INTRODUCTION

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AN INITIAL BOUT OF EXERCISE with muscle lengthening (eccentric contractions) causes muscle damage, a loss of voluntary and tetanic forces, a reduction in range of motion, a loss of muscle proteins, and muscle soreness (1, 6, 7, 13, 21). Peculiarly, these symptoms are substantially moderated or are even absent when the same exercise is repeated after recovery from the initial bout (3, 7) and a "rapid adaptation" occurs (1, 2). Concentric exercise is not associated with such rapid adaptation (7). It has been suggested (2) that muscle damage after an initial bout of exercise occurs because of the high forces and strain (16, 19). Some researchers hypothesized that a repetition of eccentric exercise makes the myofibrils, extracellular matrix, cytoskeleton, and cell membranes more resistant, providing a morphological mechanism for rapid adaptation (2, 6). However, this hypothesis was not directly tested in prior studies. Thus one purpose of the present work was to answer the question: does myofibrillar disruption reoccur after the repetition of an initial bout of eccentric exercise? If it does, then other than morphological factors must mediate the rapid-adaptation phenomenon.

It has been suggested that the rapid-adaptation process may in part be neurally mediated through a more complete activation of the motor unit pool associated with learning to perform the task (2, 7). The muscle activation hypothesis is favored because it is unlikely that hypertrophy of undamaged muscle fibers would occur in one session. Hence, if muscle forces are restored and myofibrillar disruption is still present, a more complete muscle activation could conceivably mediate force restoration (7). Thus the second purpose was to test the hypothesis that neural adaptation aids force recovery in the process of rapid adaptation.

	METHODS

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Subjects and Design

Table 1 illustrates the general study design that was similar to the study of Golden and Dudley (7), with the following differences. Based on the results of that study, we used only one experimental group (6 men and 6 women) and a control group (3 men and 3 women). Postexercise follow-ups were done on days 2, 4, and 7. We used a total of six criterion measures (Table 1) compared with strength as the only criterion measure in that study. In brief, the present study involved baseline testing for the criterion measures followed by the first exercise bout. After a 2-wk hiatus, the baseline testing, the exercise bout, and the follow-up testing were repeated. One experimental group was sufficient because concentric exercise is not associated with rapid adaptation (2) but it is associated with different neural mechanisms of compensating for fatigue (11, 25).

Every subject was evaluated for every criterion measure, except that the control group did not exercise and did not have muscle samples taken. In addition, as illustrated in Table 1, muscle samples were taken in a staircase fashion from subjects in the experimental group. Such design required only two samples from the same subject. Two days after the baseline biopsy, subjects were tested for muscle soreness, patellar reflex, blood sampling, isokinetic strength with electromyogram (EMG), and 1 repetition maximum (RM) strength. Three days after the first biopsy, the first exercise bout was done. Two weeks later, the entire first protocol was repeated.

Procedures

Isokinetic strength and EMG. Subjects underwent maximal effort isometric and isokinetic concentric and eccentric quadriceps strength testing of the left leg on a Kin-Com dynamometer (500H, Chattecx, Chattanooga, TN). Subjects performed 3 min of stretching of the leg muscles and were seated on the dynamometer's seat. The hip angle was ~1.57 rads, and arms were folded in front of the chest. Shoulder straps, a lap belt, a knee strap, and an ankle cuff were used to limit extraneous movements. The center of the knee joint was aligned with the center of the dynamometer's power shaft. The anatomical zero was set at a knee angle of 3.14 rads. The length of the lever arm and the mass of the leg were individually determined. Force was measured by the strain gauges embedded in the ankle cuff. Force corrected for the gravitational effects of leg mass was computed by the dynamometer's software. Familiarization with the dynamometer included two trials of 50, 75, and 90% intensity of isometric and dynamic contractions separated by 1 min of rest.

Maximal isometric force was measured at a knee angle of 2.36 rads. Subjects performed two maximal-effort 5-s trials with 1 min of rest between trials. Subjects also performed two maximal-effort concentric and eccentric quadriceps muscle contractions at 1.04 rad/s. To reduce fatigue, subjects did not exercise the hamstrings so the operator returned the lever arm to the starting position after each quadriceps action. There was 1 min of rest between contraction modes (isometric, concentric, eccentric). The order of contraction modes was randomized between subjects. The concentric-eccentric force-angle curves were digitized at 2.36 rads. The higher of the two trials was used in the statistical analysis.

Surface EMG activity was recorded from the vastus lateralis. The skin surface was cleaned with alcohol. One box electrode with a built-in preamplifier (Motion Control, Salt Lake City, UT), powered by 9-V batteries, was placed axially, taped, and ace-bandaged on the muscle belly. The electrode had a common mode rejection ratio of 105 dB, a bandwidth of 8 Hz-28 kHz, quiescent current of 0.12 mA, and a direct current input impedance of 1 M.

The force and the goniometer signals from the dynamometer's analog-to-digital board and the EMG signal were input to a digital adapter (model 4000A, Vetter, Rebersburg, PA) sampled at 80 MHz. The adapter was connected to a modified video recorder (JVC, HR-D86OU, model 500C, Vetter). Data from the videotape were transferred through a 12-bit analog-to-digital board (Data Translation, model 2801A, Marlboro, MA). The Myosoft software (Noraxon, Scottsdale, AZ) package was used to store and digitize the data.

Before digitization, the direct EMG signal was inspected, and if movement artifacts (4.2% of all tracings) were present, another representative segment of the data was digitized that was artifact free. When necessary, the tracing was adjusted for baseline shift. Peak root mean square (RMS) of the direct EMG data was obtained using a 20-ms window. Across all channels, the first marker was placed at peak force, and a second marker was placed 250 ms before the first marker. Within this 250-ms window, the highest RMS value was taken as peak EMG (µV), and the average over the 250-ms window was taken as an average EMG (µV · s). Eccentric and concentric EMG activities were normalized to isometric EMG to avoid errors due to electrode placement.

Patellar tendon tap reflex measurements were taken in a quiet room with the subjects keeping their eyes closed. Subjects sat on the seat of the Kin-Com dynamometer with hips at 1.74 rads and knees at 1.57 rads. A reflex hammer was suspended on a pendulum that was manually released from the horizontal. The hard rubber edge of the hammer contacted the patellar tendon at 90° angle. Six to eight practice trials were done to identify the position that resulted in maximal reflex amplitude. Data were collected for six test trials: three trials in the relaxed state and three trials when subjects pressed the palms together in front of the chest (Jendrassik maneuver) (5, 12). The relaxed and Jendrassik trials were systematically rotated among subjects. There was a 30-s pause between tendon percussions. Reflex response was recorded with a preamplified surface box electrode as described above. Maximal reflex amplitude was determined on the oscilloscope's calibrated grid background.

Muscle biopsy and electron microscopy. Percutaneous muscle biopsy was performed under local anesthesia (3 ml of 1% lidocaine). A ~50-mg sample was obtained with a 5-mm Bergström needle from the distal portion of left vastus lateralis by using the suction method. The repeat sample was taken at ~2-3 cm proximal to the first sample at the same depth of ~4-5 cm. The specimen was dissected from visible fat and connective tissue. The samples were fixed in 2.5% glutaraldehyde 0.1 M sodium phosphate buffer, pH 7.4, within 1-2 min of sampling. The samples were washed overnight in a 0.1 M sodium phosphate buffer, postfixed in 2% osmium tetroxide, dehydrated through graded ethanol series, and embedded in an Epon/Aradite mixture. Three tissue blocks were prepared from each biopsy sample. From each block, three semithin sections (1 µm) were cut on a Reichter Ultramicrotom (Reichter Optische Werke, Vienna, Austria) and prepared for examination in a Jeol 1200EX transmission electron microscope (Jeol USA, Peabody, MA).

Quantification of damage was done for three subjects at day 2 after bouts 1 and 2, respectively (Table 1). Three images of photo negatives from one randomly selected section of each block were captured with a Northan light box (Imaging Research, Ontario, Canada) system through a Sierra Scientific CCD camera. The images were digitized (Macintosh IICi, NIH Image 1.58 software) for total number of pixels and the number of pixels showing damage. Damage was quantified by averaging percent damage from each of the three pictures.

Blood sampling. Five-milliliter blood samples were drawn from an antecubital vein. Blood samples were analyzed for serum creatine kinase (CK) by using reagent kits that included controls and standards (see Ref. 9; Sigma Diagnostics, St. Louis, MO). All determinations were done in triplicate, and the averaged value was used in the statistical analysis.

Muscle soreness. Muscle soreness was evaluated on a subjective basis by using a questionnaire with a scale from 0 (not sore at all) to 10 (extremely sore). Subjects rated muscle soreness of the anterior, posterior, medial, and lateral aspects of the thigh. The criterion score was the average of the four sites.

Exercise bouts 1 and 2. Subjects performed 10 sets of 10 repetitions of eccentric quadriceps muscle actions by lowering weights that corresponded to ~80% of isotonic eccentric maximum. The weight was manually lifted by an operator in preparation for the next repetition. To minimize fatigue, there were 3 min of rest between sets. If a subject failed to perform 10 repetitions, the number of repetitions was reduced to 7 or, if the subject was unable to do 7 repetitions, some weight was removed to achieve 10 repetitions (7). For bout 2, each subject used the same weights and repetitions that were administered during bout 1.

Statistical Analyses

The experimental and control groups were not different in any measurements at baseline. The control group did not change significantly on the repeated measures compared with baseline. Thus the experimental group's data were expressed as a percentage of the mean of the control group, and the dependent variables were analyzed with an analysis of variance with repeated measures on time (10 levels), followed by Tukey's post hoc contrast. The level of significance was set at P < 0.05.

	RESULTS

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Maximal isotonic concentric and eccentric forces were 65 ± 7 and 81 ± 12 kg, respectively, in the experimental group and 63 ± 6 and 78 ± 10, respectively, in the control group at baseline. The time-by-mode interaction was not significant (F = 1.2, P = 0.87), but there was a significant time main effect (F = 22.7, P = 0.0001). Figure 1A shows that exercise bout 1 resulted in a similar decrease in eccentric and concentric isotonic forces immediately postexercise, an average of 11% of the control group (P > 0.05). Two days after bout 1, isotonic concentric force declined 58% and eccentric force declined 39% (P < 0.05). By day 7, isotonic strength returned to control levels. After bout 2, the greatest isotonic concentric or eccentric force decline was 3% of control (P > 0.05).

View larger version (20K): [in this window] [in a new window]   Fig. 1.   Changes in isotonic eccentric () and concentric () (A) and isokinetic eccentric (1.04 rad/s; ), isometric (), and isokinetic concentric (1.04 rad/s; ) strength following 2 bouts of eccentric exercise (B). BL, Baseline; IP, immediately postexercise; D, day. Data are expressed as %mean of control group in both panels. Bars are ±SD. * Significant change compared with BL.

Maximal isokinetic eccentric, isometric, and isokinetic concentric forces were 626 ± 26, 552 ± 23, and 441 ± 15 N, respectively, in the experimental group and 616 ± 20, 555 ± 13, and 445 ± 19 N, respectively, in the control group at baseline. There was no significant time-by-contraction mode interaction (F = 1.5, P = 0.239). Figure 1B shows that the average decline in isometric, isokinetic eccentric, and concentric knee extension strength at 1.04 rad/s averaged ~10% (P > 0.05) immediately after bout 1 and ~37% at day 2 (P < 0.05). By day 7, there was almost a complete recovery in these forces to baseline. After bout 2, the largest decrease was 4%. There were no significant differences in any of the variables at baseline before bouts 1 and 2.

Maximal patellar reflex amplitude with and without Jendrassik maneuver averaged 22.1 ± 4.9 and 11.6 ± 3.4 µV, respectively, in the experimental group and 21.4 ± 4.4 and 9.9 ± 3.0 µV, respectively, in the control group at baseline. There was a significant time-by-tap type (Jendrassik vs. non-Jendrassik) interaction (F = 11.2, P = 0.0001). Figure 2A shows that reflex amplitude increased more with Jendrassik maneuver (25%, P < 0.05) than without it (15%) immediately after bout 1 and remained significantly elevated under both conditions until day 7 (P < 0.05). Immediately and 2 days after bout 2, reflex amplitude with Jendrassik facilitation increased significantly more (P < 0.05) than without facilitation and remained significantly (P < 0.05) elevated above baseline through day 7.

View larger version (22K): [in this window] [in a new window]   Fig. 2.   A: patellar tendon reflex amplitude evoked with () and without () Jendrassik facilitation before and after 2 bouts of eccentric exercise. Data are expressed as %mean of control group. B: changes in vastus lateralis surface EMG during concentric () and eccentric () in experimental group and during concentric () and eccentric () in control group. Data are expressed as %isometric EMG. Vertical bars are ±SD. * Significant change compared with BL; ** significantly greater change than in other condition;  significantly greater than eccentric EMG.

Figure 2B shows the changes in surface EMG activity of isokinetic eccentric and concentric contractions at 1.04 rad/s normalized for isometric EMG activity. Peak EMG activity of the vastus lateralis was significantly greater by ~20% during concentric than during eccentric contractions at each time point (P < 0.05). After bout 1, EMG activity for concentric and eccentric contractions decreased significantly (P < 0.05) immediately after exercise, remained significantly depressed at days 2 and 4, and returned to baseline by day 7. After bout 2, there were no significant changes in EMG activity of the vastus lateralis.

Muscle soreness increased (on the scale from 1 to 10) in the experimental group from zero at baseline to 5.2 ± 1.7 at day 2 after bout 1, and it decreased to 1.3 ± 0.9 by day 7. After bout 2, the highest soreness rating was 2 in one subject. Serum CK levels were similar in the experimental and control groups and averaged 34.7 ± 11.0 and 42.9 ± 7.0 IU/l at baseline, respectively. CK levels increased to 45, 220, 164% (all P < 0.05), and 22% of control immediately and 2, 4, and 7 days after bout 1. After bout 2, CK levels did not change (range 31-42 IU/l).

Electron micrographs of the vastus lateralis revealed a normal pattern in all subjects at baseline. Figure 3 shows longitudinal sections at baseline (Fig. 3A), at day 2 (Fig. 3B), and at day 7 (Fig. 3C). Samples from all three subjects at day 2 after bout 1 showed substantial disorganization of the myofilaments, misalignment of the adjacent sarcomeres, widening of Z lines, and Z-line streaming (Fig. 3B). Longitudinal sections at day 7 after bout 1 revealed normal patterns in all three subjects (Fig. 3C).

View larger version (149K): [in this window] [in a new window]   View larger version (235K): [in this window] [in a new window]   Fig. 3.   Longitudinal sections of human vastus lateralis before (A) and 2 (B) and 7 days (C) after bout 1 of 10 sets of 10 repetitions of eccentric quadriceps exercise. Longitudinal sections are also shown before (D), 2 days (E and F, for 2 different subjects), and 7 days after (G) bout 2 of 10 sets of 10 repetitions of eccentric exercise done 2 wk after bout 1. Note in B the myofilament disorganization and extensive damage and in C the complete recovery. Magnification ×10,000 in all panels. Calibration bar in A is 500 nm and refers to all panels.

Two weeks later at baseline before bout 2 (Fig. 3D), a normal pattern was apparent, suggesting a complete recovery from bout 1. At day 2 after bout 2, two of three subjects revealed myofibrillar disruption and Z-line streaming (Fig. 3E). In one of three subjects at day 2 after bout 2, white infiltrations appeared mostly at the M line, suggesting perhaps regeneration, remodeling, or leftover damage (Fig. 3F). At day 7 after bout 2, longitudinal sections from all three subjects appeared normal (Fig. 3G).

At day 2 after bout 1, all three subjects revealed severe myofibrillar disruption so that 65 ± 12% of the pixels in the micrographs showed damage. At day 2 after bout 2, two of three subjects showed damage so that 23 ± 4% of the pixels were associated with damaged tissue.

	DISCUSSION

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The present results suggest that, when one allows for a complete recovery between repeated bouts of eccentric exercise, a rapid adaptation occurs, as observed in some prior studies (3, 7). This rapid adaptation was associated with minimal changes in CK levels, soreness, and muscle force (3). The mechanism of this rapid adaptation is unclear. The present study provided some new insights into this adaptive mechanism by showing that, despite an almost complete force recovery (Fig. 1), damage was still present (Fig. 3E) after the second bout of eccentric exercise in two of the three subjects for whom muscle biopsy samples were available.

Two days after one bout of eccentric or concentric exercise of the biceps muscle, a partial recovery of voluntary and electromyostimulation forces was reported, with muscle fiber disruption at the same level as seen immediately after exercise (6). The present study expands on these findings (6) and findings of other studies that investigated the rapid-adaptation phenomenon (1, 3, 13) by qualitatively examining muscle fiber morphology after a second bout of eccentric exercise. With only ~5% mean deficit in voluntary force compared with baseline, two of three subjects showed myofibrillar disruption at day 2 after bout 2 (Fig. 3E). The force deficit in these two subjects was +6 and +1% (actual increase). In the third subject, who showed no myofibrillar disruption, the force deficit was +3%. Subjects at this time reported virtually no soreness, and CK levels were similar to baseline, indicating no damage. Although CK has been used extensively as a marker for muscle damage (9), in this study, CK levels were normal with damage present. In addition, the present data contradict the findings of Mair et al. (18). These authors, using 70 eccentric contractions of the quadriceps, reported no significant elevation in myosin heavy chain levels within 1, 3, or 4 days after the second bout of exercise. Perhaps the extent of myofibrillar disruption was little in that study, and myosin heavy chain levels did not reflect the damage.

In view of recovery of force and CK levels after repeated eccentric exercise, an observation of myofibrillar disruption in two of three subjects is somewhat surprising. We cannot be absolutely certain that the myofibrillar disruption at day 2 after bout 2 (Fig. 3E) was not remnant damage caused by bout 1, because muscle regeneration or repair may occur in previously damaged fibers within up to 20 days (13). However, it is not likely that the damage after bout 2 was remnant from bout 1 because the longitudinal samples were completely normal at day 7 after bout 1 and, again, 2 wk later at baseline before bout 2. We thus interpret the less extensive focal damage at day 2 after bout 2 as de nouveau myofibrillar disruption. Perhaps the damage occurred to inherently weak muscle fibers unable to withstand the strain associated with bout 2 (2). Although such explanations have generally been applied to damage resulting from an initial eccentric exercise bout (2), damage may reoccur when one starts to exercise after a hiatus (6), even in trained subjects (23). It is also possible that the damage was associated with sarcomere length heterogeneity (14). Because sarcomeres are stretched in a nonuniform fashion (20), some sarcomeres may not be capable of resisting muscle lengthening beyond thick- and thin-filament overlap. Alternatively, the damage is part of myofibrillar reorganization to form new muscle fibers.

If damage is present in some subjects while forces are normal at day 2 after bout 2, some mechanism must compensate for the damage that would otherwise reduce the force-producing capacity of muscle. Some authors alluded to the possibility that during rapid adaptation the nature of compensation may be neural (2, 7). We addressed this issue by measuring surface EMG during maximal voluntary contractions and assessing afferent integrity by patellar tendon tap reflex. Muscle activation as measured with surface EMG of the vastus lateralis paralleled force loss after bout 1: as force decreased by 30-50%, so did EMG by 30-40% (Fig. 2B). After bout 2, EMG activity again paralleled force production: as force remained unchanged, so did EMG. Because in some subjects there were damaged muscle fibers and yet forces were normal, subjects may have learned to activate motor units normally not used during eccentric actions (7, 28).

In contrast to the decline in EMG during voluntary effort, immediately after bout 1 there was a ~25% increase in patellar tendon reflex amplitude, and this elevated reflex activity was maintained up to day 7. A sharp, up to ~35%, elevation was also noted after bout 2 (Fig. 2A). An elevation of reflex amplitude after exercise is not unusual (8, 11) and could be associated with the type of exercise or fatigue level. Most often such increase is interpreted as a compensation for fatigue (10, 15). A reduction in reflex amplitude has also been observed after isometric contractions (17). In a prior study (27), muscle fibers were found to be capable of conducting action potentials despite damage of the muscle fiber membranes. Thus myofibrillar disruption does not seem to be related to changes in reflex excitability (11). We are uncertain whether this reflex enhancement was caused in part by fatigue and metabolites. Some of the increase in reflex amplitude immediately after exercise could be related to fatigue. Although we did not measure any blood metabolites or muscle electrolytes other than CK, it is likely that a decrease in pH or an increase in lactate concentration would have decreased and not increased reflex amplitude because of sensitization of groups III and IV afferents (5). Interestingly, reflex amplitude increased more with than without Jendrassik facilitation after exercise (Fig. 2A). Reflex augmentation by the Jendrassik maneuver may occur (4) as proprioceptive afferent impulses from the contracting muscles are transmitted to supraspinal centers that, in turn, facilitate the reflex action, possibly through the reticulospinal pathway, although intersegmental facilitation is also possible (26). An increase in such reflex would reflect either a greater central facilitation associated with exercise as part of "neural adaptation" to exercise (22), or an enhanced afferent sensitivity, or both. Most likely then, an increase in the myotatic reflex loop sensitivity may be related to increased excitatory input from the muscle spindles (facilitation) due to repetitive stretching during exercise and/or augmented supraspinal influence (11).

There are several factors that limit the conclusions of the present work. One may question the adequacy of a single-muscle biopsy to assess exercise-induced damage in light of data suggesting that damage may occur because of the biopsy itself (23). However, in the present work, the second sample was taken more than an inch away from the first sample, minimizing the chance of observing damage remnant from the first biopsy. In addition, the damage after both exercise bouts contained Z-line streaming and myofibrillar disruption (Fig. 3), which are indicators of damage caused by exercise (13, 16, 23, 24), not by a biopsy (23). Another drawback of the present work is that the extent of damage to the whole muscle was not determined from the biopsy samples. Only the extent of myofibrillar disruption within specimens was assessed as the number of pixels showing disruption in relation to the total number of pixels, a conceptually similar method used in prior studies (6, 23). Estimation of the extent of damage in the entire muscle would not have been feasible even by repeated biopsies of the same muscle. To this aim, radiographic evidence will have to be collected, as it was done previously (13). Another limitation was the small sample size for the morphological data. Two of three subjects showed myofibrillar disruption at day 2 after bout 2, suggesting individual variability in the responses to eccentric exercise. Because we had no biopsy samples from the remaining subjects, it is not possible to tell whether damage was present or absent in those subjects. However, because force recovered to normal levels in all subjects, the only conclusion that can be drawn is that force recovery after a second bout of exercise is independent of cell disruption. Clearly, sample size will have to be increased in future studies to assess the extent of individual variability in the morphological responses to eccentric exercise.

In summary, results of this study suggest that a bout of eccentric exercise with the quadriceps muscle causes myofibrillar disruption, enhanced monosynaptic reflex activity, and a reduction in muscle force and EMG activity. When the same bout is repeated after a full recovery, myofibrillar disruptionat least in some subjectsdoes reappear without neuromuscular performance being compromised. Thus the results suggest that the rapid force recovery following eccentric exercise is mediated, at least in part, by neural factors and that this recovery may occur independently of cell disruption.