Human muscle biopsies from biceps brachii muscle were taken under sterile conditions from DM1 (n=3) and DM2 (n=4) patients enrolled in Ilalian DM Registry and from age matched subjects with no sign of neuromuscular disease (n=4) used as controls. The diagnosis of DM was based upon the clinical diagnostic criteria set by the International Consortium for Myotonic Dystrophy.³⁷ DM2 diagnosis was performed by fluorescence in situ hybridization on muscle frozen sections using a (CAGG)₅ probe as previously reported by Cardani et al.³⁸ to verify the presence of ribonuclear inclusions. DM1 and DM2 genotyping were performed on genomic DNA extracted from peripheral blood leukocytes as previously described.^39-41

Biceps brachii biopsies were fresh-frozen in isopentane cooled in liquid nitrogen. Histopathological analysis was performed on serial sections (8 µm) processed for routine histological and histochemical stainings. A standard myofibrillar ATPase staining protocol was used after preincubation at pH 4.3, 4.6, and 10.4.⁴² Immunohistochemical staining was performed on serial sections (6 µm) air-dried and rehydrated in phosphate buffer pH 7.4 (PBS). Non-specific binding sites were blocked with normal goat serum (NGS; Dako, Glostrup, Denmark) at a dilution 1:20 in PBS containing 2% bovine serum albumin (BSA; Sigma-Aldrich, St. Louis, MO, USA) for 20 min at room temperature. Sections were then incubated for 1 h at room temperature with mouse monoclonal primary antibodies against two different myosin heavy chain (MHC): MHCfast, diluited 1:400 in PBS+2%BSA (Sigma-Aldrich); MHCslow, diluited 1:400 in PBS+2%BSA (Sigma-Aldrich). After washing in PBS 3 times for 5 min (3x5 min), sections were incubated for 1 h at room temperature with goat antimouse biotinylated secondary antibody diluted 1:300 in PBS+2%BSA. After washing in PBS 3x5 min, sections were incubated for 30 min with Vectastain ABC complex (Vector Laboratories, Burlingame, CA, USA) and then with 3,3’-Diaminobenzidine (DAB) and hydrogen peroxide for 20 min. Nuclei were counter-stained with Mayer’s hematoxylin. Quantitative evaluation of fiber diameter was made as described previously by Vihola et al.¹³ with Scion Image (Scion Corporation, Frederick, MD, USA) on images taken with a light microscope (1600x, original magnification). The size of muscle fibers was assessed by measuring the smallest fiber diameter and all data were elaborated using Microcal Origin (Microcal Software Inc., Northampton, MA, USA). The metahistograms were normalized to normal mean diameter for men and women. Relative atrophy and hypertrophy factors were also calculated.

The human satellite cells were isolated from muscle biopsies as reported by Cardani et al.⁴³ Myoblasts were grown in HAM’s F10 medium (Sigma-Aldrich) supplemented with 15% FBS (Euroclone), 0.5 mg/mL albumin from bovine serum (BSA, Sigma-Aldrich), 0.5 mg/mL fetuin (Sigma-Aldrich), 0.39 µg/mL dexamethasone, 10 ng/mL epidermal growth factor, 0.05 mg/mL insulin, 3 mg/mL glucose, 100 U/mL penicillin and 100 µg/mL streptomycin (proliferative medium). For this study, cells from DM and control patients were plated at a density of 85,000 cells per 60 mm dishes or 50,000 cells per 35 mm dishes. Myogenic purity was evaluated by immunofluorescence using desmin as marker (see below). The percentage of desmin positive myoblasts was calculated as the number of positive cells vs the total number of cells observed. All starting cell populations used in this study had a myogenic purity higher than 80%. Moreover, the myogenic purity was monitored every 5 passages (7 days per passage for a total of 35 days) until senescence was reached. At least 100 cells were counted in at least 10 different optical fields randomly chosen.

Cells from DM patients and controls were plated in triplicate at the density 50,000 per 35 mm dishes and, after 7 days, were trypsinized, counted with trypan blue and then replated at the same density (I passage; early stage). At each passage, every 7 days, the proliferative capacity was evaluated as mean population doubling (MPD) using the formula log (N/n)/log2 where N is the number of cells counted and n is the number of cells initially plated. Cultures were considered to be in senescence when cells have arrested their growth for 3 consecutive passages (late stage). As biomarker for replicative senescence, SA-β-gal, a β-galactosidase activity detectable in cultured cells undergoing replicative senescence but absent from proliferating cells, was evaluated according to Dimri et al.⁴⁴ on myoblast at early and late stages of proliferation. Quantification of SA-βGal positive cells was done by counting blue stained cells in 5 randomly selected fields and expressing the percentage of positive cells respective to the total number of cells visualized in each field.

Myoblasts at early and late stages were plated at a high density in 35 mm dishes and, when they reached 80% of confluence, proliferative medium was replaced by differentiative medium (DMEM/High Glucose supplemented with 7% FBS, in presence of 100 U/mL penicillin, 100 µg/mL streptomycin and 0.01 mg/mL insulin) to allow myoblasts fusion. This condition of plating at high density prior to inducing differentiation was chosen in order to eliminate any bias in the quantification of fusion, which could be decreased by the poor migration of senescent cells. After 5 days in differentiative medium (T5), myotubes were analyzed in term of protein expression (MHC-fast myosin and myogenin, a myogenic factor involved in myoblast differentiation; see below) and fusion index to evaluate myoblasts differentiative capacity. Fusion index was determined as number of nuclei in multinucleated myotubes expressed as a percentage of the total number of nuclei. At least 100 nuclei were counted in at least 10 different optical fields randomly chosen.

Immunofluorescence has been performed on proliferating myoblasts at early stage, on senescent myoblasts at late stage and on myotubes obtained from myoblasts at early and late stages. Myoblast and myotube (T5) cultures were fixed in 4% paraformaldehyde for 15 min at 4°C. After fixation, cells were washed several times in PBS and then permeabilized in 0.4% Triton X-100 in PBS for 5 min. After washing in PBS, non-specific binding sites were blocked with NGS (Dako-Cytomation) at a dilution 1:20 in PBS+2% BSA for 20 min at room temperature. Myoblast and myotubes were then incubated respectively with a primary antibody mouse monoclonal anti-desmin (CD33, Dako, 1:100 in PBS+2%BSA) and a mouse monoclonal anti MHC-fast (Sigma-Aldrich, 1:600 in PBS+2%BSA) for 1 h at room temperature. After washing in PBS 3x5 min, cells were incubated for 1 h at room temperature with secondary antibodies (goat anti mouse Alexa 488-labeled; Molecular Probes, Eugene, OR, USA; 1:400 in PBS+2%BSA). After washing in PBS 3x5 min, nuclei were stained with 165 nM 4,6-diamidino-2-phenylindole (DAPI). Cells were finally mounted with Mowiol and examined using a fluorescence microscope (LEICA FW4000).

During the in vitro ageing, the expression of different proteins involved in proliferation and/or in differentiation was analyzed by western blot (WB) on myoblasts or on myotubes (T5) protein extracts. WB analyses were performed on proliferating (early stage) and non-proliferating (late stage) myoblasts and on myotubes obtained from myoblasts at early and late stage. Whole cell extracts were prepared by the addition to the plates of lysis buffer [50 mM Tris–HCl, pH 7.4 (TBS), 200 mM NaCl, 1% NP-40, 1 mM phenylmethylsulfonyl fluoride, 0.1% SDS, 0.5% sodium deoxycholate, 10 mM b-glycerophosphate, 10 mM sodium fluoride, 1 mM sodium vanadate, Protease inhibitor cocktail (Sigma Aldrich)] and rubbed with a cell scraper. After incubating on ice for 15 min, samples were centrifuged at 5700g for 15 min at 4°C, and supernatant was collected and stored at -80°C. Pellets were suspended in 50 mM TBS with 5% SDS and stored at -80°C. Protein concentration in each sample was determined by using BCA assay kit (Pierce, Rockford, IL, USA). Eight µg of protein for each sample was loaded per lane and electrophoresed on 8% or 15% sodium dodecyl sulfate-polyacrylamide gels, and than transferred to nitrocellulose membranes (Bio-Rad Laboratories). After blocking non specific sites in TBS containing 5% BSA for 30 min at 42°C, membranes were incubated overnight at 4°C with different primary antibodies diluted in TBS+5%BSA+0.1%Tween20 for mouse monoclonal antibodies, and in TBS+5%BSA+ 0.3%Tween20 for rabbit polyclonal antibodies. The expression of desmin (1:500; Clone D33; Dako-Cytomation), MyoD (1 µg/mL; Clone 5.2F; Sigma-Aldrich), p16 (1:200; Clone H-156; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and PCNA (1:5000; Clone PC10; Dako-Cytomation) was analyzed in myoblast cultures, while MHCf (1:5000; Sigma-Aldrich) and myogenin (1 µg/mL; Clone F12B; Sigma-Aldrich) were analyzed in myotubes cultures. After several washes in TBS+0.2%Tween20, membranes were incubated with horseradish peroxidase conjugated goat anti-mouse or anti-rabbit secondary antibodies diluted respectively 1:5000 and 1:10,000 in TBS+5% BSA+0.2%Tween20 (Jackson Immuno Research Laboratories, West Grove, PA, USA). Membranes were washed and immune complexes were detected using the ECL detection system (Amersham Pharmacia Biotech, Piscataway, NJ, USA). Blots were performed in triplicate.

Genomic DNA has been extracted from 1x10⁶ DM1 (n=2), DM2 (n=4) and control (n=3) myoblasts at early and late stages with FlexiGene kit (QIAGEN, Hilden, Germany), following the manufacture’s instructions. The quantification of telomere lengths has been assessed through Southern blot analysis of terminal restriction fragments (TRF) using the TAGGG Telomere Length Assay (Roche). Briefly, 2 µg of genomic DNA from each sample were digested with HinfI and RsaI enzymes, digested fragments were resolved by gel electrophoresis in a 0.8% agarose gel and transferred onto a nylon membrane. The blotted DNA fragments were hybridized to a digoxigenin (DIG)-labeled (TTAGGG)₄ probe and incubated with a anti DIG-specific antibody covalently coupled to alkaline-phosphate. Metabolized CDP-Star substrate is then detected by chemiluminescence indicating the location of the TRF on the blot. The average TRF length was determined by comparing the location of the TRF on the blot relative to a molecular weight standard. The TRF signal has been scanned and digitalized by STORM 860 (Molecular Dynamics, Sunnyvale, CA, USA) and ImageQuant software. Mean TRF lengths have been calculated for each sample on three independent gels, as reported previously by Kimura et al.⁴⁵ The number of cell division being known, the shortening velocity of telomeres has been determined, and it has been calculated whether in DM cells the telomere loss is higher than in controls.

To determine significance between two groups, comparisons were made using an unpaired Student’s t-test. The groups were considered statistically different at P<0.05.

This study was performed on DM1 and DM2 age-matched patients compared to subjects with no sign of neuromuscular disease used as controls. Although the number of patients analyzed in this study was small, DM1 and DM2 groups were homogeneous: the DM1 cohort was represented by 3 classical adult patients mildly affected with a range of CTG repeat expansion of 361.6±102.8 (E2) and the MIRS was 3-4, the DM2 cohort was represented by 4 patients with classical PROMM phenotype and fluctuating myotonia, except for patient #3 (Table 1).

Analysis of muscle sections immunostained for MHC fast or slow myosin allows us to detect and measure fibers smaller than 5 µm, including all nuclear clump fibers which are recognizable by the presence of a thin rim of immunoreaction around the nuclei (Figure 1 A,B). All patients used in this study show an increase of fibre atrophy. In particular, an increase of both type 1 and type 2 fiber atrophy is present in DM1 patients and a prevalent type 2 fibre atrophy is evident in DM2 patients. Hypertrophic type 1 and type 2 fibers were also present in DM patients (Figure 1C). The evaluation of atrophy (AF) and hypertrophy (HF) factors for each patient group are reported in Figure 1D.

The myogenic purity of cell preparations was evaluated from the percentage of cells expressing desmin. Immunofluorescence assay revealed that the cell preparations used in this study were quite similar in terms of the desmin expression. The myogenic purity was around 75-80% throughout the experimental procedures (data not shown). Also MyoD protein expression indicated that the myogenic purity remained similar in the early and late stages both in DM and control cultures (Figure 2 A,B).

The in vitro proliferative capacity of myoblasts obtained from skeletal muscle biopsies of 3 DM1 and 4 DM2 patients was compared to that of myoblasts obtained from 4 age-matched unaffected individuals used as controls. As shown in Figure 2C, an interindividual variability was evident in both DM1 and DM2 patient group, however the average proliferative lifespan of the DM1 and DM2 myoblasts was reduced by 50% and 36% respectively as compared to that observed in control cells. Moreover, DM1 (9 passages) and DM2 (10 passages) myoblasts stopped dividing earlier compared with control cultures (15 passages) (Figure 2C). Myoblasts from DM1 and DM2 patients showed a clear reduction of the maximum number of divisions compared to those observed in controls cells (Figure 2D). Analysis of PCNA expression showed a significant reduction of protein expression in the late passages compared to the early stages both in DM and control cells (Figure 2E,F). The expression of PCNA in the late passages indicated that the majority of DM and control cells were arrested in G1 phase of the cell cycle.

To evaluate if a mechanism similar to senescence was responsible for the early proliferative growth arrest of DM myoblasts, we analyzed biomarkers usually observed in senescent cells. At early passages DM and control cells were actively dividing with rare or no senescent cells being detected but as the cells reached their late passage, the number of senescent cells positive for SA-β gal increased dramatically in both DM and control cells (Figure 3). Moreover, DM and control myoblasts at the late stages of proliferation showed a flattened morphology with an enlarged cytoplasm and extended cytosolic processes, a morphology reminiscent of that observed when myoblasts reach a senescent state (Figure 4).

We examined the expression of p16 protein at the beginning and at the end of DM lifespan to verify if this mechanism was also involved in the premature proliferative arrest observed in DM2 myoblasts in vitro. Our analysis indicate that p16 was overexpressed in DM1 cells both at the early and late stages compared to controls, while p16 protein expression appeared to be similar in DM2 and control myoblasts at both stages analyzed (Figure 5 A,B). Critically short telomeres trigger loss of cell viability in tissues, which has been related to alteration of tissue function and loss of regeneration tissue capabilities. Since it has been observed that DM1 myoblasts stopped dividing prematurely independently of telomere shortening,^34,35 we have analyzed telomere loss induced by in vitro aging in satellite-cell-derived myoblasts, isolated from muscles of DM1 (n=2), DM2 (n=4) patients and from age-matched control subjects (n=3). As shown in Figure 5C, satellite cells isolated from DM1 patients stopped growing prematurely with telomeres longer than controls (respectively 11.4±1.1 Kb and 9.5±0.3 Kb), differently from what observed in DM2 myoblasts which stop dividing with a median telomere length of 8.3±1.7 Kb even if a wide variability in mean TRF length can be observed in DM samples. It should be noted that the two DM2 cultures (#2 and #3, Figure 2C) showing the most premature growth arrest also showed the shortest telomere length at the early stages probably indicating an excessive proliferation in vivo before their isolation (data not shown). Moreover these two DM2 patients also show the highest values of atrophy factor in vivo (Figure 1C). As shown in Figure 5D, DM2 satellite cells has a clear two-fold decrease in the amount of telomeric DNA at every cell passage compared to controls whereas in DM1 cells the mean bp loss per division is 30% lower than controls.

Myoblasts at early and late stages of proliferation were induced to fuse into myotubes during 5 days of differentiation (T5) in order to evaluate their differentiative capacity. Although the senescent cells were still able to form myotubes, they appeared to be significantly smaller than those formed by the young cells, which also formed branched myotubes not observable in senescent cultures (Figure 6 A-F). As observed in Figure 6G, a significant reduction in fusion index is observable both in DM and control myoblasts which has reached a proliferative arrest compared to proliferating myoblasts. This reduction appears to be more evident in senescent myoblasts from DM1 patients who also show higher value of atrophy factor compared to DM2. Moreover, the expression of myogenin appeared to be decreased both in DM and control myotubes obtained from non-proliferating myoblasts as compared to those obtained from proliferating myoblasts (Figure 6 H,I).