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Plot shows over-represented pathways for genes up-regulated in 6 m GRMD MHG. Statistical significance (smaller p values = larger numbers = greater significance) is shown on the x-axis, and numbers of genes altered per pathway is shown to the right of each bar. (DOCX 32 kb)

Functional analysis results of GRMD-induced gene alterations. MHG = medial head of the gastrocnemius, LV = left ventricle, B-H p val = Benjamini & Hochberg-corrected p value, No. = number of differentially expressed genes in the given functional category, ns = not significant, Dash (“-”) indicates not identified in the analysis. (DOCX 16 kb)

Plot shows over-represented pathways for genes altered in 6 m (light blue bars, top) or 12 m (dark blue bars, bottom) GRMD LV. Statistical significance (smaller p values = larger numbers = greater significance) is shown on the x-axis, and numbers of genes altered per pathway is shown to the right of each bar. AMPK signaling pathway genes are shown below the corresponding pathway bar, colored based on directionality (red for up-regulation and green for down-regulation, respectively in GRMD versus normal dogs). (DOCX 26 kb)

Hierarchical clustering of 5,835 significantly altered transcripts is shown. Rows represent individual samples, labeled according to group, and columns represent individual transcripts. Bright red, bright blue and gray represent highest, lowest and median normalized and scaled signal intensity values, respectively. (DOCX 116Â kb)

Plot shows over-represented pathways for genes up-regulated in 6 m GRMD MHG. Statistical significance (smaller p values = larger numbers = greater significance) is shown on the x-axis, and numbers of genes altered per pathway is shown to the right of each bar. (DOCX 32 kb)

Plot shows over-represented pathways for genes altered in 6 m (light blue bars, top) or 12 m (dark blue bars, bottom) GRMD LV. Statistical significance (smaller p values = larger numbers = greater significance) is shown on the x-axis, and numbers of genes altered per pathway is shown to the right of each bar. AMPK signaling pathway genes are shown below the corresponding pathway bar, colored based on directionality (red for up-regulation and green for down-regulation, respectively in GRMD versus normal dogs). (DOCX 26 kb)

Functional analysis results of GRMD-induced gene alterations. MHG = medial head of the gastrocnemius, LV = left ventricle, B-H p val = Benjamini & Hochberg-corrected p value, No. = number of differentially expressed genes in the given functional category, ns = not significant, Dash (“-”) indicates not identified in the analysis. (DOCX 16 kb)

Plot shows over-represented pathways for genes down-regulated in 6 m GRMD MHG. Statistical significance (smaller p values = larger numbers = greater significance) is shown on the x-axis, and numbers of genes altered per pathway is shown to the right of each bar. (DOCX 35 kb)

Plot shows over-represented pathways for genes up- (red bars) or down-regulated (green bars) in 12 m GRMD MHG. Statistical significance (smaller p values = larger numbers = greater significance) is shown on the x-axis, and numbers of genes altered per pathway is shown to the right of each bar. (DOCX 30 kb)

Plot shows over-represented pathways for genes down-regulated in 6 m GRMD MHG. Statistical significance (smaller p values = larger numbers = greater significance) is shown on the x-axis, and numbers of genes altered per pathway is shown to the right of each bar. (DOCX 35 kb)

Volcano plot is shown highlighting gene expression differences between dystrophic left ventricle (LV) and medial head of the gastrocnemius (MHG) skeletal muscle of golden retriever muscular dystrophy (GRMD) dogs at 6 months. The y-axis displays p values, and fold-differences are plotted on the x-axis for comparison of GRMD LV versus MHG (LV/MHG). Each circle represents an individual probe set (gene) and is colored based on fold-difference (blue for genes with lower expression in GRMD MHG and higher expression in LV, red for genes with higher expression values in LV and lower in MHG, and gray for no change), as shown in the legend. Orange lines mark cut-off values (p value 2). Official gene symbols are indicated to highlight major expression differences between LV and MHG. (PPTX 136 kb)

Plot shows over-represented pathways for genes up- (red bars) or down-regulated (green bars) in 12 m GRMD MHG. Statistical significance (smaller p values = larger numbers = greater significance) is shown on the x-axis, and numbers of genes altered per pathway is shown to the right of each bar. (DOCX 30 kb)

Background Myostatin (Mstn) is a negative regulator of muscle growth whose inhibition promotes muscle growth and regeneration. Dystrophin-deficient mdx mice in which myostatin is knocked out or inhibited postnatally have a less severe phenotype with greater total mass and strength and less fibrosis and fatty replacement of muscles than mdx mice with wild-type myostatin expression. Dogs with golden retriever muscular dystrophy (GRMD) have previously been noted to have increased muscle mass and reduced fibrosis after systemic postnatal myostatin inhibition. Based partly on these results, myostatin inhibitors are in development for use in human muscular dystrophies. However, persisting concerns regarding the effects of long-term and profound myostatin inhibition will not be easily or imminently answered in clinical trials. Methods To address these concerns, we developed a canine (GRippet) model by crossbreeding dystrophin-deficient GRMD dogs with Mstn-heterozygous (Mstn +/−) whippets. A total of four GRippets (dystrophic and Mstn +/−), three GRMD (dystrophic and Mstn wild-type) dogs, and three non-dystrophic controls from two litters were evaluated. Results Myostatin messenger ribonucleic acid (mRNA) and protein levels were downregulated in both GRMD and GRippet dogs. GRippets had more severe postural changes and larger (more restricted) maximal joint flexion angles, apparently due to further exaggeration of disproportionate effects on muscle size. Flexors such as the cranial sartorius were more hypertrophied on magnetic resonance imaging (MRI) in the GRippets, while extensors, including the quadriceps femoris, underwent greater atrophy. Myostatin protein levels negatively correlated with relative cranial sartorius muscle cross-sectional area on MRI, supporting a role in disproportionate muscle size. Activin receptor type IIB (ActRIIB) expression was higher in dystrophic versus control dogs, consistent with physiologic feedback between myostatin and ActRIIB. However, there was no differential expression between GRMD and GRippet dogs. Satellite cell exhaustion was not observed in GRippets up to 3 years of age. Conclusions Partial myostatin loss may exaggerate selective muscle hypertrophy or atrophy/hypoplasia in GRMD dogs and worsen contractures. While muscle imbalance is not a feature of myostatin inhibition in mdx mice, findings in a larger animal model could translate to human experience with myostatin inhibitors. Published version

Table S2. T2 mapping values (Mean ± SD) in non-dystrophic control, GRMD (Mstn +/+ ), and GRippet (Mstn +/− ) Dogs (mean ± SD). (DOCX 369 kb)

Table S1. MRI volumes in non-dystrophic control, GRMD (Mstn +/+ ), and GRippet (Mstn +/â ) Dogs (Meanâ Âąâ SD). (DOCX 954 kb)

Table S3. Histopathologic, morphometric, and molecular findings in non-dystrophic control, GRMD (Mstn +/+ ), and GRippet (Mstn +/â ) dogs. (DOCX 403 kb)