Transcriptomic profiling of skeletal muscle in the DMDmdx rat model of Duchenne muscular dystrophy

Abstract

Duchenne muscular dystrophy (DMD) is a severe X-linked recessive disorder caused by a mutation in the Dmd gene, leading to progressive muscle degradation, increasing weakness, and typically resulting in death before the third decade of life. To investigate the pathobiology of DMD, this study employed the Sprague-Dawley Dmd-mutated rat model (DMD^mdx) and analyzed gene expression profiles and pathological molecular pathways. The methods used included histopathological, biochemical, and transcriptomic analyses of dystrophic skeletal muscle from DMD^mdx and wild-type (WT) individuals. Histological analysis of skeletal muscle tissue from DMD^mdx rats revealed multifocal necrosis, fibrosis, and inflammation, whereas WT rats displayed normal muscle architecture. Biochemical analysis revealed significant alterations in plasma markers of muscle damage and metabolism in DMD^mdx rats compared to WT controls, including elevated AST, ALT, ALP, CPK, and LDH levels. Additionally, oxidative status measurements showed reduced antioxidant capacity and increased lipid peroxidation in dystrophic skeletal muscle, as evidenced by lower TAS, GR, GPx, and SOD activities and higher TBARS levels. RNA-seq analysis identified 3,615 differentially expressed genes between the two groups, associated with muscle contraction, extracellular matrix (ECM) organization, and cytoskeleton organization. Notably, Dmd, Actc1, Col6a1, and Mmp2 were significantly downregulated. Gene ontology and pathway enrichment analyses indicated dystrophic changes in skeletal muscle, disruptions in calcium homeostasis, and alterations in actin cytoskeleton regulation. KEGG and Reactome pathway analyses revealed upregulation of the MAPK signaling and immune system pathways and downregulation of the ECM organization pathway. These findings support the hypothesis that targeting complex intracellular signaling pathways in DMD may represent a promising therapeutic strategy. Given that the DMD^mdx rat model closely mimics human DMD pathology compared to other animal models, it offers a more realistic platform for studying the molecular mechanisms of the disease and improving the translational potential of therapeutic approaches.

Keywords: Duchenne muscular dystrophy (DMD); Dystrophin gene mutation; MAPK signaling pathway, DMD mdx rat model; RNA-seq gene expression analysis; Skeletal muscle degeneration.

Fig. 1

Histopathological features in the biceps femoris muscle of DMD^mdx and WT rats: A) multifocal necrosis of myofibers (asterisk) and inflammatory infiltrates with a predominance of mononuclear cells (arrows), DMD^mdx, H&E stain, 400x; B) satellite cells at the periphery of myofibers (arrows) in DMD^mdx rat, indicating myofiber regeneration, H&E stain, 400x; C) marked variation in myofiber size, DMD^mdx, H&E stain, 400x; D) interstitial hyperplasia of fibrous connective tissue (arrows) in DMD^mdx, PSR stain, 100x; E) normal muscle fibers of WT, H&E stain, 100x, F) normal wild-type muscle of WT, PSR stain, 100x. DMD^mdx – Sprague-Dawley DMD^mdx rats; WT – Sprague-Dawley wild-type rats; H&E – hematoxylin and eosin; PSR – Picrosirius red.

Fig. 2

Biceps femoris muscle fiber diameter (µm) (A) and the average fibrous connective tissue and muscle fiber area in WT and DMD^mdx rats (percentage) (B). DMD^mdx muscle structure, PSR stain, 40x (C) and WT muscle structure, PSR stain, 40x (D). DMD^mdx – Sprague-Dawley DMD^mdx rats; WT – Sprague-Dawley wild-type rats; PSR – Picrosirius red. The dots represent the mean; the boxes – the standard error; the whiskers – the standard deviation. All comparisons between DMD^mdx and WT rats are statistically significant (p < 0.05; n = 7 per genotype).

Fig. 3

The biochemical analysis of blood plasma. CPK – creatine phosphokinase; LDH – lactate dehydrogenase; ALT – alanine aminotransferase; DMD^mdx – Sprague-Dawley DMD^mdx rats; WT – Sprague-Dawley wild-type rats. The dots represent the mean; the boxes – the standard error; the whiskers – the standard deviation. All comparisons between DMD^mdx and WT rats are statistically significant (p < 0.05; n = 7 per genotype).

Fig. 4

The antioxidative status parameters. TAS – total antioxidant status; GSH – glutathione; TBARS – thiobarbituric acid reactive substances; SOD – superoxide dismutase; DMD^mdx – Sprague-Dawley DMD^mdx rats; WT – Sprague-Dawley wild-type rats. The dots represent the mean; the boxes – the standard error; the whiskers – the standard deviation. All comparisons between DMD^mdx and WT rats are statistically significant (p < 0.05; n = 7 per genotype).

Fig. 5

The distribution of DEGs between the DMD^mdx and WT rat biceps femoris muscle samples: A) The volcano plot with gene distribution. The log2 fold change (log2FC) is shown on the x-axis, while the -log10 (p-value) is shown on the y-axis. The genes on the left side are downregulated in DMD^mdx rats compared to the WT, while genes on the right are significantly upregulated. The vertical dashed lines represent a threshold of differential expression standard value log2FC > |0.3|. The horizontal lines represent the threshold for statistical significance (P < 0.05; n = 4 per genotype). B) Top 10 upregulated and downregulated genes in DMD^mdx rats compared to WT. Ranking according to the log2 fold change (log2FC). Significantly upregulated genes are colored green, while significantly downregulated genes are colored red. Each bar displays the level of expression change and the p-value. DMD^mdx – Sprague-Dawley DMD^mdx rats; WT – Sprague-Dawley wild-type rats; DEGs – differentially expressed genes.

Fig. 6

Top 15 enriched Gene Ontology (GO) terms categorized into Biological Processes (BP, red), Cellular Components (CC, blue), and Molecular Functions (MF, green), identified using DEGs in DMD^mdx vs. WT rat biceps femoris muscle samples. DMD^mdx – Sprague-Dawley DMD^mdx rats; WT – Sprague-Dawley wild-type rats; DEGs – differentially expressed genes.

Fig. 7

Pathway analysis of identified DEGs in DMD^mdx vs. WT rat biceps femoris muscle samples using A) KEGG and B) Reactome. KEGG pathways are sorted by the number of genes. The left column lists the target pathways, while the right column categorizes them according to their assigned KEGG classifications. Reactome pathways are sorted based on the p-value. DMD^mdx – Sprague-Dawley DMD^mdx rats; WT – Sprague-Dawley wild-type rats; KEGG – Kyoto Encyclopedia of Genes and Genomes DEGs – differentially expressed genes.

Fig. 8

Relevance network of identified DEGs. Nodes in the network represent genes, while the expression status is indicated through color coding, where downregulated genes appear red and upregulated genes show green. Blue squares identify DMD-related biological processes.