Myotonic dystrophy (DM) is caused by a (CTG)(n) expansion in the 3′-untranslated region of DMPK gene. Mutant transcripts are retained in nuclear RNA foci, which sequester RNA binding proteins thereby misregulating the alternative splicing. Controversy still surrounds the pathogenesis of the DM1 muscle distress, characterized by myotonia, weakness and wasting with distal muscle atrophy. Eight primary human cell lines from adult-onset (DM1) and congenital (cDM1) patients, (CTG)(n) range 90-1800, were successfully differentiated into aneural-immature and contracting-innervated-mature myotubes. Morphological, immunohistochemical, RT-PCR and western blotting analyses of several markers of myogenesis indicated that in vitro differentiation-maturation of DM1 myotubes was comparable to age-matched controls. In all pathological muscle cells, (CTG)(n) expansions were confirmed by long PCR and RNA fluorescence in situ hybridization. Moreover, the DM1 myotubes showed the splicing alteration of insulin receptor and muscleblind-like 1 (MBNL1) genes associated with the DM1 phenotype. Considerable myotube loss and atrophy of 15-day-differentiated DM1 myotubes indicated activated catabolic pathways, as confirmed by the presence of apoptotic (caspase-3 activation, cytochrome c release, chromatin fragmentation) and autophagic (P62LC3) markers. Z-VAD treatment significantly reduced the decrease in myonuclei number and in average width in 15-day-differentiated DM1 myotubes. We thus propose that the muscle wasting typical in DM1 is due to impairment of muscle mass maintenance- regeneration, through premature apoptotic-autophagic activation, rather than altered myogenesis.
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Acknowledgements. This work was supported by grants from ‘Progetti Ricerca di Interesse Nazionale-Ministero Istruzione-Università-Ricerca grant number 2005064759’ to L Vergani; ‘Progetti di Eccellenza’ Fondazione Cariparo, 2008–09 to C Angelini; ‘Association Franc¸aise contre les Myopathies, grant number 13360’ to C Angelini; and ‘Telethon grant number GGP07250’ to G Novelli. AM was supported by the University of Padua. Muscle samples were provided by Telethon Biobank no. GTF05003.