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Keywords:

  • aging;
  • human;
  • satellite cells;
  • skeletal muscle;
  • telomere

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgments
  8. References

In this study, we have investigated the consequences of aging on the regenerative capacity of human skeletal muscle by evaluating two parameters: (i) variation in telomere length which was used to evaluate the in vivo turn-over and (ii) the proportion of satellite cells calculated as compared to the total number of nuclei in a muscle fibre. Two skeletal muscles which have different types of innervation were analysed: the biceps brachii, a limb muscle, and the masseter, a masticatory muscle. The biopsies were obtained from two groups: young adults (23 ± 1.15 years old) and aged adults (74 ± 4.25 years old). Our results showed that during adult life, minimum telomere lengths and mean telomere lengths remained stable in the two muscles. The mean number of myonuclei per fibre was lower in the biceps brachii than in the masseter but no significant change was observed in either muscle with increasing age. However, the number of satellite cells, expressed as a proportion of myonuclei, decreased with age in both muscles. Therefore, normal aging of skeletal muscle in vivo is reflected by the number of satellite cells available for regeneration, but not by the mean number of myonuclei per fibre or by telomere lengths. We conclude that a decrease in regenerative capacity with age may be partially explained by a reduced availability of satellite cells.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgments
  8. References

Skeletal muscle is a post-mitotic tissue composed of multinucleated myofibres, elements that arise from the fusion of mononucleated myoblasts during development (Hawke & Garry, 2001). In the adult muscle there is a population of mitotically quiescent cells sequestered between the basal lamina and the plasma membrane of the myofibres (Mauro, 1961). These cells are called satellite cells and once activated they proliferate to form new myofibres in response to injury or disease in the adult muscle (Schmalbruch, 1991; Bischoff & Heintz, 1994; Hawke & Garry, 2001). Therefore, satellite cells determine the regenerative capacity of skeletal muscle after injury, surgery or in neuromuscular diseases involving muscle degeneration such as muscular dystrophies. Among the parameters involved in muscle regeneration, the number of available satellite cells, their proliferative capacity, which depends on previous cycles of regeneration since the proliferation of human somatic cells is limited (Hayflick, 1965; Decary et al., 1997), and the speed at which the general response to injury will be induced will all influence the recovery. While an evaluation of the regenerative history can be carried out by measuring telomere lengths, the number of available satellite cells can be counted directly on biopsies.

Telomeres are repeated DNA sequences located at the end of all eukaryotic chromosomes. They play an important role in their function and structure preventing aberrant recombination and end-chromosome degradation (Orr-Weaver et al., 1981; Haber & Thorburn, 1984). In mammals, they consist of short repeated non-coding DNA sequences (TTAGGG)n, which in human are 5–20 kb in length (de Lange et al., 1990; Harley et al., 1990; Klapper et al., 2001). During DNA replication, DNA polymerase is unable to copy the 3′ terminal segment of each DNA strand. This results in chromosome shortening at each round of cell division (Harley et al., 1990). In somatic cells, telomere length decreases regularly with cell division. In vivo, a decrease in the length of telomeric DNA with aging has been demonstrated in many human mitotic tissues, such as the liver (Aikata et al., 2000; Takubo et al., 2000), peripheral blood lymphocytes (Vaziri et al., 1993), the skin (Lindsey et al., 1991; Friedrich et al., 2000), the large bowel (Nakamura et al., 2000), the kidneys (Melk et al., 2000), leucocytes and synovial tissue (Friedrich et al., 2000). In skeletal muscles, since nuclei are added to muscle fibres at various times during fibre formation and regeneration, the mean telomere length value reflects this heterogeneity while the minimum value will correspond to nuclei which have undergone the most divisions, i.e. the most recent nuclei incorporated. The length of telomeric DNA has thus been proposed to be a good indicator of the regenerative history. Using this approach on biopsies of human quadriceps muscles, a small decrease (11 bp per year) in the minimum telomere lengths was observed between 9 months and 86 years of age (Decary et al., 1997) whereas a dramatic decrease (187 bp per year) was observed in the muscles of children suffering from muscular dystrophy (Decary et al., 2000).

The proportion of satellite cells in skeletal muscle has been studied in several species by two different techniques, electron microscopy and immunolabelling with antibodies which react specifically with satellite cells. An antibody directed against N-CAM (CD56) initially called Leu-19 (Schubert et al., 1989) was found to label satellite cells in normal and diseased muscle (Schubert et al., 1989). In several previous studies, we have used this specific labelling to quantify satellite cell numbers in human subjects (Kadi & Thornell, 2000).

How this pool of satellite cells evolves during normal aging in human skeletal muscle is still controversial. In humans, satellite cell proportions were estimated at 15% of all the myonuclei at birth, 6–10% at 2 years of age, and only 4% in the adult by electron microscopy (Schmalbruch & Hellhammer, 1976; Fardeau & Tomé, 1981). For aged subjects, this value varied between 0.6 and 3.4% in independent studies (Ishimoto et al., 1983; Schmalbruch & Hellhammer, 1976; Roth et al., 2000).

In the present study, we were interested in seeing whether there was a decrease in regenerative capacity of human skeletal muscle with age, using minimum and mean telomere lengths as a marker of regenerative history, and the number of satellite cells (expressed as a percentage of the total muscle nuclei) as one of the parameters involved in the regenerative potential. These two parameters were studied in the same individuals in a limb (biceps brachii) and a masticatory muscle (masseter) since previous studies have shown distinct quantitative and qualitative modifications occurring with age in these two muscles (Larsson et al., 1978; Klitgaard et al., 1990; Lexell, 1995; Monemi et al., 1999). The age of the subjects ranged between 20 and 83 years. Our results demonstrate that no significant decrease was observed in the telomere length in adult masseter and biceps muscles between 20 and 83 years of age. In contrast, in these muscles the number of satellite cells decreased with age whereas the mean number of myonuclei per fibre remained constant.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgments
  8. References

Telomere length analysis in adult human skeletal muscle

Figure 1 is a representative Southern Blot of telomeric DNA obtained from adult muscles. The values for the minimum and mean telomere lengths measured in biceps and masseter muscles from young and aged adults are shown in Table 1 (average values and standard deviation). There was no significant difference in both the minimum and the mean telomere length values in biopsies that we obtained from the anterior, posterior or deep part of the young masseter muscle. As a consequence, data from these samples were pooled.

image

Figure 1. TRF length analysis of human skeletal muscle from two groups of adults: young adults (23 ± 1.15 years old) and aged adults (74 ± 4.25 years old). Representative autoradiogram showing the size distribution of telomere lengths (TRF) from skeletal muscles (B: biceps, AM: Anterior masseter, PM: Posterior masseter and DM: Deep masseter). The age of each donor is represented. Molecular weight standards are indicated in kbp.

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Table 1.  The minimum and mean telomere length in biceps and masseter (AM: anterior; PM: posterior and DM: deep) for young and aged adults. The values given are mean ± standard deviation
 BicepsMasseter
Young adultsAged adultsYoung adultsAged adults
Minimum telomere length (kbp)7.01 ± 0.927.06 ± 0.92   7.51 ± 0.89 (AM) 7.73 ± 0.35 (PM) 7.23 ± 0.44 (DM) 7.35 ± 0.847.37 ± 0.71
Mean telomere length (kbp)10.78 ± 1.1011.38 ± 1.00   11.60 ± 1.30(AM) 12.07 ± 0.75 (PM) 11.35 ± 0.38(DM) 11.28 ± 1.1011.79 ± 0.72

No significant difference in the minimum telomere length values was shown in DNA between young adults (20–28 years old) and aged adults (58–83 years old) either in the biceps or in the masseter. Furthermore, the difference which we have observed in the minimum telomere lengths between the biceps and masseter was not significant. When all minimum telomere length values were pooled, regardless of the muscle considered, we observed a non-significant decrease in the minimum telomere length, of approximately 0.73 ± 4.41 bp per year during muscle aging in healthy individuals (Fig. 2).

image

Figure 2. Minimum telomere length of human skeletal muscle (biceps and masseter) as a function of age. Graphic representation of the minimum telomere length calculated from the combined data for the biceps and the masseter muscles of young and aged adults plotted against age. Each point is the mean value from three independent Southern analyses.

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Mean number of myonuclei per fibre and percentage of satellite cells in human skeletal muscles

Satellite cells were visualized using an antibody directed against N-CAM and all nuclei (myonuclei and satellite cell nuclei) were revealed by Mayer's Hematoxylin counterstaining. A typical example of myonuclei and satellite cell visualization in young (Fig. 3A,C) and aged adult (Fig. 3B,D) masseter and biceps muscles is shown in Fig. 3. Nuclei localized within the cell boundary of a muscle fibre were counted as myonuclei and nuclei which were in cells expressing N-CAM (brown rim on Fig. 3) were counted as satellite cell nuclei. The mean number of satellite cells and myonuclei per fibre cross-section and the proportion of satellite cells are shown in Table 2. It should be noted that very similar results were obtained using an antibody raised against M-Cadherin (data not shown).

image

Figure 3. Detection of myonuclei and satellite cells in human skeletal muscles. Cross-section of biceps (A; young adult; B: aged adult) and masseter (C: young adult, D: aged adult) muscles stained with a monoclonal antibody against N-CAM and counterstained with Mayer's haematoxylin. The arrows indicate satellite cells stained positively for N-CAM (peroxidase, brown) whereas the arrowheads give examples of myonuclei (blue).

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Table 2.  The mean number of myonuclei and satellite cells per fibre cross-section and the proportion of satellite cells in biceps and masseter (AM: anterior; PM: posterior and DM: deep) for young and aged adults. The values given are mean ± standard deviation
 BicepsMasseter
Young adultsAged adultsYoung adultsAged adults
  1. Significantly different in adult vs. aged adults (***P < 0.001). Significantly different in biceps vs. masseter in same age group ([***]P < 0.001; [****]P < 0.0001).

Mean number of myonuclei2.54 ± 0.86[****]2.62 ± 0.48[****]   1.80 ± 0.261.908 ± 0.22
per fibre cross-section   (AM) 1.93 ± 0.25 (PM) 1.68 ± 0.21 (DM) 1.80 ± 0.30 
Proportion of4.17 ± 0.52[***]1.44 ± 0.74***   5.89 ± 1.001.77 ± 0.70***
satellite cells (%)   (AM) 5.20 ± 1.03  (PM) 6.24 ± 0.95 (DM) 6.22 ± 0.81 
Mean number of satellite0.103 ± 0.0340.039 ± 0.022***   0.115 ± 0.0280.034 ± 0.012***
cells per fibre cross-section  (AM) 0.107 ± 0.028(PM) 0.116 ± 0.028(DM) 0.122 ± 0.033 

Number of myonuclei in the masseter and biceps with increasing age

The mean number of nuclei per fibre cross-section was determined in young and aged adults in both the biceps and the masseter muscles (Table 2). The values obtained for the different regions of the young adult masseter were similar.

In the biceps, the mean number of myonuclei per fibre was 2.54 ± 0.86 in young adults and 2.62 ± 0.48 in aged adults. In the masseter, the mean number of myonuclei per fibre was 1.80 ± 0.24 and 1.91 ± 0.22, respectively, in young and aged adults. The mean number of myonuclei per fibre remained constant with increasing age both in the biceps and in the masseter. The biceps muscle had more myonuclei per fibre as compared with the masseter muscle, and this increase was significant in both young and aged adults.

Proportion of satellite cells in masseter and biceps with increasing age

The proportion of satellite cells and their mean number per fibre were determined in both muscles in young and aged adults (Table 2). In the biceps, the proportion of satellite cells showed a significant decrease with age, representing 4.17% of the total nuclei in young adults and only 1.44% in aged adults. Similarly in the masseter, the proportion of satellite cells was significantly higher in young adults (5.89 ± 1.00%) than in aged adults (1.77 ± 0.70%), as shown in Fig. 4.

image

Figure 4. Proportion of satellite cells from young adults (23 ± 1.15 years old) and aged adults (74 ± 4.25 years old).The proportion of satellite cells from the biceps and the masseters muscle of young and aged adults. Data for young masseter muscles are the combined values obtained from the three different parts (anterior, posterior and deep). The data were expressed as mean ± standard deviation (***P < 0.001).

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This was also the case for the mean number of satellite cells per fibre cross-section: 0.103 ± 0.034 for the young biceps vs. 0.039 ± 0.022 for the old biceps and 0.115 ± 0.028 for the young masseter vs. 0.034 ± 0.012 for the old masseter. The mean number of satellite cells per fibre cross-section was therefore always higher in young adults as compared to aged adults, and in both the biceps and in the masseter muscles.

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgments
  8. References

During human aging, a functional decline and a loss of muscle mass are the result of many molecular and cellular changes and involve progressive denervation (Luff, 1998) with loss of motor units (Larsson, 1998) and reduction in the speed of contraction (Bilodeau et al., 2001), decreased capillarization (Degens, 1998), evidence of increased oxidative stress (Squier, 2001), altered equilibrium of growth factors and hormones involved in the maintenance of muscle function (Ferrannini et al., 1996) and a decrease in muscle strength and muscle atrophy (Fano et al., 2001). Studies on human limb muscles have related this progressive atrophy to both a decrease in fibre diameter and a loss of muscle fibres (Larsson et al., 1978; Lexell et al., 1988). Myosin heavy chain (MHC) isoform expression is also modified with age: while slow MHC is reduced and fast and fetal MHC are increased in aged masseter muscles; the reverse is observed in aged biceps (Monemi et al., 1999) and in other limb muscles (Klitgaard et al., 1990). This suggests that these age-related changes most probably reflect the adaptative response of the different muscles to modified functional demands associated with the aging process.

In the present study, we asked whether a decrease in the regenerative potential of human skeletal muscle with increasing age could be reflected in either the telomere length, which is an indicator of the regenerative history, or the number of satellite cells available at a given time. Two muscles have been investigated, the biceps and the masseter, which age differently in terms of MHC expression (Monemi et al., 1999), in two groups of adults: one with an average age of 23 ± 1.15 years (young) and the other with an average age of 74 ± 4.25 years (aged).

In humans as well as other organisms, telomere shortening occurs during the replicative aging of normal somatic cells both in vitro and in vivo (Goyns, 2002) and telomere length has been proposed as a biomarker for somatic cell turnover. Telomere shortening can be influenced by factors such as oxidative stress, which may partly explain variations in telomere loss between different tissues (Campisi et al., 2001). Although skeletal muscle is generally considered a post-mitotic tissue, we have shown that extensive regeneration through incorporation of newly formed myonuclei, such as occurs in muscular dystrophies, can be clearly demonstrated by measuring minimum telomere length. This parameter relates to the population of nuclei which have undergone the most divisions, including those myonuclei incorporated during the last mitotic cycles associated with muscle regeneration (Decary et al., 2000). In this study, we observed no significant difference in the minimum or mean telomere lengths in either the biceps or the masseter muscles from young and aged adults. This would suggest that introduction of new myonuclei originating from satellite cells, either through nuclear turn-over or through focal regeneration, is limited in these two groups of muscles and cannot be detected using this technique. In the muscular dystrophies, the significant decrease in telomere length can be explained by the fact that during the dystrophic process, fibres are continually degenerating and being repaired or replaced. In contrast, the stability of the telomere length described in this study indicates that there is only a limited nuclear turnover in both of these muscles during normal aging. These results are in agreement with those we obtained concerning the proliferative capacity of human satellite cells with aging (Decary et al., 1997), where we showed that satellite cells could still achieve 15–20 divisions even when isolated from the oldest subjects we investigated. This is also in agreement with the recent findings of Carlson et al. (2001) who showed that the EDL muscles of very old rats still possess a normal capacity for regeneration, even when near the end of their lifespan, given that the conditions for regeneration are favourable. Therefore, telomere shortening, which is significantly increased only when extensive degeneration occurs, may be a good indicator of pathological situations beyond normal aging.

Although skeletal muscle is mainly a post-mitotic tissue it also possesses a population of cells, satellite cells, which can be activated and proliferate in response to increased load or damage. The number of cells available at a given time in this compartment will determine the regenerative potential of the muscle. Satellite cells can be easily identified in human skeletal muscle using an antibody directed against N-CAM, an adhesion molecule which has been established as a reliable marker for quiescent satellite cells (Cashman et al., 1987; Maier & Bornemann, 1999; Kadi & Thornell, 2000; Renault et al., 2002). It has been shown that the number of N-CAM-positive muscle fibres increases following age-related denervation in the rat (Kalliainen et al., 2002). However, the fact that we are counting only the nuclei of N-CAM-positive cells situated outside the muscle fibre but within the basal lamina eliminates any possible bias due to an increase in N-CAM expression.

Using this marker, we have quantified the number of satellite cells present in muscles of the two groups of subjects: young and aged adults. In the young adult biceps and masseter, we found 4.17–5.89% of the nuclei to be satellite cells. These results are in close agreement with previous ultrastructural studies which have estimated the proportion of satellite cells to be between 4 and 5% in young adult muscle (Fardeau & Tomé, 1981; Watkins & Cullen, 1988). Conflicting results using the same technique were recently published by Roth et al. (2000). Our observations using N-CAM labelling and light microscopy allowed much larger samples to be analysed, thus giving results which are more representative of the variability within the section and between individuals. Our results are again in agreement with those published by Fardeau & Tomé (1981) and Watkins & Cullen (1988).

The proportion of satellite cells found in the muscles of the aged subjects was significantly lower (1.44–1.77%) than in young subjects, both in the biceps and the masseter. These results were also in agreement with the majority of previous studies which found a proportion of between 0.6 and 3.4% using electron microscopy (Schmalbruch & Hellhammer, 1976; Ishimoto et al., 1983; Roth et al., 2000). The decrease in satellite cell number was significantly greater (P < 0.05) in the masseter muscle (0.08 ± 0.008% per year on average) in comparison to the biceps (0.05 ± 0.0005% per year). It should be noted that there is a higher proportion of satellite cells in young adult masseter compared to young adult biceps, whereas the proportion was similar in both muscles in aged adults.

Two previous studies carried out on muscle aging showed a decrease in the number of muscle fibres during aging (Larsson et al., 1978; Lexell et al., 1988). However, a loss of fibres would be expected to result in a decrease in both myonuclei and satellite cells, and cannot explain the selective loss of satellite cells which we and others have observed with increasing age. The loss of satellite cells during aging could be due to a defect in the restoration of the quiescent satellite cell pool. Quiescent satellite cells are activated in response to insult and damage (Schmalbruch, 1991; Bischoff & Heintz, 1994), and, after proliferation, the majority of their progeny enter into terminal differentiation to form new muscle fibres or repair damaged fibres, while a minority will restore the pool of quiescent satellite cells. This mechanism for the maintenance of a pool of reserve cells implies an asymmetric division during the regeneration process. One daughter cell will proliferate extensively while the other daughter cell will return to quiescence. This mechanism limits the decrease in regenerative potential at each round of regeneration. A decrease with age in the proportion of satellite cells which are able to return to the quiescent state and restore the satellite cell pool could result from a dysfunction of the mechanism of asymmetric division, which may explain the decrease in satellite cell number which we have observed in the aged subjects.

Finally, the decrease in numbers of satellite cells is probably due to a continued low level of turnover of the satellite cells which occurs during normal growth and repair. This would progressively exhaust the proliferative capacity of some of these cells. When satellite cells come to the end of their proliferative lifespan, they can no longer be activated and the size of the reserve pool would decrease. A previous study (Decary et al., 1997; Renault et al., 2000) supports this hypothesis by showing that satellite cells isolated from muscle biopsies of aged donors have a limited proliferative capacity. In addition, the numbers of these cells which were unable to proliferate in culture increased with age.

These results confirm that a decrease in regenerative potential as demonstrated by a decrease in the satellite cell pool is not accompanied by an excessive turnover of myonuclei, since the length of the telomeric restriction fragments remained constant. The decline in the number of satellite cells with age could be due to a deregulation in the mechanisms which control asymmetric cell division to restore the satellite cell reserve pool and/or a death of satellite cells which have reached the end of their proliferative lifespan. In addition, myonuclear function may be defective in aged muscles and consequently more nuclei may be required to maintain effective transcription.

Experimental procedures

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgments
  8. References

Source of human skeletal muscle

Masseter and biceps muscle biopsies were obtained during autopsy (Eriksson et al., 1980), in accordance with the French and Swedish legislation on ethical rules, and showed no signs of neuromuscular diseases. The investigation was approved by Socialstyrelsen, the National Board of Health and Welfare, Stockholm, Sweden. They were dissected to remove connective tissue and fat. Masseter and biceps muscle biopsies were obtained from the same patients: young adult males (20, 21, 22, 23, 24 and 28 years old; mean age: 23 ± 1.15 years) and aged adult males (58, 64, 78, 79, 82 and 83 years old; mean age: 74 ± 4.25 years) (Table 3).

Table 3.  Muscle origins
Young adults
Age (years)202122232428
 SexMMMMMM
 Type of muscle
 Biceps (B)Biceps (B)Biceps (B)Biceps (B)Biceps (B)Biceps (B)
 Anterior Masseter (AM)Anterior Masseter (AM)Anterior Masseter (AM)Anterior Masseter (AM)Anterior Masseter (AM) 
 Posterior Masseter (PM)Posterior Masseter (PM)Posterior Masseter (PM)Posterior Masseter (PM)Posterior Masseter (PM) 
 Deep Masseter (DM)Deep Masseter (DM)Deep Masseter (DM)Deep Masseter (DM)Deep Masseter (DM) 
Aged adults
 Age (years)586478798283
 SexMMMMMM
 Type of muscle
 Biceps (B)Biceps (B)Biceps (B)Biceps (B) Biceps (B)
 Posterior Masseter (PM)Deep Masseter (DM)Posterior Masseter (PM)Anterior Masseter (AM)Anterior Masseter (AM)Anterior Masseter (AM)

In five young adults (20–24 years old), three different parts of the masseter were analysed: anterior, posterior and deep. The masseter is divided into a superficial and a deep (DM) portion. The superficial portion is composed of an anterior (AM) and a posterior part (PM). Only the biceps biopsy was analysed for the 28-year-old donor and only the masseter biopsy was analysed for the 82-year-old donor. Muscle biopsies were frozen in precooled isopentane and stored at −80 °C. Serial sections (5 µm) were obtained using a cryostat microtome.

Isolation of genomic DNA from muscle samples

Thirty milligrams of muscle was digested in 650 µL of proteinase K digestion buffer (100 mm NaCl, 10 mm tris HCl pH 8, 100 mm EDTA pH 8, 1% Triton X-100) containing 0.1 mg proteinase K (20 U mL−1) (LifeTechnologies, Cergy-Pontoise, France) and incubated in a shaking water bath overnight at 55 °C. The nucleic acid was extracted with Phenol/chloroform/isoamyl alcohol (25 : 24 : 1 (vol:vol:vol)). DNA was precipitated with ammonium acetate 7.5 m and ethanol (vol:vol) at 100 °C. DNA was resuspended and stored at 4 °C in TE buffer (10 mm Tris-HCl, 1 mm EDTA pH 8).

Telomere length analysis

DNA samples obtained from muscle tissue were digested with the restriction enzyme HinfI (Biolabs, Ozyme, Saint-Quentin en Yvelines, France) for 4 h at 37 °C. This enzyme generates telomere restriction fragments (TRFs), which contain the TTAGGG tandem-repeat sequence and a constant subtelomeric fragment (Decary et al., 1997). An undigested DNA sample was used to verify the absence of DNA degradation (data not shown). Three micrograms of digested genomic DNA and 32P-DNA ladder (1 kb and HMW, Life Technologies) were resolved by electrophoresis in 0.7% agarose gels. To avoid loss of high-molecular-weight DNA, gels were dried, denatured and then neutralized. The telomere restriction fragments (TRFs) were detected by hybridization to a 32P-TTAGGG probe. The hybridization was performed directly on the dried gel as already described (Decary et al., 2000). The exposure times varied from 96 to 120 h and the signal was intensified on X-ray film (BioMax, Kodak) with a BioMax transcreen (Kodak, EIS, Massy, France).

The signal responses were analysed by a computer-assisted system using NIH Image 1.62 (densitometric data of one-dimensional gels) and ProFit (densitometric profils analysis) software. The mean and minimum values of telomere length (in kbp) was determined three times for each sample on three independent gels. The mean telomere length (L) was calculated by integrating the signal intensity above background over the entire TRF distribution as a function of TRF length using the formula: L = Σ(ODiLi)/Σ(ODi) where ODi and Li are the signal intensity and TRF length, respectively, at position i on the gel image (Harley et al., 1990; Vaziri et al., 1993). To determine the minimum telomere length in a homogeneous way for all samples, the densitometric profile of TRF length was integrated over the distance of migration. The minimum telomere length corresponds to 95% of this integration.

Determination of the percentage of satellite cells

Cryostat sections (5 µm) were mounted on glass coverslips and air dried at room temperature. For immunohistochemistry, sections were incubated at 4 °C overnight with a monoclonal antibody against N-CAM/CD56 (dilution 1 : 100) (Leu-19, Becton Dickinson, Le Pont de Claix, France) (Schubert et al., 1989; Muller-Felber et al., 1993; Kadi & Thornell, 2000). The specific antibody binding was revealed by the peroxidase antiperoxidase (PAP) method using diaminobenzidine (DAB) as chromogen in the presence of hydrogen peroxide. Finally, sections were counterstained with Mayer's Hematoxylin to reveal all nuclei. Negative controls (i.e. without primary antibody) confirmed the specificity of N-CAM staining. The analysis of sections was performed using a light microscope connected to a computerized image acquisition system (IBAS, Kontron Elektronic GmbH). For each muscle sample, at least 200 fibres were quantified within randomly chosen sections delimited by a constant frame. Nuclei localized within the cell boundary of a muscle fibre were counted as myonuclei and nuclei which localized within cells positive for N-CAM were counted as satellite cell nuclei. The ratio between the number of satellite cell nuclei and the total number of nuclei (myonuclei and nuclei of satellite cells) was defined as the proportion of satellite cells.

Statistical analysis

Data are presented as mean and standard deviation. The three parts of the masseter obtained from each young adult were pooled for stastistical analysis, after first determining that no differences existed between the different parts. Possible differences in minimum and mean telomere length, satellite cell proportion and mean number of satellite cells and myonuclei per fibre data were analysed using a two-way anova (age and muscle as independent variables) (Prism, San Diego, USA). When the anova revealed a significant effect, differences were tested using the t-test. Significance of variation with age was analysed by linear regression with slope and correlation coefficient (r). The P value tested the null hypothesis that the slope was 0 and all P values below 0.05 were considered statistically significant.

Acknowledgments

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgments
  8. References

We thank Margaretha Enerstedt, Mona Lindström and Elodie Ponsot for excellent technical assistance. This work was supported by the Association Française contre les Myopathies (AFM), the Association de Recherche contre le Cancer (ARC), The Duchenne Parent Project (The Netherlands), the University Pierre et Marie Curie (Paris 6), the CNRS, the European community (Aging Muscle; QLRT-1999-020304), the Swedish Medical Research council 12x-3934, and the Medical Faculty of Umeå University.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgments
  8. References