Introduction

Myosin heavy chain (MHC) isoforms are the main contractile proteins in skeletal muscle that determine their functional properties, such as the maximal and optimal velocity of shortening (⁵), peak power output (¹⁷), the rate of force development (²⁷), tension cost (²⁸), and mechanical efficiency (⁴³). Moreover, classification of the muscle fibers is based on MHC isoform distribution and the myofibrillar protein isoforms changes in response to variety of signals are the basis of fiber-type transitions (³⁹).

In recent years, great progress has been made in elucidating the molecular basis of the skeletal muscle fiber–type transformations (^3,44). It was demonstrated that calcium-dependent signaling pathways, involving CaN-NFAT (calcineurin-nuclear factor of activated T cells) and CaMK-HDAC-MEF-2 (calcium/calmodulin-dependent protein kinase-histone deacetylase-myocyte enhancer factor-2) pathways play a key role in muscle fiber remodeling (^6,35). Moreover, other cellular signaling molecules, such as mitogen-activated protein kinase (MAPK) (⁴⁵), myostatin (²⁹), or forkhead box O1 (FOXO1) transcription factor (⁵⁴) can also be involved. Obviously, confirmation of the signaling pathways operating in muscle fiber transition in humans is required because most of them were determined in animal models. Nevertheless, knowing these putative cellular mechanisms is helpful to link them to intra- or extramuscular signals that may influence skeletal muscle fiber–type switching.

The gonadal hormones has been recognized as one of the factors contributing to fiber-type transitions (³⁹), but little is known about the effects of the gonadal hormones and the anabolic-catabolic hormone balance on human skeletal muscle fiber–type transformation. This is somewhat surprising because significant effect of testosterone (T) on muscle fiber–type composition in animals was reported by Gutmann et al. (²²), as early as 1970. They demonstrated that postnatal T treatment in the female guinea pigs led to a shift in temporal muscle fiber–type (from type 1 to type 2) and account for male-like characteristics of this muscle in female animals. This influence of T on muscle fiber–type differentiation was supported later, both in the sexually dimorphic muscles (³³) and in other skeletal muscles of experimental animals (^31,41).

In human studies, potential influence of T on the MHC or fiber-type composition has been only rarely mentioned. Pette and Staron (⁴⁰), for example, suggested that gender differences in relative concentrations of MHC isoform (a greater area occupied by type 2A fibers in men compared with women), which were earlier found by Staron et al. (⁴⁶), may be T-dependent. Similarly, Glenmark et al. (¹⁸) stated that a possible explanation for the observed increase in the proportion of type 2 fibers from adolescence to adulthood in men was androgen hormone exposure during maturation. There is also a training study where a positive correlation between serum T and the percentage of type 2a and 2x was noticed (⁴⁷). Moreover, during recent years, it has become evident that the anabolic activity of T on skeletal muscle tissue is exerted not only through the classical androgen receptor but also through the rapid nongenomic actions (¹²). Because these rapid effects are mediated through the multiple cellular signaling pathways (e.g., Ca²⁺-dependent pathways, MAPK cascade, PI3K-Akt pathway), which may be also involved in muscle fiber transformations, the possible role of T in this process cannot be ruled out.

On the other hand, the role of physiologically elevated T as an anabolic and hypertrophic signal has been recently questioned (⁵¹), and this issue is now being debated (^50,52). Nevertheless, evidence that T and anabolic-catabolic hormone balance may affect the human MHC isoform expression has been neither confirmed nor refuted so far. Because it is well known that athletes from different sports are characterized by different MHC isoforms, we studied both MHC composition and basal serum T concentration in the 2 groups of athletes with different training background and in a control group consisted of young untrained men. Moreover, we have also examined basal free testosterone (fT) and bioavailable testosterone (bio-T) concentrations and basal T:C and fT:C ratio (C indicates cortisol) in the untrained group because all these variables reflect the anabolic-catabolic hormone balance that possibly may influence MHC composition.

Methods

Experimental Approach to the Problem

All participants underwent a muscle biopsy from the vastus lateralis muscle and blood testing to analyze basal serum T concentration. Moreover, the subjects performed a maximal incremental cycle exercise test to determine V[Combining Dot Above]O₂peak. Basic blood tests (hemoglobin, hematocrit, erythrocyte and leukocyte count, sodium, potassium, and albumin) were performed to evaluate health status of all studied subjects. Additionally, in half of the subjects (n = 15, untrained subjects), basal sex hormone–binding globulin (SHBG) and C concentrations were measured and fT and bio-T concentration were calculated to determine the basal gonadal hormone profile and the relationship between MHC-2 and basal anabolic-catabolic hormone ratio in the body (T:C and fT:C ratio).

The subjects were familiarized with all procedures before testing, and they were instructed to refrain from physical activity, alcohol, and caffeine-containing beverages within 48 hours before the examination. Moreover, the blood tests were performed after overnight fasting in the morning hours (7.30–8.00 AM), whereas maximal exercise test and muscle biopsy were conducted 2 hours after standard meal in early afternoon (1–4 pm) in all subjects. Each test was performed on separate days in a defined order (blood testing, maximal exercise test, and muscle biopsy), with at least 2 days rest in between.

Subjects

All participants were informed about the purpose of the study and signed a written consent to the experimental procedure, which was approved by the local ethical committee and conducted in accordance with the Declaration of Helsinki. A total of 30 men participated in this study. They were assigned to 3 groups according to their training background: untrained, but physically active students, group U (n = 15); sub-elite endurance-trained athletes, group E (n = 10); and sub-elite sprint/strength-trained athletes, group S (n = 5). The basic anthropometric characteristics of subjects in the 3 groups are presented in Table 1.

Group E consisted of 6 middle and long distance runners, 2 cross-country skiers, and 2 cyclists, and group S was composed of 3 track and field jumpers (2 long jumpers and 1 high jumper) and 2 karate fighters. The E group had an average of 6.8 years of training experience, whereas the S group had an average of 4.8 years. All the athletes involved in this study regularly competed in their sports on national and international levels. The study was conducted in the end of March, at the time when athletes had finished general preparation phase of training dedicated mainly to improvement of endurance in their specialty, before the start of the competition season.

Incremental Exercise Test

A maximal incremental exercise test was performed on a cycloergometer (Ergoline, Ergoline GmbH, Bitz, Germany) to determine V[Combining Dot Above]O₂peak. The exercise test started with a 6-minute rest in a sitting position on the ergometer, which was intended to collect baseline cardiorespiratory variables and was followed by gradual increases of power output by 30 W every 3 minutes. The test was terminated when the subjects could not continue cycling at the required pedaling rate (60 rpm) and power output (⁵⁵). During the test, gas exchange variables were measured continuously using breath-by-breath gas analysis system (Oxycon Champion Jaeger, Mijnhardt B.V., Bunnik the Netherlands). Before each test, the volume sensor was calibrated using a calibration syringe of known volume, and the gas analyzer was calibrated using gas mixture of known concentration [as described previously by Zoladz et al. (⁵⁶)].

Muscle Biopsy

Muscle biopsies were obtained under local anesthesia (1% lidocaine) from the vastus lateralis m. quadricipitis femoris approximately 15 cm above the upper margin of patella with a 2-mm diameter needle biopsy (pro-Mag I 2.2; MDTECH). The specimens were frozen immediately and stored in liquid nitrogen until further analyses.

Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis

Muscle biopsies (8–28 mg) were minced with scissors and ultrasonicated on ice (UP 50H Sonicator; Dr. Hielscher GmbH) with 150–200 μl of buffer containing 62.5 mM Tris [pH 6.8], 10% glycerol, 2.5% sodium dodecyl sulfate. Samples were centrifuged and supernatants assayed for protein with Bicinchoninic Acid Protein Assay Reagent (Sigma) and bovine serum albumin as a standard. To the remaining samples, 2-mercaptoethanol was added to 2.5% final concentration. According to Carraro and Catani (⁸), the sodium dodecyl sulfate polyacrylamide gel electrophoresis of myosin extract was performed with 3% stacking and 6% separating gels containing 37.5% glycerol at 60 V for 30 minutes followed by 180 V for 3 hours. Densitometric analysis of protein bands was performed using the Fotodyne Incorporated video camera and Gel Pro Analyzer computer software. Relative amounts of MHC protein were expressed in optical density units.

Blood Collection

Blood samples (15 ml) were drawn from the antecubital vein. Samples were taken at rest between 7:30 and 8:00 AM after overnight fasting. Blood for serum sodium [Na⁺], potassium [K⁺], albumin (Alb), T, and SHBG concentrations was collected into plain tubes and left to clot for a minimum of 30 minutes at room temperature and then centrifuged at 4,000 rpm for 5 minutes. Blood for plasma cortisol assay was collected in plain tubes containing EDTA. Then, samples were centrifuged at 3,000 rpm for 10 minutes at 4° C. Both serum and plasma were stored at minus 40° C until further analysis.

Blood Analysis

Hemoglobin (Hb), hematocrit (Hct), red blood cell (RBC), and white blood cell (WBC) count were determined using the Advia 2120 automatic hematological analyzer (Siemens, Dublin, Ireland). Serum [Na⁺] and [K⁺] concentrations were determined using the Ciba Corning 480 flame photometer (Ciba-Corning Diagnostics Ltd., Halstead, United Kingdom). Moreover, to further calculate the fT, serum (Alb) was evaluated using agarose gel electrophoresis (Paragon System; Beckman, Fullerton, CA, USA).

All hormone measurements were performed in duplicate, and serum T and SHBG were determined by electrochemiluminescence immunoassay using the Elecsys 2010 analyzer (Hitachi, Tokyo, Japan). The intra- and interassay coefficient of variation (CV) for these assays was <4% and <8%, respectively. Plasma C concentration was measured by chemiluminescence immunoassay using the ADVIA Centaur analyzer (Bayer Diagnostics, Leverkusen, Germany) and the intra and interassay CV for cortisol was 2.1% and 3.3%, respectively.

Moreover, fT was calculated using the standard method of Vermeulen et al. (⁴⁹) in which T concentration is corrected for both SHBG and Alb levels as follows: fT = ([T] − (N × [fT]))/(K_t), where fT, T, and SHBG are free testosterone, total testosterone, and sex hormone–binding globulin concentrations, respectively, N = K_aC_a + 1, where K_a is the association constant of albumin for T and C_a is the albumin concentration, whereas K_t is the association constant of SHBG for T at 37° C. Bio-T (often referred as non–SHBG-bound T) was calculated as a sum of fT and aT (Alb-bound testosterone). As T bound to albumin is a nonspecifically bound T fraction and as it is linearly related to fT, it was evaluated as follows: aT = K_aC_a × fT.

Statistical Analyses

In this study, the data are presented as mean ± SD, and a value of p ≤ 0.05 is considered statistically significant. Comparisons between groups were made using Kruskal-Wallis test followed by 2-tailed Mann-Whitney U-test if significant differences were detected. Moreover, Cohen's d (effect size) was calculated as a measure of the magnitude of group differences that is independent of sample size. Cohen's d value of 0.2, 0.5, and >0.8 are commonly used markers of a small, medium, and large effect, respectively. The correlations between MHC-2 and the anabolic-catabolic hormone ratio and between MHC-2 and aerobic capacity indices were evaluated using the Spearman's correlation coefficient. Analyses were performed using STATISTICA 8.0 software (StatSoft, Inc., 2007, Tulsa, OK, USA).

Results

Baseline Characteristics of the Subjects

Despite different training background, basic anthropometric characteristics (Table 1) and routine blood test parameters (Table 2) of the 3 studied groups were similar. The only significant differences were found in RBC count being higher in U than S group (Mann-Whitney, p < 0.01, Cohen's d = 1.87, 95% confidence interval [CI] 0.70–3.03) and in Alb concentration being lower in E comparing to U (Mann-Whitney, p < 0.02, Cohen's d = 1.66, 95% CI 0.73–2.58) and S subjects (Mann-Whitney, p < 0.02, Cohen's d = 1.53, 95% CI 0.32–2.73).

Myosin Heavy Chain Content and Aerobic Physical Fitness

Essential distinction between all 3 groups was seen in the content of MHC isoforms (Kruskal-Wallis: H = 18.58, p < 0.001, Figure 1). As it could be expected, group E showed the lowest proportion of MHC-2 and the highest proportion of MHC-1 (37.5 ± 13.80% and 62.5 ± 13.80%, respectively, for MHC-2 and MHC-1), whereas S showed the highest proportion of MHC-2 and the lowest of MHC-1 (69.4 ± 2.39% and 30.6 ± 2.39%, respectively, for MHC-2 and MHC-1; Figure 1).

Moreover, V[Combining Dot Above]O₂peak, expressed both in absolute and relative terms, and power output observed at the V[Combining Dot Above]O₂peak (POmax) differ significantly between the 3 groups (Kruskal-Wallis: H = 9.77, p < 0.01; H = 16.12, p < 0.001; and H = 16.00, p < 0.01 for V[Combining Dot Above]O₂peak in absolute and relative terms and POmax, respectively). An intergroup comparison showed that they were significantly higher in the E group than in subjects from the S and U groups (Table 3).

We also found significant negative correlations between MHC-2 and V[Combining Dot Above]O₂peak (Spearman r = −0.49, p < 0.01) and MHC-2 and POmax (Spearman r = −0.69, p < 0.01) for all study subjects (N = 30; U, E, and S groups together).

Myosin Heavy Chain Content and Anabolic-Catabolic Balance

We have found no significant differences in basal serum T concentration between the 3 study groups (18.84 ± 5.73 nmol·L⁻¹, 18.60 ± 5.73 nmol·L⁻¹, and 20.73 ± 4.06 nmol·L⁻¹ for U, E, and S groups, respectively; Kruskal-Wallis: H = 1.11, p > 0.5). The type 2 MHC content appeared to be related to the basal anabolic-catabolic hormone balance because it was shown that MHC-2 is positively correlated with basal fT:C ratio (Spearman r = 0.63, p = 0.01) in the U group (Figure 2).

Discussion

In this study, we have shown that despite considerable dissimilarity in MHC content between endurance trained, sprint/strength-trained, and untrained men, there are no differences between them in regard to basal T concentration (see Figure 1 and Results). These results are also consistent with our recent study (²¹), where we compared top-class sprinters (higher performance level and larger group of athletes, n = 16, than S group in this study) with untrained subjects. Although the MHC composition was not determined in that article, it is reasonable to assume that sprinters with their personal best times in a 100-m run ranged from 10.17 to 10.71 seconds had a significantly higher proportion of the fast-contracting type 2 MHC isoforms in their muscles than untrained men (¹). Despite this expected difference, we found that top-class sprinters do not have a higher basal gonadal hormone concentration than a control group composed of young, healthy untrained men.

On the other hand, data showing that trained athletes and untrained subjects differ in terms of MHC isoforms composition but not in terms of their basal T concentration may seem a little surprising. Several reports demonstrated (^4,7,37) that T correlates with explosive power performance and running speed (^4,9), that is, performance parameters generally related to a higher percentage of type 2 muscle fibers (^1,43). Taking it into consideration, one might expect (in opposition to our findings) that a higher percentage of type 2 fibers is linked with higher basal T concentration in men.

Animal studies seem to confirm the relation between T and MHC isoform/muscle fiber–type composition (^31,33,41). Testosterone treatment resulted in an increase of skeletal muscle expression of the fast MHC isoform both in intact (⁴¹) and gonadectomized animals (³¹). Additionally, castration led to a decrease in proportion of type 2 fibers of the rat gastrocnemius muscle (³¹) and to inhibition of the expression of fast type 2 MHC isoform in sexually dimorphic masseter muscle of a rabbit (¹⁴) and the temporalis muscle of a guinea pig (³³). On the other hand, the androgen influence on MHC expression of the skeletal muscle (other than sexually dimorphic ones) in animal models was also questioned (³⁸), and Prezant et al. (⁴¹) suggested that the results of T treatment depend both on the basal androgen level and treatment duration. Indeed, it should be noted, that these animal experimental studies often involved supraphysiological changes in the T concentration (castration and/or large doses of T supplementation), which does not occur in the body in physiological conditions.

In humans, the impact of androgens on MHC expression/muscle fiber–type composition is even more ambiguous because the data on this topic are scarce. Admittedly, there are reports indicating that T influences the fractional synthesis rate of MHC in humans (²), but, to our best knowledge, there are only 2 previous studies reporting positive correlation between serum T concentration and percentage of the type 2 muscle fiber area in humans (^36,47). In the study by Mero et al. (³⁶), this relationship, however, was presented in very young athletes (boys aged 11–13 years). Circumpubertal age may explain these results because it was shown that the proportion of type 2 fibers markedly increases in men from adolescence to adulthood (^18,32). One possible explanation for these changes given by Glenmark et al. (¹⁸) was androgen hormone exposure during the transition from adolescence to adulthood. Having in mind the well-known age differences in T secretion [in adulthood, T concentration could be even 50% higher than in adolescence (²³)], it is very tempting to suspect a relation between an increase in type 2 fibers proportion in the skeletal muscles and augmented T secretion. Moreover, Staron et al. (⁴⁷) showed that this relationship may be found not only during adolescence. In their study, increased T concentration corresponded to increased percentage of type 2 fibers in the early phase of physical training performed by previously untrained men.

The subjects who belonged to the trained groups in our study have already had a long history of regular training (>5 years of training experience), and this fact may explain a lack of association between T concentration and MHC composition. Knowing the fact that T exerts its effects on skeletal muscle both via genomic and nongenomic action (¹²), one may assume that basal T has a weak genomic effect on skeletal muscle fiber–type transitions, but in situation of its enhance secretion/availability (e.g., T treatment, maturation, and early phase of strength training), it may influence skeletal muscle differentiation via other signaling pathways and nongenomic action. It was recently showed that T activates MAPK cascade (²⁶), stimulates intracellular calcium release and Ca²⁺-dependent pathways (¹⁵), and affects key regulatory factors for muscle differentiation because myostatin (³⁰), insulinlike growth factor 1 (¹⁶), and FOXO1 transcription factor (⁴²), which may all influence the skeletal muscle fiber–type transition. Since some of these signaling pathways were linked to fast-to-slow and other to slow-to-fast transitions in MHC isoform expression and a complete picture of the fiber-type switching remains to be fully elucidated, the contribution of T in increased MHC-2 expression cannot be neither confirmed nor excluded at this point.

Nevertheless, the findings of this study suggest that MHC could be related to the basal anabolic-catabolic hormonal balance, that is, fT:C ratio rather than to basal T concentration alone because we demonstrated that there is a significant correlation between MHC-2 content and fT:C ratio in the U group (Figure 2). It may also hold true for the aforementioned increase in type 2 fibers during adolescence (³⁶) and during early phase of strength training (⁴⁷). In the latter study, an increase in T concentration was accompanied by a decrease in C concentration, and it indicates a higher T:C and fT:C ratio. One may also expect that fT:C ratio is elevated during adolescence as an indication of increased anabolic activity (¹³). The importance of anabolic-catabolic balance for skeletal muscle differentiation was also determined at the molecular level (^42,53). These studies found that T inhibits glucocorticoid-induced stimulation of catabolic-type responses (i.e., decrease in the activity of p38 MAPK and PI3K/Akt/mTOR signaling pathway) and demonstrated opposite effects of T and C in their cellular actions.

However, the results indicating a relationship between MHC content and basal anabolic-catabolic hormonal balance are difficult to compare with other studies because there is almost no data in the literature concerning this issue. It was noticed, however, that enhanced anabolic hormonal profile (as expressed by higher basal T:C and fT:C ratio) correlates with higher muscle strength and/or sprint performance (^11,25), which in turn was often related to a higher percentage of type 2 muscle fibers (^10,19,34). These findings, together with the results of our study, may suggest that enhanced anabolic hormonal profile corresponds to a higher proportion of MHC-2 content.

We are also aware of the limitations of this study. The main limitation arises from single-point T measurement in the 2 groups of athletes. Although in physically inactive population single-point T measurement may be a reliable marker of its mean annual level (⁴⁸), it is well known that T concentration during exercise training changes in response to the training workloads [for discussion of this point, see (^20,21)]. Furthermore, heavy endurance training can also lead to exercise-induced hypogonadism (²⁴), but in this study, no signs of hypogonadal conditions in athletes as judged based on serum T level were observed. Nevertheless, it is possible, that the lack of difference in T level between the trained and untrained subjects in this study comes from the 4-month period of heavy training loads performed by the E and S groups. Simultaneously, the training-induced short-term alteration in T level would also influence the relationship between T and MHC-2 content presented in this study.

Practical Applications

In conclusion, we have shown in an untrained group of subjects a positive correlation between MHC-2 content in the vastus lateralis muscle and basal fT:C ratio; therefore, one may infer that a better anabolic hormonal profile characterizes individuals with high content of type 2 muscle fibers. Moreover, we have found no differences in basal T concentration between untrained, endurance-trained, and sprint/strength-trained subjects. This indicates that basal T concentration alone cannot be regarded as an index of adaptation to physical training. We postulate that more valuable index of adaptation to physical training could be a measurement of anabolic-catabolic hormone profile, which in view of our observation might be also involved in the mechanisms determining MHC content in human skeletal muscles.

Acknowledgments

This study was supported by funds from the University School of Physical Education (Krakow, Poland) for the statutory research in 2008 for the Department of Physiology and Biochemistry. This study was performed in the Laboratory of the Department of Muscle Physiology, Chair of Physiology and Biochemistry, Faculty of Rehabilitation, University School of Physical Education in Krakow, Poland.