specific biomarker, missing data was replaced with the mean of the relevant genotype and age-group for that biomarker. Boxes extend from the 25 th to 75 th percentile, with a line within the box at the median value. Each point represents an individual sample, and whiskers show the minimum and maximum results for that age-group. Asterisks denote the level of significance of a difference between genotypes based on linear mixed model analysis: ****P<0.0001. Letters a, b and c denote statistically significant differences (P<0.05) in the mean within either the DE50-MD (grey letters) or WT (black letters) genotypes: means sharing a letter are not significantly different within each genotype group. (CK: creatine kinase; WT: wild type.)

Article Snippet: Membranes were blocked in 10% milk powder (Marvel) in phosphate buffered saline (PBS, Fisher Scientific, #BR0014G)/Tween 0.05% (Sigma, #P9416) for one hour at room temperature, before incubation with a polyclonal rabbit anti-human MYOM3 antibody (Proteintech, #17692-1-AP, 1:1000 dilution) and a polyclonal rabbit anti-canine albumin antibody (as internal loading control, Biorbyt Ltd, #orb242465; 1:1,000,000 dilution) overnight at 4 °C.

Techniques: Clinical Proteomics, Activity Assay, Concentration Assay, Biomarker Discovery

Journal: Wellcome Open Research

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

doi: 10.12688/wellcomeopenres.17398.2

Figure Lengend Snippet: Sample size calculations. N required to show with sufficient power (0.8) an improvement in a DE50-MD biomarker towards WT levels with any given treatment. Sample sizes were calculated for the principal component analysis output (performed on blood-borne CK activity, MYOM3 quantity, MSTN concentration, and miR-1, -133a and -206 relative quantity) and for each individual biomarker that was significantly elevated in DE50-MD compared to WT blood samples for dogs aged 3–18 months. (CK: creatine kinase; MSTN: myostatin; MYOM3: myomesin-3; WT: wild type.)

Article Snippet: Membranes were blocked in 10% milk powder (Marvel) in phosphate buffered saline (PBS, Fisher Scientific, #BR0014G)/Tween 0.05% (Sigma, #P9416) for one hour at room temperature, before incubation with a polyclonal rabbit anti-human MYOM3 antibody (Proteintech, #17692-1-AP, 1:1000 dilution) and a polyclonal rabbit anti-canine albumin antibody (as internal loading control, Biorbyt Ltd, #orb242465; 1:1,000,000 dilution) overnight at 4 °C.

Techniques: Biomarker Discovery, Activity Assay, Concentration Assay