Muscular dystrophy in the mdx mouse is a severe myopathy compounded by hypotrophy, hypertrophy and hyperplasia

Abstract

BackgroundPreclinical testing of potential therapies for Duchenne muscular dystrophy (DMD) is conducted predominantly of the mdx mouse. But lack of a detailed quantitative description of the pathology of this animal limits our ability to evaluate the effectiveness of putative therapies or their relevance to DMD.MethodsAccordingly, we have measured the main cellular components of muscle growth and regeneration over the period of postnatal growth and early pathology in mdx and wild-type (WT) mice; phalloidin binding is used as a measure of fibre size, myonuclear counts and BrdU labelling as records of myogenic activity.ResultsWe confirm a two-phase postnatal growth pattern in WT muscle: first, increase in myonuclear number over weeks 1 to 3, then expansion of myonuclear domain. Mdx muscle growth lags behind that of WT prior to overt signs of pathology. Fibres are smaller, with fewer myonuclei and smaller myonuclear domains. Moreover, satellite cells are more readily detached from mdx than WT muscle fibres. At 3 weeks, mdx muscles enter a phase of florid myonecrosis, accompanied by concurrent regeneration of an intensity that results in complete replacement of pre-existing muscle over the succeeding 3 to 4 weeks.Both WT and mdx muscles attain maximum size by 12 to 14 weeks, mdx muscle fibres being up to 50% larger than those of WT as they become increasingly branched. Mdx muscle fibres also become hypernucleated, containing twice as many myonuclei per sarcoplasmic volume, as those of WT, the excess corresponding to the number of centrally placed myonuclei.ConclusionsThe best-known consequence of lack of dystrophin that is common to DMD and the mdx mouse is the conspicuous necrosis and regeneration of muscle fibres. We present protocols for measuring this in terms both of loss of muscle nuclei previously labelled with BrdU and of the intensity of myonuclear labelling with BrdU administered during the regeneration period. Both measurements can be used to assess the efficacy of putative antinecrotic agents. We also show that lack of dystrophin is associated with a number of previously unsuspected abnormalities of muscle fibre structure and function that do not appear to be directly associated with myonecrosis.

Impeding Transcription of Expanded Microsatellite Repeats by Deactivated Cas9

“…Because myotonia is measured by assaying individual bundles of fibers, we hypothesized that some regions of muscle may be rescued and not others, and that any potential change in isoform composition in these regions may be diluted in analyses of bulk tissue. This is consistent with a roughly ∼2.5-fold increase in myonuclei per fiber over the first 4 weeks of post-natal development, which could also dilute the proportion of myonuclei containing AAV episomes (Duddy et al, 2015). AAV6-delivered dSaCas9 distribution in EDL fibers revealed mosaic expression, with rare fibers showing region-specific nuclear signal for dCas9 (Fig.…”

“…Our approach used systemically delivered AAV, which revealed regional variation in transduction efficiency, likely because NLS-tagged Cas9 protein remained restricted to myonuclear domains transduced by AAV episomes, and many new myonuclei are recruited to fibers throughout postnatal muscle development (Duddy et al, 2015). Previous studies with AAV-mediated gene therapy typically evaluate expression of proteins that can spread throughout muscle fibers, precluding measurement of transduction efficiency to all myonuclei.…”

Concurrent Label-Free Mass Spectrometric Analysis of Dystrophin Isoform Dp427 and the Myofibrosis Marker Collagen in Crude Extracts from mdx-4cv Skeletal Muscles

“…, the almost complete loss of the full-length Dp427 isoform of dystrophin is the underlying pathobiochemical defect that triggers progressive fiber degeneration [73], a crucial secondary alteration in dystrophinopathy is presented by extensive myofibrosis [74]. Thus, in addition to central nucleation, rounded fibers, altered myofiber sizes, inflammation, fiber branching, necrosis, and fatty deposition [75], the progressive accumulation of extracellular matrix components plays a major role in contractile weakness [76]. Due to a hyperactive connective tissue, collagens and associated proteins of the extracellular matrix accumulate in dystrophic muscles causing tissue scarring and the loss of skeletal muscle elasticity [77].…”

MicroRNA expression patterns in post-natal mouse skeletal muscle development

“…In the first 3 weeks, muscle precursor cells (also known as myoblasts or satellite cells) proliferate and fuse with the rapidly elongating myofibres to provide new myonuclei (hyperplasia) (Fig. 1a) [26, 27]. From 3 to 8 weeks of age, rapid hypertrophy results in a 3-fold increase in the myofibre size [26].…”

“…From 3 to 8 weeks of age, rapid hypertrophy results in a 3-fold increase in the myofibre size [26]. This increase in sarcoplasmic volume continues until at least 14 to 28 weeks of age [27]. Early autoradiography studies tracking the in vivo kinetics of myoblast proliferation and fusion in healthy muscles of mice aged 6–8 weeks confirmed that most satellite cells were quiescent at this age [28].…”