Expansion of revertant fibers in dystrophic mdx muscles reflects activity of muscle precursor cells and serves as an index of muscle regeneration

Abstract

Duchenne muscular dystrophy and the mdx mouse myopathies reflect a lack of dystrophin in muscles. However, both contain sporadic clusters of revertant fibers (RFs) that express dystrophin. RF clusters expand in size with age in mdx mice. To test the hypothesis that the expansion of clusters is achieved through the process of muscle degeneration and regeneration, we analyzed muscles of mdx mice in which degeneration and regeneration were inhibited by the expression of micro-dystrophins or utrophin transgenes. Postnatal RF expansion was diminished in direct correlation to the protective effect of the transgene expression. Similarly, expansion of RFs was inhibited when muscle regeneration was blocked by irradiation. However, in irradiated muscles, irradiation-tolerant quiescent muscle precursor cells reactivated by notexin effectively restored RF expansion. Our observations demonstrate that revertant events occur initially within a subset of muscle precursor cells. The proliferation of these cells, as part of the regeneration process, leads to the expansion of RF clusters within degenerating muscles. This expansion of revertant clusters depicts the cumulative history of regeneration, thus providing a useful index for functional evaluation of therapies that counteract muscle degeneration.

Comparative Analysis of Antisense Oligonucleotide Sequences for Targeted Skipping of Exon 51 During Dystrophin Pre-mRNA Splicing in Human Muscle

“…At the same time, interstitial mutations at the DMD locus that maintain the dystrophin mRNA open reading frame give rise to internally deleted but semifunctional dystrophins and the milder Becker muscular dystrophy (BMD) (Hoffman et al, 1987;Monaco et al, 1988). Internally truncated but at least partially functional dystrophins are also expressed in so-called revertant fibers, individual dystrophin-positive fibers found in 50% of DMD patients and in mdx mice (Nicholson et al, 1989;Hoffman et al, 1990;Burrow et al, 1991;Fanin et al, 1992;Sherratt et al, 1993;Yokota et al, 2006). Revertant fibers arise via some alternative splicing mechanism occurring within dystrophin pre-mRNAs, skipping of frame-shifting exons to remove protein-truncating mutations, and restoration of the dystrophin open reading frame (Lu et al, 2000).…”

Cell Transplantation and “Stem Cell Therapy” in the Treatment of Myopathies: Many Promises in Mice, Few Realities in Humans

“…These graft-derived satellite cells would be able to participate later in muscle hypertrophy and regeneration. Perhaps the amount of myofibers expressing dystrophin in DMD patients transplanted with normal myoblasts may increase over time, provided that there is a phenomenon similar to that reported in mdx mice, that is, the expansion of clusters of myofibers expressing revertant dystrophin [30].…”

Fetal Microchimeric Cells in a Fetus-Treats-Its-Mother Paradigm Do Not Contribute to Dystrophin Production in Serially ParousmdxFemales

“…Moreover, we did not find FMCs robustly expressing proteins associated with muscle differentiation or integration. Despite using notexin to amplify the muscle regeneration and stimulate production of dystrophin [32], FMCs expressing dystrophin were never found (0/16). Furthermore, only one FMC (1/24) was detected within the basal lamina.…”

“…We therefore evaluated the efficacy of wild-type FMCs as opposed to dystrophin-deficient FMCs to contribute to dystrophin production in mdx females. Notexin-treated TA muscles were chosen for this investigation, as the day-7 timepoint reflects the expected peak for the production of new appropriately localized dystrophin protein [26,32], and mdx muscles previously transplanted with young satellite cells have been shown to have donor-derived dystrophin + ve fibers 7 days after notexin treatment [33]. Individual dystrophin + ve fibers were counted and normalized to the total number of fibers present in each field of view.…”