Uniform sarcolemmal dystrophin expression is required to prevent extracellular microRNA release and improve dystrophic pathology.

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-Xist mice that express variable amounts of dystrophin from birth as a consequence of skewed X-chromosome inactivation. miRNA profiling was performed in mdx-Xist 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-Xist 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-Xist mice regardless of dystrophin expression. PPMO-treated muscles exhibited a uniform pattern of dystrophin localization and were devoid of regenerating fibres, whereas mdx-Xist 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.