Normal myogenesis and increased apoptosis in myotonic dystrophy type-1 muscle cells

Abstract

Myotonic dystrophy (DM) is caused by a (CTG) n expansion in the 3 0 -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 (P62/LC3) 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 maintenanceregeneration, through premature apoptotic-autophagic activation, rather than altered myogenesis. Myotonic dystrophy (DM) is a multi-systemic disorder caused by two different microsatellite expansions in non-coding regions. Together, these two mutations affect 1 out of 8000 individuals and represent the most common form of muscular dystrophy in adults. DM1 and DM2 have common symptoms such as myotonia, muscle weakness and early cataract development. 1,2 Although DM1 and DM2 initially affect different muscles (distal versus proximal), histological analysis of the muscular tissues shows common aspects such as central nucleation. The classic form of DM1 is characterized by muscle distress with myotonia, progressive muscle weakness and wasting. Atrophy has also been reported, occurring preferentially in type-1 fibers in DM1 and in type-2 in DM2. 3 DM1 but not DM2 also presents a congenital form (cDM1), characterized by a high neonatal mortality and symptoms such as hypotonia, mental retardation and respiratory distress. 4,5 DM1 is associated with an unstable (CTG) n trinucleotide expansion located in the 3 0 -untranslated (3 0 -UTR) region of the DM protein kinase (DMPK) gene on chromosome 19q13.3. The mutant DMPK transcript, containing the expanded (CTG) n sequence, accumulates in discrete nuclear foci able to sequester various nuclear factors such as RNAbinding proteins or splicing regulators, causing different and highly variable downstream deleterious effects. 2,6...

Targeting deregulated AMPK/mTORC1 pathways improves muscle function in myotonic dystrophy type I

“…On the basis of the abnormal splicing and protein trafficking of the insulin receptor in metabolic tissues in DM1 (15,16), it has also been hypothesized that PKB/Akt-mTORC1 may be less responsive to insulin. However, results regarding the activation state of PKB/Akt signaling in DM1 human muscle biopsies or cells are conflicting (18,19). In our experiments, we did not detect changes in PKB/Akt activation or in the expression of Insr in HSA LR muscle, suggesting that AMPK/mTORC1 deregulation is independent of insulin receptor deficiency.…”

“…The involvement of autophagy in DM1 was largely deduced from the presence of autophagic vesicles and/or accumulation of autophagic markers in DM1 cells, but usually without dynamic measurement of the autophagic flux (13,14,17,19,36,37,49). In our study, we combined several methods to establish that mild changes in autophagic markers in muscle from HSA LR mice are caused by autophagic flux limitation during the degradation steps.…”

“…In parallel, perturbation in the PKB/Akt pathway may arise from altered expression of the insulin receptor, which correlates with glucose intolerance in DM1 patients (15). Although PKB/Akt deregulation has been reported in Dmpk-deficient mice (16), in DM1 flies (13), and in DM1 human neural stem cells (17), contradictory results have been obtained in human muscle cells (18,19).…”

Generation and Use of Cultured Human Primary Myotubes

“…As such primary skeletal muscle myotubes have multiple research applications including but not limited to; 1) the study of the effects of myopathies and systemic metabolic diseases on skeletal muscle function, 2) tissue regeneration and renewal for tissue engineering purposes, 3) gene therapy and 4) drug screening (Berggren et al, 2007;Chen et al, 2005;Kessler et al, 1996;Loro et al, 2010;McAinch & Cameron-Smith, 2009;McAinch et al, 2006a;McAinch et al, 2006b;McAinch et al, 2007;Stern-Straeter et al, 2008).…”

Lactate levels affect motor performance in MD 1

“…15 Recent studies evidenced the higher presence of reactive oxygen species (ROS) that suggests a failure in the removal system of toxic proteins that interfere with mitochondria function, inducing the release of pro-apoptotic factors. 16 Mitochondria have the major role in energetic homeostasis, thus determining ATP availability in the cells. Indeed, their dysfunction will be unable to meet cellular ATP demands, thus compromising the cellular ability to adapt to physiological stress.…”