Molecular Basis for Impaired Muscle Differentiation in Myotonic Dystrophy

Timchenko, Nikolai A.; Iakova, Polina; Cai, Zong-Jin; Smith, James R.; Timchenko, Lubov

doi:10.1128/mcb.21.20.6927-6938.2001

Cited by 173 publications

(209 citation statements)

References 42 publications

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“…The strong reduction of the mutated DMPK transcripts by the (CUG)13 antisense could explain the restored differentiation of the infected DM1 myoblasts. This possibility is also supported by the recent report of Timchenko et al 33 showing that alterations in the activity of the CUGBP1 by the mutant DMPK transcripts causes disruption of p21-dependent control of cell cycle arrest, rendering DM1 myoblasts incapable of withdrawing from the cell cycle and fuse to form myotubes. We demonstrated that the misregulation of the CUG-binding protein in DM1 myoblasts was corrected in the antisense-infected DM1 myoblasts, indicating that molecular alterations induced by the mutated DMPK transcript were reversed by the expression of the (CUG) 13 antisense in DM1 cells.…”

Section: Targeting Of Mutant Dmpk Transcripts D Furling Et Almentioning

confidence: 53%

“…Proteins from normal, DM1 and DM1 myoblasts infected with recombinant retrovirus expressing antisense CUG repeats were incubated with radioactive RNA for 30 min at room temperature, and then subjected to UV treatment for 5 min as described. 33 The samples were treated with RNase A and then baded on 10% polyacrylamide gel containing 0.1% SDS. After electrophoresis, proteins were transferred onto nitrocellulose membrane, and subjected to radioautography.…”

Section: Uv-crosslinking Analysismentioning

confidence: 99%

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Viral vector producing antisense RNA restores myotonic dystrophy myoblast functions

Furling

Doucet²,

Ma³

et al. 2003

Gene Ther

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Myotonic dystrophy (DM1) is caused by the expansion of a trinucleotide repeat (CTG) located in the 3 0 untranslated region of the myotonic dystrophy protein kinase gene, for which currently there is no effective treatment. The data available suggest that misregulation of RNA homeostasis may play a major role in DM1 muscle pathogenesis. This indicates that the specific targeting of the mutant DMPK transcripts is essential to raise the rationale basis for the development of a specific gene therapy for DM1. We have produced a retrovirus which expresses a 149-bp antisense RNA complementary to the (CUG)13 repeats and to the 110-bp region following the repeats sequence to increase the specificity. This construct was introduced into human DM1 myoblasts, resulting in a preferential decrease in mutant DMPK transcripts, and effective restoration of human DM1 myoblast functions such as myoblast fusion and the uptake of glucose. It was previously shown that delay of muscle differentiation and insulin resistance in DM1 are associated with misregulation of CUGBP1 protein levels. The analysis of CUGBP1 levels and activity in DM1 cells expressing the antisense RNA indicated a correction of CUGBP1 expression in infected DM1 cells. We therefore show that current antisense RNA delivered in vitro using a retrovirus is not only capable of inhibiting mutant DMPK transcripts, but also can ameliorate dystrophic muscle pathology at the cellular levels. Gene Therapy (2003) 10, 795-802.

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Section: Targeting Of Mutant Dmpk Transcripts D Furling Et Almentioning

confidence: 53%

Section: Uv-crosslinking Analysismentioning

confidence: 99%

Viral vector producing antisense RNA restores myotonic dystrophy myoblast functions

Furling

Doucet²,

Ma³

et al. 2003

Gene Ther

View full text Add to dashboard Cite

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“…Therefore, it seems that proliferation and differentiation are intrinsic features of the individual samples, and are not correlated with their disease status. In this respect, the HIBM primary myoblast cultures differ from those derived from myotonic dystrophy and HD patients, 18 that showed lower differentiation index than controls, and from congenital myotonic dystrophy (CMD), 19 and Duchenne muscular dystrophy (DMD) cells, 20,21 in which both proliferation and differentiation abilities are damaged. Thus, in these pathologies, basic biological functions of muscle satellite cells in culture are impaired.…”

Section: Discussionmentioning

confidence: 99%

Characterization of hereditary inclusion body myopathy myoblasts: possible primary impairment of apoptotic events

et al. 2007

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Hereditary inclusion body myopathy (HIBM) is a unique muscular disorder caused by mutations in the UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE) gene. GNE encodes a bi-functional enzyme acting in the biosynthetic pathway of sialic acid. Since the underlying myopathological mechanism leading to the disease phenotype is poorly understood, we have established human myoblasts cultures, derived from HIBM satellite cells carrying the homozygous M712T mutation, and identified cellular and molecular characteristics of these cells. HIBM and control myoblasts showed similar heterogeneous patterns of proliferation and differentiation. Upon apoptosis induction, phosphatidylserine externalization was similar in HIBM and controls. In contrast, the active forms of caspase-3 and -9 were strongly enhanced in most HIBM cultures compared to controls, while pAkt, downregulated in controls, remained high in HIBM cells. These results could indicate impaired apoptotic signaling in HIBM cells. Since satellite cells enable partial regeneration of the post-mitotic muscle tissue, these altered processes could contribute to the muscle mass loss seen in patients. The identification of survival defects in HIBM affected muscle cells could disclose new functions for GNE in muscle cells.

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“…16 The most commonly accepted theory is that (CTG) n expansion could affect differentiation of DM myoblasts by interfering with the signals leading to the withdrawal of cell cycle and the shift toward the differentiation program. 26,32,33 However, it should be noted that the majority of the data on in vitro differentiation of DM muscle cells derive from studies on DM1 fibroblasts converted into skeletal muscle, 18,19 from primary muscle cell cultures obtained from cDM1 fetuses 31 or from mouse muscle cells. 14,15,17,33,34 No studies focusing on muscle differentiation of human primary DM1 myoblasts cultures obtained from adult muscle DM1 biopsies are available, underlining the need to work with a model as close as possible to human regenerating skeletal muscle.…”

mentioning

confidence: 99%

“…18,19 Use of human DM1 primary myoblasts cultures has been rare, owing to limited availability, and has resulted in contradictory findings. [20][21][22][23][24][25][26][27][28][29][30][31] Various experimental models have been used to speculate about the possible effects of the DM1 mutation on the myogenic process and thus find explanation for the severe skeletal muscle immaturity and wasting of DM muscle. 16 The most commonly accepted theory is that (CTG) n expansion could affect differentiation of DM myoblasts by interfering with the signals leading to the withdrawal of cell cycle and the shift toward the differentiation program.…”

mentioning

confidence: 99%

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

et al. 2010

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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...

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Molecular Basis for Impaired Muscle Differentiation in Myotonic Dystrophy

Cited by 173 publications

References 42 publications

Viral vector producing antisense RNA restores myotonic dystrophy myoblast functions

Viral vector producing antisense RNA restores myotonic dystrophy myoblast functions

Characterization of hereditary inclusion body myopathy myoblasts: possible primary impairment of apoptotic events

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

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