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... Show moreBackground 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(Delta hs) mice that express variable amounts of dystrophin from birth as a consequence of skewed X-chromosome inactivation. miRNA profiling was performed in mdx-Xist(Delta hs) 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-miRNA abundance observed following PPMO treatment was not recapitulated in the high dystrophin-expressing mdx-Xist(Delta hs) group, despite these animals expressing similar amounts of total dystrophin protein (similar to 37% of wild-type levels). Instead, ex-miRNAs were present at high levels in mdx-Xist(Delta hs) mice regardless of dystrophin expression. PPMO-treated muscles exhibited a uniform pattern of dystrophin localization and were devoid of regenerating fibres, whereas mdx-Xist(Delta hs) 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. Show less
Kogelman, B.; Putker, K.; Hulsker, M.; Tanganyika-de Winter, C.; Weerd, L. van der; Aartsma-Rus, A.; Putten, M. van 2018
Duchenne muscular dystrophy is a severe muscle wasting disease, characterized by a severely reduced lifespan in which cardiomyopathy is one of the leading causes of death. Multiple therapies aiming... Show moreDuchenne muscular dystrophy is a severe muscle wasting disease, characterized by a severely reduced lifespan in which cardiomyopathy is one of the leading causes of death. Multiple therapies aiming at dystrophin restoration have been approved. It is anticipated that these therapies will maintain muscle function for longer and extend the ambulatory period, which in turn will increase the cardiac workload which could be detrimental for cardiac function. We investigated the effects of voluntary running exercise in combination with low dystrophin levels on function and pathology of skeletal muscle and heart. We divided 15.5-month old female mdx (no dystrophin), mdx-Xist(Delta hs) (varying low dystrophin levels) and wild type mice (BL10-WT and Xist(Delta hs)-WT) to either a sedentary or voluntary wheel running regime and assessed muscle function at 17.5 months of age. Thereafter, a cardiac MRI was obtained, and muscle and heart histopathology were assessed. We show that voluntary exercise is beneficial to skeletal muscle and heart function in dystrophic mice while not affecting muscle pathology. Low amounts of dystrophin further improve skeletal muscle and cardiac function. These findings suggest that voluntary exercise may be beneficial for skeletal muscle and heart in DMD patients, especially in conjunction with low amounts of dystrophin. Show less
Veltrop, M.; Vliet, L. van; Hulsker, M.; Claassens, J.; Brouwers, C.; Breukel, C.; ... ; Putten, M. van 2018
