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Athletes with Exercise-Associated Fatigue Have Abnormally Short Muscle DNA Telomeres


Medicine & Science in Sports & Exercise: September 2003 - Volume 35 - Issue 9 - p 1524-1528
doi: 10.1249/01.MSS.0000084522.14168.49
BASIC SCIENCES: Original Investigations

COLLINS, M., V. RENAULT, L. A. GROBLER, A. ST CLAIR GIBSON, M. I. LAMBERT, E. W. DERMAN, G. S. BUTLER-BROWNE, T. D. NOAKES, and V. MOULY. Athletes with Exercise-Associated Fatigue Have Abnormally Short Muscle DNA Telomeres. Med. Sci. Sports Exerc., Vol. 35, No. 9, pp. 1524–1528, 2003.

Introduction/Purpose  Although the beneficial health effects of regular moderate exercise are well established, there is substantial evidence that the heavy training and racing carried out by endurance athletes can cause skeletal muscle damage. This damage is repaired by satellite cells that can undergo a finite number of cell divisions. In this study, we have compared a marker of skeletal muscle regeneration of athletes with exercise-associated chronic fatigue, a condition labeled the “fatigued athlete myopathic syndrome” (FAMS), with healthy asymptomatic age- and mileage-matched control endurance athletes.

Methods Muscle biopsies of the vastus lateralis were obtained from 13 patients diagnosed with FAMS and from 13 healthy control subjects. DNA was extracted from the muscle samples and their telomeric restriction fragment (TRF) or telomere lengths were measured by Southern blot analysis.

Results All 13 symptomatic athletes reported a progressive decline in athletic performance, decreased ability to tolerate high mileage training, and excessive muscular fatigue during exercise. The minimum value of TRF lengths (4.0 ± 1.8 kb) measured on the DNA from vastus lateralis biopsies from these athletes were significantly shorter than those from 13 age- and mileage-matched control athletes (5.4 ± 0.6 kb, P < 0.05). Three of the FAMS patients had extremely short telomeres (1.0 ± 0.3 kb). The minimum TRF lengths of the remaining 10 symptomatic athletes (4.9 ± 0.5 kb, P < 0.05) were also significantly shorter that those of the control athletes.

Conclusion  These findings suggest that skeletal muscle from symptomatic athletes with FAMS show extensive regeneration which most probably results from more frequent bouts of satellite cell proliferation in response to recurrent training- and racing-induced muscle injury.

1UCT/MRC Research Unit for Exercise Science and Sports Medicine, Department of Human Biology, University of Cape Town, SOUTH AFRICA; and

2CNRS UMR 7000, Medical Faculty, University of Paris, FRANCE

Address for correspondence: Dr. M. Collins, UCT/MRC Research Unit for Exercise Science and Sports Medicine, Department of Human Biology, University of Cape Town, P. O. Box 115, Newlands, 7725, South Africa; E-mail:

Submitted for publication February 2003.

Accepted for publication May 2003.

Prolonged, demanding, weight-bearing exercise has been reported to cause both acute and more chronic muscle damage (reviewed in 12). Although adult skeletal muscles are made up of highly differentiated, elongated multinucleated postmitotic cells, they also contain a small population of quiescent mononucleated satellite cells that are located between the sarcolemma and the basement membrane (13). These cells are responsible for both muscle growth and repair. After skeletal muscle damage, the satellite cells proliferate and then fuse to repair or replace the damaged fibers. Some of these mononucleated cells will return to quiescence to restore the population of satellite cells (3). With age, there is a progressive reduction in both the proliferative capacity and the total number of satellite cells (15,16). The proliferative capacity of the satellite cells is limited, in part, by the loss of telomeric sequences, which are specialized DNA-protein structures at the end of the chromosomes (1). During each somatic cell division, a small piece of telomeric DNA is lost. Once the telomeric DNA becomes too short a DNA damage signal is initiated causing the cell to become senescent and stop dividing, thus limiting the regenerative capacity of the cell (6). Since the proliferating satellite cells will lose a small piece of telomeric DNA during each replicative cycle, the newly incorporated DNA in the repaired fiber will have shorter telomeres (5). Thus, the number of cycles of satellite cell proliferation as well as their remaining regenerative capacity can be determined indirectly by measuring the telomeric length or, more specifically, the minimum telomeric restriction fragment (TRF) lengths of DNA from skeletal muscle biopsies (5,16).

Athletes suffering from exercise-related chronic fatigue are ideally suited for studying any potential long-term detrimental effects of high-volume endurance training. These athletes have a long history of very high training loads and participation in competitions. They demonstrated a precipitous decline in performance which is not related to ordinary aging, they present with complaints of chronic fatigue dominated by skeletal muscle symptoms including excessive delayed-onset-muscle-soreness, muscle stiffness, weakness, and tenderness as well as a failure to adapt to training. Typically they have sought the help of many clinicians without success. Clinical characteristics and recognized skeletal muscle pathological features, including those of the muscular dystrophies and mitochondrial myopathies, are also excluded in these athletes. The athletes who fulfill these criteria have previously been described as suffering from the “fatigued athlete myopathic syndrome” (FAMS) (7,20,21).

In this study, we have compared the lengths of the TRF in DNA isolated from biopsies of vastus lateralis muscles sampled from endurance athletes suffering from FAMS with those from control athletes, matched for age and historical training volume. We postulate that the skeletal muscle symptoms of the athletes suffering from FAMS might be caused by extensive muscle regeneration.

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Approval for this study was obtained from the Research and Ethics Committee of the Faculty of Health Sciences, University of Cape Town. Thirteen athletes diagnosed with FAMS and 13 healthy age-matched athletes were recruited for this study over a 3-yr period (7). The FAMS patients were recruited from the Sports Medicine Clinic at the Sports Science Institute of South Africa, Cape Town, South Africa. All the FAMS athletes had a history of high-volume training over several years, a history of chronic or excessive exercise-related fatigue, decreased physical performance during exercise and a clinical profile which was dominated by skeletal muscle symptoms including excessive delayed onset muscle soreness after exercise, stiffness, tenderness and skeletal muscle cramps (7). Control subjects (CON), matched for age and training volume and displaying none of the FAMS symptoms were recruited from local running clubs and the Sports Science Institute of South Africa. Athletes in the FAMS group were matched for the level of training before the onset of symptoms with individuals in the control group. Both the FAMS and control subjects were examined by a physician to exclude the presence of identifiable organic disease that would explain the athlete’s symptoms.

Before participation in the trial, all subjects completed informed consent forms as well as extensive questionnaires detailing their sporting and training history, current training and racing performance, and medical status. In addition, symptomatic athletes also described their medical, injury, training and running histories both before, and after the onset of symptoms.

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Exercise tolerance and skeletal muscle function.

Each subject’s maximal oxygen uptake (V̇O2peak) was determined during a treadmill test of increasing intensity according to a previously described method (18). Maximum voluntary contraction (MVC) of the knee extensors was measured with a Kin-Com isokinetic dynamometer (Chattanooga Group, Inc. Chattanooga, TN) as previously described (19).

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Skeletal muscle biopsy and fiber typing.

A percutaneous needle muscle biopsy sample was obtained from the vastus lateralis muscle of all subjects. Muscle fiber type proportions were determined using the myofibrillar ATPase method (8).

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Telomere length analysis.

A portion of the sample was rapidly frozen in liquid nitrogen and stored at −80°C for subsequent DNA extraction and TRF length determination as previously described (4,5,16). For total genomic DNA extraction, at least 10 mg of the muscle biopsy was ground to a powder in liquid nitrogen and digested overnight at 55°C with gentle agitation in 650 μL of proteinase K digestion buffer (10 mM Tris-Cl, pH 8.0; 100 mM EDTA, pH 8.0; 100 mM NaCl; 1% Triton X-100) containing 20 units·mL−1 proteinase K. The digest was extracted twice with 1 volume of 25:24:1 (vol:vol:vol) phenol/chloroform/isoamyl alcohol and the DNA precipitated with 1 volume of a 1:4 (vol:vol) 7.5 M ammonium acetate and 100% ethanol mixture, washed with 70% ethanol, resuspended in TE buffer (10 mM Tris-Cl, pH 8.0; 1 mM EDTA, pH 8.0) and stored at 4°C. The extracted DNA samples were resolved on agarose gels to verify the absence of any DNA degradation during the biopsy, storage or extraction processes (Fig. 1, top left panel). The intact genomic DNA was digested for 4 h at 37°C with the restriction enzyme, Hinf I, to generate a smear of DNA fragments containing TRF with different lengths of the TTAGGG repeat sequence and a subtelomeric fragment of non-TTAGGG DNA with a constant length (2). Mean and minimum TRF lengths were determined by Southern blot analysis as described by Decary et al. (5) and Renault et al. (16). Briefly, 3 μg of the digested DNA, together with a high molecular weight and 1 kb 32P-labeled DNA ladders, were resolved by electrophoresis on 0.7% agarose gels. The gels were dried, denatured, then neutralized and the TRF were detected directly in the dried treated gels by hybridization to a 32P-labeled (TTAGGG)4 probe, followed by exposed to x-ray film (BioMax, Kodak, EIS, Massy, France) with a BioMax transcreen (Kodak, EIS, Massy, France). Because the TRF lengths from a tissue range in size, mean and minimum TRF lengths were determined as previously described (10,22). The signal responses were analyzed 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 value of telomere length (in kilobase pairs) 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(ODi·Li)/Σ(ODi), where ODi and Li are the signal intensity and TRF length, respectively, at position i on the gel image (10,22). To determine the minimum value of telomere length in a homogenous way for all samples, the densitometric profile of the TRF length was integrated over the distance of migration. The minimum telomere length corresponds to 95% of this integration.



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Statistical analysis.

Data were analyzed using the STATISTICA version 5.5 (StatSoft Inc., Tulsa, OK) statistical program. Where applicable, data were presented as means ± standard deviations (SD). Pearson’s chi-square analysis was used to analyze differences between the genders of the FAMS and CON groups. A one-way analysis of variance was used to determine any significant differences between the subject characteristics, the training history, maximum voluntary contraction, V̇O2peak, fiber type proportions and the telomere lengths of the FAMS and CON groups. A dependent t-test was used to determine any significant differences between the training history of the FAMS group before and after the onset of FAMS. Statistical significance was accepted when P < 0.05.

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Subject characteristics and training history.

The FAMS group comprised 10 male and 3 female athletes. Included were 11 distance runners, a triathlete and an international squash player who had trained at a high level. Sporting achievement ranged from club to international level. The control group consisted of nine male and four female healthy asymptomatic athletes comprising 11 distance runners, a triathlete, and an endurance cyclist.

Subjects in each group were individually matched for gender, age, height and weight and for V̇O2peak and quadriceps isometric MVC (Table 1). Both groups also had a similar proportion of Type I skeletal muscle fibers. With the exception of a single FAMS athlete whose skeletal muscles comprised 91% Type I fibers, the range of Type I fiber type proportions was similar in the FAMS (31–67%) and CON (37–60%) groups.



The FAMS athletes showed a significant decrease in the training load (km·h−1, km·wk−1, and d·wk−1) and in their 5-km personal best times once they developed symptoms (Table 2).



Both the FAMS and CON athletes, who were endurance runners, ran more than 40 km continuously during training and/or racing at similar ages (FAMS: 27.6 ± 10.6 yr (N = 11); CON: 24.5 ± 9.0 yr (N = 10)) and continued to do so for a similar numbers of years (FAMS: 11.8 ± 5.6 yr (N = 11); CON: 14.2 ± 6.9 yr (N = 9)). The current training volume of the CON athletes was very similar to the training volume of the FAMS athletes before the onset of their symptoms (Table 3) but was more than the current training of the FAMS group after the onset of symptoms (Table 2).



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Telomere lengths.

The minimum TRF lengths in the vastus lateralis muscle of the FAMS athletes (FAMS all) were significantly shorter than those of the CON subjects (P = 0.017) (Table 4). Even when corrected for age, the minimum TRF lengths remained significantly shorter in the FAMS athletes (data not shown). There was no significant difference in the mean (P = 0.052) TRF lengths of the FAMS and CON athletes (Table 4).



Three of the FAMS athletes (Fig. 1 and FAMS path in Table 4) had significantly shorter mean and minimum TRF lengths than the remaining 10 FAMS subjects (FAMS nonpath in Table 4). When the data of these three subjects were excluded, the minimum TRF lengths of the remaining 10 FAMS athletes were still significantly shorter than those of the CON group (P = 0.043) (Table 4).

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The novel finding of this study was that the minimum TRF lengths of DNA from the vastus lateralis muscle of symptomatic athletes (4.0 ± 1.8 kb) were significantly shorter than those of the CON subjects (5.4 ± 0.6 kb). Decary et al. (5) have reported that the minimum TRF length of the vastus lateralis muscle decreases by about 13 bp per year from birth to 86 yr of age. When corrected for age, the minimum TRF lengths remained significantly shorter in the FAMS athletes, implying that, at least in the thigh muscles, muscle regeneration was occurring at a faster rate than in the control subjects. This suggests, that in an attempt to repair repeated bouts of exercise-induced muscle damage, the satellite cells of the FAMS patients have undergone more frequent rounds of replication, resulting in extensive regeneration of those muscles that are involved in repeated eccentric muscle contractions during running.

Indeed, histological studies of samples from these same FAMS patients have shown a significantly greater incidence of fiber size variation and the presence of internal nuclei than in controls. Ragged-red fibers, or any of the other histological signs of muscular dystrophies or mitochondrial myopathies, were not present in any of the samples. Because the presence of internal nuclei is a histological marker of muscle regeneration, the greater number of pathological changes in the FAMS patients is another indication that the damaged muscle in these patients may have reflected an accelerated aging process due to extensive regeneration (23,24).

Decary et al. (4) reported that the muscles of patients with Duchenne muscular dystrophy and limb girdle muscular dystrophy 2C have a 14-fold accelerated rate of telomeric DNA loss of 187 bp·yr−1. Although not to the same extent, these findings are similar to those we report in athletes with FAMS.

The minimum TRF lengths of the control endurance athletes were also slightly shorter than previously reported values for a sedentary population (5). This suggests that, although the control endurance athletes did not present with any of the FAMS symptoms, they nevertheless also showed evidence for extensive regeneration in their thigh muscles.

Close inspection of the maximal, mean and minimum TRF lengths of the FAMS athletes showed that 3 of the 13 athletes had significantly shorter telomeres. The mean and minimum TRF lengths of these three athletes were significantly shorter than those of the control group. Because skeletal muscle is a postmitotic tissue, mean and minimum TRF lengths should remain constant throughout its life (5). However, both mean and minimal TRF lengths in these three athletes were significantly shorter than the remaining 10 FAMS or control athletes; an alternative or additional mechanism, other than an accelerated satellite cell replication, could be implicated in telomere shortening. Because of its high guanine and cytosine content, which is a major target of reactive oxygen species (17), telomeric DNA may be more susceptible to oxidative damage. Telomeric sequences are also susceptible to breakages (11). During exercise, there is an increase in metabolism and the production of reactive oxygen species, such as superoxide anions, hydrogen peroxide, and hydroxyl radicals. Poulsen et al. (14) have shown that 30 d of extreme endurance exercise caused a significant increase in DNA modification. Exercise would only be expected to increase the rate of oxidative damage to DNA and other cellular structures if the capacity of the antioxidant defense mechanisms is exceeded. Dufaux et al. (9) have demonstrated a large interindividual variation in the oxidative stress experienced by moderately trained athletes after a 2.5-h run. It is therefore tempting to speculate that certain athletes are more susceptible to exercise-induced oxidative damage to their telomeres. However, one of these athletes had previously been diagnosed with anorexia, while a second had Type II diabetes. There is no evidence that either of these conditions is associated with accelerated telomere shortening in skeletal muscle. However, the effects of these conditions on premature telomere shortening remains to be investigated.

Finally, although the FAMS athletes had short telomeres their V̇O2peak and MVC were no different to the controls, suggesting that their short-duration muscle function was not affected in contrast to endurance activities.

In conclusion, the telomere lengths of DNA isolated from muscle biopsies from endurance athletes presenting with FAMS was significantly shorter than those of control athletes who did not have symptoms. These findings suggest that skeletal muscle from FAMS athletes shows extensive regeneration, which likely results from more frequent cycles of satellite cell proliferation induced by repeated bouts of muscle damage in these symptomatic athletes.

A preliminary report of this work has been published in abstract form (Collins, M., G. S. Butler-Browne, L. Grobler, A. St Clair Gibson, M. I. Lambert, E. W. Derman, and T. D. Noakes. (2001) Abnormal telomere lengths in athletes diagnosed with fatigued athlete myopathy syndrome. S. Afr. J. Sports Med. Sept: 33–34, 2001).

This study was supported in part by funds from the University of Cape Town, the South African Medical Research Council, Discovery Health, the Association Française contre les Myopathies (AFM), the University Pierre et Marie Curie (Paris 6), the CNRS, and the European community (Ageing Muscle, QLRT-1999-020304).

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