Throughout the last decade, researchers have investigated the impact of gender on exercise-induced muscle damage and associated postdamage intramuscular responses. Although some debate remains toward the impact of gender, evidence exists to suggest that gender may impact the physiological response to damaging exercise (27). The primary female sex hormone, 17β-estradiol (E2), centers this debate and is purported to have potent antioxidant properties and subsequent impact on the degree of exercise-induced muscle damage, in addition to the postexercise inflammatory and repair processes (11). Limited investigations, however, have reported on the nature to which E2 can attenuate muscle damage and antioxidative activities after a bout of eccentric muscle contractions.
Using animal and human models, the impact of E2 on markers of inflammation and muscle damage during continuous bouts of moderate-intensity aerobic exercise (30-90 min at 60-70% V˙O2max) has been reported. In men, an 8-d supplementation period with E2 had no impact on inflammatory responses to acute exercise (35). In women, however, an attenuation of circulating IL-6 (a proinflammatory cytokine) was found to be dependent of the menstrual cycle phase and oral contraceptive use after an acute cycling exercise (34). Similarly, Carter et al. (5) concluded that serum creatine kinase (CK) was significantly reduced 72 h after 30 min of downhill running in women taking oral contraceptives and tested during their midluteal phase (high E2) when compared with eumenorrheic women not taking oral contraceptives and tested during their midfollicular phase (low E2) (5). Furthermore, Tiidus et al. (33) reported significantly lower levels of intramuscular neutrophils and myeloperoxidase in addition to an 80% reduction in proteolytic calpain activity in E2-supplemented rats after intense treadmill running.
The impact of a protective and antioxidative E2 after damaging eccentric muscle contractions is less clear. Recent reviews outline the relationship between eccentric muscle contractions and muscle damage (11), which typically include an acute loss and recovery of muscle strength, sarcolemmal disruption, increased myocellular protein and enzyme release, changes in calcium balance, and acute-phase neutrophil and inflammatory responses (27,38). In addition, after exposing nine men to a single damaging bout of eccentric muscle contractions, Willoughby et al. (37,38) reported significant increases in both the mRNA and protein content of HSP-72 and major components of the ubiquitin proteolytic pathway (e.g., ubiquitin, E2-conjugating enzyme, 20S proteosome) as well as caspase-3 activity, whereas in other investigations, his research group reported significant increases in inflammatory markers (e.g., IL-6, cortisol), proteolytic activity, and cellular apoptosis (caspase-3 activity) although total DNA content decreased, which corresponds to other findings in the literature on muscle damage (27). As it stands, the ability of E2 to mitigate changes in exercise-induced muscle damage, in addition to various intracellular events (i.e., oxidative stress, proteolysis, apoptosis), is largely unknown.
In the only study that investigated the impact of E2 on the changes in myostatin, a stress-responsive myokine known to be a catabolic regulator of skeletal muscle, Willoughby and Wilborn (39) found that, while men (low E2) experienced significant increases, women during their midluteal phase (high E2) experienced significant reductions in mRNA levels of myostatin and serum levels of its propeptide 24 h after a bout of eccentric contractions. In a related fashion, Stupka et al. (27) also used eccentric exercise to determine gender differences in muscle inflammation. They concluded that men, when compared with women, had greater amounts of tissue infiltrated with leukocytes in addition to a significantly increased amount of skeletal muscle cells with bcl-2 activity, an indicator of mitochondrial apoptosis (27). The literature on E2 and oxidative stress during exercise is lacking and nonconclusive. For example, Case et al. (6) found no differences between genders in peroxidation levels of LDL after a half marathon. In contrast, resting levels of antioxidant markers have been shown to increase after only a few bouts of intense exercise training (23). In addition, Tiidus (32), after a series of studies, concluded that normal or vitamin E-depleted female rats were not as susceptible to oxidative stress during exercise as male rats and that E2 may be responsible for this interaction.
Consequently, the objectives of the present study were to determine whether higher circulating levels of E2 in women altered the changes in exercise-induced muscle damage, oxidative stress, mitochondrial apoptosis, and myofibrillar protein content after a damaging bout of eccentric muscle contractions. It was hypothesized that due to the higher circulating levels of E2, women would exhibit a diminished response of exercise-induced muscle damage, oxidative stress, and apoptosis after the exercise bout when compared with men.
Eight men (20.6 ± 1.5 yr, 175 ± 11 cm, 82.2 ± 12.3 kg) and eight women (21.5 ± 3.2 yr, 160 ± 4 cm, 57.7 ± 8.1 kg) served as participants in this study (Table 1). All participants were recreationally active but had not consistently performed resistance training for at least 6 months before beginning the study. All women were self-reported as eumenorrheic and were not using any form of oral contraceptive. Only those individuals stratified as low risk according to the American College of Sports Medicine criteria were allowed to participate (36). Eligible participants signed university institutional review board-approved informed consent documents, and all experimental procedures were also in accordance with the ethical considerations of the Declaration of Helsinki.
A minimum of 5 d before the eccentric exercise bout, participants completed a familiarization session and one testing session in which their maximal dynamic knee extensor strength of the dominant leg was determined. Before testing, a standardized warm-up consisting of a 10-min ride on a bicycle ergometer (Monark 828E, Varberg, Sweden) was completed at a work rate of 360 kg·m·min−1. Maximum dynamic strength using an isotonic knee extension machine (Universal, Cedar Rapids, IA) was assessed using the one-repetition maximum (1RM) according to previously described procedures (36). To prevent ensuing fatigue from repeated maximal attempts, a goal of no more than five trials was set for all 1RM testing sessions throughout the study (37). Subsequently, all participants were able to achieve their 1RM within five trials, and the mean ± SD number of trials was 3.25 ± 0.58. After 1RM testing, participants were instructed to follow their normal dietary pattern and to refrain from strenuous physical exercise for the 72 h before the eccentric exercise bout.
Body composition testing.
Body mass, fat-free mass, percent body fat, and fat mass were determined before the eccentric exercise session. Body mass (kg) was determined on a standard dual-beam balance scale (Detecto, Terre Haute, IN). Percent body fat was determined using hydrostatic weighing according to standard procedures (4). The test-retest reliability of performing these assessments on participants in our laboratory has yielded low mean coefficients of variation and high reliability for the determination of percent body fat (intraclass correlation coefficient = 1.9%, r = 0.93).
Eccentric exercise bout.
To control for natural variations in E2 levels throughout the menstrual cycle, each female participant was age-matched with a male participant, and both performed the exercise bout on the same day which corresponded with each female's midluteal phase (days 21-23) of her 28-d menstrual cycle (5). Participants reported to the laboratory for the eccentric exercise bout after an 8-h fast and completed the same warm-up used during 1RM determination in addition to completing one set of 10 repetitions at 50% 1RM on a knee extension machine. On the basis of previous guidelines, each participant then completed seven sets of 10 repetitions at 120% of their 1RM, using eccentric contractions of the dominant knee extensors (37). For each repetition and with each participant's knee at full extension (180°), a load equivalent to 150% of 1RM was released by the study investigators. Participants were instructed to maximally extend their knee against the resistance. To ensure an adequate and consistent effort between participants, verbal commands to terminate each repetition were given by investigators to each participant. Study investigators then manually lifted the weight back to the starting point to prevent any effort by the participants during the concentric phase of the repetition. Each repetition lasted approximately 2-3 s, with 15 s of rest between each repetition, whereas each of the seven sets was separated by 3 min of rest. Subsequently, the entire exercise bout lasted approximately 45 min.
Perceived muscle soreness.
As a subjective indicator of the severity of muscle injury, perceived soreness was assessed along a 10-cm scale (0 = no soreness, 10 = extreme soreness) (30). Participants rated their level of soreness before and 6, 24, 48, and 72 h after exercise by drawing an intersecting line across the continuum line extending from 0 to 10. The distance of each mark was measured from 0, and the measurement was used as the perceived soreness level.
Venous blood sampling and muscle biopsies.
Venous blood samples were obtained immediately before the eccentric exercise bout from an antecubital vein into a 10-mL collection tube using a standard vacutainer apparatus. Blood samples were also obtained at 6, 24, 48, and 72 h after exercise and were allowed to stand at room temperature for 10 min and then centrifuged at 800g for 10 min. The serum was removed and was frozen at −80°C for later analysis.
Percutaneous muscle biopsies were obtained from the vastus lateralis immediately before and 6 and 24 h after the eccentric exercise bout. In conjunction with standard needle biopsy collection procedures, muscle samples were extracted under local anesthesia of 2% xylocaine with epinephrine using a fine needle aspiration procedure using a 16-gauge × 10-cm Tru-Core soft-tissue microbiopsy needle (AngioTech, Gainesville, FL). Previous investigations have suggested that a "repeated biopsy" effect may confound findings (13). Although no data exist on similar intramuscular markers, this muscle collection approach was used as an attempt to minimize scarring, invasiveness, and discomfort attributed to traditional needle muscle biopsy procedures. Hayot et al. (10) did, however, report that the expression of citrate synthase, phosphofructokinase, and myosin heavy chain was similar when compared to the traditional Bergstrom needle muscle biopsy procedure while participants reported less pain and discomfort. All muscle samples were taken from the middle portion of the vastus lateralis at the midway point between the patella and the greater trochanter of the femur at a depth between 1 and 2 cm. Using these procedures, extracted muscle tissue averaged 9.54 ± 2.04 mg for all participants. Subsequent biopsies were taken from approximately the same location by using the previous biopsy puncture location and depth markings on the needle. Each successive puncture was made approximately 1.0 cm to the former from medial to lateral (37). After sampling, muscle specimens were immediately frozen in liquid nitrogen and then stored at −80°C for later analysis.
Serum hormone and protein quantitation.
Serum levels of lactate dehydrogenase (LDH) were assessed using a DADE Dimension RXL clinical chemistry analyzer (Dade-Dehring, Inc, Newark, DE). The analyzer was calibrated daily using liquid assay multiqual (Bio-Rad, Hercules, CA), and two levels of quality control with known concentrations were performed.
Serum concentrations of E2 were analyzed using a commercially available enzyme-linked immunosorbent assay (ELISA) kit (Diagnostic Systems Laboratories, Webster, TX). Serum levels of total superoxide dismutase (Cu/Zn-SOD) and 8-isoprostane (8-iso) were determined using commercially available enzyme immunosorbent assay (EIA) kits (Cayman Chemical, Ann Arbor, MI). All measures were made in duplicate with a microplate reader (Wallac Victor 1420; Perkin Elmer, Boston, MA) at a wavelength of 450 nm for all ELISA, whereas 405 nm was used for each EIA, respectively. Standard curves were generated for all measures using commercially developed standards with specific antigens with reported r values in the range of 0.948-0.999. Intra-assay coefficients of variation for each assay were determined for each duplicate for all participants and resulted in coefficients that ranged from 1.64% to 5.4%.
DNA and protein isolation.
All muscle samples were first separated and weighed before being isolated for total DNA and protein using the Tri-Reagent (Sigma Chemical Co., St. Louis, MO) procedure according to Chomczynski (7). Total DNA content was determined from the remaining interphase of the initial total RNA isolation procedure with 100% ethanol, 0.1 mmol·L−1 sodium citrate, and 8 mmol·L−1 sodium hydroxide. Total protein remaining after the total RNA isolation procedure was isolated with isopropanol, ethanol, and 0.3 mmol·L−1 guanidine hydrochloride.
Intramuscular protein quantitation.
Intramuscular concentrations of bax, bcl-2, and cytochrome c were analyzed using commercially available EIA kits (Assay Designs, Ann Arbor, MI). All measures were made in duplicate with a microplate reader (Wallac Victor 1420; Perkin Elmer) at 405 nm according to the manufacturer's guidelines. Standard curves were generated for all measures using commercially developed standards with specific antigens with reported r values in the range of 0.988-0.998 and with intra-assay coefficients of variation ranging from 2.7% to 6.3%. Detection of cell death was done with a commercially available ELISA kit (Roche Laboratories, Indianapolis, IN) by determining, in duplicate, the changes in optical density at 455 nm, which was directed against the presence of cytoplasmic mono- and oligonucleosomes known to be generated after cytoplasmic apoptotic activity.
Total DNA content and myofibrillar protein quantitation.
Total DNA concentration was determined at OD260 and expressed relative to muscle wet weight (w.w.) (1), whereby each optical density (OD) unit is equivalent to 50 μg DNA·mL−1. Myofibrillar protein was further isolated with repeated incubations in 0.1% SDS at 50°C and was separated by centrifugation. Myofibrillar protein content was determined spectrophotometrically on the basis of the Bradford method at a wavelength of 595 nm (3). A standard curve was generated (r = 0.98, P < 0.001) using bovine serum albumin (Bio-Rad), and myofibrillar protein was quantified relative to muscle w.w. (37).
Separate two-way (gender × test) factorial ANOVA with repeated measures on test were used to determine differences between our main effects and any interactions. Tukey post hoc procedures were used to locate significant differences among testing sessions and interactions. When significant noninteractive, between-gender effects were found, simple pairwise comparisons were used for each respective gender to determine any differences between tests. Body composition data at the beginning of study were analyzed using independent t-tests. To protect against Type I error, the conservative Hunyh-Feldt Epsilon correction factors was used to evaluate the observed within-group F ratios when the sphericity assumption was not met. A probability level of ≤0.05 was adopted throughout to determine significance; statistical power was estimated at 0.40 for a large effect size of 0.80.
A significant difference between genders for body mass (P = 0.01), fat-free mass (P = 0.02), and percent body fat (P = 0.03) was observed. No differences, however, were found between genders for fat mass (P = 0.94; Table 1).
No significant gender × test interactions were found (P = 0.85). A significant main effect for gender was found (P < 0.001), indicating women to have higher levels of E2 than men. In addition, no significant main effect for test was found (P = 0.93), indicating that E2 levels were not impacted by the eccentric contractions (Table 2).
Changes in strength, soreness, and serum levels of LDH were assessed as indicators of muscle damage. Significant main effects for test (P < 0.001) and gender (P < 0.001) and a gender × test interaction (P = 0.03) were found for strength levels. As expected, men had significantly higher levels of strength when compared with women at all time points. Men experienced a sharp decline in strength until 24 h after exercise and tended to be different than women (P = 0.07), which then returned to baseline levels by 72 h after exercise (Table 2). Regarding muscle soreness, a significant (P = 0.01) gender × test interaction effect was found in addition to significant main effects for gender (P < 0.001) and test (P < 0.001). Men reported significantly higher levels of soreness at 24, 48, and 72 h after exercise when compared to women. In women, soreness levels peaked at 6 h and were significantly greater than baseline at 6 and 24 h after exercise, whereas in men, soreness levels peaked at 24 h and were significantly greater than baseline at 6, 24, and 48 h after exercise (Fig. 1). No significant gender × test interactions (P = 0.76) or main effects for test (P = 0.54) and gender (P = 0.28) were found in serum levels of LDH (Table 2).
Serum levels of SOD and 8-iso were assessed as indicators of oxidative stress. For either measure, no significant gender × test interactions were found (SOD: P = 0.84; 8-iso: P = 0.34). In addition, no significant main effect for test was found for 8-iso (P = 0.91); however, a significant trend for SOD to increase was exhibited (P = 0.064), which was largely attributed to increased serum SOD levels in females through the 72-h time point when compared with men. A significant main effect for gender (P < 0.001) was found for SOD levels, suggesting that the levels in women were greater than men at all time points (Fig. 2). Interestingly, SOD levels in men experienced a sharp decline after 6 h, which was not seen in women (P < 0.05). Concurrently, a significant main effect for gender was also found for circulating levels of 8-iso (P < 0.001), with higher levels in men when compared with women (Fig. 3).
Mitochondrial and cytoplasmic apoptosis.
Skeletal muscle protein contents of bax, bcl-2, bax/bcl-2 ratio, and cytochrome c were assessed to determine changes in mitochondrial apoptosis in addition to a global cytoplasmic assessment of cell death. No significant gender × test interactions were found for changes in bax (P = 0.52); however, bax protein levels in both genders similarly increased (P = 0.01) across testing sessions. For both genders, bax protein levels at 6 (P = 0.04) and 24 h after exercise (P < 0.01) were significantly greater than baseline levels (Table 2). A significant gender × test interaction (P = 0.03) revealed that bcl-2 protein levels in men were significantly greater than women at baseline and 6 h after exercise, although bcl-2 expression increased at 24 h after exercise and was no longer significantly different. Follow-up pairwise comparisons in men revealed no change in bcl-2 protein levels over time (P = 0.91), whereas in women, bcl-2 protein levels at 24 h were significantly greater than those at baseline (P = 0.005) and tended to be greater than the levels at 6 h (P = 0.10; Fig. 4). The ratio of bax and bcl-2 protein (bax/bcl-2) is commonly used as a marker for mitochondrial apoptosis (18,19). Using these data, no significant test (P = 0.49) or gender (P = 0.14) effects were found, although a significant gender × test interaction was found (P = 0.02). Using a one-way ANOVA at baseline, bax/bcl-2 levels were significantly higher in women with no gender differences at 6 and 24 h after exercise. When each gender was analyzed over time, women's bax/bcl-2 levels were significantly lower after 6 h (P = 0.03) but returned to baseline levels after 24 h (P = 0.17), whereas in men, bax/bcl-2 levels were significantly increased after 24 h when compared to baseline (Figs. 5 and 6). No significant changes (P > 0.05) were noted for cytochrome c (Table 2). In addition to assessing mitochondrial apoptosis, an additional cytoplasmic-specific assessment of cell death revealed no significant gender × test interaction (P = 0.57) in conjunction with no main effect for test (P = 0.36); however, a significant gender effect (P < 0.001) was found. Separate one-way ANOVA revealed that men had significantly higher levels of cell death at baseline (P = 0.04) and at 6 (P = 0.002) and 24 h after exercise (P = 0.02; Fig. 7).
Total DNA content.
Total DNA content in muscle samples was assessed as an additional marker of cellular degradation. No gender × test significant interactions were reported (P = 0.87) in addition to no main effect for gender (P = 0.12). A significant main effect for test was found (P < 0.001). In comparison to baseline, DNA content for both genders was significantly decreased at 6 (P < 0.01) and 24 h after exercise (P < 0.001), whereas the DNA content at 24 h after exercise was significantly lower (P < 0.05) from levels found at 6 h after exercise (Table 2).
Myofibrillar protein content.
Myofibrillar protein levels were assessed as a corollary measure to the changes in mitochondrial and cellular integrity. No significant (P = 0.52) gender × test interactions were found in addition to there being no main effect for gender (P = 0.21). Significant main effects for test revealed that protein levels at 24 h after exercise were significantly lower than baseline levels (P = 0.02) and tended to be lower than those levels found at 6 h after exercise (P = 0.06; Table 2).
Gender differences in muscle damage have been reported in the animal and human literature; however, such differences are inconclusive when discussing oxidative stress, apoptosis, proteolysis, and inflammation (11). This apparent gender difference in muscle damage is attributed to the female sex hormone, E2 (32). This study examined the gender differences in healthy men and eumenorrheic women during their midluteal phase for muscle damage, oxidative stress, and apoptosis in response to eccentric exercise. Results from the present study provide evidence of gender differences in muscle damage (e.g., strength decrements, soreness, serum LDH) and intramuscular apoptosis (e.g., bax, bcl-2, cytochrome c, and total DNA content) but lack gender differences in oxidative stress (e.g., serum levels of Cu/Zn SOD and 8-iso).
Several markers of muscle damage are commonly reported, including changes in strength, soreness, z-disk streaming, and serum levels of CK and LDH (5,12,27,38). The notion of gender differences for muscle damage, however, remains equivocal. For example, no gender differences in CK were found by Clarkson (8), whereas other investigators have reported no differences in force decrements after a single session of eccentric exercise (28). Other investigators, however, have found significantly lower levels of CK and LDH at rest and after exercise in human (26) and animal models (12), in addition to different granulocyte numbers (27) and myofibrillar disruptions (9). Nonetheless, the changes in strength (gender × time, P = 0.03) and soreness (gender × time, P < 0.001) in the present study do provide evidence of a gender difference. When compared to women, strength levels in men decreased sharply after 24 h (Table 2), whereas soreness levels for men were significantly higher at 24, 48, and 72 h after exercise (Fig. 1). Changes in plasma levels of LDH, however, exhibited no change. Although not hypothesized, similar findings for CK and LDH have often been attributed to the high intersubject variability that exists for these measures (16,27). Although somewhat undetermined, studies have attributed this inherent variability to the actual muscle damage that occurs between subjects in addition to other physiological factors such as membrane permeability (16) and previous bouts of similar exercise (28,38). However, this last point is not relevant because all participants were excluded if they had prior resistance training experience. Although the present study suggests potential for a gender-specific response, further research is needed to delineate the mechanism responsible. Serum levels of E2 in the present study were significantly greater in women at all times points (Table 2); however, recent findings continue to complicate the suggestion that E2 may play a significant role (35).
Unlike muscle damage, little evidence has discussed the impact of gender on exercise-induced changes in oxidative stress. For example, no gender difference was reported after a half marathon (6), but Dernbach et al. (9) did report exercising females to have lower oxidative stress levels at all time points when compared to equally trained males; however, acute response data from this study did not support a gender difference. The ability of E2 to have antioxidant properties (11), in addition to membrane-stabilizing properties, has led many investigators to suggest that the presence of E2 may influence the extent to which oxidative stress develops (31). In addition, ovariectomized rats with E2 replaced had significantly lower levels of oxidative stress and neutrophil infiltration when compared to placebo or non-E2-replaced animals (29), and a combined in vivo and in vitro model in laboratory rats suggested that the presence of E2 was responsible for significantly less lipid peroxidation (17).
Although no gender or exercise differences were found in the present study, women were found to have significantly higher levels of SOD and lower levels of 8-iso at all time points (Figs. 2 and 3, respectively), which could be due to differences in muscle mass quantity, although no such data exist to support this possibility. Although no causality can be drawn, this finding does provide additional evidence to support a protective effect for women. Although the data remain equivocal, similar findings have been found for serum levels of CK in human (26) and animal (12) data. Furthermore, an attenuated CK response in women has been linked to the antioxidative properties of E2 (30) and that plasma CK activity and plasma levels of lipid peroxidation were lower in female versus male rowers (9).
Possibly, the dietary intake of our participants did not provide adequate levels of antioxidants, thereby precluding any changes in the oxidative stress measures taken; however, no dietary intake measures were made in the present investigation. The likelihood of this, however, is minimal because studies that have reported on low dietary intake of known antioxidants (vitamin E/tocopherols) have still resulted in measurable changes in sera, whereas studies that have administered high levels of these same antioxidants have reported no changes in oxidative stress after exercise (40). The lack of any measurable change in oxidative stress does raise questions as previous investigations have reported changes (40); however, others have not (14). Although work by Bloomer (2) has established a macromolecule-specific response, a recent investigation by Michailidis et al. (14) has clearly illustrated that sampling time associated with antioxidant enzymes is crucial. Evidence from this study makes it highly possible that any serum-based changes in oxidative stress that may have been elicited from our exercise session were largely cleared from the serum by our 6-h time point. Nonetheless, results from the present study lack evidence to support a gender difference or an ability of oxidative stress to acutely change after eccentric exercise.
Research investigating the changes in apoptosis after various forms of exercise has increased (25). Limited data that have investigated any gender differences or the impact of E2 are available. Of the available data, an attenuated inflammatory response in women and a greater induction of intramuscular bcl-2 in men have been shown (27), although Navalta et al. (15) did not find any impact of gender or of menstrual cycle phase on serum levels of lymphocyte apoptosis after maximal exercise. To evaluate changes in apoptosis, intramuscular levels of bax, bcl-2, cytochrome c, and total DNA content, and a cytoplasmic assessment of cell death were determined in the present study. Although results from the present study failed to identify gender differences in bax, total DNA content, cytochrome c, and cell death, significant gender × exercise interactions were found for bcl-2 and the bax/bcl-2 ratio (Figs. 4-6). In a similar fashion to our findings, Stupka et al. (27) reported significantly higher levels of bcl-2 in men at rest. The lower baseline levels in women increased 48 h after exercise, whereas values in men stayed the same. These findings are also in accordance with available animal data that have reported an increase in bcl-2 mRNA and protein content after several weeks of endurance training (24,25). It stands to reason that an additional biopsy collection at 48 h in the present study as was completed in the Stupka study may have led to an even greater increase in the bcl-2 levels in women; however, this is merely speculative (Fig. 4). Nonetheless, the interactive nature of our findings does support the potential for a gender difference for intramuscular levels of bcl-2; however, the mechanism behind this relationship is still unknown.
Although no gender difference was found, bax protein levels in the present study were increased over baseline levels at both 6 and 24 h after eccentric exercise (Table 2). Bax is a proapoptotic mitochondrial protein found in skeletal muscle (20), and in animal studies, it has been shown to increase after several weeks of endurance training (24,25); however, its changes in human skeletal muscle after exercise are currently not available. In this regard, the ratio of bax (proapoptotis) and bcl-2 (antiapoptosis) has been used in the literature as a general marker that reflects to which the cell is experiencing apoptotic conditions (18,19). When expressed in this fashion, an interactive effect for gender was found, with bax/bcl-2 levels being significantly higher in women at baseline and similar levels between genders at 6 and 24 h after exercise (Figs. 5 and 6). Although these changes are largely explained by the increases in bcl-2 levels in women, the underlying mechanism is still misunderstood. To date, no human studies have reported on the changes in the bax/bcl-2 ratio in conjunction with exercise and only limited animal data are available. In this regard, previous investigations using healthy animal models have reported increases in the bax/bcl-2 ratio 16 h after exercise (18,19). This finding contrasts with the decreased bax/bcl-2 ratio that Song et al. (25) reported in aging laboratory animals after 12 wk of treadmill exercise; however, the differences in exercise stimulus and age of animals between that and the present study are clear. No changes in cytochrome c were reported in the present study (Table 2); however, this finding is not surprising because cytochrome c is a ubiquitous cellular protein that primarily translocates between cytosolic and mitochondrial origins. Lastly, the decreases in total DNA and myofibrillar protein content (Table 2) and changes in cell death (Fig. 7) after eccentric exercise in both genders provide additional evidence for apoptosis and muscle proteolysis (37). Although skeletal muscle proteolysis was not investigated in the present study, previous findings have suggested that eccentric exercise can increase ubiquitin proteolytic expression, which is responsible for the majority of myofibrillar protein breakdown and has been linked to increased DNA fragmentation during eccentric exercise (22). In addition, other investigations have linked E2 concentration and the estrogen receptor content to altered proteolytic expression (21). Collectively, our findings suggest evidence of gender differences in apoptotic mechanisms in response to an eccentric bout of exercise. Although the role of E2 in this gender difference is an obvious consideration, other mechanistic studies need to be completed to more fully elucidate this finding. The available data continue to provide support that acute exercise training can impact apoptotic expression in many tissues, but the exact mechanism is still unknown. Increases in glucocorticoid secretion and reactive oxygen species may be important factors when researching the related mechanisms. In this regard, Willoughby et al. (37) reported increases in cortisol 6, 24, and 48 h after a single bout of eccentric exercise. They also reported an increase in the mRNA and protein levels of several components of the ubiquitin proteolytic pathway (e.g., ubiquitin, E2-conjugating enzyme, and 20S proteosome) in addition to the caspase-3 enzyme activity at 6 and 24 h after eccentric exercise. Nonetheless, additional studies need to be conducted, which can provide a more definitive link in humans between acute exercise stress and apoptotic induction.
Gender differences after eccentric-induced exercise may be related to changes in apoptotic activity secondary to changes in muscle damage and associated inflammatory cascades. Further, these findings provide additional data to explore further the potential for greater endogenous protection against oxidative stress in women in addition to changes in both specific and global indicators of cell death and apoptosis. Future studies should attempt to explore changes within women throughout their menstrual cycle and any impact gender has on the relationship of various inflammatory markers and estrogen receptors in skeletal muscle after eccentric exercise. These types of studies would have great relevance to women taking oral contraceptives or hormone replacement therapy in relation to the regenerative capacity of their skeletal muscle.
The authors thank all participants for their effort and dedication to the protocol. The results of the present study do not constitute endorsement by ACSM.
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Keywords:©2008The American College of Sports Medicine
ESTROGEN; ECCENTRIC EXERCISE; MUSCLE DAMAGE; CELL DEATH; ANTIOXIDANT