Uniform sarcolemmal dystrophin expression is required to prevent extracellular microRNA release and improve
Tirsa L E van Westering1, Yulia Lomonosova1,2, Anna M L Coenen-Stass1
1Department of Physiology, Anatomy and Genetics, University of Oxford, South Parks Road, Oxford, UK.
Background:
Duchenne muscular dystrophy (DMD) is a fatal muscle-wasting disorder caused by genetic loss of dystrophin protein. Extracellular microRNAs (ex-miRNAs) are putative, minimally invasive biomarkers of DMD. Specific ex-miRNAs (e.g. miR-1, miR-133a, miR-206, and miR-483) are highly up-regulated in the serum of DMD patients and dystrophic animal models and are restored to wild-type levels following exon skipping-mediated dystrophin rescue in mdx mice. As such, ex-miRNAs are promising pharmacodynamic biomarkers of exon skipping efficacy. Here, we aimed to determine the degree to which ex-miRNA levels reflect the underlying level of dystrophin protein expression in dystrophic muscle.
Methods:
Candidate ex-miRNA biomarker levels were investigated in mdx mice in which dystrophin was restored with peptide-PMO (PPMO) exon skipping conjugates and in mdx-XistΔhs mice that express variable amounts of dystrophin from birth as a consequence of skewed X-chromosome inactivation. miRNA profiling was performed in mdx-XistΔhs mice using the FirePlex methodology and key results validated by small RNA TaqMan RT-qPCR. The muscles from each animal model were further characterized by dystrophin western blot and immunofluorescence staining.
Results:
The restoration of ex-myomiR abundance observed following PPMO treatment was not recapitulated in the high dystrophin-expressing mdx-XistΔhs group, despite these animals expressing similar amounts of total dystrophin protein (~37% of wild-type levels). Instead, ex-miRNAs were present at high levels in mdx-XistΔhs mice regardless of dystrophin expression. PPMO-treated muscles exhibited a uniform pattern of dystrophin localization and were devoid of regenerating fibres, whereas mdx-XistΔhs muscles showed non-homogeneous dystrophin staining and sporadic regenerating foci.
Conclusions:
Uniform dystrophin expression is required to prevent ex-miRNA release, stabilize myofiber turnover, and attenuate pathology in dystrophic muscle.
Insights
Extracellular microRNAs (ex-miRNAs) are not reliable biomarkers for Duchenne muscular dystrophy (DMD) when dystrophin protein expression is uneven. Uniform dystrophin expression is crucial for stabilizing muscle fibers and reducing disease severity in DMD.
Area of Science:
- Biochemistry
- Genetics
- Molecular Biology
Background:
- Duchenne muscular dystrophy (DMD) is a fatal genetic disorder characterized by the loss of dystrophin protein.
- Extracellular microRNAs (ex-miRNAs) are being investigated as potential minimally invasive biomarkers for DMD.
- Certain ex-miRNAs are elevated in DMD patients and animal models and decrease with successful dystrophin restoration.
Purpose of the Study:
- To investigate the relationship between ex-miRNA levels and dystrophin protein expression in dystrophic muscle.
- To assess the utility of ex-miRNAs as pharmacodynamic biomarkers for exon skipping therapies.
Main Methods:
- Studied ex-miRNA levels in mdx mice treated with peptide-PMO (PPMO) exon skipping conjugates.
- Analyzed ex-miRNA profiles in mdx-XistΔhs mice with variable dystrophin expression.
- Validated miRNA levels using RT-qPCR and assessed dystrophin protein via Western blot and immunofluorescence.
Main Results:
- Ex-miRNA restoration was not observed in mdx-XistΔhs mice with high, but non-uniform, dystrophin expression (~37% WT).
- Ex-miRNAs remained elevated in mdx-XistΔhs mice irrespective of dystrophin levels.
- PPMO treatment led to uniform dystrophin localization and reduced muscle regeneration, unlike the non-homogeneous staining in mdx-XistΔhs mice.
Conclusions:
- Uniform dystrophin expression is necessary to inhibit ex-miRNA release.
- Consistent dystrophin levels are key for stabilizing myofiber turnover and mitigating DMD pathology.
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