Despite serious and irreversible side effects, the use of anabolic steroids is a widespread phenomenon not only among athletes who want to improve their performance but also in a large part of the population who simply desire to improve their appearance (51).
Recently, Bhasin et al. (6) provided a clear demonstration of the myotrophic effects of anabolic steroids. The administration of 600 mg of testosterone per week for 10 wk to untrained men produced an increase in muscle strength and in the cross-sectional area of the quadriceps as measured by magnetic resonance imaging. These effects were further enhanced when testosterone treatment was combined with strength training. It was concluded that supra physiological doses of testosterone, especially when combined with strength training, increase both muscle size and strength in normal men (6). However, no morphological analysis was performed. Such an analysis is essential for the discovery of the mechanisms involved in the myotrophic action of anabolic steroids. In previous morphological studies on subjects using anabolic steroids, no increase in muscle fiber area was seen despite an increase in protein synthesis and muscle mass (20,30).
There are two mechanisms involved in the enlargement of skeletal muscles: the hypertrophy of individual muscle fibers and muscle fiber hyperplasia (3,4,19). An increase of the size of muscle fibers reflects a greater protein synthesis resulting from an increase in the mRNA synthesis (18). The increased amount of mRNA could be attributed to either an elevation of the nuclear density or the transcription per nucleus. In this regard, in animal studies, Giddings and Gonyea (17) and Winchester and Gonyea (49) observed an elevation of the myonuclear number in hypertrophied muscles. Evidences for muscle fiber hyperplasia have been provided by the identification of small-sized muscle fibers containing a myosin isoform characteristically expressed during the embryonic stage of muscle development (27) or expressing the embryonic myosin heavy chain mRNA (50).
The myotrophic action of anabolic steroids has been explained by an increase in the rate of protein synthesis and a decrease in the protein breakdown (9), an increase in the rate of gene transcription assessed by RNA polymerase activity (36) and by an increase in the number of myonuclei in the rat levator ani muscle (16,25).
The trapezius muscle, which has been previously characterized according to its muscle fiber type composition, capillary supply, and mitochondrial pattern (31,32), is frequently affected by work-related myalgia (21). In our laboratory, we are interested to gain insight into the adaptational capacity of this muscle in relation to different conditions.
The aim of the present study was to carry out a comprehensive investigation of the trapezius muscle of high-level power lifters who reported the use of more than one steroid at a time, over a period of several years, and at doses that were far beyond therapeutic levels. This group was compared to a group of high-level power lifters with no drug history. On the basis of muscle fiber-type frequency and fiber area, as well as the evaluation of the number of myonuclei and satellite cells and fibers expressing developmental proteins, we have determined the possible mechanisms of increase in muscle size induced by anabolic steroids.
Nineteen power lifters participated in the present study. All were highly competitive and participated in Swedish National and International competitions. We were able to perform this study because one of the authors (Anders Eriksson), a European champion in power lifting (Oldstad, Germany, 1988) and Nordic champion (Denmark, 1988 and Finland, 1990), has personal contacts with the Swedish elite power lifters. The athletes participated regularly in International competitions (World championship, European championship, and Swedish championship in power events). All the athletes had similar training regimen. They trained regularly four to six times a week, 2–3 h per session. The sessions consisted in four to seven sets of exercises and 3 to 12 repetitions per set.
Among the 19 power lifters, nine subjects (31 ± 3 yr, 109 ± 15 kg) have reported the use of a wide variety of high doses of anabolic steroids (steroid users) for a period of 9 ± 3.3 yr. Testosterone (100–500 mg·wk−1) was used in combination to a variety of anabolic steroids (nandrolone, stanozolol, Primobolan, oxymetholone, Mastoron, Proviron, and Durobolan). These subjects have been individually interviewed regarding their steroid usage, and they reported the amount and the type of drugs by themselves. The steroid regimen included both “staking,” or simultaneous use of several types at high doses, and “cycling,” a drug-free period followed by times when the doses and the types of drugs taken were increased to a maximum to anticipate peak performance. In addition, four subjects had taken insulin-like growth factor-1, and one subject had taken growth hormone. Two of them have been caught in regular drug testing.
The 10 other power lifters (27 ± 7 yr, 98 ± 21 kg) have never used these substances, and they were selected from the same club where they signed a contract that committed them never to use any drugs. Four of them participated voluntarily as controls in an other project aiming to find more effective methods to detect drugs.
The performance in bench press was 188 ± 35 kg in the reported steroid users and 183 ± 28 kg in the nonsteroid users. The characteristics of these athletes, which are summarized in Table 1, are comparable to those given for best athletes in the world (28). This work was approved by the Ethical Committee of Umeå University. Written consent in accordance with the policy statement regarding the use of human subjects was obtained from all the subjects.
Biopsy forceps were used to take biopsies from the upper part of the trapezius muscle (descending I), which is one of the muscles used in strength training. The samples were mounted in embedding medium (Tissue tekR, Miles Laboratories, Naperville, IL) and quickly frozen in propane chilled in liquid nitrogen. The samples were stored at −80°C until analysis.
Serial cross-sections 5–10 μm thick were cut at about −20°C. Histochemical analysis of myofibrillar adenosine triphosphatase (mATPase) was performed after preincubations at the acidic pHs 4.3 and 4.6 and the alkaline pH 10.4, and myosin heavy chain expression was assessed using well-characterized monoclonal antibodies (mAb) specific to human slow myosin heavy chain (mAb A4.840) and slow and fast A myosin heavy chain (mAb N2.261) (22). Six muscle fiber types were delineated as described previously (26). The Type I fibers (slow twitch, oxidative fibers) were stable in the acid ranges but labile at pH 10.4 and strongly stained with the mAb A4.840. Type IIA fibers (fast twitch, oxidative glycolitic fibers) displayed a reversed pattern and were strongly stained with the mAb N2.261. Fibers that were stable at pH 10.4 and 4.6 but labile at pH 4.3 were classified as either Type IIB (fast twitch glycolitic fibers) or Type IIAB, depending on their staining intensities. Type IIAB fibers were stained intermediately between IIA and IIB fiber types at pH 4.6 and weakly stained with the mAb N2.261, whereas Type IIB fibers were unstained with the mAb N2.261. Fibers that were stained at all pHs were classified either as intermediate stained fibers (IM) and as Type IIC fibers. Type IIC fibers were strongly stained with the mAb N2.261 and moderately stained with the mAb A4.840 and Type IM fibers exhibited the inverse staining pattern. To delineate the circumference of each fiber type, sections were stained with a mAb against the laminin alpha chains (4C7) (48).
Sections were also stained with mAbs against embryonic (F1 651) and neonatal (MHCn NCL) myosin heavy chains isoforms (11,14), and Leu 19 antigen, identical to the neural cell adhesion molecule (40). Embryonic and neonatal myosin heavy chains represent developmental isoforms of the myosin molecule, and Leu 19 antigen is a cell-cell recognition molecule expressed during the early stages of fiber formation and in satellite cells (40). The antibodies were purchased from the American Type Culture Collection, Novocastra, and Becton Dickinson.
To assess the myonuclear content and the frequency of satellite cells, we have developed a method that allowed easy distinction between a true nucleus and a nucleus of a satellite cell on muscle fiber cross sections. In sections immunostained with the mAb against the Leu 19 antigen and counter stained with Mayer’s hematoxylin, the nuclei of satellite cells were blue surrounded by a brown rim. The myonuclear population was evaluated on two different sections at different levels of the fiber length. Satellite cell frequency was calculated in each subject as follows: satellite cell number/(myonuclear number + satellite cell number) × 100. For the visualization of primary antibody binding, the peroxidase-antiperoxidase method or labeling with fluorescein isothiocyanate (FITC) was used (45). Briefly, the sections were pretreated in bovine serum albumin and incubated in unlabeled primary antibody for 1 h at 37°C. The sections were then rinsed in 0.1 M phosphate buffer and incubated in the secondary antibody raised against the primary antibody for 30 min followed by incubation in peroxidase-antiperoxidase complex and revealed in a solution containing diaminobenzidine and hydrogen peroxidase. In the sections stained with FITC, the nuclei were visualized using a mounting medium containing 4′,6-diamidino-2-phenylindole (DAPI) from Vector laboratories. The analysis of sections was performed using a light microscope (Zeiss Axiophot) connected to a computerized image analysis system (IBAS, Kontron Elektronic GMBH). An average of 200 fibers were photographed and used for the determination of fiber types, fiber area, capillarization, myonuclear number, and frequency of satellite cells. The muscle fibers stained with developmental myosin were counted on the whole cross-section and expressed as a proportion of the total number of fibers.
Data are presented as mean and standard deviations. The statistical significance of the differences between the two groups was determined using a t-test for unpaired data. The statistical power analysis for each t-test was estimated according to MSSE guidelines. For alpha = 0.05, the power of all tests ranged between 0.70 and 0.99. P values < 0.05 were considered statistically significant. The correlation coefficient (r) was used to determine the degree of relationship between two variables. Due to their rare occurrence, Type IIAB, IIC, and IM fibers were not included in the statistical analysis.
Fiber type and fiber area.
The main fiber types in the trapezius muscles of both groups were Type I and Type IIA (53.5 ± 6% Type I and 42.4 ± 7% Type IIA in the reported steroid-using and 55 ± 8.5% Type I and 39 ± 7.5% Type IIA in the nonsteroid-using group). The additional fibers were of Type IIAB, IIC and IM; no Type IIB fibers were observed. There were no statistical differences in the proportion of Type I and IIA fibers between the two groups. The mean fiber area of both Type I and IIA fibers in the reported steroid-using group (Type I: 8979 ± 1948 μm2 and Type IIA: 10672 ± 2533 μm2) was significantly larger than that in nonsteroid-using group (Type I: 5695 ± 863 μm2 and Type IIA: 7997 ± 2136 μm2;P < 0.05;Figs. 1 and 2). Moreover, we observed a greater increase in the area of Type I than Type II muscle fibers.
The number of myonuclei and the frequency of central nuclei and satellite cells.
The mean number of nuclei in Type I and Type II fibers was significantly higher (P < 0.05) in the reported steroid-using athletes than in the nonsteroid-using athletes (4.8 ± 1.2 in Type I and 5.7 ± 1.4 in Type II in the reported steroid-using and 3.9 ± 1.1 in Type I and 5 ± 1.7 in Type II in the nonsteroid-using group). There was a greater increase in nuclei number in the Type I fibers than in the Type II fibers: 23% compared with 14%. Likewise, we found a strong correlation between the mean number of nuclei and the mean fiber area in each subject (r = 0.8 and P < 0.01;Fig. 3). There was a nearly fivefold increase in the proportion of fibers with central nuclei (P < 0.05) in the reported steroid-using athletes in comparison with the other athletes (25 ± 12.5% in the steroid-using and 5.1 ± 2.8% in the nonsteroid-using). In the reported steroid-using group, central nuclei were detected equally in Type I and Type II fibers, whereas, in the nonsteroid-using group, they were found mostly in Type II fibers and were found in Type I fibers only in two subjects.
The frequency of satellite cells did not differ between the two groups. Likewise, satellite cells were equally distributed in Type I and Type II fibers in both groups (Type I: 6.2 ± 1.4%, Type II: 7.3 ± 2.6% in the reported steroid-using and Type I: 67 ± 1.7%, Type II: 6.8 ± 2% in the nonsteroid-using group).
Fibers expressing developmental myosin isoforms and LEU 19 antigen.
Fibers expressing the markers of recent myogenesis were found in all subjects from both groups (Fig. 4). However, their proportion was significantly higher (P < 0.05) in the reported steroid-using group (7.7 ± 4% embryonic and neonatal positive fibers and 5 ± 2.3% Leu 19 positive fibers) compared with the nonsteroid-using group (2.9 ± 1.5% embryonic and neonatal positive fibers and 2.5 ± 1.4% Leu 19 positive fibers).
This is the first study that confirms at the muscle fiber level, the previously observed increase in muscle mass after testosterone treatment (6): the mean muscle fiber area was significantly increased in the steroid users group. Furthermore, it was possible to identify two mechanisms by which anabolic steroids induced muscular growth in strength-trained athletes: the self-administration of steroids induced a significant increase in the myonuclear number and enhanced the proportion of newly formed fibers. Additionally, we showed that the pool of satellite cells was not decreased.
The satellite cells would be the major source of the additional nuclei seen in the hypertrophied muscle fibers, because it is known that the myonuclei in mature fibers are unable to undergo mitosis (34,39). A recent study showed that the hypertrophy of mature rat extensor digitorum longus muscle cannot occur unless satellite cells are able to reproduce and contribute nuclei to the overloaded muscle fibers (37). The greater activation of satellite cells demonstrated in steroid-using athletes is supported by recent immunohistochemical demonstration of androgenic receptors on satellite cells (12). The DNA unit or nuclear domain concept is defined as the theoretical volume of cytoplasm controlled by each diploid nucleus (10). An implication of this concept is that the size of a cell depends on the myonuclear number (46). Because each myonucleus has the same amount of DNA, the intake of anabolic steroids induced an increase in the DNA content, necessary to sustain the protein synthesis in the extremely hypertrophied muscle cells. This phenomenon is strongly supported by the linear relationship between mean nuclear number and mean fiber area in the two groups. The increase in the myonuclear number in the anabolic steroid users is in accordance with the results of Galavazi and Szirmai (16) and Joubert and Tobin (25) showing that muscle fiber hypertrophy in rat levator ani muscle was induced by testosterone and was characterized by increases in fiber diameter and in the number of myonuclei. Furthermore, Joubert and Tobin (25) investigating the origin of the new myonuclei in the same muscle demonstrated an increase in satellite cell proliferation leading to the formation of new myonuclei.
Enhanced satellite cells stimulation would provide more nuclei to the muscle fibers. Because androgen receptors are located in the myonucleus (23,38,47), the increased nuclear number would also give rise to an elevation of the number of androgen binding sites, thus making the muscle more susceptible to the anabolic compounds.
The present study showed that there was no difference in the number of satellite cells between the two groups. Because the pool of stem cells remained unchanged, we conclude that the capacity of muscle fiber repair after injury is not altered by using anabolic steroids.
Fibers expressing developmental proteins were observed in both groups and suggested the formation of new fibers. Furthermore, anabolic steroids enhanced the proportion of newly formed fibers. In animal models, the formation of new muscle fibers after exercise was suggested by using specific markers expressed during various events of muscle fiber development, for review see (4). In this study, the first line of evidence for the formation of new muscle fibers is provided by the expression of the embryonic and neonatal myosin in the small-sized muscle fibers. The embryonic and neonatal myosin are the major isoforms of the myosin molecule expressed during early stages of fiber formation (5,8). The second line of evidence is provided by the expression of Leu 19 antigen, a glycoprotein which is found to be expressed in satellite cells (24,40), developing myotubes and newly formed fibers (13,33,35,40). Several factors have been proposed to trigger satellite cell activation leading to the formation of new muscle fibers: substances released after muscle fiber degeneration, denervation, ischemic injury, or transplantation (7). The subjects in the present study being elite power lifters, their training as such might have induced muscular micro injuries that could have triggered the formation of new muscle fibers. The enhancement of new fiber formation in the doped group might, in addition, reflect the direct action of anabolic steroids on satellite cells as discussed above.
This study showed that the proportion of slow and fast fibers was not affected by the use of anabolic steroids. However, it is important to note that the trapezius muscles of these athletes are characterized by a higher proportion of Type IIA fibers and a lower proportion of Type I fibers than the trapezius muscles of control subjects (64% Type I and 26% Type IIA) (26). This is in accordance with studies performed on limb muscles and showing an increase in the proportion of IIA fibers with strength training (41,44). In addition, in the trapezius muscles of these athletes, Type IIAB fibers were very rare and there were not any IIB fibers, which is in accordance with several studies showing that strength training is associated with a decrease in the number of MHC IIB-containing fibers; it has been proposed that IIB fibers represent a pool of fibers that transform into IIA fibers if they are recruited often enough (1,2,15,29,41–44).
In conclusion, from the view of sports medicine, the increase in nuclear content might reflect a long-standing doping effect. Taking anabolic steroids for a period dopes the muscles with more nuclei, which can be used to synthesize more proteins upon future strength training. Although all the subjects in this study are high-level power lifters with similar performances, the possibility of a genetic endowment contributing to the differences observed between the two groups cannot be excluded.
We thank A-K. Nordlund and U. Ranggård for excellent technical assistance and Drs. G. S. Butler-Browne and M. Price for the critical reading of this manuscript.
This study was supported by grants from the Swedish Medical Research Council (12 x 3934), the Medical Faculty of Umeå University, the Department of Musculoskeletal Research, National Institute for Working Life, Umeå, and the Association Française Contre les Myopathies (to Dr. G. S. Butler-Browne).
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