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Cultured Myoblasts from Patients Affected by Myotonic Dystrophy Type 2 Exhibit Senescence-Related Features

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Cultured Myoblasts from Patients Affected by Myotonic Dystrophy Type 2 Exhibit Senescence-Related Features European Journal of Histochemistry 2011; volume 55:e26 Cultured myoblasts from Introduction Correspondence: Manuela Malatesta, Diparti - patients affected by myotonic mento di Scienze Neurologiche, Neuropsico - dystrophy type 2 exhibit Myotonic dystrophies (DMs) are autosomal logiche, Morfologiche e Motorie, Sezione di senescence-related features: dominant disorders exhibiting a variety of Anatomia e Istologia, University of Verona, Italy. ultrastructural evidence multisystemic features among which muscular E-mail: [email protected] dystrophy, myotonia, dilated cardiomyopathy, Key words: myotonic dystrophy type 2 - DM2, cardiac conduction defects, cerebral abnormal - M. Malatesta, 1 M. Giagnacovo, 2 myoblasts, RNA processing, electron microscopy, ities, insulin-resistance, and cataracts; 1-3 in 3 3 4 immunocytochemistry. L.V. Renna, R. Cardani, G. Meola, addition, disease-specific serological abnor - 2 C. Pellicciari malities are present, such as hyperglycemia Acknowledgments: the authors would like to 1Dipartimento di Scienze Neurologiche, and increased levels of gamma-glutamyltrans - thank Mrs. Paola Veneroni for her skilful techni - Neuropsicologiche, Morfologiche e ferase, 4,5 hypotestosteronism, and decreased cal assistance. Marzia Giagnacovo is a PhD stu - dent in receipt of a fellowship from the Dottorato levels of IgG and IgM immunoglobulin. 6 The Motorie, Sezione di Anatomia e Istologia, di Ricerca in Biologia Cellulare (University of University of Verona; more frequent and severe DM form is the Pavia). The authors would also express their 2Dipartimento di Biologia Animale, DM1-Steinert’s disease (OMIM 160900), gratitude to Prof. T.E. Martin for kindly providing Laboratorio di Biologia Cellulare, which affects 1:8000 individuals worldwide, the anti-hnRNP core protein antibody. University of Pavia; and is caused by an expanded (CTG)n nucleotide sequence in the 3’ untranslated Received for publication: 27 July 2011. 3Dipartimento di Biologia molecolare e region of the Dystrophia Myotonic Protein Accepted for publication: 5 September 2011. Biotecnologie, University of Milan, and Kinase (DMPK) gene (OMIM 605377) on chro - This work is licensed under a Creative Commons Centro per lo Studio delle Malattie mosome 19q13. 7-9 In 1999, a second DM form Neuromuscolari – CMN, Milan; Attribution NonCommercial 3.0 License (CC BY- displaying a milder clinical phenotype has NC 3.0). 4 Dipartimento di Neurologia, IRCCS been identified: 10 it has been essentially Policlinico San Donato, University of described in European Countries and has been ©Copyright M. Malatesta et al., 2011 Milan, Italy called DM2 (OMIM 602688); it is much less Licensee PAGEPress, Italy frequent than DM1 since it affects 1:20000 European Journal of Histochemistry 2011; 55:e26 doi:10.4081/ejh.2011.e26 individuals, although a recent genotypic study on Finnish patients 11 suggested that DM2 fre - Abstract quency could be much higher than previously estimated; it is caused by the expansion of the common cellular mechanisms responsible for Myotonic dystrophy type 2 (DM2) is an auto - tetranucleotidic repeat (CCTG)n in the first the loss of muscle mass, strength and function somal dominant disorder caused by the expan - intron of the Zinc Finger Protein (ZNF-9) gene typical of sarcopenia in aged individuals, 23,24 as 12 13 sion of the tetranucleotidic repeat (CCTG)n in (OMIM 116955) on chromosome 3q21. well as for the muscle alterations caused by the first intron of the Zinc Finger Protein-9 Due to its less severe phenotype, diagnosis DM2, which include fibre atrophy-hypertrophy, gene. In DM2 tissues, the expanded mutant of DM2 is sometimes difficult and may be made increased number of centrally located nuclei, transcripts accumulate in nuclear focal aggre - relatively later, compared to DM1; to face this and the presence of fibres with nuclear gates where splicing factors are sequestered, difficulty, the localization of ribonucleoprotein clumps. 3 thus affecting mRNA processing. Interestingly, (RNP)-enriched domains (called foci ) in DM2 A common hypothesis proposed to explain the ultrastructural alterations in the splicing cell nuclei has been recently proposed as a skeletal muscle wasting in both sarcopenia machinery observed in the myonuclei of DM2 rapid and reliable marker for recognizing sub - and myotonic dystrophy implies a decreased skeletal muscles are reminiscent of the jects affected by this pathology. 14,15 In fact, in efficiency of muscle regeneration due to hin - nuclear changes occurring in age-related mus - tissues of DM2 patients, the expanded-CCUG- dered activation, proliferation and/or differen - cle atrophy. Here, we investigated in vitro containing transcripts are retained in the cell 25-28 structural and functional features of satellite nucleus and accumulate in the form of focal tiation capability of satellite cells. cell-derived myoblasts from biceps brachii , in aggregates. 12 These nuclear foci of mutant In vitro myoblast cultures derived from the attempt to investigate cell senescence mRNA specifically sequester splicing factors human satellite cells provide a suitable and indices in DM2 patients by ultrastructural cyto - among which muscleblind-like (MBNL) pro - unique model for studying DM2 muscular pre - chemistry. We observed that in satellite cell- teins, 14-17 which are essential for the alternative cursor cells, and can be used to elucidate the derived DM2 myoblasts, cell-senescence alter - splicing for muscle cell proteins, 18 and the RNP- molecular and cellular mechanisms involved in ations such as cytoplasmic vacuolization, containing complexes called snRNPs and the pathogenesis of this disease. 29,30 reduction of the proteosynthetic apparatus, hnRNPs: 19 this leads to the depletion and/or In this work we have investigated some accumulation of heterochromatin and impair - loss of function of these nuclear regulators. 20 structural and functional features of myoblasts ment of the pre-mRNA maturation pathways In a recent study, the distribution of nuclear obtained from biceps brachii biopsies in the occur earlier than in myoblasts from healthy RNP-containing structures and molecular fac - attempt to detect cell senescence traits in DM2 patients. These results, together with prelimi - tors responsible for pre-mRNA transcription patients in comparison with healthy control nary in vitro observations on the early onset of and maturation has been described in myonu - subjects. To do this, satellite-cell-derived defective structural features in DM2 myoblast clei of dystrophic skeletal muscles, 21 providing myoblasts were grown in vitro , and underwent derived-myotubes, suggest that the regenera - evidence for alterations of post-transcriptional several passages in culture: for sake of simplic - tion capability of DM2 satellite cells may be pre-mRNA events as much as it occurs in the ity, myoblasts at early (2 nd and 3 rd ) passages impaired, thus contributing to the muscular myonuclei of skeletal muscles from old rats. 22 were defined as young in culture , while those at dystrophy in DM2 patients. These findings open interesting perspectives late (14 th to 17 th ) passages were defined as for comparative studies aimed at detecting senescing . We have applied cytochemical and [page 136 ] [European Journal of Histochemistry 2011; 55:e26] Original paper immunocytochemical techniques at light and Light microscopy were considered. electron microscopy as proved valuable tools for In order to provide in situ evidence for the For fluorescence cytochemistry, cells on studying neuromuscular diseases, especially presence of senescent myoblasts, -galactosi - glass coverslips were fixed with 4% formalde - b when cell nuclear functions are involved. 31-33 In dase was detected according to Dimri et al .41 In hyde in PBS for 15 min at 4°C, post-fixed with fact, in eukaryotic cells, maturation of mRNA brief, samples were washed in PBS and fixed 70% ethanol at -20°C for 30 min, and rehydrat - implies that the primary transcripts (pre- with 2% paraformaldehyde and 0.2% glu - ed in PBS at room temperature (RT). mRNAs) undergo several transformation steps taraldehyde in 0.1 M PBS, pH 7.4, for 7 min at To specifically label myoblasts, the slides in the spliceosome, a molecular complex com - RT. After, cells were incubated for 18 h at 37°C were incubated for 60 min at RT with a mono - posed of five small nuclear RNPs (snRNPs), in fresh senescence associated SA- -gal stain - clonal antibody recognizing desmin (Dako, b many non-snRNP splicing factors, and regulat - ing solution [1 mg/mL 5-bromo-4-chloro-3- Glostrup, Denmark) diluted 1:100 in PBS con - ing factors. 34,35 The structural counterpart of inolyl-b-D-galactoside (X-gal) in dimethylfor - taining 0.1% BSA and 0.05% Tween-20, and mamide (20 mg/mL stock); 40 mM citric such macromolecular complexes is represented then incubated for 60 min at RT with an Alexa acid/sodium phosphate, pH 6.0; 5 mM potassi - by RNP-containing structures named perichro - 488-conjugated anti-mouse IgG (Molecular um ferrocyanide; 150 mM NaCl; 2 mM MgCl ]. matin fibrils, perichromatin granules and Probes, Invitrogen), washed in PBS, counter - 2 interchromatin granules: the correct intranu - stained for DNA with Hoechst 33258 (Sigma- After 2 washings in PBS, cells were mounted in clear distribution and molecular composition of Aldrich; 1 μg/mL for 5 min), and mounted in a drop of Mowiol. these structures is essential for mRNA matura - Mowiol (Calbiochem, Milan, Italy). 500 tion, and their ectopic reorganization is a
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