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 reli - desmin-positive cells per patient and passage 36-39 Transmission electron microscopy able sign of impaired nuclear activity. were scored for BrdU-positivity. nd rd th To estimate the S-phase fraction of Myoblasts at early (2 - 3 ) and late (14 to th myoblasts in the cell cultures, experiments of 17 ) passages (as defined after BrdU-incorpo - BrdU incorporation were performed. Briefly, ration experiments, see Results) were fixed in Materials and Methods cells were pulse-labeled with 40 µM BrdU the flasks and then collected by scraping. For (Sigma) for 2 h at 37°C, fixed with 70% conventional morphology, cells were fixed with 2.5% glutaraldehyde and 2% paraformaldehyde Sample collection ethanol for 30 min at -20°C, and incubated for 20 min at RT in 2 N HCl, to denature DNA par - in 0.1 M Sörensen phosphate buffer at 4°C for Samples of biceps brachii muscles were tially; after neutralization with 0.1 M sodium 1 h, washed, post-fixed with 1% OsO 4 at 4°C for taken from male adult patients affected by tetraborate (pH 8.2) for 3 min, the slides were 2 h, dehydrated with acetone and embedded in DM2 (three subjects, aged 46-55) as well as washed in PBS, permeabilized for 15 min in Epon. For immunoelectron microscopy, cells from three male healthy donors (aged 46-50), PBS containing 0.1% BSA and 0.05% Tween-20, were fixed with a mixture of 4% paraformalde - after informed consent. The experimental pro - and incubated with the monoclonal antibody hyde and 0.2% glutaraldehyde in 0.1 M tocols have been approved by the Ethical recognizing the myoblast-specific desmin, Sörensen phosphate buffer at 4°C for 1 h, Committee of the IRCCS Policlinico San revealed with an Alexa 568-conjugated anti- washed, treated with NH 4Cl 0.5 M in PBS, Donato. All the subjects were in the adulthood mouse IgG (Molecular Probes), as reported dehydrated with ethanol and embedded in LR range, and this allowed excluding the influ - above; after two washings with PBS, the slides White resin. ence of possible aging-related changes in were then incubated for 1 h with an FITC-con - Ultrathin sections from Epon-embedded nuclear features. The histological diagnosis jugated anti-BrdU mouse monoclonal antibody samples were conventionally stained with was performed on serial sections processed for (Clone b44; BD Biosciences, Beckton uranyl acetate and lead citrate. routine histological or histochemical staining, Dickinson Italia, Milan, Italy) diluted 1:20. The Ultrathin sections from LR White-embedded based on the clinical criteria set by the slides were finally counterstained for DNA samples were placed on Formvar-carbon coated International Consortium for Myotonic Dystro - with Hoechst 33258 (0.1 μg/mL for 10 min), to nickel grids and processed for immunocyto - phies. 40 The biopsies were trimmed of blood be finally mounted with Mowiol (Calbiochem). chemistry. The sections were treated with the vessels, fat and connective tissues, and rinsed For fluorescence microscopy, an Olympus following probes: mouse monoclonal antibod - in phosphate-buffered saline (PBS); satellite BX51 microscope equipped with a 100W mer - ies directed against the phosphorylated, acti - cells were isolated as reported in Cardani et cury lamp was used, under the following condi - vated form of RNA polymerase II (Research al .29 and placed in HAM’s F10 medium (Sigma- tions: 330-385 nm excitation filter (excf), 400 Diagnostic Inc., Flanders, NJ, USA) or the Aldrich, Buchs, Switzerland) supplemented nm dichroic mirror (dm), and 420 nm long- (Sm)snRNP (small nuclear RNP) core protein with 15% fetal bovine serum (Gibco, pass filter, for Hoechst 33258; 450-480 nm excf, (Abcam, Cambridge, MA, USA); rabbit poly - Invitrogen, S. Giuliano Milanese, Italy), 0.5 500 nm dm, and 515-550 nm band-pass filter clonal antibodies directed against the cleavage mg/mL albumin from bovine serum (BSA, for FITC; 540 nm excf, 580 nm dm, and 620 nm stimulation factors CStF; 42 chicken antibodies Sigma-Aldrich), 0.5 mg/mL fetuin, 0.39 µg/mL long-pass filter, for Alexa 568. Digital images directed against the heterogeneous nuclear dexamethasone, 10 ng/mL epidermal growth were recorded with an Olympus Magnifire dig - RNP (hnRNP) core protein. 43 Sections were factor, 0.05 mg/mL insulin, 3 mg/mL glucose, ital camera system, and stored on a PC by the floated for 3 min on normal goat serum (NGS) 100 U/mL penicillin and 100 µg/mL strepto - Olympus software, for processing and printing. diluted 1:100 in PBS and then incubated for mycin (Sigma-Aldrich). The myoblasts The same Olympus BX51 microscope previ - 17 h at 4°C with the primary antibodies dilut - obtained by this procedure were propagated in ously described was also used for phase con - ed with PBS containing 0.1% bovine serum plastic flasks at 37°C in a humidified 95% air / trast microscopy, under the appropriate condi - albumin (Fluka) and 0.05% Tween 20. After 5% CO 2 atmosphere. For transmission electron tions; in this case, micrographs were taken rinsing, sections were floated on NGS and microscopy, myoblasts were grown in flasks, with an Olympus Camedia 5050 digital camera. were then allowed to react for 30 min at RT whereas for light microscopy the myoblasts The percentage of myoblasts containing vac - with the appropriate secondary (either 6nm- or were planted on glass coverslips in multiwell uoles was estimated on 25 randomly selected 12nm-gold-conjugated) antibodies (Jackson plates (all the plastics were from Euroclone, microscope fields per sample using a 40x ImmunoResearch Laboratories Inc., Suffolk, Milan, Italy). objective lens; at least 500 cells per sample UK) diluted 1:10 in PBS. Finally, the sections

[European Journal of Histochemistry 2011; 55:e26] [page 137 ] Original paper were rinsed and air-dried. As controls, some desmin-positive myoblasts had an S-phase myoblasts. Consistently, the percentage of grids were incubated without the primary anti - fraction ranging from 20-26% in both control beta-galactosidase positive cells was higher, body and then processed as described above. and DM2 cultures, as estimated after BrdU- with a much stronger intensity of the final To reduce contrast and selectively incorporation experiments. Myoblasts were reaction products, in senescing DM2 than in reveal nuclear RNP constituents, the sections considered as senescing at late (14 th to 17 th ) healthy myoblasts (about 90 and 80% of posi - were bleached by the EDTA method 44 and passages when the percentage of BrdU-posi - tive cells, respectively). observed in a Philips Morgagni TEM operating tive, S-phase cells dropped to less than 5% and At light microscopy (Figure 1), myoblasts at 80kV and equipped with a Megaview II cam - 2%, in control and DM2 myoblasts respectively. from both control and DM2 patients appeared era for digital image acquisition. This suggests that the decrease in the S-phase as thin elongated cells with a centrally located Quantitative assessment of the immunola - fraction is faster in DM2 than in control nucleus. Phase contrast microscopy showed belling was carried out by estimating the gold grain density over the nucleoplasmic region on sections treated in the same run. As for anti- snRNP antibody, the labelling density was eval - uated also on interchromatin granule clusters. The grain densities were measured on fifteen randomly selected electron micrographs (x 22,000) of myonuclei from each patient by using a computerized image analysis system (AnalySIS Image processing, Soft Imaging System GmbH, Munster, Germany). For back - ground evaluation, samples treated in the absence of primary antibody were considered. Gold grains present over the selected compart - ments were counted and the labelling density was expressed as the number of gold grains/ μm2. In addition, the percentage of the nuclear surface occupied by heterochromatin and the perichromatin granules density (PG number/ μm2 of ) were calculated by using the same image analysis system. Statistical comparisons were performed by the two-way ANOVA test to evaluate the influ - ence of two factors: the presence of the dys - trophic condition (we called this factor dystro - phy , in Tables 1 and 2) and the increasing pas - sages in culture ( cell senescing in vitro , in Tables 1 and 2); in addition, the interaction term between these two factors was also con - sidered. The one-way ANOVA test was used to compare the immunolabelling density on interchromatin granules between myoblasts samples from dystrophic or healthy patients at early and late passage. Statistical significance was set at P<0.05.

Results

In primary culture from skeletal muscles, a fraction of fibroblasts is always present and myoblasts can be recognized by their immunopositivity for the muscle specific pro - tein, desmin (Figure 1a, a’). In the cultures used in the present investigation, the myo - genic index was always fairly high and ranged Figure 1. Phase contrast and fluorescence micrographs of myoblasts from healthy (a,a’ from about 60% at the 2 nd -3 rd passage to more and b,b’) and DM2 patients (c,c’ and d,d’) at early (a and c) and late (b and d) passages in culture, after immunolabeling for desmin. In a’, the myoblast (arrow) may be distin - than 90% at the 14 th -17 th passage, without sig - guished from the fibroblast for its thin and elongated shape, and the immunopositivity nificant difference between control and DM2 for desmin (green fluorescence). Nuclear DNA was counterstained with Hoechst 33258 cell populations. (blue fluorescence). Note the cytoplasmic (arrowheads) in DM2 myoblasts (c,d). At low (2 nd -3 rd ) passages from the explant, Scale bar: 20 µm.

[page 138 ] [European Journal of Histochemistry 2011; 55:e26] Original paper that about 15% of young DM2 myoblasts exhib - showed a reticular structure. cantly influence the labelling values for all the ited small roundish vesicles in their Observation of EDTA-stained samples parameters analysed; moreover, the interac - (Figure 1c), which were on the contrary rarely showed that, in all patients, the usual RNP tion term was always significant indicating a found in the controls (less than 4%) (Figure structural constituents involved in pre-mRNA relationship between the changes during in 1a). At late passages, vacuolization of senesc - transcription and processing were evident in vitro senescence and the healthy or diseased ing DM2 myoblasts became much more appar - the nucleoplasm: perichromatin fibrils and origin of the cells; in fact, DM2 was always ent (about 30% of myoblasts contained vac - perichromatin granules were mostly distrib - associated with lower levels for all the parame - uoles), with large portions of the cytoplasm uted in the perichromatin region, and inter - ters analysed (Table 2). Anti-snRNP labelling often occupied by huge vacuoles and devoid of chromatin granules formed clusters in the density on interchromatin granule clusters desmin (Figure 1d); about 10% of the interchromatin space. 36,37 Immunolabelling was significantly higher in young than in myoblasts in senescing controls (Figure 1b) experiments showed a similar intranuclear senescing control myoblasts (120.53±10.04 vs also showed small vacuoles in the cytoplasm, distribution of polymerase II, hnRNPs, snRNPs 48.49±3.01 gold grains/µm 2, respectively; although there was never evidence of the and cleavage factor CStF in control and DM2 P<0.001); conversely, in DM2 myoblasts the extensive cytoplasm degeneration found in myoblasts at both early and late passages. values were similar at early and late passages senescing DM2 myoblasts. At electron Activated polymerase II, hnRNPs and CStF (67.86±7.98 vs 69.59±6.92 gold grains/µm 2, microscopy, myoblasts were identified by their were specifically associated with perichro - respectively; P=0.889). Background values elongated shape and the features matin fibrils (Figures 5 and 6), which repre - were negligible in all immunolabelling experi - already described in literature. 45-49 sent the form in situ of pre-mRNA transcrip - ments ( not shown ). In the cytoplasm of young control myoblasts tion, splicing and 3’-end processing, 36,37 while (Figure 2 a-c), ovoid or elongated mitochon - snRNPs were restricted to perichromatin fib - dria, large amounts of free , well rils and interchromatin granules, which repre - developed rough and sent the storage, assembly and phosphoryla - Discussion numerous Golgi complexes were observed; tion sites for transcription and splicing factors conversely, , residual bodies and vac - (Figure 6). 51,52 The multifactorial phenotype of DM2, con - uoles were only seldom found. Myoblast nuclei Quantitative evaluation of the immunola - sisting of muscle weakness and atrophy, and were generally characterised by a thin hete - belling (Figure 7) revealed that both factors, the wide spectrum of extramuscular manifes - rochromatin rim just beneath the nuclear dystrophy and cell senescing in vitro , signifi - tations strongly support the hypothesis of a envelope and one reticular with many fibrillar centres and abundant dense fib - rillar component (the latter component is the site of pre-rRNA transcription and splicing) (for a recent review on nucleolar components, see 50 ). Senescing control myoblasts (Figure 2 d-f) were even more elongated than at early passages. In the cytoplasm, the belonging to the proteosynthetic apparatus and the mitochondria were abundantly pres - ent; in addition, glycogen clusters, residual bodies and vacuoles - even of large size - were frequently observed. Myoblast nuclei showed increased heterochromatin amounts along the nuclear border, while the nucleoli appeared unchanged in comparison to early passages. Young DM2 myoblasts (Figure 3 a-c) were quite similar to the controls, apart from the occurrence of differently sized vacuoles in the cytoplasm, and larger amounts of heterochro - matin and higher density of perichromatin granules in the nucleus (Figure 4 and Table 1, for the statistical analysis). Senescing DM2 myoblasts at late passages (Figure 3 d-f) had very elongated shapes and one elongated nucleus with an often irregular border. In the cytoplasm many vacuoles and Figure 2. Young (a-c) and senescing (d-f) healthy control myoblasts. (a) The myoblast large electron lucent areas were abundantly shows an elongated shape and one ovoid nucleus with a reticular nucleolus (Nu) and present; mitochondria, free ribosomes, rough scarce heterochromatin. In the cytoplasm, rough endoplasmic reticulum (arrows) and a endoplasmic reticulum and Golgi complexes Golgi complex (g) are visible. Scale bar: 1 µm. (b,c) Cytoplasmic details showing free ribo - were markedly reduced in comparison to the somes (asterisk), well developed rough endoplasmic reticulum (arrows), numerous Golgi early passages, while glycogen clusters and complexes (g) and ovoid mitochondria (m). Vacuoles (v) are quite scarce. Bars: 2 µm. (d) The myoblast shows a very elongated shape and one elongated nucleus with scarce hete - residual bodies were abundant and ubiqui - rochromatin. In the cytoplasm, rough endoplasmic reticulum (arrows), residual bodies (r) tously distributed. Myoblast nuclei frequently and vacuoles (v) are visible. Scale bar: 2 µm. (e,f) Cytoplasmic details showing free ribo - showed irregular shapes and evident hete - somes (asterisk), ovoid mitochondria (m), a Golgi complex (g), a residual body (r) and rochromatin clumps, and most of the nucleoli clustered glycogen granules (star). Scale bars: 1 µm.

[European Journal of Histochemistry 2011; 55:e26] [page 139 ] Original paper

multisystemic syndrome caused by a general suggests a common mechanistic basis for both the presence of several vacuoles in the cyto - alteration of the pre-mRNA post-transcription - skeletal muscle aging and dystrophy. plasm which could be related to a dysregula - al pathway. 53-55 However, the cellular mecha - Consistent with this hypothesis, the mor - tion of the protein degradation systems in nisms leading to the highly variable clinical pho-functional features of myoblasts from these cells. 60 Therefore, cytoplasmic and manifestations in the different tissues of DM2 healthy and DM2 patients at different passages nuclear features clearly indicate a high pro - patients have not been clarified yet. In particu - in culture provided experimental evidence for teosynthetic rate in both control and DM2 lar, at the skeletal muscle level, no explanation the more pronounced occurrence of senes - myoblasts at early compared to late passages; for the muscle wasting has so far been found. cence-related events in satellite cells of DM2- in particular, the large amount of euchromatin It has been hypothesised that, in dystrophic affected muscles. At early culture passages, and the presence of reticular nucleoli with a muscle tissue, the myogenic potential is when cell proliferation rate is high, both con - large dense fibrillar component and numerous altered, probably in relation to a hindered trol and DM2 myoblasts show the typical mor - regenerative capability of satellite cells. 25,28,56-59 phological features of growing myoblasts pre - Interestingly, a similar hypothesis has been viously described by several authors, i.e. cells also proposed to explain sarcopenia, i.e. the characterised by elongated shapes, cytoplasm age-related loss of muscle mass and func - rich in free ribosomes, rough endoplasmic tion; 23,24 in addition, recent data on dystrophic reticulum and Golgi complexes, nuclei with skeletal muscle myonuclei 21 have demonstrat - scarce heterochromatin and prominent nucle - ed alterations of mRNA pathways reminiscent oli. 45-49 At this culture stage, the only distinc - of those observed during aging. 22 This strongly tive morphological feature of DM2 myoblasts is

Figure 4. Mean ± SE values of heterochro - matin percentage and perichromatin gran - ule density (PG number/ µm2 of nucleo - plasm) in myoblasts from healthy and DM2 patients (n=45).

Table 1. Two-way ANOVA test of the mor - phometric evaluation of heterochromatin percentage and perichromatin granule density (PG number/ μm2 of nucleoplasm) in myoblasts: factor Dystrophy (healthy and DM2), factor Cell senescing in vitro (early and late passage), and interaction Figure 3. Young (a-c) and senescing (d-f) DM2 myoblasts. (a) The myoblast shows an elon - term ( Dystrophy - Cell senescing in vitro ). gated shape and one ovoid nucleus with a reticular nucleolus (Nu) and scarce heterochro - Heterochromatin % matin. In the cytoplasm, rough endoplasmic reticulum (arrows), a Golgi complex (g) and many vacuoles (v) are visible. Scale bar: 2 µm. (b,c) Cytoplasmic details showing well Dystrophy P<0.001 developed rough endoplasmic reticulum (arrows), Golgi complexes (g), free ribosomes, elongated mitochondria (m), and many vacuoles with heterogeneous content (v). Cell senescing in vitro P=0.031 Cytoplasmic electron lucent areas (stars) sometimes occur. Scale bars: 1 µm. (d) The Interaction term P=0.042 myoblast shows a very elongated shape and one irregularly shaped nucleus with evident heterochromatin. In the cytoplasm, well developed Golgi complexes (g), and vacuoles (v) PG density are visible. Scale bar: 2 µm. (e) Large cytoplasmic regions are filled with heterogeneous vacuoles (v) or appear as electron lucent areas (stars). Scale bar: 2 µm. (f) Cytoplasmic Dystrophy P=0.063 detail showing rough endoplasmic reticulum (arrow), Golgi complexes (g), glycogen clus - Cell senescing in vitro P=0.001 ters (star) and numerous vacuoles (v). Scale bar: 1 µm. Interaction term P=0.001

[page 140 ] [European Journal of Histochemistry 2011; 55:e26] Original paper fibrillar centres are suggestive of a high meta - bolic activity. 50 However, the ultrastructural immunocytochemical analyses revealed that DM2 myoblasts are relatively less active than the controls: the immunolabeling for activated polymerase I (which is necessary to synthesize pre-mRNA), for hnRNPs and snRNPs (which are essential for pre-mRNA co-transcriptional splicing 34,35 ), and for CStF (involved in the mat - uration of 3’ ends 61 ), were all significantly lower in DM2 than in control myoblasts. Accordingly, morphometrical evaluations demonstrated an increase in the amount of heterochromatin in DM2 myoblasts, consistent with the already described reduction in protein synthesis. 60 A similar decrease in the factors involved in pre-mRNA transcription and maturation, and a parallel increase in the amount of heterochro - matin have been found in senescing control myoblasts, when the proliferation rate decreased. However, no significant change in perichromatin granule density has been found in senescing control myoblasts: these granules represent storage and/or transport sites for 36 Figure 5. Myoblasts from healthy (a,b) and DM2 (c,d) patients at early (a,c) and late (b,d) spliced (pre-)mRNA and their steady number passages. Anti-polymerase II (6 nm) and anti-CStF (12 nm) antibodies: both probes indicates unperturbed RNA pathways. specifically label perichromatin fibrils (arrowheads), while the interchromatin granules Moreover, these control myoblasts are charac - (ig) are devoid of signal. Arrows indicate perichromatin granules. Ch: bleached hetero - terised by the presence of residual bodies, chromatin. Scale bars: 0.25 µm. many vacuoles and glycogen clusters: these are typical features of aging cells, where the cyto - plasmic degradation systems undergo dysfunc - tion. 62 Thus, DM2 myoblasts at early culture passages (when they proliferate at a similar rate as the controls) already show some mor - phological and functional features found in healthy myoblasts at late culture passage only. The cytoplasmic and nuclear features typical of cells senescing in culture are definitely more pronounced in DM2 myoblasts at late cul - ture passage than in the corresponding healthy controls. The amount of vacuoles and residual bodies is remarkably high, and large cytoplasmic regions appear as empty , likely due to degradation phenomena; the cell nuclei are often irregular in shape and contain many heterochromatin clumps; the factors involved in pre-mRNA transcription and processing are further reduced in comparison to senescing healthy myoblasts. It should be also noted that snRNPs, usually occurring on perichromatin fibrils and interchromatin granules, show an accumulation in the interchromatin granules which has not been observed in senescing healthy myoblasts. Intranuclear accumulation of factors involved in pre-mRNA maturation has been previously observed in different aging tissues 63-65 including skeletal muscle, 22,66 and this has been considered as a conse - Figure 6. Myoblasts from healthy (a,b) and DM2 (c,d) patients at early (a,c) and late (b,d) quence of altered pre-mRNA processing and/or passages. Anti-snRNP (6 nm) and anti-hnRNP (12 nm) antibodies: both probes label impaired intranuclear or nucleus-to-cytoplasm perichromatin fibrils (arrowheads); in addition, the anti-snRNP antibody strongly labels transport. Accordingly, in senescing DM2 interchromatin granules (ig). Arrows indicate perichromatin granules. Ch: bleached myoblasts a significant increase in perichro - hetero chromatin. Scale bars: 0.25 µm.

[European Journal of Histochemistry 2011; 55:e26] [page 141 ] Original paper

matin granule amount has been found; this nificant differences exist for some cytological phenomenon is known to occur when RNA pro - and functional characteristics in the myoblasts cessing is physiologically or experimentally from healthy and DM2 patients, despite their impaired 39,67,68 as well as during aging. 22,63-66,69 similarities in proliferation rate and age in cul - Taken together, the results of the present study ture. Interestingly, recent preliminary observa - demonstrate that satellite cell-derived DM2 tions on the in vitro differentiation of DM2 myoblasts are characterised by senescence- myoblasts showed an early structural disorgan - related features mainly consisting in the early ization of the derived myotubes, especially the appearance of cytological alterations and cytoskeletal apparatus, which may affect their impairment of the pre-mRNA maturation path - functionality and survival. 73 ways. It is possible that the sequestration of Further studies are however needed to splicing factors in the foci of DM2 myoblasts 14-19 investigate in vitro myoblast differentiation may hamper the functionality of the whole and myotube survival in DM2 patients using splicing machinery; this would consequently both morphological and biomolecular analyses reduce, in dystrophic muscles, the capability of such as those previously used to characterise satellite cells to positively respond to anabolic the differentiation process of C2C12 myoblast stimuli. Interestingly, experimental therapeu - cell line. 74 Moreover, studies on the occurrence tic approaches for DM1 aimed at reducing of senescence traits in both satellite cells and intranuclear sequestration of splicing factors myofibres in DM2 muscle biopsies 75 would pro - demonstrated that the restoration of normal vide important clues in the attempt to charac - splicing processes represents a key passage for terize the cellular mechanisms at the basis of alleviating disease symptoms. 70,71 muscle wasting in DM2 myotonic dystrophy. It has been reported that alteration of the RNA pathways does not affect the fusion process, so that cultured DM2 myoblasts are able to differentiate into myotubes as much as References myoblasts from healthy subjects do. 29,72 Similarly, cultured myoblasts from aged 1. Meola G, Moxley RT 3rd. Myotonic dystro - healthy subjects are able to form myotubes in phy type 2 and related myotonic disorders. 49 culture. This would suggest that the capabili - J Neurol 2004;251:1173-82. ty of cultured myoblasts to proliferate and dif - 2. Bachinski LL, Udd B, Meola G, Sansone V, ferentiate is maintained in aged and dystroph - Bassez G, Eymard B, et al. Confirmation of ic subjects. However, the present ultrastructur - the type 2 myotonic dystrophy (CCTG)n al and cytochemical data demonstrate that sig - expansion mutation in patients with proxi - mal myotonic myopathy/proximal myotonic Table 2. Two-way ANOVA test of the dystrophy of different European origins: a immunolabelling densities (gold single shared haplotype indicates an grains/µm 2) of polymerase II, snRNPs, ancestral founder effect. Am J Hum Genet hnRNPs and CStF in myoblast nucleo - 2003;73:835-48. plasm: factor Dystrophy (healthy and DM2), factor Cell senescing in vitro (early 3. Vihola A, Bassez G, Meola G, Zhang S, and late passage), and interaction term Haapasalo H, Paetau A, et al. Histo- (Dystrophy - Cell senescing in vitro ). pathological differences of myotonic dys - trophy type 1 (DM1) and PROMM/DM2. Polymerase II Neurology 2003;60:1854-7. Dystrophy P<0.001 4. Savkur RS, Philips AV, Cooper TA. Aberrant Cell senescing in vitro P<0.001 regulation of insulin receptor alternative Interaction term P=0.001 splicing is associated with insulin resist - ance in myotonic dystrophy. Nature Genet hnRNPs 2001;29:40-7. Dystrophy P<0.001 5. Savkur RS, Philips AV, Cooper TA, Dalton JC, Moseley ML, Ranum LP, et al. Insulin Cell senescing in vitro P=0.002 receptor splicing alteration in myotonic Interaction term P<0.043 dystrophy type 2. Am J Hum Genet 2004;74: snRNPs 1309-13. 6. Day JW, Ricker K, Jacobsen JF, Rasmussen Dystrophy P<0.001 LJ, Dick KA, Kress W, et al. Myotonic dystro - Cell senescing in vitro P<0.001 phy type 2: molecular, diagnostic and clini - Figure 7. Quantitative immunoelectron Interaction term P<0.001 cal spectrum. Neurology 2003;60:657-64. microscopy of myoblast nuclei from healthy CStF 7. Brook JD, McCurrach ME, Harley HG, and DM2 patients (n=45). Each bar repre - Buckler AJ, Church D, Aburatani H, et al. sents the mean ± SE values of the labelling Dystrophy P<0.001 density (gold grains/µm 2) for different pre- Molecular basis of myotonic dystrophy: mRNA processing factors in the nucleoplas - Cell senescing in vitro P=0.027 expansion of a trinucleotide (CTG) repeat mic region. Interaction term P=0.017 at the 3’ end of a transcript encoding a pro -

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