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The Effects of Regular Strength Training on Telomere Length in Human Skeletal Muscle

KADI, FAWZI1; PONSOT, ELODIE2; PIEHL-AULIN, KARIN2; MACKEY, ABIGAIL3; KJAER, MICHAEL3; OSKARSSON, EVA2; HOLM, LARS3

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Medicine & Science in Sports & Exercise: January 2008 - Volume 40 - Issue 1 - p 82-87
doi: 10.1249/mss.0b013e3181596695
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Abstract

Telomeres are noncodant repetitive sequences (TTAGGG)n located at the end of chromosomes. The length of telomeres is an important measure of the replicative history of the cell and also an indicator of its proliferative potential, because in cell cultures chromosomal telomeres shorten with each round of cell division (2,10,16,26). One reason for this shortening is the so-called end replication problem attributable to the inability of the DNA polymerase to completely replicate the ends of the linear molecules. However, recent in vivo studies suggest that cell replication is not the only factor accountable for the regulation of telomere length, and that external factors also can affect telomeres. In this respect, it has been shown that telomere shortening is stress dependent and that it occurs at higher rates in cells with low antioxidative defense (24). The fact that telomeres are influenced by external factors has recently been shown in a study on the effects of chronic stress on telomere length (9). Caregiving mothers (mothers of chronically ill children) with the highest levels of perceived stress have a significantly lower telomere length in peripheral blood mononuclear cells than do control mothers (mothers of healthy children) (9). It has also been shown that mood disorders are associated with accelerated telomere shortening (25), and it is known that telomere shortening is not an irreversible process, because of the existence of telomerase, an enzyme capable of extending telomeres. New evidence suggests that the regulation of telomere and telomerase in vivo is more complex than what has been described in cell culture systems (23). Telomerase has been demonstrated in many tissues, including heart myocytes and skeletal muscle (15,20). Thus, it now seems likely that telomeres are subject to various regulations in many body systems to maintain tissue integrity (23).

Skeletal muscle satellite cells are undifferentiated myogenic precursors able to reenter the cell cycle to 1) provide new myonuclei during postnatal growth, 2) generate new muscle fibers, or 3) produce new satellite cells (13). Recent studies have demonstrated the important role played by satellite cells in the adaptation of skeletal muscle to strength training (13). Satellite cell proliferation can contribute to the acquisition of new myonuclei in hypertrophying muscle fibers and to the repair of segmental muscle injuries caused by excessive loading (1,11,14,17,18). It has also been shown that the proliferation of satellite cells in response to exercise allows the renewal of their own pool (3,5,8,12,14,18,22). The increase in CyclinD1 mRNA levels, together with the increase in the number of satellite cells during resistance training, is indicative of the occurrence of extensive cell divisions (14). Moreover, recent studies on the acute effects of strength training demonstrate the capacity of satellite cells to proliferate very rapidly in exercised muscle. Significant increases in the proportion of satellite cells have been detected in young men at 4 and 8 d after a single bout of maximal exercise (5), and a remarkable increase in satellite cell number (141% increase) has been observed as early as 24 h after 92 maximal eccentric contractions in young men (8).

The significant recruitment of satellite cells in the adaptive process of skeletal muscle to exercise raises the important question of whether the regular practice of strength training affects skeletal muscle DNA telomere length. To our knowledge, the influence of regular strength training on skeletal muscle telomere length of healthy athletes has never been documented. There is only one report on skeletal muscle DNA telomere length in endurance athletes suffering from exercise-associated chronic fatigue, a condition labeled fatigued athlete myopathic syndrome (FAMS) (4). A severe reduction in skeletal muscle DNA telomere length has been shown in FAMS patients compared with a healthy, asymptomatic, age-matched control group.

Given the above discussion, the aim of the present study was to assess whether the practice of strength training for several years affects skeletal muscle DNA telomere length. This was achieved by comparison of skeletal muscle telomere length from a population of power lifters regularly involved in strength training against that of a population of healthy, active individuals.

METHODS

Our study aims to answer the question of whether long-term practice of sports activities where muscles are put under great strain affects the rate of telomere shortening in skeletal muscle. To assess the effects of long-term power lifting on telomere length in skeletal muscle, we have studied a population of athletes who regularly trained for several years, and we compared them with a population of healthy, active individuals with no history of strength training.

Subjects

Seven power lifters (PL group) (28.5 ± 6.6 yr, 100.2 ±15.5 kg, 178 ± 5 cm) and seven healthy control individuals (C group) (24.1 ± 2.1 yr, 77.9 ± 15.8 kg, 180 ± 6 cm) with no history of strength training participated in the study. All power lifters participated in Swedish national competitions. They practiced power lifting for a period of 8 ± 3 yr and competitive power lifting for a period of 7 ± 3 yr. They had a training frequency of three to four sessions per week, corresponding to a mean of 7 h of training per week. Subjects in the control group did not participate in any regular sport activities, and they exercised less than once a week. They were active in that most did cycling and walking for transportation. The study was approved by the ethics committees of Uppsala and Copenhagen (M-255, 2003 and KF01-171/04), and written informed consent conforming to the standards set by the Declaration of Helsinki was obtained from all subjects.

Procedures

Evaluation of telomere length in skeletal muscle.

Mean and minimum telomere lengths were determined, using the Southern blot protocol specifically adapted for the study of human skeletal muscle (19) and previously described by Decary et al. (6,7), Collins et al. (4), and Renault et al. (21).

Isolation of genomic DNA.

Muscle biopsies were obtained from the vastus lateralis muscle. The biopsies were rapidly frozen in liquid nitrogen and stored at −80°C for subsequent DNA extraction and telomere restriction fragment (TRF)-length determination. For total genomic DNA extraction, 8-10 mg of the muscle biopsy was digested overnight at 55°C, with gentle agitation in 500 μL of digestion buffer (100 mM NaCl; 10 mM Tris-Cl, pH 8.0; 100 mM EDTA, pH8; 1% Triton X-100) containing 40 U·mL−1 of proteinase K. The digest was extracted with one volume of 25:24:1 (vol:vol:vol) phenol/chloroform/isoamyl alcohol. The DNA was precipitated with one volume of 0.5:2 (vol:vol) 7.5 M ammonium acetate and ethanol 100%, washed with 70% ethanol, resuspended in TE buffer (10 mM Tris-Cl, pH 8.0; 1 mM EDTA, pH 7.5), and stored at 4°C (4,7).

Telomere length analysis.

The intact genomic DNA obtained from muscle tissue samples was digested for 4 h at 37°C with the restriction enzyme Hinf I (New England Biolabs). A smear of DNA fragments containing TRF with different lengths is generated (2). The undigested, extracted DNA samples were resolved on agarose gels to verify the absence of any DNA degradation. Mean and minimum TRF lengths were determined, using Southern blot analysis (7). Three micrograms of the digested DNA, together with two DNA ladders (1 kb plus and high molecular weight, invitrogen), were resolved, using electrophoresis in 0.7% agarose gels (+4°C, 90 V, 20 h), as previously described (4,6,7,23). The gels were dried, denatured, and then neutralized. TRF were detected by hybridization to a 32P-labeled (TTAGGG)4 probe. Hybridization was performed directly on the dried gels, which were then exposed to x-ray film (BioMax MS, Kodak) with a BioMax transcreen (BioMax MS, Kodak). A representative Southern blot of telomeric skeletal muscle DNA is shown in Figure 1. The signals were analyzed, using a computer-assisted system (Scion image 4.03 software, Scion Corporation) as previously described (19). The intensity of the signal is plotted as a function of the distance of migration, to determine the migration distances corresponding to the mean and the minimum TRF lengths. TRF length is then obtained by converting the migration distance (cm) into kbp, using the migration profile of low-molecular weight (range: 1-12 kb) and high-molecular weight (range 8-19kb) ladders. The signal obtained is made of an ascending slope up to the plateau intensity, followed by a descending slope down to the baseline signal intensity. As shown in Figure 1, for each sample, both the ascending and the descending parts were modelized, using a sixth-degree polynomial equation with a coefficient of correlation of 0.99. Using the equations of the ascending and the descending slopes, several parameters were determined (Fig. 1):

FIGURE 1
FIGURE 1:
Telomere length assay. A, Representative autoradiogram showing the size distribution of TRF from skeletal muscle; molecular weight standards are indicated in kilobase pairs. B, Determination of the mean TRF length (L mean) and minimum TRF length (L mini), using a sixth-degree polynomial equation. A max, maximal signal amplitude; I 0, 2.5% of A max added to baseline signal intensity. TRF length is then obtained by converting the migration distance (cm) into kilobase pairs, using the migration profile of low-molecular weight (range: 1-12 kb) and high-molecular weight (range: 8-19 kb) ladders.
  1. Amax: maximal signal amplitude from the baseline signal to the plateau intensity.
  2. I0: corresponds to 2.5% of Amax added to the baseline signal intensity.
  3. Lmean: mean TRF length, which corresponds to the midpoint of the segment formed by the intersection of the ascending and the descending linear slopes with the plateau intensity. Lmean comprises TRF of the majority of postmitotic myonuclei, which were incorporated into muscle fibers from birth and, consequently, have undergone only few mitoses before differentiation.
  4. Lmini: minimum TRF length, which corresponds to the intersection point between the linear slope of the descending part with I0. Lmini comprises TRF from satellite cells and also from the myonuclei newly incorporated during the last mitotic cycles.

To improve the precision of the measurements, we have determined the Lmean and Lmini three times, on three independent gels, with a coefficient of variation of 3.6%.

Statistical Analysis

Data are presented as means ± standard deviation (SD). The statistical analysis was performed by using the Sigmastat 3.0 software (SPSS, Chicago, IL). The statistical power analysis was estimated according to MSSE® guidelines. For an alpha level of 0.05, the power of the tests ranged between 0.62 and 0.67. Differences in skeletal muscle telomere length between groups were analyzed, using a one-way analysis of variance (ANOVA). Pearson linear regression analysis was used to study the relationship between two variables. Differences were considered to be significant for P < 0.05 and to be a tendency for P < 0.10.

RESULTS

Mean personal records in squat, bench press, and deadlift were 292.5 ± 42, 184 ± 25, and 292.5 ± 34 kg, respectively. The performance in squat was highly associated with deadlift (r = 0.97, P = 0.0002), whereas the association between bench press and both squat (r = 0.84, P = 0.02) and deadlift (r = 0.82, P = 0.02) was slightly weaker.

The Lmean of skeletal muscle DNA in PL (11.7 ± 1.5 kbp) tended to be higher (P = 0.07) than in C (10.5 ± 0.5 kbp) (Fig. 2). Similarly, the Lmean tended to be higher in PL (5.3± 0.7 kbp) compared with C (4.7 ± 0.3 kbp) (P = 0.09) (Fig. 2). Because there was variability in both Lmean and Lmini in PL, and because the personal records of the power lifters also varied, we reasoned that the performance in power lifting might have a stronger association with telomere length than simply belonging to the PL group. Interestingly, within the PL group, the Lmini of vastus lateralis DNA was inversely correlated to the individual records in squat (r = −0.86; P = 0.01) and deadlift (r = −0.88; P = 0.01) (Fig. 3). The heavier the load lifted, the shorter the power lifter's skeletal muscle DNA minimum telomere length. The Lmean of vastus lateralis also tended to be inversely correlated to the personal records in deadlift (r = −0.7; P = 0.07) but not to squat (r = −0.64; P = 0.1). Skeletal muscle DNA TRF length was not associated with the number of training sessions per week, the number of years of power lifting practice, or the number of years of competitive power lifting. When both groups were taken together, we found no relationship between the age of the subjects and skeletal muscle DNA TRF length.

FIGURE 2
FIGURE 2:
Minimum (A) and mean (B) TRF length of vastus lateralis muscles from power lifters (PL) and controls (C).
FIGURE 3
FIGURE 3:
Relationships between the minimum TRF length of skeletal muscle in power lifters and their personal records in squat (A), deadlift (B), and bench press (C).

DISCUSSION

According to Collins et al. (4), a population of athletes suffering from exercise-associated chronic fatigue had extremely short skeletal muscle DNA Lmini. This important report raises the question of whether the regular practice of sports at a high level is accompanied by deleterious effects on telomere length in skeletal muscle. To our knowledge, the exact influence of regular strength training on skeletal muscle telomeric DNA has never been addressed. Our study shows for the first time that at a group level, regular power lifting is not associated with an abnormal shortening of skeletal muscle DNA telomere length. On the contrary, skeletal muscle telomeres of power lifters tended to be longer than those in a population of active subjects with no history of strength training. We also have demonstrated, for the first time, the existence of an inverse relationship between skeletal muscle minimum telomere length and the personal records in squat and deadlift.

In our study, the Lmean and Lmini of skeletal muscle DNA in both PL and C were within the range of values previously recorded in healthy populations (4,7,21). The mean telomere length is a parameter frequently used to investigate telomere length in many tissues under normal and pathological conditions. However, in a postmitotic tissue such as skeletal muscle, it has been shown that the Lmean is not an appropriate parameter to evaluate the small loss of telomeric DNA. The mean telomere length comprises TRF of the majority of postmitotic myonuclei, which had been incorporated into the muscle fibers from birth and, consequently, have undergone only few mitotic divisions before differentiation. On the contrary, the minimum telomere length comprises TRF from satellite cells and also from the myonuclei newly incorporated during the last mitotic cycles (7). Thus, the minimum telomere length gives information on the low rate of telomere shortening, which occurs in normal muscle as a result of satellite cell recruitment and myonuclear replacement or during muscle regeneration (7). For instance, using the Lmini, dramatic shortening of telomeres has been shown to occur in children suffering from muscular dystrophies (−187 bp per year) (6).

Our findings indicate that at a group level, the Lmini tended to be higher in the power lifters compared with the subjects with no history of regular strength training. This observation clearly shows that long-term practice of power lifting is not associated with an abnormal shortening of telomeres such as that seen in the athletes suffering from exercise-associated chronic fatigue, or FAMS (4). It is noteworthy that the minimum telomere length in highly trained subjects actually tended to be longer than in subjects with no history of strength training. Although outside the scope of this study, both genetic and nongenetic factors might explain this observation. First, the existence of particular performance genes or the occurrence of genetic predispositions to sports-related injuries has been frequently discussed in the field of sports. In this respect, the possibility that the higher telomere length in the athlete population is part of their genetic predispositions cannot be excluded. Second, the existence of in vivo regulatory mechanisms able to influence the length of telomeres also cannot be excluded. In this respect, recent studies clearly have demonstrated the disparity between the regulation of telomere length in vivo and in vitro (23). Third, the extent of injury/regeneration cycles in skeletal muscle of these athletes might be much lower than that seen in pathological conditions. Finally, the handling of the biopsy includes the removal of surrounding fat and connective tissue. However, the occurrence of segmental fibrosis within the muscle fiber cannot be ruled out. Therefore, it should be emphasized that the possible occurrence of connective tissue cells in skeletal muscle of athletes who might suffer from inflammation/fibrosis might also affect telomere length in vastus lateralis.

Interestingly, our findings also indicate that the performance in power lifting (squat and deadlift) is inversely correlated to the Lmini in vastus lateralis. This suggests that the achievement of a high level of performance in power lifting is associated with a higher level of satellite cell recruitment. Thus, although the minimum telomere length in the athlete population remains within normal physiological ranges, it seems that the heavier the load put on muscles, the shorter the Lmini of the muscles in question. Thus, it can be hypothesized that when the exercise-induced satellite cell recruitment exceeds a given threshold, the telomere length positive regulation becomes ineffective. Interestingly, the number of years of practice and the number of years of competitive power lifting were not associated with minimum telomere length.

It is suggested that within a muscle fiber, each myonucleus is responsible for protein synthesis over a finite volume of cytoplasm-a concept named the myonuclear domain. Repeated bouts of resistance exercise induce increases in the number of domains (enhancement of the myonuclear number) as existing myonuclei become unable to sustain further enlargement of the cytosolic area (1,12,13,17,18). As for the Lmini, the Lmean of skeletal muscle in PL was within normal physiological ranges and tended to be inversely correlated to the personal records in power lifting. However, contrary to the strong associations found between Lmini and performance level, Lmean was only weakly associated with personal records. This might be explained by the fact that a skeletal muscle fiber is a multinucleated syncytium, where the newly incorporated myonuclei during the course of muscle adaptation to strength training are drowned within the thousands of myonuclei already present in the muscle fiber.

Our results might indicate that the newly recruited myonuclei are beginning to have an influence on the Lmean of vastus lateralis in this population of power lifters.

Practical application.

An alarming report has indicated that endurance athletes with exercise-associated fatigue have abnormally short skeletal muscle DNA telomeres (4). This would suggest a dramatic decrease in the capacity to repair severe or mild muscle injuries that would occur during training or in daily activities. Our data clearly show that the practice of regular strength training for several years is not associated with abnormal telomere shortening. For coaches and athletes, our data indicate that the regular practice of strength training would not impair the capacity of muscle repair, despite the regular occurrence of cycles of muscle fiber injury/repair during training. However, the question of whether the occurrence of an overtraining syndrome negatively affects telomere length remains to be assessed. Finally, it should be emphasized that the data in this study were obtained in young males performing strength training for a period of 7 ± 3 yr. Therefore, the results may not be applicable to a longer period of strength training, older individuals, or females.

In conclusion, at a group level, skeletal muscle DNA telomere length in power lifters remains within the range of values found in subjects with no history of regular strength training. Thus, well-designed strength training protocols where athletes are free from symptoms of overtraining do not lead to deleterious effects on telomeres. The inverse relationship between Lmini and personal records indicates that the maximum load that can be sustained by skeletal muscle, rather than the total amount of training, is an important factor influencing satellite cell recruitment in the course of skeletal muscle adaptation to strength training. The existence of regulatory mechanisms allowing the control of telomere length in vivo cannot be excluded. Further investigations are needed to better understand the regulation of telomere length in vivo under normal physiological conditions.

This study was supported by grants from the Swedish National Center for Research in Sports (74/06, 101/07, and 159/07).

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Keywords:

RESISTANCE EXERCISE; HYPERTROPHY; MUSCLE DNA; MYONUCLEI; SATELLITE CELLS; REGENERATION

© 2008 American College of Sports Medicine