MOLECULAR BASIS OF SKELETAL MUSCLE ATROPHY IN MYOTONIC DYSTROPHY

Myotonic dystrophy (DM) is an autosomal dominant multisystemic disorder characterized by a variety of multisystemic features including myotonia, muscular dystrophy, cardiac dysfunctions, cataracts and insulin-resistance. DM1 is caused by an expanded (CTG)n in the 3\u2019 UTR of the DMPK gene, while DM2 is caused by the expansion of a (CCTG)n repeat in the intron 1 of the CNBP gene. In both forms, the mutant transcripts accumulate in nuclear foci altering the function of some alternative splicing regulators which are necessary for the physiological processing of mRNAs. However, the downstream pathways by which these RNA binding proteins cause skeletal muscle alteration are not well understood. For these reasons the aim of my PhD project was to analyze the molecular mechanisms behind DM skeletal muscle atrophy. In the first part of my PhD we have performed different studies to better define the molecular pathogenesis of DM. In particular, we have analysed the histopathological and biomolecular features of skeletal muscle biopsies from a cohort of DM1 and DM2 patients presenting different phenotypes. The results indicated that the splicing and muscle pathological alterations observed are related to the clinical DM1 and DM2 phenotype and that CUGBP1 seems to play a role only in DM1, confirming that the molecular pathomechanism of DM is more complex than the one actually suggested. These data were confirmed by the analysis of two different biopsies obtained from 5 DM2 patients that showed that morphological alterations evolve more rapidly over time than the molecular changes suggesting that the molecular mechanisms that drive to skeletal muscle atrophy are still unclear and that these features cannot be explained only by spliceopathy. For all these reasons we decided to analyse DM satellite cells activity in vitro. Satellite cells are the muscle fibre precursor cells and our data indicated that both DM1 and DM2 skeletal muscle cells have lower proliferative capability than control myoblasts. Moreover, the premature proliferative growth arrest observed in DM cells appears to be caused by an overexpression of p16 in DM1 muscle cells, while DM2 muscle cells stop dividing with telomeres shorter than controls, suggesting that in these cells the signaling involved in premature senescence depend on a telomere-driven pathway. Finally, we decided to analyze the insulin pathway which is involved in the regulation of skeletal muscle atrophy. Our data have shown that DM1 and DM2 cells exhibit a lower glucose uptake and a lower proteins activation after 10 nM insulin stimulation when compared to controls suggesting that also this pathway could play a role in the molecular mechanisms that drive skeletal muscle atrophy in DM patients. In conclusion, we have shown that the molecular mechanisms behind skeletal muscle atrophy in DM1 and DM2 patients are more complicated than that previously suggested and further analysis are necessary to understand why skeletal muscle atrophy affect mainly type 1 fibres in DM1 patients, while on the contrary it affects selectively type 2 fibres in DM2 patients

Cultured myoblasts from patients affected by myotonic dystrophy type 2 exhibit senescence-related features: ultrastructural evidence

Myotonic dystrophy type 2 (DM2) is an autosomal dominant disorder caused by the expansion of the tetranucleotidic repeat (CCTG)n in the first intron of the Zinc Finger Protein-9 gene. In DM2 tissues, the expanded mutant transcripts accumulate in nuclear focal aggregates where splicing factors are sequestered, thus affecting mRNA processing. Interestingly, the ultrastructural alterations in the splicing machinery observed in the myonuclei of DM2 skeletal muscles are reminiscent of the nuclear changes occurring in age-related muscle atrophy. Here, we investigated in vitro structural and functional features of satellite cell-derived myoblasts from biceps brachii, in the attempt to investigate cell senescence indices in DM2 patients by ultrastructural cytochemistry. We observed that in satellite cell-derived DM2 myoblasts, cell-senescence alterations such as cytoplasmic vacuolization, reduction of the proteosynthetic apparatus, accumulation of heterochromatin and impairment of the pre-mRNA maturation pathways occur earlier than in myoblasts from healthy patients. These results, together with preliminary in vitro observations on the early onset of defective structural features in DM2 myoblast derived-myotubes, suggest that the regeneration capability of DM2 satellite cells may be impaired, thus contributing to the muscular dystrophy in DM2 patients

RNA transcription and maturation in skeletal muscle cells are similarly impaired in myotonic dystrophy and sarcopenia : the ultrastructural evidence

In recent years, histochemistry at light and electron microscopy has increasingly been applied to investigate basic mechanisms of skeletal muscle diseases; in particular, the study in situ of skeletal muscle cell nuclei proved to be crucial for elucidating some pathogenetic mechanisms of skeletal muscle wasting in myotonic dystrophy (DM) and sarcopenia. DM is an autosomal dominant disorder whose multisystemic features originate form nucleotide expansions: (CTG)n in the dystrophy myotonic protein kinase (DMPK) gene on chromosome 19q13 in DM type 1 (DM1), or (CCTG)n in intron 1 of the CNBP gene (previously know as zinc finger 9 gene, ZNF9) on chromosome 3q21 in DM type 2 (DM2). Sarcopenia is an age-related condition characterized by the decline of muscle mass, strength and function, whose causes are still poorly known and probably manifold (e.g., altered levels of anabolic hormones and inflammatory mediators, impairment of proteolytic and autophagic pathways, mitochondrial or neuromuscular dysfunction, loss of satellite cells). Interestingly, skeletal muscles in both DM and sarcopenia show myofibre atrophy, fibre size variability and centrally located nuclei, as well as a reduced satellite cells' effectiveness. Based on ex vivo and in vitro studies, we have demonstrated that both myofibres and satellite cells of DM and sarcopenic muscles exhibit a massive nuclear rearrangement of the structural and molecular factors responsible for pre-mRNA transcription and maturation: the impairment in the pre-mRNA post transcriptional pathways would thus account for the aging-reminiscent muscle phenotype of DM patients suggesting that the skeletal muscle wasting observed in DM and sarcopenia may result from similar cellular mechanisms

Invecchiamento precoce delle cellule satelliti: suo possibile ruolo nell\u2019atrofia muscolare dei pazienti affetti da distrofia miotonica.

Il lavoro descrive l'invecchiamento precoce delle cellule satelliti derivate da biopsie di pazienti affetti da distrofia miotonica valutato in vitro e discute il suo possibile ruolo nell\u2019atrofia muscolare tipica di questa patologia