Eur J Appl Physiol (1992) 65:258-264 European Journal of

Applied

Physiology and Occupational Physiology © Springer-Verlag 1992

Assessment of skeletal muscle damage in successive biopsies from strength-trained and untrained men and women Robert S. Staron 1'2, Robert S. Hikida 1, Thomas F. Murray 2, Marcia M. Nelson 1, Peter Johnson 2'3, and Fredrick Hagerman 1 t Department of Zoologicaland BiomedicalSciences, 2 College of Osteopathic Medicine, 3Department of Chemistry, Ohio University, Athens, OH 45701, USA Accepted April 13, 1992

Summary. The effects of repeated biopsy sampling on muscle morphology was qualitatively and quantitatively assessed in strength-trained and untrained men and women. College-age men (13) and women (8) resistance trained twice a week for 8 weeks. A progressive resistance-training program was performed consisting of squats, leg presses, and leg extensions. Nontraining men (7) and women (5) served as controls. Muscle biopsy specimens and fasting bloods were obtained at the beginning and every 2 weeks and histochemical, biochemical, and ultrastructural methods were employed to assess the type and amount of damage. Except for a few scattered atrophic fibers in 2 of the 33 biopsy samples, all initial specimens were normal. In contrast, many of the subsequent biopsy samples from both untrained and resistance-trained men and women contained evidence of damage. Ultrastructural analysis confirmed that degenerative-regenerative processes were occurring in both groups. However, training subjects had a four-fold greater number of damaged fibers than nontraining subjects (8.53°7o vs 2.08O7o). In addition, only biopsy samples from training individuals contained fibers with internal disorganization (e.g., Z-line streaming, myofibrillar disruption). Calpain II levels in the biopsy samples and serum creatine kinase activity were not significantly affected supporting the light and electron microscopic observations that most of the damaged fibers were normal in appearance except for their small diameter. In summary, focal damage induced by the biopsy procedure is not completely repaired after 2 weeks and could affect the results, particularly cross-sectional area measurements. Moreover, resistance training appears to cause additional damage to the muscle and may delay repair of the biopsied region. Key words: Resistance training - Skeletal muscle - Fiber types - Muscle biopsy - Damage

Correspondence to: R. S. Staron

Introduction Our past studies have observed skeletal muscle damage in resistance-trained women (Staron et al. 1990, 1991a), in which 7 of 24 post-training muscle biopsy samples contained evidence of degeneration-regeneration (Staron et al. 1990). No damage was found in any of the pretraining biopsy samples. Although efforts were made to extract the post-training biopsy samples in proximity to the pre-biopsy samples, the 20-week time period between the pre- and post-biopsies was considered of sufficient length for the traumatized area to heal (Fisher et al. 1990). A follow-up study (Staron et al. 1991a) of shorter duration (6 weeks) also revealed varying amounts of muscle damage in 8 of 13 biopsy samples after resistance training. Exercise-induced fiber damage in human muscle has been previously reported. Aerobic (Frid6n et al. 1983a; Hikida et al. 1983; Apple et al. 1987) and anaerobic (Frid6n et al. 1981, 1988) exercises have been shown to result in muscle damage. Because the eccentric component of muscle contraction induces the greatest amount of damage (Newham et al. 1983; Clarkson et al. 1986), resistance training has a large potential to injure the muscle. Indeed, recent evidence suggests that lifting weights does cause muscle damage in humans (Paul et al. 1989; Pivarnik et al. 1989; Stauber et al. 1990; Hikida et al. 1991). In training studies utilizing successive muscle biopsies, it is not known how much damage is done by the percutaneous needle biopsy technique and/or the exercise activity. To our knowledge, no study has investigated the events that occur in human muscle after a biopsy sample is extracted. Our most recent training study gave us an opportunity to compare damage induced by the biopsy procedure with damage induced by the biopsy procedure combined with resistance training in men and women. The purpose of the present investigation was to qualitatively and quantitatively assess the amount and type of skeletal muscle damage caused by resistance training and the biopsy procedure utilizing a series of successive biopsy samples. Some of the data

259 have been r e p o r t e d in abstract f o r m (Nelson et al. 1991; S t a r o n et al. 1991b).

Methods

Subjects. Thirty-five individuals (21 men and 14 women) volunteered to participate in the present investigation. All subjects signed informed consent documents and approval was given by the Ohio University Institutional Review Board prior to beginning the study. One man and 1 woman dropped out and 33 individuals completed the study. The training group consisted of 13 men [mean (SD) age, height, mass: 23.5 (3.2) years, 1.77 (0.08) m, 82.6 (17.5) kg] and 8 women [20.6 (1.5) years, 1.66 (0.05) m, 60.4 (5.8) kg]. Of the remaining 12 individuals, 7 men [20.7 (1.4) years; 1.80 (0.09) m; 76.9 (13.8) kg] and 5 women [20.6 (1.6) years, 1.61 (0.01) m, 68.8 (12.8) kg] were physically active, but were not involved in any regular form of exercise and served as controls. Training subjects were closely supervised at all times. Training protocol. The training protocol was similar to our pre-

sy each (weeks 3 and 9). Therefore, 161 biopsy samples were analyzed in the present investigation. Attempts were made to extract tissue from approximately the same location each time using the pre-biopsy sc~tr and depth markings on the needle. To ensure adequate sample sizes, large biopsy samples were obtained using a double-chop method (Staron et al. 1990, 1991a) combined with suction (Evans et al. 1982).

Light microscopy. The frozen biopsy samples were serially sectioned (12 ~tm thick) at - 2 0 ° C for histochemical and histological analyses. Routine myofibrillar adenosine triphosphatase (mATPase) histochemical analysis was performed using pre-incubation pH values of 4.3, 4.6, and 10.4 (Brooke and Kaiser 1970) to determine the muscle fiber type composition. Six fiber types (I, IC, IIAC, IIA, IIAB, and IIB) were distinguished based on their staining intensities (Staron and Pette 1986; Staron 1991). In addition, serial transverse sections were assayed for nicotinamide adenine dinucleotide activity and histological examination was performed on sections stained with hematoxylin and eosin.

Electron microscopy. The sample that was removed for ultrastruc-

vious resistance studies (Staron et al. 1990, 1991a). Briefly, the training period consisted of 1 week pre-conditioning and orientation followed by 8 weeks of high-intensity training. Three basic" lower limb exercises for the quadriceps femoris muscle group (leg presses, squats, and leg extensions) were performed twice a week (Monday and Friday) with every other Wednesday used for maximal dynamic strength testing. Workouts consisted of two warmup sets followed by three sets to failure of either 6-8 repetitions (Mondays) or 10-12 repetitions (Fridays) for each of the three exercises. The weights were progressively increased to maintain this range of repetitions/set.

tural analysis was fixed for 2 h in a modified Karnovsky's solution (2070 paraformaldehyde and 1070glutaraldehyde in 0.1 mol.1-1 cacodylate buffer, 0.15 mol.1-1 sucrose, pH 7.1). The tissues were then rinsed overnight in cacodylate-buffered sucrose, post-fixed in osmium tetroxide for 1 h at 4° C, dehydrated in an ethanol series, and embedded in Epon and Araldite. Longitudinal and transverse sections were cut using diamond knives on a Reichert Om U2 ultramicrotome, mounted on copper grids, contrasted with uranyl acetate and lead citrate, and examined in a Zeiss EM 109 electron microscope. For this part of the study, only biopsy samples from weeks 1, 5, and 9 from control (6 men, 3 women) and training (8 men, 2 women) individuals were investigated.

Serum analysis. After 12 h of fasting, 10-15 ml of blood was

Determination of muscle calpain H levels. Calcium-activated neu-

drawn from the median cubital vein before the initial biopsy and every 2 weeks (on Friday mornings) during the high-intensity training (24 h prior to each biopsy). The whole blood was spun down and 4-5 ml of serum was removed and stored in 1-ml aliquots at - 70° C. Serum creatine kinase (CK) and one of its isoenzymes (CK-MB) were determined (in duplicate) in a Kodak DT-60 Ektachem analyzer using reagent kits (controls and standards).

tral protease (Calpain II; Zaidi and Narahara 1989) was analyzed in the muscle biopsy samples by the use of the enzyme-linked immunoadsorbent assay (ELISA) procedure as described by Hussain et al. (1987). The units of titer in the assays were expressed as optical density units.nag-1 of extracted protein. The mouse monoclonal antibody to hamster calpain II (Johnson and Hammer 1988) was used for the assays. This antibody, which cross-reacts with human calpain II, is specific for an epitope in the large subunit of the enzyme and does not recognize calpain I. Muscle samples were extracted according to the procedures of Reichman et al. (1983). Briefly, 50-~tl samples of the solubilized protein fraction containing 0.5 Iig of protein (as determined by Bio-Rad Bradford protein assay) were used in duplicate wells on the assay plate. Because of the inherent variability of the ELISA absorbance readings between different plates for the same sample, complete sampie sets from the same subjects were always run on the same ELISA plate. Results were expressed as a percentage of the average ELISA readings for "zero-time" (prior to the experimental regimen) group of subject samples.

Muscle biopsies. Muscle biopsy samples (80-160mg) were extracted from the superficial portion of the vastus lateralis muscle by the percutaneous needle biopsy technique of Bergstr6m (1962). The muscle samples were removed from the needle and divided into two pieces. A small piece was processed for electron microscopy, and the larger portion was oriented in tragacanth gum, immediately frozen in isopentane cooled by liquid nitrogen to - 159° C, and stored at - 7 0 ° C. Biopsy specimens were taken prior to the pre-conditioning and orientation week (week 1) and every 2 weeks during the 8-week high-intensity training (weeks 3, 5, 7, and 9). Control subjects gave biopsy samples at these same time periods. Two training men and 2 training women missed one biop-

Table 1. Appearance of atrophic and degenerating fibers

Week:

1

3

5

7

9

Total

s/sg pfa lg tf

0/0/2/0 0/0/0/0 0/0/0/0 0/0/0/0

5/1/2/0 0/0/1/0 0/0/1/3 0/0/1/0

3/2/6/1 0/0/1/0 0/0/2/2 0/1/0/0

3/1/5/3 1/0/2/0 0/0/3/1 0/0/0/0

5/1/5/4 0/0/1/0 1/2/2/1 1/1/0/0

16/5/18/8 1/0/5/0 1/2/8/7 1/2/1/0

Values given are the number of biopsy samples at each time period which contained evidence of damage in control men/control women/training men/training women, s/sg, Scattered/small group atrophic or degenerating fibers; pfa, perifascicular atrophy; lg, large group atrophy (greater than 5°70 of the total number of fibers in a biopsy sample); tf, targetoid fibers. Some biopsy specimens contained more than one type of damage. Total, Total number of post-biopsy samples containing a specific type of damage

260

Stat&tical analys&. The statistical package for the biomedical sciences (BMDP) was utilized for all statistical analyses. A repeated-measures two-way analysis of variance (ANOVA) was used to analyze serum CK, CK-MB, and calpain II content in control and trained men and women. Significant differences between the means were determined by a modified Tukey's post hoc test. Differences were considered significant at P < 0.05.

Ultrastructural analysis With the exception of 1 biopsy sample, the initial biopsy samples (week 1) from both control and training individuals contained no ultrastructural evidence of damage. One initial biopsy sample from a control man did

Results

Light microscopy A mean (S) of 42789 (1617) fibers was examined at each time point. Atrophic fibers and fibers which were apparently degenerating were considered damaged. Muscle samples taken at the beginning of the study (from both control and training groups) contained little evidence of damage (0.04% of the total fibers) (Tables 1, 2). The few scattered, atrophic fibers found in these initial biopsy samples (Fig. la) were from 2 subjects (both training men). Evidence of damage in subsequent biopsy specimens consisted of three basic types: (1) scattered or small group atrophy-degeneration (Fig. lb), (2) perifascicular atrophy-degeneration (Fig. lc, d), and (3) large group atrophy-degeneration (affecting more than 5% of the total number of fibers in the muscle samples) (Fig. le, f). Occasionally, targetoid fibers were also found within some biopsy samples (Fig. lf). Large group atrophy was more prevalent in the biopsy samples of training individuals (both men and women) than controls. Indeed, large group atrophy from 4 biopsy specimens (each at a different time period) accounted for the large number of damaged fibers found in the muscles of training women (Tables 1, 2). The affected areas from these 4 samples composed 36°70, 45%, 74°70, and 100% of the biopsy samples. The total number of affected fibers (mostly atrophic), not including the pre-biopsy specimens, was four-fold higher for the training groups (men and women) than the control groups, 8.53% vs 2.08%, respectively. Damage did not appear to be specific for any particular fiber type, but degenerating fibers were often stable, to varying degrees, throughout the pH range for mATPase histochemistry (C fibers). It is not known if these fibers were actual C fibers (co-expressing myosin heavy chains I and IIa) or fibers expressing (or co-expressing) developmental myosin heavy chains. Table 2. Frequency of atrophic and degenerating fibers

Week:

1

3

5

7

9

Total

Controls Men Women

0.00 0.00

0.23 0.03

0.76 0.57

3.37 0.06

6.62 3.09

3.02 0.88

Trained Men Women

0.11 0.00

1.29 11.32

11.17 16.58

5.03 16.86

2.37

6.29

4.88 12.79

Values given represent the percentage of damaged fibers within the biopsy samples. Total, Total percentage of damaged fibers from samples 1-4

Fig. l a - f . Micrographs of muscle cross sections assayed for myofibrillar adenosine triphosphatase activity after pre-incubation at pH 4.6. Samples from: a training man before training (a) and a training man after 4 weeks of high-intensity training (b) demonstrating the typical appearance of scattered atrophic fibers (single arrow) and small group atrophy (double arrows) (bar = 50 gm), 2 training men after 6 weeks of training showing examples of perifascicular atrophy (c) and degeneration (d) (bar= 200 gm), and a training woman (e) and control man (f) at the conclusion of the study showing examples of large group atrophy-degeneration (bar = 200 gm). Note the unusual targetoid fibers present in f (ar-

rowheads)

261

Fig. 2a-e. For legend see page 262

262

Fig. 2a-e. Electron micrographs from control and trained subjects, a Group of small, degenerating fibers containing myofilaments and Z bodies. The thickened basal lamina is duplicated around each fiber (arrowheads) in this fifth biopsy sample (week 9) from a control subject. X6120; bar=2 ~tm. b A filamentous mass (F) in the subsarcolemmal region of a control subject (week 5). X14400; bar= 1 ~tm. e Sample from a control subject (week 9) showing an atrophic fiber (A) between two normal fibers. Note the large amount of interstitial fibrous connective tissue. X1013; bar= 10 Ixm. d Sample from a training subject (week 5) with a macrophage in the interstitial space. X5780; bar=2 ~tm. e Area of extensive Z-line streaming (S) and myofibril disruption in a sample from a subject trained for 7 weeks. A relatively normal region of the fiber is shown at the right. X16400; bar= 1 ~tm contain a few fibers with apparent breaks in the sarcolemma and signs of degeneration. On the other hand, repeated muscle specimens taken in proximity induced obvious signs of degeneration and regeneration. Damage was observed in one third of the control biopsy samples examined from week 5 and two thirds f r o m week 9. No difference was found between the muscle samples from control men and women. Degeneration and regeneration were c o m m o n in the muscles samples f r o m both the control and trained subjects at weeks 5 and 9. A m o n g the controls, 2 samples, apparently close to prior biopsies, contained areas of extensive damage. One of these samples also contained a population of muscle fibers that appeared to be degenerating and was surrounded by duplicated basal laminae (Fig. 2a). Although the duplication of basal laminae usually indicates regeneration, these fibers did not resemble myotubes. Cell infiltrates (Fig. 2d) were also found in some biopsy samples indicating possible inflammation and repair. Evidence of regeneration and growth of intracellular components included honeycomb proliferations of transverse tubules, tubular aggre-

gates, and subsarcolemmal masses of microfilaments (Fig. 2b). As was observed by light microscopy, m a n y fibers were normal in appearance except for their small diameter (Fig. 2c). In addition to the changes described above, muscle samples f r o m the individuals undergoing high-intensity resistance training showed signs of damage not observed in the control subjects. The most c o m m o n intracellular change found in the trained muscle was Z-line streaming (Fig. 2e). The streaming often occupied large segments, sometimes affecting more than half of the fiber crosssectional area. In some cases, the Z-lines of adjacent sarcomeres would become disjointed and cause the myofibrils to fragment into small, disorganized segments (Fig. 2e). Overall, the changes induced by exercise appeared to consist mainly of the disruption of the myofibrillar pattern rather than degeneration.

S e r u m C K a n d C K - M B c o n t e n t a n d calpain H analysis

Resting levels of total serum CK were variable within all groups. Although no significant differences were found for any group over time, the highest serum CK levels were found in the training men after 2 weeks of highintensity training (Table 3a). Overall, the mean value obtained for the women (both control and training) was significantly lower than the mean value for the men [107(86) units.1 -~ vs 241(237) units.l-X]. In addition, the mean CK value obtained for the controls (both men and women) was significantly lower than for the training individuals [141 (92) units.l-~ and 204(229) units. 1-1, respectively]. The CK-MB levels showed similar trends. No differences were found for any group over time (Table 3b). However, the mean CK-MB value obtained for the women (control and training) was sig-

Table 3. Table showing activity of serum creatine kinase (CK) and isoenzyme of creatine kinase (CK-MB); calpain II content in postbiopsy samples is also shown Week:

1

3

5

7

9

91 (19) 287 (319) 126 (52) 520 (446)

149 (145) 166 (60) 93 (29) 201 (137)

116 176 87 202

a. Total CK in the serum

Control women Control men Training women Training men

176 121 75 193

(146) (28) (32) (149)

(72) (111) (35) (175)

75 (19) 162 (79) 134 (134) 275 (207)

b. CK-MB content in the serum

Control women Control men Training women Training men

6.2 9.2 8.9 10.0

(1.1) (3.1) (2.8) (2.8)

2.9 9.1 9.4 9.6

(2.5) (3.9) (3.8) (4.2)

4.2 7.9 5.4 6.5

(2.4) (5.1) (2.0) (2.8)

3.2 6.9 7.8 7.3

(1.8) (1.8) (2.1) (1.8)

5.2 6.3 6.9 7.9

(1.5) (3.8) (3.0) (2.7)

c. Calpain H content in the post-biopsy samples

Control women Training women Control men Training men

91 117 101 110

(25) (31) (13) (23)

99 109 114 102

(27) (21) (25) (20)

136 105 113 96

(25) (20) (16) (25)

98 127 118 103

(17) (44) (28) (21)

Values for total CK and CK-MB activity are given as units. 1- 1 (SD). For calpain II content, data are presented as the percent change (SD) from pre-values (week 1), and the units of titer in the assay are expressed as optical density units.mg -1 of extracted protein

263 nificantly lower than the value for the men [6.3(3.3) units' 1- 1 compared with 8.2 (3.5) units" 1- a]. No significant differences in calpain II levels were found between any of the groups or over time (Table 3c).

Discussion Sporadic physical exercise, particularly with a large eccentric component, can cause muscle injury and postexercise soreness (Clarkson and Tremblay 1988). As such, it is not surprising that weight-lifting exercises have been used to induce muscle damage in men (Staub e r e t al. 1990) and women (Ebbeling and Clarkson 1990). Although recovery from exercise-induced injuries can be slow, taking 1 week or longer, the initial bout can have a protective effect if the exercise is repeated during (or shortly after) the recovery phase (Byrnes et al. 1985; Clarkson et al. 1987; Clarkson and Tremblay 1988; Ebbeling and Clarkson 1990). Indeed, prior w.eight training appears to diminish the extent of damage (Frid6n et al. 1983b; Paul et al. 1989). However, muscle damage has been observed in biopsy specimens taken from women after both long-term (20 weeks) and short-term (6 weeks) resistance-training programs (Staron et al. 1990, 1991a). Because of intramuscular variations in fiber-type composition from superficial to deep and proximal to distal (Blomstrand and Ekblom 1982; Lexell et al. 1985), it is desirable to obtain repeated muscle samples from the same general region of a muscle. Although, the insertion of a biopsy needle and extraction of muscle tissue induce focal damage, the events occurring in human muscle after a biopsy have not been documented. However, it is apparent that the damage caused by the percutaneous biopsy procedure is detectable for at least several weeks (present investigation) and perhaps longer, particularly in training individuals (Staron et al. 1990, 1991a). The current study does not specifically answer the question as to how much damage was caused by the resistance training versus the muscle biopsy procedure. Most of the damaged fibers observed in the present study appear to be the result of the biopsy procedure. The presence of necrotic fibers with coagulated myofilaments and open membranes, as well as duplicated basal laminae and undeveloped myofibrils, suggest recent damage. Less recent damage may be reflected in areas of small fibers surrounded by normal fibers. The areas of large and small group atrophy most likely represent fibers that have been regenerating and are normal except for their diameter. Repeated biopsy samples taken in proximity to a prior one appear to exacerbate the area of damage. Repeated sampling in combination with high-intensity weight training appear to increase the damage. The four-fold greater number of damaged (atrophic and degenerating) fibers found in the specimens from trained muscles compared to the control muscle biopsy specimens suggests that, in addition to the biopsy procedure, resistance training delays regeneration a n d / o r causes ad-

ditional damage. As has been previously reported (Friden et al. 1981; Newham et al. 1983), the exercise-induced damage appears to primarily consist of focal areas of intracellular disorganization (Z-line streaming and myofibrillar disruption). During high-intensity resistance training, the shear forces pulling at the Z-lines may cause them to smear and rupture, perhaps contributing to myofibrillar proliferation (Goldspink 1970; Shear and Goldspink 1971). It is not surprising that the serum CK levels did not reflect the observed damage in the current study. Focal damage induced by the biopsy procedure causes only a slight elevation in serum CK levels (absolute increases of 50-160 u n i t s . l - I ) which peaks 24 h after a biopsy (Hikida et al. 1991). In addition, most of the "damage" observed in the biopsy samples from both training and control subjects consisted of atrophic and not degenerating fibers. In support of these data, no change was found in calpain II levels. Indeed, even the samples that contained extensive regions of atrophic fibers (large group atrophy) had no significant increases in calpain II levels. The differences found between men and women for CK and CK-MB may re related to the effect of estradiol on some membrane property (Amelink et a1.1990) or to differences in clearance rates (van der Meulen et al. 1991). In summary, care must be taken when extracting successive muscle biopsy samples in proximity to one another. Focal damage induced by the biopsy procedure is not completely repaired after 2 weeks and perhaps longer. Resistance training appears to cause additional damage primarily consisting of myofibrillar disorganization, but may also delay repair of the biopsied site. As such, muscle damage could affect the results, particularly in fiber cross-sectional area determinations. Therefore, although it is important to obtain repeated samples from the same general area of a muscle, efforts should be made to avoid the exact site of a previous biopsy.

Acknowledgements. The authors thank the Ohio University College of Osteopathic Medicine photographic and graphic departments for help with the figures and tables, Dr. Jeffrey Falkel for assistance with blood withdrawal, Ms. Janet L. Hammer for technical assistance, Mr. Daniel Karapondo for assistance with the statistics and training, and Drs. Kerry Ragg and Gerald Noga for physical screening of the subjects. A very special thanks goes to all the men and women who volunteered for this study. This study was supported in part by a grant from the National Strength and Conditioning Association. References Amelink GJ, Koot RW, Erich WBM, Van Gijn J, B~trPR (1990) Sex-linked variation in creatine kinase release, and its dependence on oestradiol, can be demonstrated in an in vitro rat skeletal muscle preparation. Acta Physiol Scand 138:115-124 Apple FS, Rogers MA, Casal DC, Lewis L, Ivy JL, Lampe JW (1987) Skeletal muscle creatine kinase MB alterations in women marathon runners. Eur J Appl Physiol 56:49-52 BergstrOm J (1962) Muscle electrolytes in man. Scand J Clin Lab Invest 14 [Suppl 68] : 1-110 Blomstrand E, Ekblom B (1982) The needle biopsy technique for fibre type determination in human skeletal muscle - a methodological study. Acta Physiol Scand 116: 437-442

264 Brooke MH, Kaiser KK (1970) Three "myosin ATPase" systems: the nature of their pH lability and sulfhydryl dependence. J Histochem Cytochem 18 : 670-672 Byrnes WC, Clarkson PM, White JS, Hsieh SS, Frykman PN, Maughan RK (1985) Delayed onset muscle soreness following repeated bouts of downhill running. J Appl Physiol 59:710715 Clarkson PM, Tremblay I (1988) Rapid adaptation to exercise induced muscle damage. J Appl Physiol 65 : 1-6 Clarkson PM, Byrnes WC, McCormick KM, Turcotte LP, White JS (1986) Muscle soreness and serum creatine kinase activity following isometric, eccentric, and concentric exercise. Int J Sports Med 7 : 152-155 Clarkson PM, Byrnes WC, Gillisson E, Harper E (1987) Adaptation to exercise-induced muscle damage. Clin Sci 73 : 383-386 Ebbeling CB, Clarkson PM (1990) Muscle adaptation prior to recovery following eccentric exercise. Eur J Appl Physiol 60 : 2631 Evans WJ, Pinney SD, Young VR (1982) Suction applied to a muscle biopsy maximizes sample size. Med Sci Sports Exerc 14:101 Fisher BD, Baracos VE, Shnitka TK, Mendryk SW, Reid DC (1990) Ultrastructural events following acute muscle trauma. Med Sci Sports Exerc 22:185-193 Frid6n J, Sj6str0m M, Ekblom B (1981) A morphological study of delayed muscle soreness. Experientia 37:506-507 Frid6n J, Sj6strOm M, Ekblom B (1983a) Myofibrillar damage following intense eccentric exercise in man. Int J Sports Med 4:170-176 Frid6n J, Seger J, Sj6str6m M, Ekblom B (1983b) Adaptive response in human skeletal muscle subjected to prolonged eccentric training. Int J Sports Med 4:177-183 Frid6n J, Seger J, Ekblom B (1988) Sublethal muscle fibre injuries after high-tension anaerobic exercise. Eur J Appl Physiol 57 : 360-368 Goldspink G (1970) The proliferation of myofibrils during muscle fibre growth. J Cell Sci 6 : 593-603 Hikida RS, Staron RS, Hagerman FC, Sherman WM, Costill DL (1983) Muscle fiber necrosis associated with human marathon runners. J Neurol Sci 59:185-203 Hikida RS, Staron RS, Hagerman FC, Leonardi M, Gilders R, Falkel J, Murray TF, Appell K (1991) Serum creatine kinase activity and its changes due to a muscle biopsy. Clin Physiol 11:51-59 Hussain H, Dudley GA, Johnson P (1987) Effects of denervation on calpain and ealpastatin in hamster skeletal muscles. Exp Neurol 97 : 635-643 Johnson P, Hammer JL (1988) Calpain and calpastatin levels in

dystrophic hamster skeletal muscles. Int J Biochem 20:12271230 Lexell J, Taylor C, SjOstr0m M (1985) Analysis of sampling errors in biopsy techniques using data from whole muscle cross sections. J Appl Physiol 59:1228-1235 Nelson MM, Hikida RS, Staron RS (1991) Effects of repeated muscle biopsy sampling on the morphology of human skeletal muscle. Anat Rec 229:63A Newham D J, McPhail G, Mills KR, Edwards RHT (1983) Ultrastructural changes after concentric and eccentric contractions of human muscle. J Neurol Sci 61 : 109-122 Paul GL, DeLany JP, Snook JT, Seifert JG, Kirby TE (1989) Serum and urinary markers of skeletal muscle tissue damage after weight lifting exercise. Eur J Appl Physiol 58 : 786-790 Pivarnik JM, Hickson JF, Wolinsky IRA (1989) Urinary 3-methylhistidine excretion increases with repeated weight training exercise. Med Sci Sports Exerc 21:283-287 Reichman H, Srihar, Pette D (1983) Ipsi- and contralateral fibre transformations by cross-reinnervation. A principle of symmetry. Pfliigers Arch 397:202-208 Shear CR, Goldspink G (1971) Structural and physiological changes associated with the growth of avian fast and slow muscle. J Morphol 135:351-372 Staron RS (1991) Correlation between myofibrillar ATPase activity and myosin heavy chain composition in single human muscle fibers. Histochemistry 96 : 21-24 Staron RS, Pette D (1986) Correlation between myofibrillar ATPase activity and myosin heavy chain composition in rabbit muscle fibers. Histochemistry 86:19-23 Staron RS, Malicky ES, Leonardi M J, Falkel JE, Hagerman FC, Dudley GA (1990) Muscle hypertrophy and fast fiber type conversions in heavy resistance-trained women. Eur J Appl Physiol 60:71-79 Staron RS, Leonardi M J, Karapondo D, Malicky ES, Falkel JE, Hagerman FC, Hikida RS (1991a) Strength and skeletal muscle adaptations in heavy resistance-trained women after detraining and retraining. J Appl Physiol 70:631-640 Staron R, Hikida R, Murray T, Falkel J (1991b) Assessment of skeletal muscle damage in heavy resistance-trained men and women. Med Sci Sports Exerc 23 :S145 Stauber WT, Clarkson PM, Fritz VK, Evans WJ (1990) Extracellular matrix disruption and pain after eccentric muscle action. J Appl Physiol 69:868-874 Van der Meulen JH, Kuipers H, Drukker J (1991) Relationship between exercise-induced muscle damage and enzyme release in rats. J Appl Physiol 71:999-1004 Zaidi SIM, Narahara HT (1989) Degradation of skeletal muscle plasma membrane proteins by calpain. J Membr Biol 110:209216