Pig Models Reveal MicroRNA Networks Driving Duchenne and Becker Muscular Dystrophy

Duchenne muscular dystrophy is one of the most devastating genetic diseases a child can inherit, and yet the molecular choreography that unfolds inside failing muscle has remained only partly mapped. Now, a team working with genetically tailored pigs has charted, in unprecedented detail, the regulatory networks of microRNAs and messenger RNAs that distinguish severe Duchenne disease from its milder Becker counterpart. The study, published in the Journal of Cachexia, Sarcopenia and Muscle, offers both a deeper mechanistic understanding of dystrophinopathies and a shortlist of microRNAs that could serve as biomarkers or therapeutic targets for patients.

Duchenne and Becker muscular dystrophies are X-linked disorders caused by mutations in the dystrophin gene, one of the largest protein-coding genes in mammals. Duchenne affects roughly 4.8 individuals per 100,000 people and typically arises from deletions that disrupt the reading frame, abolishing dystrophin production entirely. The result is progressive muscle degeneration and premature death from cardiorespiratory failure. Becker disease, affecting about 1.5 per 100,000, stems from in-frame mutations that preserve a shortened but partially functional dystrophin protein, producing a far gentler clinical course. A particularly important pathogenic mutation is the deletion of exon 52, which is amenable to exon-skipping therapies that target exon 51 or 53 to restore the reading frame.

Mouse models have long dominated dystrophy research, but they poorly predict human disease progression. The mdx mouse shows only a mild phenotype with robust regeneration and minimal cardiac involvement, while the Golden Retriever muscular dystrophy dog, though clinically closer to humans, suffers from phenotypic variability, long generation intervals and small litter sizes. Pigs occupy a valuable middle ground: their muscle structure and biology closely resemble human muscle, their body size permits realistic dosing and delivery studies, and modern genome editing allows the creation of large, genetically homogeneous cohorts. Using somatic cell nuclear transfer, the researchers previously generated pigs lacking dystrophin exon 52, which develop dystrophin deficiency, impaired mobility and progressive muscle pathology with lifespans of three to nine months. They then deleted exon 51 in those same cells, restoring the reading frame and creating a Becker-like line that expresses dystrophin in skeletal muscle and myocardium with markedly improved pathology.

For the new study, the team analysed triceps brachii biopsies from thirteen animals at 3.5 months of age: four Duchenne-model pigs, four Becker-model pigs and five wild-type controls. They prepared stranded total RNA sequencing libraries and small RNA libraries, sequenced them on an Illumina NextSeq 1000 platform, and aligned the reads to the pig reference genome. Differential expression analysis flagged genes and microRNAs with at least a twofold change and an adjusted p-value of 0.05 or below. To move from correlation to regulation, the researchers predicted microRNA targets across 3′ untranslated regions, 5′ untranslated regions and coding sequences using stringent hybridization criteria, then retained only pairs showing significant inverse correlations between microRNA abundance and messenger RNA expression, consistent with the canonical repressive role of microRNAs.

The messenger RNA results were striking. In Duchenne muscle, 1,440 genes were upregulated and 487 downregulated relative to wild type, while Becker muscle showed 1,804 upregulated and only 155 downregulated genes. Crucially, most changes were condition-specific: 1,282 genes were upregulated only in Duchenne, 1,646 only in Becker, and just 158 were shared. Genes uniquely downregulated in Duchenne muscle were enriched for muscle cell differentiation, sarcomere structure, contractile fibre components and metabolic regulation, including insulin signalling and lipolysis. Genes uniquely upregulated in Duchenne pointed to a fierce immune activation, with enrichment in complement cascades, cytokine signalling, chemokine pathways and phagocytosis. Quantitative PCR confirmed key inflammatory changes, with IL10 and TGFB elevated in Duchenne but not Becker muscle.

On the microRNA front, the team detected 457 microRNAs, of which 34 were significantly dysregulated in Duchenne versus wild type, 57 in Becker versus wild type and 55 in the direct Becker-versus-Duchenne comparison. Hierarchical clustering again placed Duchenne samples in a distinct cluster while Becker samples often grouped with wild type, mirroring the milder phenotype. The resulting microRNA-messenger RNA networks were vast: in the Becker-versus-Duchenne comparison alone, 24 upregulated microRNAs were linked to 875 downregulated target genes through 3,875 unique interactions, while 31 downregulated microRNAs targeted 568 upregulated genes through 2,675 pairs. Enrichment analysis of these targets implicated chemokine signalling, phagosome function, neurogenesis, apoptosis, insulin and glucagon signalling, and synaptic signalling, suggesting that exon skipping reshapes developmental, metabolic and inflammatory regulatory circuits.

Two microRNAs emerged as headline candidates. ssc-miR-296-3p was upregulated exclusively in Duchenne muscle and predicted to target 228 downregulated genes enriched in muscle structure development, skeletal system development, fatty acid metabolism and hormone signalling, positioning it as a potential driver of Duchenne pathology and a pig-specific biomarker. ssc-miR-423-5p, by contrast, was upregulated only in Becker muscle, targeting 67 genes involved in endothelial cell proliferation, tissue development, calcium signalling and cytokine receptor interaction, a profile more consistent with tissue and vascular homeostasis than direct muscle regulation. Neither microRNA had previously been linked directly to human muscular dystrophies, making both novel findings.

Familiar suspects also appeared. miR-199a-5p, previously implicated in dystrophic muscle pathogenesis and elevated in exosomes from Duchenne muscle fibroblasts, was strongly upregulated in Duchenne pigs and predicted to target 77 downregulated genes, while miR-199b, known to repress porcine muscle satellite cell proliferation through a JAG1-NOTCH1 feedback loop, targeted 22 genes. A cross-species comparison with published human datasets covering ten neuromuscular disorders identified 35 microRNAs dysregulated in both the porcine data and human data, underscoring shared molecular mechanisms. Intriguingly, several microRNAs known for protective or anti-atrophic effects, including miR-199a-3p, miR-23a, miR-223, miR-146b and miR-224, were also upregulated in Duchenne muscle, possibly reflecting compensatory feedback against wasting and fibrosis. In Becker muscle, the greater number of dysregulated microRNAs may reflect active regeneration and remodelling, whereas advanced degeneration in Duchenne may simplify the regulatory landscape.

The functional implications align with earlier work showing severely reduced muscle force in Duchenne pigs and partial, incomplete recovery in Becker animals. The constitutive genomic deletion in the Becker-like pigs provides uniform reframing across all muscle fibres, representing an upper bound of molecular rescue that FDA-approved exon-skipping therapies, which achieve low and variable dystrophin restoration in patients, do not reach. Exon 51 skipping is applicable to roughly 14 percent of Duchenne patients, particularly those with deletions involving exon 50 or 52, so the pig model offers a realistic ceiling for what such interventions could accomplish at the molecular level. The authors caution that whole-muscle biopsies may partly reflect shifts in cellular composition, such as inflammatory infiltration, and that the predicted microRNA-messenger RNA interactions require functional validation to establish causality; future single-nucleus RNA sequencing should help disentangle cell-type-specific contributions.

Even with those caveats, the study delivers a resource with immediate translational value. Sixteen microRNAs were specifically upregulated in Duchenne relative to both Becker and wild type, and eighteen were upregulated specifically in Becker, each with distinct target gene sets and pathway signatures. These condition-specific signatures could support non-invasive disease stratification, monitoring of exon-skipping therapy efficacy and the design of microRNA-based interventions. Combined with earlier demonstrations that these pig models support non-invasive imaging of muscle fibrosis and in vivo CRISPR/Cas9 genome editing, the new microRNA-messenger RNA atlas strengthens the case that tailored porcine dystrophinopathy models can bridge the persistent gap between encouraging rodent studies and the harsh realities of human therapeutic development.

Subject of Research: MicroRNA-mRNA regulatory networks in skeletal muscle of genetically engineered pig models of Duchenne and Becker muscular dystrophy

Article Title: MicroRNA–mRNA Networks in Skeletal Muscle of Tailored Pig Models for Dystrophinopathies

Article References: Reschke, S., Graf, A., Hadlich, F., Jaudas, F., Fröhlich, T., Stirm, M., Klymiuk, N., Krebs, S., Wolf, E., & Ali, A. (2026). MicroRNA–mRNA Networks in Skeletal Muscle of Tailored Pig Models for Dystrophinopathies. Journal of Cachexia, Sarcopenia and Muscle, 17(5), Article e70337. https://doi.org/10.1002/jcsm.70337

Image Credits: AI Generated

DOI: 10.1002/jcsm.70337

Keywords: Duchenne muscular dystrophy, Becker muscular dystrophy, microRNA, dystrophin, exon skipping, pig models, skeletal muscle, RNA sequencing, biomarkers, gene regulation, muscle degeneration, porcine models