Résumé :
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The diagnosis of muscular diseases, or the assessment of the functional benefit of gene or cell therapies, often remains difficult. Duchenne muscular dystrophy results from the lack of dystrophin. This protein mediates part of a physical link between the cytoskeleton and the extracellular matrix of muscle fibers, and its absence leads to muscle dystrophic pattern. We have shown that the absence of dystrophin affects the elasticity of individual fibers within the muscle tissue, as probed using atomic force microscopy (AFM), providing a sensitive and quantitative assay of the properties of normal and dystrophic myofibers at the nanometric scale (Puttini et al., Mol Ther 2009 :17 ;19-25). AFM was shown to provide a sensitive and quantitative description of the resistance to deformation of normal and dystrophic myofibers within live muscle tissues explanted from Duchenne mdx mice. Utrophin expression, or the rescue of dystrophin expression, by gene therapy approaches led to the functional recovery of treated dystrophic muscle fibers, as probed using AFM and by whole-muscle strength measurements. This indicated that the presence or absence of a functional dystrophin protein can be directly assessed within live muscle fibers. Comparison of muscles treated with viral or non-viral gene therapy vectors indicated that the efficacy of the gene transfer approaches could be distinguished with a single myofiber resolution. Full correction of the resistance to deformation in nearly all of the muscle fibers was observed upon treatment with an adenoviral gene therapy vector. Having shown that AFM can provide a quantitative assessment of the expression of muscle proteins and of the muscular function in animal models, we assessed myofiber resistance to deformation in the context of various human muscular myopathies. We observed that various forms of human muscle disorders could also be detected using AFM assays of small frozen biopsies. Interestingly, in biopsies analysed from the affected adults of a Nemaline myopathy family we even detected anomalies in the less affected patients. This approach may thereby allow an estimation of the proportion of muscle fibers with abnormal properties, and also of the severity of the effect. Overall, we conclude that AFM allows a functional and sensitive assessment of live cells at the nanometer scale within the context of the biological tissue. Thus, this method may complement current diagnosis tools for known and unknown muscle human diseases, in research and in a clinical context.
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