Effects of Step Exercise on Muscle Damage and Muscle Ca²⁺ Content in Men and Women

Abstract

Fredsted, A, Clausen, T, Overgaard, K. Effects of step exercise on muscle damage and muscle Ca²⁺ content in men and women. J Strength Cond Res 22: 1136-1146, 2008-Eccentric exercise often produces severe muscle damage, whereas concentric exercise of a similar load elicits a minor degree of muscle damage. The cellular events initiating muscle damage are thought to include an increase in cytosolic Ca²⁺. It was hypothesized that eccentric muscle activity in humans would lead to a larger degree of cell damage and increased intracellular Ca²⁺ accumulation in skeletal muscle than concentric activity would. Furthermore, possible differences between men and women in muscle damage were investigated following step exercise. Thirty-three healthy subjects (18 men and 15 women) participated in a 30-minute step exercise protocol involving concentric contractions with 1 leg and eccentric contractions with the other leg. Muscle Ca²⁺ content, maximal voluntary contraction (MVC), and muscle enzymes in the plasma were measured. In a subgroup of the subjects, T2 relaxation time was measured by magnetic resonance imaging. No significant changes were found in muscle Ca²⁺ content in vastus lateralis biopsy specimens in women or in men. Following step exercise, MVC decreased in both legs of both genders. The women had a significantly larger strength decrease in the eccentric leg than the men had on postexercise day 2 (p < 0.01). Plasma creatine kinase increased following step exercise, with a sevenfold higher response in women than in men on day 3 (p < 0.001). The women, but not the men, had an increase in T2 relaxation time in the eccentrically working adductor magnus muscle, peaking on day 3 (75%) (p < 0.001). In conclusion, step exercise does not lead to Ca²⁺ accumulation in the vastus lateralis but does induce muscle damage preferentially in the eccentrically working muscles, considerably more in women than in men. This indicates that gender-specific step training programs may be warranted to avoid excessive muscle damage.

Introduction

It is well known that a single bout of eccentric exercise produces more muscle damage than concentric exercise of a similar load. The degree of muscle damage depends on the type, load, and duration of eccentric exercise (⁷). A number of studies have compared pain, loss of intracellular enzymes from the muscles, and fatigue in humans after concentric and eccentric exercise.

The cellular events initiating muscle damage are thought to include an increase in cytosolic Ca²⁺ (^2,16). The elevated Ca²⁺ level may activate proteolytic enzymes, such as calpain, to digest structural elements of the cell membrane or cytoskeleton (^3,34). This destruction leads to membrane leakage, as evidenced by increased plasma levels of intracellular enzymes, such as creatine kinase (CK), myoglobin, and lactate dehydrogenase (LDH). Membrane damage also causes further unspecific entry of Ca²⁺, exaggerating the damaging process (^16,17). The authors have previously shown that when the uptake of Ca²⁺ in isolated rat skeletal muscle is increased by electrical stimulation, electroporation, or addition of the Ca²⁺ ionophore A23187, significant cell membrane damage takes place and is correlated to the uptake of, or content of, Ca²⁺ in the muscle (^15,16). Also, studies of human subjects have shown that total cellular Ca²⁺ content increases significantly in the vastus lateralis following long distance running (^32,33). In these studies, the muscle Ca²⁺ content was increased for up to 48 hours after the run.

During eccentric exercise, the sarcomeres and the membranes are stretched and possibly disrupted and lead to loss of force and increased permeability of the membrane (¹³). The initial membrane damage during eccentric exercise may originate from the combination of excitation-induced Ca²⁺ influx and an unspecific Ca²⁺ influx due to the stretch imposed upon the muscle during lengthening actions. This process is possibly in contrast to the initial damage process during concentric or isometric exercise, in which the sarcomeres are not overstretched and the membranes are most likely not disrupted. Thus, the damage process during concentric or isometric contractions must be initiated by other factors (e.g., increased influx of Ca²⁺) (⁵).

Several studies have suggested a relationship between eccentric exercise and increased intracellular Ca²⁺ concentration. Duan et al. (⁹) found that the mitochondrial Ca²⁺ concentration was elevated in rats after downhill walking and that it was inversely related to the number of intact fibers in the muscle. In another study, Lynch et al. (²⁵) found that the free intracellular Ca²⁺ concentration was increased in adult mice after downhill running. These studies support the hypothesis of Armstrong et al. (²), who proposed that eccentric exercise increased the resting intracellular Ca²⁺ concentration and resulted in the activation of Ca²⁺-sensitive degradative enzymes. This hypothesis, however, has not been examined in humans.

Newham et al. (³¹) investigated the effect of bench-stepping and found that pain and tenderness developed in the quadriceps muscle of the eccentrically working leg and that the reduction in the maximal voluntary contraction (MVC) of the knee extensors was also greater in this leg. In addition, morphological changes as a response to eccentric exercise were observed in biopsy specimens taken from the quadriceps muscle (^30,31). However, in another study, also using a bench-stepping exercise, Newham et al. (²⁹) found an increase in technetium Tc 99m pyrophosphate uptake in other parts of the eccentrically working leg (e.g., the adductors and gluteal muscles), indicating membrane damage, but this increase was not seen in the quadriceps muscle.

Magnetic resonance imaging (MRI) has been used to assess which muscles have been damaged following exercise (⁷). The T2 relaxation time, which has been interpreted as a measure of the water content of the tissue (¹¹), has been shown to increase after intense exercise and reflects exercise-induced damage (^11,24). Recently, in a study by Larsen et al. (²⁴), MRI was used to localize and quantify muscle damage in women after step exercise. In 5 of 8 subjects, a large increase in T2 relaxation time was observed in the adductor magnus muscle of the leg that had been working eccentrically. This increase in T2 relaxation time was significantly correlated to CK activity, and this correlation suggested that these 2 parameters are related to the same aspect of damage and that MRI can be used to identify muscles with membrane damage from which enzymes have leaked out into the circulation.

In most animal studies, female animals show an attenuated response to eccentric exercise compared to males (^1,7,23). This has mainly been explained by a possible protective effect of estrogen on the structural integrity of the plasma membrane (^1,23). In human studies, though, gender differences in response to exercise are less clear (^6,7). A gender-specific response to eccentric exercise has been suggested, but no clear conclusion has been reached so far, since some studies show that women are more susceptible to exercise-induced muscle damage, whereas other studies indicate that they are not (⁷). To resolve this discrepancy, the group of subjects in study was analyzed according to gender.

Based on the aforementioned findings, the authors hypothesized that eccentric exercise of 1 leg leads to a larger increase in muscle Ca²⁺ content than concentric exercise of a similar load in the other leg does. An increased Ca²⁺ load in the cells would promote the mechanisms causing muscle damage, as described above, and could thereby explain the larger damaging effects of eccentric muscle activity. In addition, due to the earlier findings from Larsen et al. (²⁴) and Newham et al. (²⁸), it was hypothesized that women would experience more muscle damage in response to step exercise than men would.

Methods

Experimental Approach to the Problem

The study was designed to investigate whether eccentric exercise would lead to a larger degree of muscle damage than concentric exercise would. Localized muscle damage in each leg was assessed by MRI technique and measurements of total muscle calcium content in addition to systemic muscle damage parameters, such as blood values of muscle enzymes and myoglobin. Both men and women were recruited in the study, since it was suggested that the degree of muscle damage is gender-specific, with women being more susceptible to develop muscle damage than men are. Subjects performed a 30-minute step exercise protocol involving eccentric exercise in 1 leg and concentric exercise in the other leg. This protocol has previously been shown to induce muscle damage in untrained women (²⁴).

Subjects

In total, 33 subjects (18 men and 15 women) were recruited mainly among the student population of the University of Aarhus in Denmark to participate in this study. Nineteen subjects were recruited in 2003 and 14 in 2006. The overall design and exercise protocol were the same in both groups, but not all parameters were measured in all subjects since new ideas came up between the 2 recruiting periods. For example, MRI and soreness were not measured in the first group of subjects and were included only in the last group of recruited subjects. Due to an incomplete series of biopsy specimens in 9 of the 33 subjects, they were excluded from the analysis of the muscle biopsy specimens. The incomplete series occurred because control biopsy specimens were taken in only 1 leg in 6 subjects and were not analyzed for dry weight in these subjects. Finally, 3 subjects felt uncomfortable with the first biopsy specimen taken and refused further biopsies. All subjects were moderately physically active, and most participated in recreational sports (Table 1). None of the subjects had participated in any strength training programs or specific eccentric exercise within the 6 months prior to the study. The subjects gave their written informed consent of participation, and the local ethical committee of the county of Aarhus approved the study.

Table 1: Characteristics of the subjects.

Procedures

The subjects performed step exercise on an adjustable bench for 30 minutes (²⁸). The bench was adjusted to be the same relative height for each subject, which was 110 % of the lower leg length. This is a commonly used height of the step (^24,29-31), which ensures eccentric work in 1 leg and concentric work in the other leg. The knee extensors of 1 leg contracted concentrically, while lifting the body of the subject during the stepping-up phase, and the knee extensors of the other leg contracted eccentrically, while lowering the body of the subject during the stepping-down phase. Both eccentric and concentric contractions lasted for about 1 second, and a metronome determined a stepping frequency of 15 cycles·min⁻¹. The subjects were instructed to try to land softly during the stepping-down phase in order to ensure that the eccentric contraction lasted throughout the whole stepping-down phase.

Oxygen Uptake Measurement and Heart Rate Monitoring

Two weeks prior to the step exercise, the subjects performed a progressive maximal cycle ergometer test (i.e., o₂max test), in which resistance was increased in steps of 30 to 75 W every 90 seconds until exhaustion. During the o₂max test, the expired air was sampled continuously, and the rates of oxygen uptake and carbon dioxide release were determined every 10 seconds by an online respiratory gas exchange analyzer (AMIS 2001; Innovision, Odense, Denmark). The o₂max tests lasted from 5 to 12 minutes. The o₂max was calculated as the maximal rate achieved over any 30-second period during the o₂max test.

The heart rate (HR) was monitored and recorded for every 5-second interval during the o₂max test with an HR monitor (Polar Accurex Plus; Polar Electro Oy, Kempele, Finland), and the highest HR measured during the period of maximal oxygen uptake for each subject was considered to be the maximal HR (HRmax). During the step exercise, the subjects were equipped with pulse monitors, and the HR was recorded for every 5-second interval.

Isometric Muscle Strength

Measurements of isometric maximal voluntary contractions (MVCs) were performed 2 times on separate days prior to the step exercise. The first measurement was done to familiarize the subjects with the test, while the second was used to establish a control value. Furthermore, the MVC was performed within 15 minutes of stepping and on days 1, 2, 3, 8, and 15 after the step exercise.

To measure MVC torque in the knee extensors, subjects were seated with the knee angle fixed at 90°, and the mechanical response was recorded by using a strain gauge transducer calibrated with standard weights. In a subgroup of the subjects (7 women and 7 men), the MVC of the adductor muscles was also examined. This measurement was accomplished with the subjects lying on their side on a bench. The upper legs were bent and inactive, whereas the lower legs were stretched. A strain gauge dynamometer was attached to the ankle, which was maximally pressed upward.

Each MVC test involved 3 trials separated by 30 seconds. The subjects were encouraged to perform their best, and the highest reading was considered the true MVC and used for further analysis. The results were normalized relatively to the pre-exercise values.

Muscle Soreness

Muscle soreness in the knee extensor region was recorded with a visual analog scale in the group of subjects recruited in 2006 (7 men and 7 women). The soreness was estimated while rising from and sitting down on a chair and evaluated by setting a mark on a 100-mm horizontal line, with 0 mm representing no soreness and 100 mm representing extreme soreness.

Blood Sampling and Analysis

Blood samples were drawn from an antecubital vein by venipuncture. Samples were taken 1 to 2 weeks prior to, immediately after, and 1, 2, 3, 8, and 15 days after the step exercise. After centrifugation, the plasma samples were stored at -20°C prior to analysis for the activity of CK and LDH and the concentration of myoglobin by standard commercial kits applied in a multianalyzer system (COBAS Integra 700; Hoffmann-La Roche Ltd., Basel, Switzerland).

Muscle Biopsies

Biopsy specimens were taken from the vastus lateralis muscle 1 week prior to the step exercise, within 30 minutes of completing the step exercise and again on postexercise days 1 and 2. The rationale for this chosen time course was based on previous observations in runners (^32,33) and from studies on rat muscles (¹⁰). In these studies, it was found that Ca²⁺ accumulation was maximal 6 to 24 hours after muscle activity. In the current study, muscle biopsy specimens were obtained from 24 of the 33 subjects (11 women and 13 men) in both legs pre, post, and on postexercise day 1. In addition, in 9 subjects of the group recruited in 2006, biopsy specimens were also obtained on postexercise day 2. The specimens were taken with the use of a conchotome, according to the technique of Dietrichson et al. (⁸). Sequential biopsy specimens from the same leg were taken about 3 cm distally or laterally apart in the vastus lateralis in order to avoid sampling the same fibers more than once. The biopsy material was immediately frozen with liquid nitrogen, and the samples were stored at -80°C until further analysis.

Water and Muscle Ca²⁺ Content

Water content was determined by weighing the samples before and after overnight drying at 60°C. Biopsy samples weighing 20 to 25 mg (i.e., wet weight) were soaked overnight in 2.5 ml 0.3 M trichloroacetic acid (TCA). In this TCA extract, Ca²⁺ content was determined by atomic absorption spectrometry, as described elsewhere (¹⁴).

Determination of Muscle Glycogen

Muscle samples were placed in a freeze dryer (Micro moduly 1.5 KRV3 vacuum pump; Edwards, UK) for a minimum of 20 hours. Following freeze drying, muscles were placed in a desiccator at room temperature for 1 hour, after which they were crushed into powder. The glycogen content was analyzed enzymatically, as described by Karlsson and Saltin (²¹).

Magnetic Resonance Imaging Protocol

A subgroup of the subjects (7 men and 7 women) underwent a series of 6 MRI scans performed 1 week prior to the step exercise and at postexercise days 1, 2, 3, 8, and 15. All imaging was performed with a Philips 1.5 T superconducting magnet (NT Intera; Philips Medical Systems, Best, The Netherlands). The subjects were placed in a supine position with the feet first in the imaging magnet. The images contained both legs and were collected by using a body coil.

Scout Scan

A T1-weighted scan was applied as an introductory scan to plan the subsequent scan. The sequence was set up with the following parameters: scan resolution, 352 × 246; field of view (FOV), 485 mm; number of slices, 18; slice thickness, 10 mm; slice gap, 10 mm; repetition time (TR), 11.52 milliseconds; echo time (TE), 6.1 milliseconds; number of signal averages (NSA), 1; and scan time, 52 seconds.

For the T2-weighted scan, the sequence was set up with these parameters: scan resolution, 352 × 352; FOV, 420 × 343 mm; number of slices, 9; slice thickness, 7 mm; slice gap, 35 mm; TR, 2.0 seconds; NSA, 1; and scan time, 7:38 minutes. Six echoes were obtained with a TE of 25, 50, 75, 100, 125, and 150 milliseconds.

Magnetic Resonance Imaging Data Analysis

One of the slices from the midthigh region (5 or 6 of 9) was used for further quantitative analysis of T2 values. T2 relaxation times from the echo sequence were calculated from intensity values in an elliptical region of interest (ROI) placed in the vastus lateralis, adductor magnus, or biceps femoralis. All ROIs were placed in the individual muscles with special care to avoid inclusion of subcutaneous fat, fascia, blood vessels, and bone structures. Three ROIs were placed in each muscle in the same slice, and the mean value of the T2 relaxation times was used for further analysis. The T2 relaxation times were calculated by using the scanner software (Cygwin).

Statistical Analyses

All values are mean ± SEM, in some cases with the range of values in parentheses. To test for differences in time series between genders, a 2-way analysis of variance was used for repeated measures followed by multiple pairwise comparisons (i.e., a Tukey test).

To test for differences between 2 groups, a Student's t-test for unpaired observations was used; p values are reported for these tests. To test for correlations between 2 parameters, a linear regression analysis was performed. The significance level and a correlation coefficient (r) are reported for the performed correlations. Statistical significances are accepted when p ≤ 0.05.

Results

Characteristics of the Subjects and Exercise Intensity

Table 1 summarizes different characteristics of the participants divided by gender. As shown, the 2 groups differ significantly in most parameters. The knee extensor torque (i.e., MVC) was significantly lower in the women, and the body weight (BW)-to-MVC ratio, reflecting the relative mechanical load during stepping, was 19% higher in the women (p < 0.05). The o₂max was significantly lower in the group of women (p < 0.001). The average HR during step exercise (step HR), in b·min⁻¹ and in percentage of HRmax, were significantly higher in the women (p < 0.01 and p < 0.05, respectively).

Muscle Ca²⁺ Content

Figure 1 shows the Ca²⁺ content of the biopsy specimens from the vastus lateralis of 24 subjects before, after, and on days 1 and 2 (9 subjects) following the step exercise. There were no significant changes in Ca²⁺ content in the eccentric leg (3.75 ± 0.11 μmol·g dry weight⁻¹ to 4.36 ± 0.48 μmol·g dry weight⁻¹; p = 0.29; n = 11 women) (4.28 ± 0.17 μmol·g dry weight⁻¹ to 4.45 ± 0.17 μmol·g dry weight⁻¹; p = 0.59; n = 13 men) or in the concentric leg (4.15 ± 0.32 μmol·g dry weight⁻¹ to 4.57 ± 0.26 μmol·g dry weight⁻¹; p = 0.12; n = 11 women) (4.23 ± 0.21 μmol·g dry weight⁻¹ to 4.29 ± 0.16 μmol·g dry weight⁻¹; p = 0.77; n = 13 men) from pre to post step exercise. No significant changes were observed on day 1 or day 2 either. Furthermore, no significant differences in Ca²⁺ content could be observed between the concentric and the eccentric legs or between genders at any time.

Figure 1: Muscle Ca²⁺ content in biopsy specimens from the vastus lateralis before (pre), 30 minutes after (post), and on days 1 and 2 after the step exercise. Values are means with error bars indicating SEM of 24 subjects (11 women and 13 men) before, 30 minutes after, and 1 day after; day 2, 9 subjects (5 women and 4 men). No significant differences were seen from pre-exercise levels or between genders.

Muscle Enzymes in Plasma

Figure 2A shows the time course of plasma CK activity in the days following the step exercise. In the women, there was a marked progressive increase in CK from 191 ± 103 U/L (range, 19-1520 U/L) at baseline to 4609 ± 2111 (range, 80-30,424 U/L) on day 2 (p < 0.001) and 7239 ± 2403 U/L (range, 73-33,486 U/L) on day 3 (p < 0.001). In the men, no significant changes were observed in CK at any time. On days 2 and 3, the increase in CK was up to sevenfold larger in women than in men (p < 0.001). Among the women, very large variations in the CK response were observed. Eleven of 15 women showed a maximal CK value in the days after step exercise, between 1000 and 33,486 U/L; the remaining 4 women had a maximal CK value below 1000 U/L (mean maximal CK, 492 ± 162 U/L). Among the men, 1 subject showed a high response to exercise, reaching 11,500 U/L on day 3. The remaining 17 men showed only smaller increases in the postexercise CK level, and all peaked below 1000 U/L (mean maximal CK, 403 ± 41 U/L). The LDH response (Figure 2B) showed the same time course as the CK response, but there was no significant gender difference in the LDH activity due to a larger background LDH level and the large individual variations in the responses to step exercise. However, a significant increase in LDH activity was observed on day 3 in the women (p < 0.001) compared to the pre-exercise value. No significant increases were observed in the men.

Figure 2: Plasma activity of creatine kinase (A) and lactate dehydrogenase (B) in women and in men before (pre), immediately after (post), and 1, 2, 3, 8, and 15 days after the step exercise. Values are means with error bars indicating 2 SEM of 18 men and 15 women. +++ Significant difference from pre-exercise level (p < 0.001) for women. $$$ Significant difference between men and women (p < 0.001).

No significant gender difference was observed in the myoglobin response to step exercise (data not shown).

Magnetic Resonance Imaging Data

Figure 3 shows an image of a cross-section of the thighs of 1 of the women on postexercise day 3. There is a clearly visible difference in the T2 intensity in the adductor magnus in the eccentric and in the concentric leg. The adductor magnus in the eccentric leg is much brighter than any of the other muscles and reflects the higher T2 signal from this muscle. Figure 4A shows that in the adductor magnus of the leg that had worked eccentrically, the mean T2 relaxation time in the women was increased by 75% on postexercise day 3, and still on day 8 T2 relaxation time was significantly elevated (p < 0.001). Mean T2 relaxation time in the women's eccentrically worked adductor magnus was significantly higher than in the men on postexercise days 2, 3 (p < 0.001), and 8 (p < 0.05). In the women's adductor magnus of the leg that had worked concentrically, mean T2 relaxation time was not significantly elevated at any time. In the vastus lateralis, in which the biopsy specimens for Ca²⁺ content were taken, T2 relaxation time showed a significant increase of 7% in the eccentric leg on day 1 (p < 0.01). No changes were observed in the women's concentrically worked vastus lateralis or in either of the biceps femoralis. As shown in Figure 4B, no increases were observed in either the eccentric or the concentric adductor magnus muscle in the men. However, in the vastus lateralis of the eccentrically worked leg, mean T2 relaxation time was significantly increased by 8% on day 1 (p < 0.001). No changes were observed in the concentric vastus lateralis or in either of the biceps femoralis in the men.

Figure 3: T2-weighted magnetic resonance image of the cross-section in the thigh region in a woman on postexercise day 3. VL = vastus lateralis; AM = adductor magnus.

Figure 4: Time courses of T2 relaxation time in adductor magnus (AM) and vastus lateralis (VL) in women (A) and in men (B) before (pre) and 1, 2, 3, 8, and 15 days after the step exercise. Values are means with error bars indicating 2 SEM of 7 men and 7 women. *** Significant difference from pre-exercise level (p < 0.001). ** Significant difference from pre-exercise level (p < 0.01).

Isometric Muscle Strength

To analyze the functional effects of muscle damage, MVC was measured in both legs of the participants. Immediately following step exercise, knee extensor MVC decreased in both the eccentric and concentric legs of the men and women. Figure 5A shows strength loss in knee extensors. In the women, the strength decreased immediately after exercise by 25% in the eccentric leg (p < 0.001) and by 12% in the concentric leg (p < 0.05). There was a significant difference between the response in the 2 legs (p < 0.01). The men also showed significant strength loss in both legs after step exercise (i.e., 17% in the eccentric leg [p < 0.001] and 11% in the concentric leg [p < 0.05]). However, in the men, there was no significant difference in strength loss between the 2 legs (p = 0.14). The women had a significantly larger strength loss in the eccentric leg than the men had on days 1, 2, and 3 after the step exercise (p < 0.05). Figure 5B shows the strength response relative to the pre-exercise value in the adductor muscles of 14 subjects. There were no differences between men and women in the concentric leg, and only in the women on days 1 and 2, strength loss in the eccentric leg almost reached statistical significance (p = 0.058 and p = 0.093, respectively).

Figure 5: Maximal voluntary contraction (MVC) of the eccentric leg or concentric leg in knee extensor muscles (A) and adductor muscles (B) before (pre), immediately after (post), and 1, 2, 3, 8, and 15 days after the step exercise. Values are the percentages of pre-exercise levels (100%) and means with error bars indicating 2 SEM of 18 men and 15 women (A) and 7 men and 7 women (B). + Significant difference from pre-exercise level (p < 0.05). +++ Significant difference from pre-exercise level (p < 0.001) for women. * Significant difference from pre-exercise level (p < 0.05) for men. *** Significant difference from pre-exercise level (p < 0.001) for men. $ Significant difference between men and women (p < 0.05).

Muscle Soreness

As shown in Figure 6, muscle soreness was most prominent in the eccentric leg on day 2. The peak values for the soreness in the eccentric leg were 66.9 ± 8.4 mm (p < 0.001) and 31.9 ± 8.5 mm (p < 0.01) in women and men, respectively. Significant differences in soreness were observed between men and women in the eccentric leg on postexercise days 1, 2, and 3. In the women, the peak value for soreness in the concentric leg was 29.1 ± 8.9 mm (day 1, p < 0.001), which was significantly different from the peak value in the eccentric leg (p < 0.01). In the men, a significant increase in soreness in the concentric leg was also observed in the men on days 1 and 2 (p < 0.05), but there was no significant difference in peak values between the eccentric and the concentric legs (p = 0.07). Soreness peaked on day 1 or 2 and was normalized on day 8 or 15.

Figure 6: Muscle soreness in the eccentric and the concentric leg of women and men before (pre), immediately after (post), and 1, 2, 3, 8, and 15 days after the step exercise. Values are means with error bars indicating 2 SEM of 7 women and 7 men. + Significant difference from pre-exercise level (p < 0.05) for women. ++ Significant difference from pre-exercise level (p < 0.01) for women. +++ Significant difference from pre-exercise level (p < 0.001) for women. * Significant difference from pre-exercise level (p < 0.05) for men. ** Significant difference from pre-exercise level (p < 0.01) for men. $ Significant difference between men and women (eccentric leg) (p < 0.05). $$ Significant difference between men and women (eccentric leg) (p < 0.01). $$$ Significant difference between men and women (eccentric leg) (p < 0.001).

Correlations

Linear regression analyses were performed to investigate correlations between plasma enzyme levels and T2 relaxation times in the subgroup of the subjects in whom T2 was measured. Figure 7 shows that on day 3 there was a strong correlation between CK activity and T2 relaxation time in the eccentric adductor magnus (r² = 0.84; p < 0.001; n = 14). Although not shown, similar correlations were found between LDH activity on day 3 and T2 relaxation time in the eccentric adductor magnus (r² = 0.72; p < 0.001; n = 14) and between myoglobin and T2 time (r² = 0.50; p < 0.01; n = 14). No significant correlations were found between release of CK and strength loss (r² = 0.007; p = 0.70) or soreness (r² = 0.13; p = 0.20) on day 3. No significant correlations were found between T2 relaxation time and strength loss (r² = 0.03; p = 0.57) or soreness (r² = 0.27; p = 0.06). To investigate if the relative intensity of the step exercise could explain some of the variability in strength loss between subjects, linear regression analyses were also performed between the BW-to-MVC ratio and eccentric strength loss on day 2 in the entire group of subjects, but no significant correlation was found (r² = 0.04; p = 0.26; n = 33). Furthermore, no significant correlation between step HR and eccentric strength loss on day 2 was found (r² = 0.07; p = 0.13; n = 33). However, a significant correlation was found between the 2 intensity parameters BW-to-MVC ratio and step HR (r² = 0.47; p < 0.001; n = 32).

Figure 7: Correlation between plasma creatine kinase level and T2 relaxation time on postexercise day 3. Analysis was performed by linear regression (r ² = 0.84; p < 0.001; n = 14). The 7 women are denoted by open symbols, and the 7 men are denoted by closed symbols.

Glycogen Content

Glycogen content (Figure 8) was measured in muscle biopsy specimens from a subgroup of the subjects (6 women and 6 men). Since no differences in the glycogen response between men and women were observed, data were pooled to 1 group. A significant drop of 22% (p < 0.05) was found in glycogen content in the concentric leg postexercise, and a drop of 14% on day 1 (p < 0.05). No significant drop was observed in the eccentric leg immediately postexercise (0.5% drop, p > 0.05) or on postexericse day 1 (9% drop, p > 0.05). A significant difference in glycogen level was observed between the concentric leg and the eccentric leg postexercise (p < 0.01).

Figure 8: Glycogen content in muscle biopsy specimens from the vastus lateralis before (pre), after (post), and on postexercise day 1. Values are means (mmol glycosyl units·kg dry weight⁻¹) with error bars indicating SEM of 12 subjects. * Significant difference from pre-exercise level (p < 0.05).

Discussion

Contrary to the hypothesis, no changes were found in muscle Ca²⁺ content in the vastus lateralis either in the eccentric or in the concentric legs in either women or men. However, a small increase was observed in T2 relaxation time in the eccentric vastus lateralis, and a significant long-lasting strength loss was found in the knee extensors of the eccentric leg in both men and women and indicated that these muscles in some degree were affected by step exercise. The study showed a clear gender difference in the responses to step exercise. With respect to muscle enzymes in plasma, a sevenfold higher increase was found in women than in men in the CK plasma level. Furthermore, the women showed larger increases in T2 relaxation time in the adductor magnus, and these increases were highly correlated to the plasma CK level. An increased loss of muscle strength was also found in the eccentric leg in women compared to that in men.

Studies in rat muscles have shown that the amount of membrane damage in muscles caused by electrically stimulated isometric contractions is related to the influx of extracellular Ca²⁺ (^16,19). In isolated rat muscles, the rate of influx is correlated to the extracellular concentration of Ca²⁺, and furthermore, the excitation-induced increased influx of Ca²⁺ leads to cellular Ca²⁺ accumulation (^14,16,26). The authors have previously shown that unaccustomed exercise in human subjects also leads to cellular Ca²⁺ accumulation in the vastus lateralis muscles (^32,33). In the current study, no increase was found in muscle Ca²⁺ content in the vastus lateralis after the step exercise protocol. This lack of finding may be due to the fact that the vastus lateralis muscle is exposed to sarcolemmal damage only to a minor extent during this type of exercise, in contrast to during normal running, in which Ca²⁺ accumulation is observed in the vastus lateralis (^32,33). The duration of the exercise may also be important for whether Ca²⁺ accumulation occurs. In vitro studies on rat muscles show that Ca²⁺ influx and Ca²⁺ accumulation increase with the duration of electrical stimulation (^12,14,26). Furthermore, no significant increase was found in Ca²⁺ content in muscle biopsy specimens from subjects running 10 km (i.e., approximately 50 minutes), but a significant increase was found after a 20-km run (i.e., approximately 90 minutes) (³²). The step exercise protocol lasted for only 30 minutes, and this duration may play a role in the lack of significant increases in Ca²⁺ accumulation shown in the current study.

By analysis of the magnetic resonance images in this study, it seems that the largest degree of cell damage was localized to the adductor magnus muscle in the eccentric leg. Unfortunately, the location and highly complex innervation of this muscle make it unsafe to excise biopsy specimens. A minor but significant increase was found in T2 relaxation time in the vastus lateralis of the eccentric leg in both women and men and indicated that some cell membrane damage had occurred in this muscle as well.

Very large increases were observed in plasma CK, with values above 1000 U/L, in 11 of 15 women, but in only 1 of 18 men. In the remaining women and in most of the men, the increases were absent or more moderate. The large increases seen in most women indicate that substantial muscle cell damage had developed during and following the step exercise. A main portion of the muscle enzymes probably originate from the adductor magnus muscles, as indicated by the MRI findings. Changes in T2 relaxation times reflect changes in the water chemistry of the tissue and have been linked to muscle membrane damage in earlier studies (^7,36,38). The current study also showed large increases in adductor magnus T2 relaxation times that were well correlated to the plasma CK level. This finding is supported by Newham et al. (²⁹), who found that following step exercise, a large influx of technetium pyrophosphate occurred in the thigh adductor magnus muscles and in the gluteal muscles. Furthermore, in a study from the authors' own group using MRI, the large CK levels in plasma following step exercise in women coincided with increased T2 relaxation times in the adductor magnus of the eccentric leg (²⁴). Together with the current results, these findings indicate that membrane damage in the adductor magnus of the eccentric leg occurs to a much greater extent than in the vastus lateralis of the knee extensors, where only small, albeit significant, increases in T2 relaxation time were observed. A possible explanation for this difference could be that the strain on the adductor magnus muscles during step exercise is relatively more intense than that on the vastus lateralis muscles. In addition, it may be speculated that the vastus lateralis muscles are more often activated in eccentric contractions during daily-life movements (e.g., sitting down), which confer an adaptation to the vastus lateralis muscles and possibly make them less prone to this type of muscle damage.

It is well known that unaccustomed exercise leads to a decrease in muscle strength in the days following the exercise (^{18,27,28,31,32,37}). The mechanism behind this prolonged strength decrease is still debated. A large part of it is likely to be related to processes such as a disruption of the triad of the EC coupling (³⁹) with slow recovery in the muscle, rather than to metabolic disturbances (²⁷). This hypothesis is supported by the observations that glycogen content was decreased only in the muscle biopsy specimens taken in the concentric leg and that no significant decrease was found in the eccentric leg. It is well known that energy cost is lower during eccentric exercise than during concentric exercise of a similar load (⁴). Nevertheless, eccentric work has been shown to cause more cell damage and loss of force than concentric work causes (^27,28,31). The current study found a larger strength decrease in the eccentric leg than in the concentric leg in the knee extensors in the women immediately after the step exercise. However, this difference was not significant in the men, possibly due to the low overall damaging effect produced by the step exercise in this group. The strength loss in the adductor muscles was not at any time significant, most likely due to a high variance and the low number of subjects performing this strength test. However, the time course and magnitude of the strength loss (i.e., tendencies) in the adductor magnus were almost similar to those in the knee extensors. Thus, regarding functional impairment caused by the step exercise, no differences were observed between adductor muscles, including the adductor magnus, and knee extensors, including the vastus lateralis.

Muscle soreness was most pronounced in the eccentric leg of the women, just as with the T2 relaxation time and strength loss. Muscle soreness showed the same delayed time course (i.e., peaking on postexercise day 2) as the parameters of muscle damage (i.e., enzyme release and T2 relaxation time), but no significant correlations were found between soreness and any of these parameters. The mechanism leading to muscle soreness is not clarified yet. It was suggested in the 1980s that exercise-induced muscle pain is due to damage to the connective tissue instead of damage to the muscle and therefore not related to muscle enzyme release (²⁰). By palpation of the damaged muscle in these studies, the soreness seemed to be located where the tendon attaches to the muscle (²⁰). No clear conclusion has been offered since then. Thus, due to the lack of correlations between soreness and any of the damage parameters in this study, the observed soreness might have originated from factors other than the damaged muscle fibers per se.

There has been much focus on the gender-specific response to different kinds of exercise. It is still not clear whether there is a gender difference in the susceptibility to muscle damage (²²). It has been shown that female rodents demonstrate attenuated responses to exercise, with less muscle damage, as indicated by indirect markers such as plasma CK, histological measurements, and inflammatory responses (^1,7,23). Explanations for gender differences have mainly been based on the hypothesis that estrogen in females may improve the structural integrity of the plasma membrane by having either a receptor-mediated effect or an antioxidant property (²³). In contrast to this hypothesis, other studies have found that estrogen is not related to indicators of muscle damage in humans (⁶). Thompson et al. (⁴⁰) found that women taking oral contraceptives did not have a lower increase in CK following step exercise. Actually, there was a tendency to have a larger increase in CK in these women than in women at their midluteal stage of the menstrual cycle. In accordance with this finding and with the results of the current study, Newham et al. (²⁸) observed large increases in plasma CK in 4 of 8 women and only in 1 of 5 men after step exercise. Other studies have not found any gender differences among humans when measuring CK activity or strength loss after eccentric exercise (i.e., elbow flexions or unilateral leg press) (^35,37). A clearly larger decrease of muscle strength and increase of muscle enzymes in plasma were found in response to step exercise in women than in men. Furthermore, a significantly higher increase was observed in T2 relaxation time in the eccentric adductor magnus in the women than in the men. It is speculated that the gender difference may be due to a higher relative workload experienced by the women than by the men in this type of exercise. The women had a higher HR during the step exercise (16%, p < 0.01) and a higher BW-to-MVC ratio (19%, p < 0.05), and the significant correlation found between these 2 parameters indicated that the women were working at a higher relative intensity in step exercise, in which their own body weight determines the load of each step. However, since no significant relationship could be found between strength loss and exercise intensity either in mechanical (i.e., BW-to-MVC ratio) or in cardiovascular (i.e., step HR) terms, it must be concluded that other factors apart from these intensity-related factors contribute to the individual and gender differences in muscle damage and reduced muscle function following step exercise. Since an adaptive response of repeated step exercise bouts has previously been seen (²⁴), it may be that adaptation to exercise performed prior to the step exercise protocol is more pronounced in men than in women. Regular exercise makes the muscles more resistant to muscle cell damage. This resistance is seen by a markedly reduced CK response after the second bout of step exercise compared to the highly elevated CK release observed after the first bout of exercise (²⁴). A possible explanation for the higher degree of damage in the adductor magnus seen in the women could be that since the women exercised at higher relative intensities, they were more prone to fatigue in the knee extensor muscles near the end of the step exercise. To compensate for this fatigue and to relieve the load on the knee extensors, the down-stepping could be performed with a higher degree of flexion of the hips, which in turn may increase the eccentric activity of the adductor magnus muscle. In support of this hypothesis, preliminary unpublished data from movement analysis of the step exercise indicated that the hip flexion during down-stepping was higher in women than in men.

In conclusion, step exercise of this specific type and duration does not induce changes in muscle Ca²⁺ content in the vastus lateralis. However, it has been shown that step exercise induces muscle damage, as evidenced by increased plasma levels of muscle enzymes and increased T2 relaxation time in specific muscles. Damage-related variables were most affected in the women, who also showed the largest degree of strength loss, especially in the eccentric adductor and knee extensor muscles. The strong correlation found between the plasma level of CK and the T2 relaxation time in the eccentric adductor magnus indicated that cell membrane damage is mostly located in this muscle during this specific exercise protocol.

Practical Applications

This study implies that when constructing a training program, specifically involving weight-bearing eccentric exercise, gender differences regarding risk of muscle damage should be taken into account. The results indicate that especially for women inexperienced with prior eccentric exercise, such exercise should be performed at low volumes in the initial phase of the exercise program to avoid unwanted pain and muscle damage and hereafter carefully progress to more intense and long-term eccentric exercise.

Although it may be prudent to use careful familiarization to weight-bearing eccentric exercise especially in women, such exercise should not be discouraged entirely, since the muscle damaging effects are not harmful in the long term, but may, in fact, protect from further muscle damage in subsequent exercise, as shown elsewhere (²⁴).

Acknowledgments

We would like to thank Prof. Thorsten Ingemann-Hansen for performing the biopsies and Steffen Ringgaard for designing the MRI scanning protocol. Vibeke Uhre and Tove Lindahl Andersen are thanked for expert technical assistance in measuring muscle Ca²⁺ content. Also, Thomas Bruun Lund and Jens Østergaard are thanked for performing the muscle strength measurements and Klavs Madsen for performing the glycogen content measurements. Finally, a special thanks to all the voluntary participants. The study was funded by grants from Danish Medical Research Council (22-04-0241) and from Aarhus Universitets Forskningsfond.