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Asynchronous Bouts of Muscle Regeneration Lead to Features of Failed Regeneration in the Muscular Dystrophies Open Access

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In many of the muscular dystrophies, age-related failure of regeneration is often seen with progressive replacement of the muscle with fibrofatty connective tissue. In Duchenne muscular dystrophy, lack of dystrophin is compatible with muscle function. However, failure in regeneration is associated with significant muscle wasting, weakness, and functional disability suggesting that dystrophin deficiency is necessary, but not sufficient for disability. The goal of this dissertation research was to develop a model that explains failed regeneration in DMD and other chronic inflammatory diseases. The mRNA profiling of 166 patient muscle biopsies from 12 disease groups showed a 56 member protein network centered on TGFâ associated with severe pathology disease groups (fibrosis and failed regeneration). Studying the second independent muscle biopsy data set of four muscle diseases (49 biopsies) showed that indeed the TGFâ network is driven by tissue fibrosis and failed regeneration. Superimposing TGFâ associated network on a 27 time point murine normal muscle regeneration series showed that these 56 members of TGFâ network are also expressed during normal regeneration process. However, the single network parsed into time-specific subnetworks, suggesting that failed regeneration may be due to inappropriate crosstalk between different temporal stages of regeneration happening within the same dystrophic microenvironment. To evaluate inappropriate crosstalk that may explain weakness and failed regeneration in muscular dystrophies, we developed an asynchronous remodeling model. According to this model, two week regeneration process was predicted to be disrupted due to inappropriate crosstalk between neighboring injured myofibers that are at different stages of regeneration. To create an experimental mouse model with focal asynchronous bouts of muscle regeneration, we induced episodes of staged degeneration and regeneration in normal (wild-type) mouse muscle using localized notexin injection at two adjacent sites in different time points during the two week regeneration window. The first set of injections was separated by four days and the second set by 10 days. Laser capture microscopy was used to isolate each region of regeneration (first bout and second bout) and the myofibers in between the staged injection sites (crosstalk areas). The mRNA profiling and immunohistochemical studies showed that the crosstalk areas become inappropriately fixed in the developmental time point by which the initial bouts were separated. Consequently, these conditions mediated a chronic inflammatory state and mitochondrial insufficiency in 4 days and a chronic pro-fibrotic state in 10 days crosstalk areas. We showed that molecular networks associated with these localized areas of pro-inflammatory states were suppressed by treatment with glucocorticoids and VBP15. In conclusion, our data suggest that neighboring asynchronous bouts create inappropriate crosstalk between cells in different stages of the muscle regeneration which results in failed regeneration and the pro-fibrotic/ pro-inflammatory states. However, successful muscle remodeling is a synchronous process where all myofibers are at the same stage of regeneration and can complete the regeneration process in an orchestrated manner. Furthermore, our data support a model for failed regeneration and pathological fibrosis conditions in muscular dystrophies. Failure in regeneration model can be generalized to chronic inflammatory states in other diseased tissues where injured cells cannot successfully regenerate. This model also introduces a novel mechanism of action for glucocorticoids in many of these disorders in that they serve to re-synchronize remodeling, much as diurnal cortisol fluctuations do in most animals.

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