Effects of exercise on plasma myosin heavy chain fragments and MRI of skeletal muscle JOHANNES MAIR, ARNOLD KOLLER, ERIKA ARTNER-DWORZAK, CHRISTIAN HAID, KLAUS WICKE, WERNER JUDMAIER, AND BERND PUSCHENDORF Departments of Medical Chemistry and Biochemistry, Sportsmedicine, Magnetic Resonance Imaging and Spectroscopy, and Orthopedics, University of Innsbruck Medical School, A-6020 Innsbruck, Austria MAIR,JOHANNES,ARNOLDKOLLER,ERIKAARTNER-DWORZAK, CHRISTIAN HAID, KLAUS WICKE, WERNERJUDMAIER, AND BERNDPUSCHENDORF. Effects of exercise on plasma myosin heavy chain fragments and MRI of skeletal muscle. J. Appl. Physiol. 72(2): 656-663,1992.-The effects of a single seriesof high-force eccentric contractions involving the quadriceps musclegroup (single leg) on plasma concentrations of muscle proteins were examined asa function of time, in the context of measurementsof torque production and magnetic resonance imaging (MRI) of the involved musclegroups. Plasma concentrations of slow-twitch skeletal (cardiac ,&type) myosin heavy chain (MHC) fragments, myoglobin, creatine kinase (CK), and cardiac troponin T were measured in blood samples of six healthy male volunteers before and 2 h after 70 eccentric contractions of the quadriceps femoris muscle. Screenings were conducted 1, 2, 3, 6, 9, and 13 days later. To visualize muscle injury, MRI of the loaded and unloaded thighs was performed 3,6, and 9 days after the eccentric exercisebout. Force generation of the knee extensors was monitored on a dynamometer (Cybex II+) parallel to blood sampling. Exercise resulted in a biphasic myoglobin release profile, delayed CK and MHC peaks. IncreasedMHC fragment concentrations of slow skeletal musclemyosin occurred in late samplesof all participants, which indicated a degradation of slow skeletal musclemyosin. Becausecardiac troponin T was within the normal range in all samples,which excluded a protein releasefrom the heart (cardiac ,&type MHC), this finding provides evidence for an injury of slow-twitch skeletal muscle fibers in responseto eccentric contractions. Muscle action revealed delayed reversible increasesin MRI signal intensities on T,-weighted imagesof the loadedvastus intermedius and deepparts of the vastus lateralis. We attributed MRI signal changesdue to edemain part to slow skeletal musclefiber injury. Muscle sorenessand the decrements in muscletorque appear to occur before muscle protein peak concentrations and maximum MRI signal intensity differences between the exercised leg and the control. exercise-inducedmuscleinjury; creatine kinase;myoglobin; cardiac troponin T; magnetic resonanceimaging
ALTHOUGH THE ADAPTABILITY ofthe
skeletalmuscleis considerable, there are certain limits. When skeletal muscle is exposed to loads to which it is not accustomed, muscle damage may occur. Muscle pain and tenderness after vigorous physical activity, especially contractions that involve stretching of active muscle (eccentric exercise), are common in sports or occupational tasks (28, 31). These symptoms are strongest 24-48 h after exercise and disappear within a few days without any residual 656
0161-7567/92 $2.00 Copyright
0
dysfunction (31). Scarring does not take place after a single exercise insult (9, 31). The repair process of the muscle may even result in an improved resistance to subsequent damage (4). Despite the familiarity of these symptoms, the exact pathophysiology underlying exercise-induced muscle injury remains uncertain. Measurement of serum creatine kinase (CK) activity is a common method for determining muscle injury. Eccentric lengthening exercise causes a large delayed increase in CK activity. However, concentric shortening exercise results in a small or even no increase in plasma CK activity. Peak values then usually occur within the first 24 h (4). Clarkson and Tremblay (3) recently hypothesized that the loss of sarcolemmal integrity accompanied by a release of CK marks the final stage of muscle fiber necrosis. An exercise-induced release of CK (a predominantly cytoplasmic enzyme), however, can be due to either temporary muscle fiber damage accompanied by membrane leakage or final death of the muscle fiber. Although animal studies on the effect of eccentric exercise reveal fiber necrosis (1,16), histological examinations of loaded muscle tissue in humans are contradictory. Jones et al. (17) reported degenerating fibers, infiltration by mononuclear cells, and eventually signs of regeneration in late biopsies of the affected muscles. Stauber et al. (32) found extracellular matrix disruption and a few necrotic muscle fibers in, human muscle biopsies obtained 48 h after eccentric exercise of the elbow flexors. However, Friden et al. (10,ll) and Newham et al. (29) found only evidence of sublethal muscle fiber injuries (distortion of the contractile and cytoskeletal material) without signs of fiber degeneration after eccentric or high-tension anaerobic exercise. Although all studies indicate that eccentric exercise causes greater injury to the muscles, questions remain. The purpose of the present study was to investigate the effects of a single series of high-force eccentric contractions involving the quadriceps muscle group on plasma concentrations of muscle proteins as a function of time and in the context of measurements of torque production and magnetic resonance imaging (MRI) of the involved muscle groups. In view of histological evidence for potential muscle fiber degeneration after eccentric exercise, we tested plasma concentrations of myosin heavy chain (MHC) fragments. MHC is a structurally bound contractile protein. An increase in plasma
1992 the American
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MHC concentrations, therefore, is indicative of membrane leakage and degradation of the contractile apparatus. Moreover, exercise is known to produce changes in the amount and distribution of water in skeletal muscle (30). Late edema has been reported after muscular exercise (2). Owing to the high sensitivity of MRI to changes in water distribution and because of a previous report of MRI in delayed-onset muscle soreness (9), sequential MRI examinations of the loaded and unloaded thighs were performed on several different days after eccentric exercise was finished to visualize the exercise-induced muscle injury. MATERIALS
AND
METHODS
Subjects
Six healthy male volunteers (physical education teacher trainees) ranging in age from 21 to 25 yr were recruited from the Department of Sports (University of Innsbruck). Subjects had no physical limitations to exercise and were not involved in any unilateral leg training. The risks and benefits of the study were explained, and written informed consent was obtained from each participant. All subjects were instructed to refrain from unaccustomed exercise during the course of the study starting 48 h before the exercise session. In these subjects, muscle soreness and pain after exercise were assessed by questionnaire (8). On a scale of 1 (normal) to 10 (very sore) subjects rated the perceived soreness of the quadriceps muscle. Exercise Regimen
Exercise was conducted in a sitting body posture on an exercise rack specially designed to elicit the required eccentric action of the quadriceps femoris (14). All subjects were tested for maximal voluntary force generation of the investigated leg with the knee held at an angle of 100° (180’ corresponds to full extension of the knee). Subjects then had to hold their knees at an angle of 150”, when a special trigger mechanism suddenly released 110% of the maximal voluntarily generated force. Subjects were instructed to straighten their knee against the pressure of this weight. Given the arrangement, they could not help bending their knee, although they tried to resist. A pulley system allowed the researcher to bring the weight to the starting position without any loading concentric exercise of the investigated leg. After warming up, each subject performed a single bout of eccentric exercise using only one leg. The exercise bout consisted of seven sets of ten eccentric contractions of the quadriceps femoris muscle group. Each contraction lasted l-2 s, with 15 s of rest between contractions. The seven sets were each separated by 3 min of rest. The function of the knee extensors was monitored by measuring the ability to generate force (concentric muscle action) by use of a computer-interfaced dynamometer (Cybex II+, Ronkomkoma, NY) before the eccentric exercise, 2 h after exercise, and 1, 2,3, 6, and 9 days later. Standardized loading of the thigh muscles was performed at angular velocities of 30, 90, and 18O”/s with three repetitions each at 90 and 18O”/s and two repetitions at 3O”/s. Each isokinetic action was followed by 2 min of rest. The subjects performed volun-
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tary maximum knee extension from 90° flexion over a range of 90’ to full extension of the knee. Peak torques (in Nm) were recorded throughout the exercise regimen at each angular velocity. The highest value achieved in the repetitions was used as the criterion score. Magnetic
Resonance
Imaging
MRI was performed on a 1.5-T supraconducting unit (magnetom, Siemens, Erlangen, FRG) with a circular polarized receive-transmit coil. Both legs (exercised and control) were tested. In a prior pilot study, we did MRI after the eccentric exercise of the quadriceps femoris muscle group and in daily follow-ups. We did not find a visable increase of the signal intensity of the exercised thigh muscles until 2 days after the eccentric exercise bout (20). Because of these findings, the MRI results of a study of delayed-onset muscle soreness (9) and the design of our study, where in every participant only one leg was loaded and the other leg can be used as a control, we decided to do MRI of the loaded and unloaded leg 3, 6, and 9 days after eccentric exercise without preexercise imaging. Coronal T,-weighted images were done with a multislice spin echo sequence [repetition time (TR) = 650 ms, echo time (TE) = 15 ms], proton density and T,-weighted images in multislice spin echo sequence (TR = 2,400 ms, TE = 15/90 ms). In both sequences additional radio-frequency pulses were used to saturate the signal from inflowing blood. Slice thickness was 6 mm. The positioning of the participants was the same in all sequential MRI examinations. T, relaxation times were estimated within regions of interest (ROI) in the loaded and unloaded thigh muscles by use of an algorithmn of the MRI system computer. The T, value was calculated from an ROI drawn on a transverse plane where the most obvious visual changes were seen (Fig. 1). The ROI was placed within the vastus lateralis muscle near the septum to the vastus intermedius muscle. An identical ROI was placed in the other leg to obtain a reference value. The same procedure was done on two slices above and below the slice of the first measurement. The mean T, value of these five calculations was used. Laboratory
Analysis
Blood collection. Blood was collected in EDTA-coated tubes (Sarstedt, Ntimbrecht, FRG). Samples were withdrawn immediately before eccentric exercise, 2 h after exercise, and 1, 2, 3, 6, 9, and 13 days later. CK activity was assayed without delay. Blood samples for MHC, myoglobin, and cardiac troponin T (TnT) measurements were immediately centrifuged, and the plasma was subsequently frozen and stored at -2OOC until assayed. Myoglobin. Myoglobin (mol wt 17,800) is an oxygenbinding sarcoplasmic protein in striated muscle fibers. Myoglobin concentrations are higher in human slowtwich fibers than in fast-twitch fibers. Its half-life in the general circulation is -10 min (19). Myoglobin was determined by a commercially available radioimmunoassay (Byk-Sangtec, Dietzenbach, FRG). The upper limit of the reference interval is 80 pg/l. CK activity. CK (mol wt 88,000) is a key enzyme of muscular metabolism that exists predominantly as a solu-
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antibodies to slow-twitch skeletal muscle myosin and ,& type cardiac MHC were identical (22). By contrast, the antibodies do not significantly react with cardiac a-type MHC or with MHC of human fast-twitch skeletal muscle fibers or any human smooth muscle. Thus the assay recognizes MHC fragments of human ,&type and slowtwitch muscle MHC very well. The monoclonal antibodies of the assay recognize MHC subfragment 2 in the whole molecule and as proteolyzed fragments. The upper limit of the reference interval (upper cutoff value) of MHC in plasma is 300 pU/l. The detection limit of the assay is 10 pU/l; 1 pU/l corresponds to 1 fig/l (22). Statistics
Nonparametric methods were used throughout. Median, interquartile range, and percentiles were calculated to describe continuous variables. The Wilcoxon signed rank test, Mann-Whitney test, and Friedman test were used for between-group comparison. P < 0.05 was considered significant. FIG. 1. Coronal slice of exercised and resting leg of proband 1 obtained 6 days after a single series of high-force eccentric contractions involving quadriceps muscle group [1.5-T magnetom (Siemens): repetition time (TR) = 2,400 ms, echo time (TE) = SO ms, slice thickness = 6 mm]. Signal intensity was increased in distinct area of loaded thigh (vastus intermedius and deep part of vastus lateralis; longitudinal extension in mm). There was no evidence of signal intensity changes within unloaded thigh.
ble sarcoplasmic protein in muscle fibers. CK is found in all types of skeletal muscle fibers in similar concentrations. Its half-life in the general circulation is -15.5 h (21). CK activities were measured at 25°C by means of an N-acetylcysteine-activated optimized ultraviolet test obtained from Merck (Darmstadt, FRG). The upper limits of the reference interval of CK is 80 U/l for men. Cardiac TnT assay. TnT (mol wt 37,000) is a structurally bound protein existing in different isotypes in heart and skeletal muscle fibers. An enzyme-linked immunosorbent assay (Boehringer Mannheim, Mannheim, FRG) developed by Katus et al. (18) was used to detect circulating cardiac TnT in plasma. The upper limit of the reference interval of cardiac TnT in plasma is 0.5 pg/l. MHC. Myosin is a hexameric structurally bound contractile protein containing four light and two heavy chains (mol wt 230,000). MHC can be cleaved into its subfragments by enzymes. The rod portion can be further degraded to form light meromyosin and subfragment 2 (mol wt 51,000) (33). Concentrations of MHC fragments were measured by an immunoradiometric assay (ERIA Diagnostics Pasteur, Marnes la Coquette, France). This sandwich assay uses a pair of monoclonal antibodies primarily raised against two different epitopes on subfragment 2 in the rod of human ventricular P-type heavy meromyosin. The antibodies used have been described in detail elsewhere (22,23). Briefly, owing to the strong structural similarity of P-type cardiac MHC and MHC of slow-twitch skeletal muscle fibers (5, 35), both antibodies react strongly with human slow-twitch skeletal MHC. The affinities of the
RESULTS
Muscle Force Generation and Muscle Soreness
Evaluation of six subjects after a single bout of 70 eccentric muscle contractions of the knee extensors revealed increases in the perceived muscle soreness of the exercised quadriceps and a decrease in the measured ability to generate force on the dynamometer. Before exercise, all subjects reported no perceived muscle soreness of the quadriceps muscles. Soreness and pain assessed by questionnaire were maximal 24-48 h after eccentric exercise and declined on the subsequent days. A strong decrease of the force generation of the exercised leg was observed 2 h and 1 day after the eccentric exercise bout. By day 13 strength was back to baseline (Fig. 2). A significant decrease in the ability to generate force at all three angular velocities on the dynamometer was recorded after 1 day (Friedman test, p < 0.05). The maximal decrease compared with baseline measurements taken before the eccentric exercise bout was observed at 3O”ls (median -22%, range -5 to -34%; Fig. 2A). There was no residual functional impairment of the loaded thigh in any participant. Values on day 13 did not differ significantly from preexercise values. Muscle Protein Release Myoglobin. Myoglobin started to rise from 2 h after the eccentric exercise bout was finished and showed a median increase 4.3 times the upper cutoff value. Peak concentrations ranged from 110 to 693 pg/l and were significantly higher than values at the outset (p = 0.0064, Friedman test). Myoglobin showed a biphasic release profile, with a first usually smaller peak occurring on the 1st day after the eccentric exercise bout. Its second peak was observed on or about the 6th-9th day, often together with CK activity and MHC fragments peaks (Fig. 3). CK. Increased plasma CK activities were mostly found from 2 h after the eccentric exercise bout was finished. CK showed a median rise 18 times the upper cutoff value.
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PLASMA
MHC AND MRI OF MUSCLE
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EXERCISE
A 300 T -3
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250
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days after the onset of experiment FIG. 3. Myoglobin concentrations after a single series of high-force eccentric contractions involving quadriceps muscle group.
240 -
C
0
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days after the onset of experiment
B
-6
loo-
-5
150
-5
T
were significantly higher than values at the outset (p = 0.0001, Friedman test). Cardiac TnT. Cardiac TnT concentrations, by contrast, did not show a significant increase (p = 0.48, Friedman test). All concentrations measured were within the reference interval, which shows that the observed muscle protein increases were exclusively from skeletal muscle damage. All biochemical marker proteins were within the reference interval at the outset in all but one participant. Although proband 3 did not complain of muscle pain or tenderness and had refrained from unaccustomed exercise before the onset of the study, increased plasma CK activity (193 U/l) and MHC concentrations (1,475 ,&J/l) reflect some muscle damage to slow-twitch muscle fibers before eccentric exercise (Figs. 4 and 5). Magnetic Resonance Imaging of the Muscle
200 Ez_ 3 i= E 150
The exercise schedule led to a delayed increase in MRI signal intensity on T,-weighted images of the loaded quadriceps, which was most apparent in the vastus intermedius and in the deep parts of the vastus lateralis (Fig. 6). The longitudinal extension of the involved muscle signal intensity changes can be seen on a coronal slice (Fig. 1, proband 1, 6th day, distances in mm). We ob-
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days after the onset of experiment FIG. 2. Force generation on a dynamometer after a single series of high-force eccentric cohtractions involving quadriceps muscle group. Numbers (inset) represent the same probands in Figs. 2-5 and 7; values at 0 represent preexercise measurements. A: strength at 3O”/s; B: strength at 9O”/s; C: strength at BOO/s.
Peak activities ranged from 215 to 1,970 U/l, were significantly higher than values at the outset (p = 0.001, Friedman test), and occurred a few days after the eccentric exercise bout (Fig. 4). MHC. Increased plasma concentrations of MHC fragments of slow-twitch skeletal muscle fiber myosin could be detected in late samples of all subjects. MHC fragments started to rise from the 2nd day after the eccentric exercise bout and showed a median increase 6.8 times the upper cutoff value. MHC peaks occurred on or about the 6thA9th day, ranged from 637 to 7,818 @U/l (Fig. 5), and
500
0 -1 0
1 2
3
4
5
6 7
8
9 10 11 12 13 14
days after the onset of experiment
4. Creatine kinase (CK) activities after a single series of highforce eccentric contractions involving quadriceps muscle group. FIG.
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660
PLASMA
MHC AND MRI OF MUSCLE
8000
1 -2
7000
-3
6000
-4 -5 -6
5
5000
;
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3000 2000 1000 0 -1 0
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2
3
4
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days after the onset of experiment FIG. 5. Myosin heavy chain (MHC) fragment concentrations after a single series of high-force eccentric contractions involving quadriceps muscle group.
served no difference in the time courses of signal intensity changes in the proximal and distal regions of the affected vastus intermedius and lateralis. Overlapping pulsation artifacts caused by arteries could be eliminated by using additional radio-frequency pulses to saturate the signal from inflowing blood. No evidence for focal hematoma or fascial herniation of the loaded muscles was found. In all but one participant (proband 2) we
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found large differences between the loaded and unloaded thighs both in the MRI signal intensities on T,-weighted images and calculated T, relaxation times. T, relaxation time changes over time of the exercised and control leg are shown in Fig. 7. T, relaxation time peaks of the exercised leg were significantly higher than values on the 3rd day (p = 0.0431). This was not true for the control leg. We never found any visible signal intensity changes or MRI signal abnormalities of the control leg. Peak MRI signal intensities of the exercised quadriceps muscle were usually observed on the 6th day after eccentric exercise bout. Thus they follow the maximal decrease in the force generation, pain, and tenderness of the exercised muscles. The difference between the peak T, relaxation times of the exercised leg and T, relaxation times of the control leg measured on the same day were significant (p = 0.0277). The exercise-induced changes of MRI signal intensity were reversible. The loaded muscles could be visualized on T,-weighted images of the thighs, whereas active and inactive muscles did not differ in signal intensities on T,-weighted images. Thus, changes in the MRI signal intensity result primarily from increases in the amount and variations of free water protons (edema) in loaded skeletal muscle (12). Serial magnetic resonance cross-sectional images of the thigh muscles of one representative participant are given in Fig. 6, which
FIG. 6. Proband 1: transverse T,-weighted images of both thighs 3 (A), 6 (B), and 9 days (C) after a single series of high-force eccentric contractions involving quadriceps muscle group [1.5-T magnetom (Siemens): TR = 2,400 ms, TE = 90 ms, slice thickness = 6 mm]. T, relaxation time was increased in distinct area of loaded thigh (vastus intermedius and deep part of vastus lateralis, arrows). There was no evidence of signal intensity changes within unloaded thigh.
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PLASMA
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-1 -2 -3 -4 -5 -6
0
i
;
4
s
i
lb
Ii
days after the onset of experiment
4
10
days after the onset of experiment FIG. 7. Time courses of T, relaxation times of exercised (A) and resting leg (B) after a single series of high-force eccentric contractions involving quadriceps muscle group.
shows a marked increase of the signal intensity of the vastus intermedius and deep parts of the vastus lateralis (proband 1, maximum 6th day after the eccentric exercise bout). In all 6 cases there was considerable individual variation in the responses to physical activity of MHC, CK and myoglobin release, MRI signal intensity changes, T, relaxation time, and decrease in force generation. There was no obvious strong correlation among these markers. DISCUSSION
Exercise-related muscle damage represents a frequent problem in training and rehabilitation. Recently, there has been increased interest in how muscles resist externally applied forces and how eccentric muscle action leads to delayed-onset muscle soreness, which appears l-2 days after the cessation of the actual exercise bout. This study examined the effec ts of a single series of highforce eccentric cant ractions involving the quadriceps muscle group of a single leg on plasma concentrations of proteins indicative of muscle injury. These markers were examined as a function of time, in the context of torque production and MRI of the involved muscles. A new approach in the present study involved determinations of slow-twitch skeletal (P-type cardiac) MHC fragment concentrations. Because cardiac TnT concentrations were within the reference interval in all samples, increased MHC fragment concentrations that were
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found in late samples of all participants indicate injury of slow-twitch skeletal muscle fibers in response to eccentric exercise. MHC fragment plasma concentrations did not rise until the 2nd day after eccentric exercise bout. There was considerable variability in the severity of the responses in different subjects. On the contrary, in most participants pathologically increased myoglobin and CK values were already found 2 h after the eccentric exercise bout was finished. Because proteins are mainly transported from the interstitium of muscle into the general circulation via the lymph because of the extremely low capillary permeability (continuous capillaries) in muscle tissue (26)) differences observed between myoglobin and CK time courses are most likely due to their different half-lives in the general circulation (19, 21). Although exercise has been shown to raise the circulating levels of soluble cytoplasmic proteins (e.g., myoglobin, CK), to our knowledge, this study is the first to report increased plasma concentrations of MHC fragments after exercise in humans. MHC is a structurally bound contractile protein and part of the thick filaments in muscle fibers. The existence of soluble cytoplasmic MHC has been controversial. Accurate estimates of the soluble cytoplasmic precursor pools for the contractile proteins have not been published except for myosin light chains (-1% of total) (15). The two monoclonal antibodies of the assay recognize epitopes on subfragment 2 in the rod of cardiac ,& type or slow-twitch skeletal heavy meromyosin. This fragment is very resistant to proteolysis because of its central location in the rod of MHC/myosin molecule and the thick filament, respectively (22). The magnitude of increase in MHC fragments in plasma indicates degradation of myofilaments and leakage of the plasma membrane. MHC peak concentrations and time courses resemble those after myocardial infarction (24). The relatively long time before the appearance of MHC in plasma (22 days) compared with other proteins is one of the most characteristic features of the catabolism of MHC after myocardial infarction or eccentric exercise. This phenomenon supports the hypothesis that MHC is exclusively located in the contractile apparatus of muscle fibers. There are at least two different but linked mechanisms that mediate protein efflux and the destruction of the contractile apparatus (6). Therefore an increase of MHC in plasma, which requires enzymatic degradation of myosin and a leaky plasma membrane, indicates a larger damage to muscle fibers than the release of cytoplasmic proteins, where only membrane leakage is required. Thus the early rise in plasma myoglobin and CK during the first 48 h after the eccentric exercise bout may be due to membrane leakage of still viable muscle fibers, whereas the later MHC peaks reflect damage and subsequent degradation of the subcellular contractile apparatus. These considerations and the morphological results obtained in previous studies of human muscle biopsies after eccentric exercise (17,32) suggest that the increase in MHC fragments observed in plasma probably indicates irreversible cell damage of some slow-twitch skeletal muscle fibers of the quadriceps femoris muscle in response to eccentric exercise. N~~comment is possible on the extent of injury in fast-twitch fibers, which has been
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described in previous reports (10, 11, 17). It cannot be excluded that the contractile appartus and the plasma membrane after muscle fiber injury are repaired without cell death. However, the time course of plasma MHC fragment concentrations resembles that of histological signs of fiber degeneration described by Jones et al. (17) in muscle biopsies obtained after eccentric exercise. Skeletal muscle damage after eccentric muscle action results from mechanical rather than metabolic factors (28). Eccentric muscle action requires less energy than comparable concentric muscle action (31). The muscle, however, develops more tension than during concentric exercise. Presumably the mechanical strain is greater than the ability of the muscle to resist, which probably results in disruption of the contractile apparatus, some myofibers, and mechanical damage of the plasma membrane and sarcoplasmic reticulum (31). Calcium leakage from the latter could induce a cascade of events leading to damage to the myofilament apparatus and degeneration of muscle fibers (6). Muscle soreness and the decrements in mu .scle torque appear to occur before muscle protein peak concentrations and maximum MRI signal intensity differences between the exercised leg and the control. It is unclear what the large strength loss 2 h after the exercise exactly reflects. It somehow has to be a combination of injury and fatigue. On the other hand, muscle soreness is also out of phase with peak plasma concentrations of proteins indicative of muscle injury. This supports the hypothesis that pain, soreness, and muscle injury occur by different mechanisms. Pain and soreness in injured muscles after eccentric exercise may result from overuse of the connective tissue around the active muscle rather than from injury to the myofiber itself (31). The time course of increases in muscular MRI signal intensity after exercise appears similar to that of MHC and CK release into blood, which means that muscle protein release and MRI signal intensity changes are related to the same aspect of muscle damage and that MRI can be used to identify injured muscles from which proteins are being released into the circulation. Moreover, the uptake of the radioisotope technetium by muscle, which is indicative for increased permeability of the cell membrane, has also been reported to be highest -1 wk after eccentric exercise (17). Delayed reversible MRI signal intensity increases were strongest on or about the 6th9th day after the eccentric exercise bout. They were most apparent in the vastus intermedius and in the deep parts of the vastus lateralis on T,-weighted images. A few days after eccentric exercise, active and inactive muscles could be clearly distinguished in all but one subject. These findings confirm prior observations (9, 20). The large signal intensity difference between T,- and T,weighted images reveals changes in the amount and distribution of free water protons (edema) as the major underlying causes of increases in MRI signal intensities. MRI studies, however, detect total tissue water change and cannot distinguish intracellular from interstitial water. The proportion of slow-twitch fibers in the whole vastus muscle is ~50% (13, 25). The distribution of different fiber types varies with .in the vastus lateral .is, mainly as a function of depth, with a predominance of
AFTER
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fast-twitch fibers at the surface and slow-twitch fibers in the deeper regions of the muscle (25). The location of MRI signal intensity changes and the increases in plasma MHC fragments of slow-twitch skeletal myosin indicate that MRI findings in our subjects are at least partly due to edema of slow-twitch fiber injury. In addition, interstitial edema from extracellular matrix disruption (32) may also contribute to the increases in MRI signal intensity on T,-weighted images. Thus, muscle fiber injury, breakdown of connective tissue, release of muscle-specific proteins, and their slow removal from the extracellular matrix via the lymph probably provide the oncotic substances necessary for the localized edema observed. From the data presented, MRI is a useful tool to visualize exercise-induced skeletal muscle injury. MRI could help avoid sampling errors in obtaining muscle biopsies after exercise. The observed increases in MRI signal intensity of the loaded thigh were due to edema, resulting in part from slow-twitch muscle fiber injury. In all participants, however, there was no residual functional impairment of the loaded quadriceps. Whether such muscle fiber injury after exercise should be regarded merely as mechanical damage or whether it might be stimulus or part of an adaptational degenerating-regenerating repair mechanism of the kind observed in higher animals (27, 34) remains to be investigated. Address for reprint requests: B. Puschendorf, Institut fur Medizinische Chemie und Biochemie, Fritz-Preglstrasse 3, A-6020 Innsbruck, Austria. Received 11 February 1991; accepted in final form 4 September 1991. REFERENCES R. B., R. W. OGILVIE, AND J. A. SCHWANIZ. Eccentric exercise-induced injury to rat skeletal muscle. J. Appl. Physiol. 54:
1. ARMSTRONG,
80-93,1983. 2. BRENDSTEP, Med. Rehabil. 3. CLARKSON, COTTE, AND
P. Late edema after muscular exercise. Arch. Phys. 43: 401-405,
1962.
P. M., W. C. BYRNES, K. M. MCCORMICK, L. P. TURJ. S. WHITE. Muscle soreness and serum creatine kinase activity following isometric, eccentric and concentric exercise.
ht. J. Sports 4. CLARKSON,
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7: 152-155,
1986.
P. M., AND I. TREMBLAY. Exercise-induced muscle damage, repair, and adaptation in humans. J. Appl. Physiol. 65:
l-6,1988. 5. DIEDERICH, K. W., AND H. P. VOSBERG.
I. EISELE, T. RIED, T. JAENICKE, P. LICHTER, Isolation and characterization of the complete human beta-myosin heavy chain gene. Hum. Genet. 81: 214-220,
1989. 6. DUNCAN,
C. J. Role of calcium in triggering rapid ultrastructural damage in muscle: a study of chemically skinned fibres. J. Cell Sci.
87: 581-594,1987. 7. DUNCAN, C. J., AND
M. J. JACKSON. Different mechanisms mediate structural changes and intracellular enzyme efflux following damage to skeletal muscle. J. Cell Sci. 87: 183-188, 1987. 8. EBBELING, C. B., AND P. M. CLARKSON. Muscle adaptation prior to recovery following eccentric exercise. Eur. J. AppZ. Physiol. Occup. Physiol. 60: 26-31, 1990. 9. FLECKENSTEIN, J. L., P. T. WEATHERALL, R. W. PARKEY, J. A. PAYNE, AND R. M. PESHOCK. Sports-related muscle injuries: evaluation with MR imaging. RadioZogy 172: 793-798, 1989. 10. FRIDEN, J., J. SEGER, AND B. EKBLOM. Sublethal muscle fiber injuries after high-tension anaerobic exercise. Eur. J. AppZ. Physiol. Occup. Physiol. 57: 360-368, 1988. 11. FRIDEN, J., M. SJ~STR~M, AND B. EKBLOM. A morphological study of delayed muscle soreness. Ecperientia BaseZ 37: 506-507, 1981. 12. FULLERTON, G. D. Physiologic basis of relaxation. In: Magnetic
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13.
14.
15.
16.
17.
18.
19.
20.
21. 22.
23.
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AND
MRI
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Resonance Imaging, edited by D. D. Stark and W. G. Bradely, Jr. St. Louis, MO: Mosby, 1988, p. 47-55. GOLLNICK, P. D., B. SJ~DIN, J. KARLSSON, E. JANSSON, AND B. SALTIN. Human soleus muscle: a comparison of fiber composition and enzyme activities with other leg muscles. pfluegers Arch. 348: 247-255,1974. HAID, C., A. KOLLER, AND W. AMBACH. Exzentrische Belastung der Beinstreckmuskulatur unter biomechanisch exakt definierten Bedingungen (Abstract). Biomed. Technik 35, Suppl. 2: 197-198, 1990. HORVATH, G., AND E. GAETJENS. Immunochemical studies on the light chains from skeletal muscle myosin. Biochim. Biophys. Actcz 263: 779-793,1972. IRINTCHEV, A., AND A. WERNIG. Muscle damage and repair in voluntarily running mice: strain and muscle differences. Cell Tissue Res. 249: 509-521,1987. JONES, D. A., D. J. NEWHAM, J. M. ROUND, AND S. E. J. TOLFREE. Experimental human muscle damage: morphological changes in relation to other indices of damage. J. Physiol. Lord. 375: 435-448, 1986. KATUS, H. A., A. REMPPIS, S. LOOSER, K. HALLERMEIER, T. SCHEFFOLD, AND W. KUBLER. Enzyme linked immunoassay of cardiac troponin T for the detection of acute myocardial infarction in patients. J. Mol. Cell. CardioZ. 21: 1349-1353, 1989. KLOCKE, F. J., D. P. COPLEY, J. A. KRAWCZYK, AND M. REICHLIN. Rapid renal clearance of immunoreactive canine plasma myoglobin. Circulation 65: 1522-1528, 1982. KOLLER, A., S. FELBER, C. HAID, K. WICKE, AND E. RAAS. Exercise myopathy: magnetic resonance imaging (Abstract). Pfluegers Arch. 415, Suppl. 1: R112,1990. LANG, H. (Editor). Creatine Kinase Isoenzymes. New York: Springer, 1981, p. l-9. LARUE, C., C. CALZOLARI, J. 0. C. LEGER, J. LEGER, AND B. PAU. Immunoradiometric assay of myosin heavy chain fragments in human plasma. CZin. Chem. 37: 78-82, 1991. LEGER, J. 0. C., B. BOWAGNET, B. PAU, R. RONCUCCI, AND J. J. LEGER. Levels of ventricular myosin fragments in human sera after myocardial infarction, determined with monoclonal antibodies to myosin heavy chains. Eur. J. CZin. Invest. 15: 422-429,1985.
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24. LEGER, J. 0. C., C. LARUE, TAO MING, C. CALZARONI, P. GAUTIER, C. MOUTON, R. GROLLEAU, P. LOUISOT, P. PUECH, B. PEPERSTRAETE, M. STAROUKINE, M. TELERMAN, B. PAU, AND J. J. LEGER. Assay of serum cardiac myosin heavy chain fragments in patients with acute myocardial infarction: determination of infarct size and long-term follow-up. Am. Heart J. 120: 781-790, 1990. 25. LEXELL, J., K. HENRIKSSON-LARSEN, AND M. SJOSTROM. Distribution of different fibre types in human skeletal muscle. Actu PhysioZ. Scud. 117: 115-122, 1983. 26. LINDENA, J., W. K~PPNER, AND I. TRAUTSCHOLD. Enzyme activities in thoracic duct lymph and plasma of anaesthetized and conscious resting and exercising dogs. Eur. J. AppZ. Physiol. Occup. Physiol. 52: 188-195, 1984. 27. MAIER, A., B. GAMBKE, AND D. PETTE. Degeneration-regeneration as a mechanism contributing to the fast to slow conversion of chronically stimulated fast-twitch rabbit muscle. Cell Tissue Res. 244: 635-643,1986. 28. NEWHAM, D. J. The consequences of eccentric contractions and their relationship to delayed onset of muscle pain. Eur. J. AppZ. Physiol. Occup. Physiol. 57: 353-359, 1988. 29. NEWHAM, D. J., G. MCPHAIL, K. R. MILLS, AND R. H. T. EDWARDS. Ultrastructural changes after concentric and eccentric contractions of human muscle. J. Neural. Sci. 61: 109-122, 1983. 30. SJOGAARD, G., AND B. SALTIN. Extraand intracellular water spaces in muscles of man at rest and with dynamic exercise. Am. J. PhysioZ. 243 (Regulatory Integrative Camp. Physiol. 12): R271R280,1982. 31. STAUBER, W. T. Eccentric action of muscles: physiology, injury and adaptation. Exercise Sport Sci. Rev. 17: 157-185, 1989. 32. STAUBER, W. T., P. M. CLARKSON, V. K. FRITZ, AND W. J. EVANS. Extracellular matrix disruption and pain after eccentric muscle action. J. AppZ. Physiol. 69: 868-874, 1990. 33. WARRICK, H. M., AND J. A. SPUDICH. Myosin structure and function in cell motility. Annu. Rev. Cell BioZ. 3: 379-421, 1987. 34. WERNIG, A., A. IRINTCHEV, AND P. WEISSHAUPT. Muscle injury, cross-sectional area and fibre type distribution in mouse soleus after intermittent running. J. Physiol. Lord. 428: 639-652, 1990. 35. YAMAUCHI-TAKIHARA, K., M. J. SOLE, J. LIEW, D. ING, AND C.-C. LIEW. Characterization of human cardiac myosin heavy chain genes. Proc. NatZ. Acad. Sci. USA 86: 3504-3508, 1989.
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ALTHOUGH THE ADAPTABILITY ofthe
skeletalmuscleis considerable, there are certain limits. When skeletal muscle is exposed to loads to which it is not accustomed, muscle damage may occur. Muscle pain and tenderness after vigorous physical activity, especially contractions that involve stretching of active muscle (eccentric exercise), are common in sports or occupational tasks (28, 31). These symptoms are strongest 24-48 h after exercise and disappear within a few days without any residual 656
0161-7567/92 $2.00 Copyright
0
dysfunction (31). Scarring does not take place after a single exercise insult (9, 31). The repair process of the muscle may even result in an improved resistance to subsequent damage (4). Despite the familiarity of these symptoms, the exact pathophysiology underlying exercise-induced muscle injury remains uncertain. Measurement of serum creatine kinase (CK) activity is a common method for determining muscle injury. Eccentric lengthening exercise causes a large delayed increase in CK activity. However, concentric shortening exercise results in a small or even no increase in plasma CK activity. Peak values then usually occur within the first 24 h (4). Clarkson and Tremblay (3) recently hypothesized that the loss of sarcolemmal integrity accompanied by a release of CK marks the final stage of muscle fiber necrosis. An exercise-induced release of CK (a predominantly cytoplasmic enzyme), however, can be due to either temporary muscle fiber damage accompanied by membrane leakage or final death of the muscle fiber. Although animal studies on the effect of eccentric exercise reveal fiber necrosis (1,16), histological examinations of loaded muscle tissue in humans are contradictory. Jones et al. (17) reported degenerating fibers, infiltration by mononuclear cells, and eventually signs of regeneration in late biopsies of the affected muscles. Stauber et al. (32) found extracellular matrix disruption and a few necrotic muscle fibers in, human muscle biopsies obtained 48 h after eccentric exercise of the elbow flexors. However, Friden et al. (10,ll) and Newham et al. (29) found only evidence of sublethal muscle fiber injuries (distortion of the contractile and cytoskeletal material) without signs of fiber degeneration after eccentric or high-tension anaerobic exercise. Although all studies indicate that eccentric exercise causes greater injury to the muscles, questions remain. The purpose of the present study was to investigate the effects of a single series of high-force eccentric contractions involving the quadriceps muscle group on plasma concentrations of muscle proteins as a function of time and in the context of measurements of torque production and magnetic resonance imaging (MRI) of the involved muscle groups. In view of histological evidence for potential muscle fiber degeneration after eccentric exercise, we tested plasma concentrations of myosin heavy chain (MHC) fragments. MHC is a structurally bound contractile protein. An increase in plasma
1992 the American
Physiological
Society
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PLASMA
MHC
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MRI
OF
MHC concentrations, therefore, is indicative of membrane leakage and degradation of the contractile apparatus. Moreover, exercise is known to produce changes in the amount and distribution of water in skeletal muscle (30). Late edema has been reported after muscular exercise (2). Owing to the high sensitivity of MRI to changes in water distribution and because of a previous report of MRI in delayed-onset muscle soreness (9), sequential MRI examinations of the loaded and unloaded thighs were performed on several different days after eccentric exercise was finished to visualize the exercise-induced muscle injury. MATERIALS
AND
METHODS
Subjects
Six healthy male volunteers (physical education teacher trainees) ranging in age from 21 to 25 yr were recruited from the Department of Sports (University of Innsbruck). Subjects had no physical limitations to exercise and were not involved in any unilateral leg training. The risks and benefits of the study were explained, and written informed consent was obtained from each participant. All subjects were instructed to refrain from unaccustomed exercise during the course of the study starting 48 h before the exercise session. In these subjects, muscle soreness and pain after exercise were assessed by questionnaire (8). On a scale of 1 (normal) to 10 (very sore) subjects rated the perceived soreness of the quadriceps muscle. Exercise Regimen
Exercise was conducted in a sitting body posture on an exercise rack specially designed to elicit the required eccentric action of the quadriceps femoris (14). All subjects were tested for maximal voluntary force generation of the investigated leg with the knee held at an angle of 100° (180’ corresponds to full extension of the knee). Subjects then had to hold their knees at an angle of 150”, when a special trigger mechanism suddenly released 110% of the maximal voluntarily generated force. Subjects were instructed to straighten their knee against the pressure of this weight. Given the arrangement, they could not help bending their knee, although they tried to resist. A pulley system allowed the researcher to bring the weight to the starting position without any loading concentric exercise of the investigated leg. After warming up, each subject performed a single bout of eccentric exercise using only one leg. The exercise bout consisted of seven sets of ten eccentric contractions of the quadriceps femoris muscle group. Each contraction lasted l-2 s, with 15 s of rest between contractions. The seven sets were each separated by 3 min of rest. The function of the knee extensors was monitored by measuring the ability to generate force (concentric muscle action) by use of a computer-interfaced dynamometer (Cybex II+, Ronkomkoma, NY) before the eccentric exercise, 2 h after exercise, and 1, 2,3, 6, and 9 days later. Standardized loading of the thigh muscles was performed at angular velocities of 30, 90, and 18O”/s with three repetitions each at 90 and 18O”/s and two repetitions at 3O”/s. Each isokinetic action was followed by 2 min of rest. The subjects performed volun-
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tary maximum knee extension from 90° flexion over a range of 90’ to full extension of the knee. Peak torques (in Nm) were recorded throughout the exercise regimen at each angular velocity. The highest value achieved in the repetitions was used as the criterion score. Magnetic
Resonance
Imaging
MRI was performed on a 1.5-T supraconducting unit (magnetom, Siemens, Erlangen, FRG) with a circular polarized receive-transmit coil. Both legs (exercised and control) were tested. In a prior pilot study, we did MRI after the eccentric exercise of the quadriceps femoris muscle group and in daily follow-ups. We did not find a visable increase of the signal intensity of the exercised thigh muscles until 2 days after the eccentric exercise bout (20). Because of these findings, the MRI results of a study of delayed-onset muscle soreness (9) and the design of our study, where in every participant only one leg was loaded and the other leg can be used as a control, we decided to do MRI of the loaded and unloaded leg 3, 6, and 9 days after eccentric exercise without preexercise imaging. Coronal T,-weighted images were done with a multislice spin echo sequence [repetition time (TR) = 650 ms, echo time (TE) = 15 ms], proton density and T,-weighted images in multislice spin echo sequence (TR = 2,400 ms, TE = 15/90 ms). In both sequences additional radio-frequency pulses were used to saturate the signal from inflowing blood. Slice thickness was 6 mm. The positioning of the participants was the same in all sequential MRI examinations. T, relaxation times were estimated within regions of interest (ROI) in the loaded and unloaded thigh muscles by use of an algorithmn of the MRI system computer. The T, value was calculated from an ROI drawn on a transverse plane where the most obvious visual changes were seen (Fig. 1). The ROI was placed within the vastus lateralis muscle near the septum to the vastus intermedius muscle. An identical ROI was placed in the other leg to obtain a reference value. The same procedure was done on two slices above and below the slice of the first measurement. The mean T, value of these five calculations was used. Laboratory
Analysis
Blood collection. Blood was collected in EDTA-coated tubes (Sarstedt, Ntimbrecht, FRG). Samples were withdrawn immediately before eccentric exercise, 2 h after exercise, and 1, 2, 3, 6, 9, and 13 days later. CK activity was assayed without delay. Blood samples for MHC, myoglobin, and cardiac troponin T (TnT) measurements were immediately centrifuged, and the plasma was subsequently frozen and stored at -2OOC until assayed. Myoglobin. Myoglobin (mol wt 17,800) is an oxygenbinding sarcoplasmic protein in striated muscle fibers. Myoglobin concentrations are higher in human slowtwich fibers than in fast-twitch fibers. Its half-life in the general circulation is -10 min (19). Myoglobin was determined by a commercially available radioimmunoassay (Byk-Sangtec, Dietzenbach, FRG). The upper limit of the reference interval is 80 pg/l. CK activity. CK (mol wt 88,000) is a key enzyme of muscular metabolism that exists predominantly as a solu-
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658
PLASMA
MHC AND MRI OF MUSCLE
AFTER
EXERCISE
antibodies to slow-twitch skeletal muscle myosin and ,& type cardiac MHC were identical (22). By contrast, the antibodies do not significantly react with cardiac a-type MHC or with MHC of human fast-twitch skeletal muscle fibers or any human smooth muscle. Thus the assay recognizes MHC fragments of human ,&type and slowtwitch muscle MHC very well. The monoclonal antibodies of the assay recognize MHC subfragment 2 in the whole molecule and as proteolyzed fragments. The upper limit of the reference interval (upper cutoff value) of MHC in plasma is 300 pU/l. The detection limit of the assay is 10 pU/l; 1 pU/l corresponds to 1 fig/l (22). Statistics
Nonparametric methods were used throughout. Median, interquartile range, and percentiles were calculated to describe continuous variables. The Wilcoxon signed rank test, Mann-Whitney test, and Friedman test were used for between-group comparison. P < 0.05 was considered significant. FIG. 1. Coronal slice of exercised and resting leg of proband 1 obtained 6 days after a single series of high-force eccentric contractions involving quadriceps muscle group [1.5-T magnetom (Siemens): repetition time (TR) = 2,400 ms, echo time (TE) = SO ms, slice thickness = 6 mm]. Signal intensity was increased in distinct area of loaded thigh (vastus intermedius and deep part of vastus lateralis; longitudinal extension in mm). There was no evidence of signal intensity changes within unloaded thigh.
ble sarcoplasmic protein in muscle fibers. CK is found in all types of skeletal muscle fibers in similar concentrations. Its half-life in the general circulation is -15.5 h (21). CK activities were measured at 25°C by means of an N-acetylcysteine-activated optimized ultraviolet test obtained from Merck (Darmstadt, FRG). The upper limits of the reference interval of CK is 80 U/l for men. Cardiac TnT assay. TnT (mol wt 37,000) is a structurally bound protein existing in different isotypes in heart and skeletal muscle fibers. An enzyme-linked immunosorbent assay (Boehringer Mannheim, Mannheim, FRG) developed by Katus et al. (18) was used to detect circulating cardiac TnT in plasma. The upper limit of the reference interval of cardiac TnT in plasma is 0.5 pg/l. MHC. Myosin is a hexameric structurally bound contractile protein containing four light and two heavy chains (mol wt 230,000). MHC can be cleaved into its subfragments by enzymes. The rod portion can be further degraded to form light meromyosin and subfragment 2 (mol wt 51,000) (33). Concentrations of MHC fragments were measured by an immunoradiometric assay (ERIA Diagnostics Pasteur, Marnes la Coquette, France). This sandwich assay uses a pair of monoclonal antibodies primarily raised against two different epitopes on subfragment 2 in the rod of human ventricular P-type heavy meromyosin. The antibodies used have been described in detail elsewhere (22,23). Briefly, owing to the strong structural similarity of P-type cardiac MHC and MHC of slow-twitch skeletal muscle fibers (5, 35), both antibodies react strongly with human slow-twitch skeletal MHC. The affinities of the
RESULTS
Muscle Force Generation and Muscle Soreness
Evaluation of six subjects after a single bout of 70 eccentric muscle contractions of the knee extensors revealed increases in the perceived muscle soreness of the exercised quadriceps and a decrease in the measured ability to generate force on the dynamometer. Before exercise, all subjects reported no perceived muscle soreness of the quadriceps muscles. Soreness and pain assessed by questionnaire were maximal 24-48 h after eccentric exercise and declined on the subsequent days. A strong decrease of the force generation of the exercised leg was observed 2 h and 1 day after the eccentric exercise bout. By day 13 strength was back to baseline (Fig. 2). A significant decrease in the ability to generate force at all three angular velocities on the dynamometer was recorded after 1 day (Friedman test, p < 0.05). The maximal decrease compared with baseline measurements taken before the eccentric exercise bout was observed at 3O”ls (median -22%, range -5 to -34%; Fig. 2A). There was no residual functional impairment of the loaded thigh in any participant. Values on day 13 did not differ significantly from preexercise values. Muscle Protein Release Myoglobin. Myoglobin started to rise from 2 h after the eccentric exercise bout was finished and showed a median increase 4.3 times the upper cutoff value. Peak concentrations ranged from 110 to 693 pg/l and were significantly higher than values at the outset (p = 0.0064, Friedman test). Myoglobin showed a biphasic release profile, with a first usually smaller peak occurring on the 1st day after the eccentric exercise bout. Its second peak was observed on or about the 6th-9th day, often together with CK activity and MHC fragments peaks (Fig. 3). CK. Increased plasma CK activities were mostly found from 2 h after the eccentric exercise bout was finished. CK showed a median rise 18 times the upper cutoff value.
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PLASMA
MHC AND MRI OF MUSCLE
AFTER
659
EXERCISE
A 300 T -3
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250
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days after the onset of experiment FIG. 3. Myoglobin concentrations after a single series of high-force eccentric contractions involving quadriceps muscle group.
240 -
C
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days after the onset of experiment
B
-6
loo-
-5
150
-5
T
were significantly higher than values at the outset (p = 0.0001, Friedman test). Cardiac TnT. Cardiac TnT concentrations, by contrast, did not show a significant increase (p = 0.48, Friedman test). All concentrations measured were within the reference interval, which shows that the observed muscle protein increases were exclusively from skeletal muscle damage. All biochemical marker proteins were within the reference interval at the outset in all but one participant. Although proband 3 did not complain of muscle pain or tenderness and had refrained from unaccustomed exercise before the onset of the study, increased plasma CK activity (193 U/l) and MHC concentrations (1,475 ,&J/l) reflect some muscle damage to slow-twitch muscle fibers before eccentric exercise (Figs. 4 and 5). Magnetic Resonance Imaging of the Muscle
200 Ez_ 3 i= E 150
The exercise schedule led to a delayed increase in MRI signal intensity on T,-weighted images of the loaded quadriceps, which was most apparent in the vastus intermedius and in the deep parts of the vastus lateralis (Fig. 6). The longitudinal extension of the involved muscle signal intensity changes can be seen on a coronal slice (Fig. 1, proband 1, 6th day, distances in mm). We ob-
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days after the onset of experiment FIG. 2. Force generation on a dynamometer after a single series of high-force eccentric cohtractions involving quadriceps muscle group. Numbers (inset) represent the same probands in Figs. 2-5 and 7; values at 0 represent preexercise measurements. A: strength at 3O”/s; B: strength at 9O”/s; C: strength at BOO/s.
Peak activities ranged from 215 to 1,970 U/l, were significantly higher than values at the outset (p = 0.001, Friedman test), and occurred a few days after the eccentric exercise bout (Fig. 4). MHC. Increased plasma concentrations of MHC fragments of slow-twitch skeletal muscle fiber myosin could be detected in late samples of all subjects. MHC fragments started to rise from the 2nd day after the eccentric exercise bout and showed a median increase 6.8 times the upper cutoff value. MHC peaks occurred on or about the 6thA9th day, ranged from 637 to 7,818 @U/l (Fig. 5), and
500
0 -1 0
1 2
3
4
5
6 7
8
9 10 11 12 13 14
days after the onset of experiment
4. Creatine kinase (CK) activities after a single series of highforce eccentric contractions involving quadriceps muscle group. FIG.
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660
PLASMA
MHC AND MRI OF MUSCLE
8000
1 -2
7000
-3
6000
-4 -5 -6
5
5000
;
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3000 2000 1000 0 -1 0
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days after the onset of experiment FIG. 5. Myosin heavy chain (MHC) fragment concentrations after a single series of high-force eccentric contractions involving quadriceps muscle group.
served no difference in the time courses of signal intensity changes in the proximal and distal regions of the affected vastus intermedius and lateralis. Overlapping pulsation artifacts caused by arteries could be eliminated by using additional radio-frequency pulses to saturate the signal from inflowing blood. No evidence for focal hematoma or fascial herniation of the loaded muscles was found. In all but one participant (proband 2) we
AFTER
EXERCISE
found large differences between the loaded and unloaded thighs both in the MRI signal intensities on T,-weighted images and calculated T, relaxation times. T, relaxation time changes over time of the exercised and control leg are shown in Fig. 7. T, relaxation time peaks of the exercised leg were significantly higher than values on the 3rd day (p = 0.0431). This was not true for the control leg. We never found any visible signal intensity changes or MRI signal abnormalities of the control leg. Peak MRI signal intensities of the exercised quadriceps muscle were usually observed on the 6th day after eccentric exercise bout. Thus they follow the maximal decrease in the force generation, pain, and tenderness of the exercised muscles. The difference between the peak T, relaxation times of the exercised leg and T, relaxation times of the control leg measured on the same day were significant (p = 0.0277). The exercise-induced changes of MRI signal intensity were reversible. The loaded muscles could be visualized on T,-weighted images of the thighs, whereas active and inactive muscles did not differ in signal intensities on T,-weighted images. Thus, changes in the MRI signal intensity result primarily from increases in the amount and variations of free water protons (edema) in loaded skeletal muscle (12). Serial magnetic resonance cross-sectional images of the thigh muscles of one representative participant are given in Fig. 6, which
FIG. 6. Proband 1: transverse T,-weighted images of both thighs 3 (A), 6 (B), and 9 days (C) after a single series of high-force eccentric contractions involving quadriceps muscle group [1.5-T magnetom (Siemens): TR = 2,400 ms, TE = 90 ms, slice thickness = 6 mm]. T, relaxation time was increased in distinct area of loaded thigh (vastus intermedius and deep part of vastus lateralis, arrows). There was no evidence of signal intensity changes within unloaded thigh.
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PLASMA
MHC
AND
MRI
OF
-1 -2 -3 -4 -5 -6
0
i
;
4
s
i
lb
Ii
days after the onset of experiment
4
10
days after the onset of experiment FIG. 7. Time courses of T, relaxation times of exercised (A) and resting leg (B) after a single series of high-force eccentric contractions involving quadriceps muscle group.
shows a marked increase of the signal intensity of the vastus intermedius and deep parts of the vastus lateralis (proband 1, maximum 6th day after the eccentric exercise bout). In all 6 cases there was considerable individual variation in the responses to physical activity of MHC, CK and myoglobin release, MRI signal intensity changes, T, relaxation time, and decrease in force generation. There was no obvious strong correlation among these markers. DISCUSSION
Exercise-related muscle damage represents a frequent problem in training and rehabilitation. Recently, there has been increased interest in how muscles resist externally applied forces and how eccentric muscle action leads to delayed-onset muscle soreness, which appears l-2 days after the cessation of the actual exercise bout. This study examined the effec ts of a single series of highforce eccentric cant ractions involving the quadriceps muscle group of a single leg on plasma concentrations of proteins indicative of muscle injury. These markers were examined as a function of time, in the context of torque production and MRI of the involved muscles. A new approach in the present study involved determinations of slow-twitch skeletal (P-type cardiac) MHC fragment concentrations. Because cardiac TnT concentrations were within the reference interval in all samples, increased MHC fragment concentrations that were
MUSCLE
AFTER
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661
found in late samples of all participants indicate injury of slow-twitch skeletal muscle fibers in response to eccentric exercise. MHC fragment plasma concentrations did not rise until the 2nd day after eccentric exercise bout. There was considerable variability in the severity of the responses in different subjects. On the contrary, in most participants pathologically increased myoglobin and CK values were already found 2 h after the eccentric exercise bout was finished. Because proteins are mainly transported from the interstitium of muscle into the general circulation via the lymph because of the extremely low capillary permeability (continuous capillaries) in muscle tissue (26)) differences observed between myoglobin and CK time courses are most likely due to their different half-lives in the general circulation (19, 21). Although exercise has been shown to raise the circulating levels of soluble cytoplasmic proteins (e.g., myoglobin, CK), to our knowledge, this study is the first to report increased plasma concentrations of MHC fragments after exercise in humans. MHC is a structurally bound contractile protein and part of the thick filaments in muscle fibers. The existence of soluble cytoplasmic MHC has been controversial. Accurate estimates of the soluble cytoplasmic precursor pools for the contractile proteins have not been published except for myosin light chains (-1% of total) (15). The two monoclonal antibodies of the assay recognize epitopes on subfragment 2 in the rod of cardiac ,& type or slow-twitch skeletal heavy meromyosin. This fragment is very resistant to proteolysis because of its central location in the rod of MHC/myosin molecule and the thick filament, respectively (22). The magnitude of increase in MHC fragments in plasma indicates degradation of myofilaments and leakage of the plasma membrane. MHC peak concentrations and time courses resemble those after myocardial infarction (24). The relatively long time before the appearance of MHC in plasma (22 days) compared with other proteins is one of the most characteristic features of the catabolism of MHC after myocardial infarction or eccentric exercise. This phenomenon supports the hypothesis that MHC is exclusively located in the contractile apparatus of muscle fibers. There are at least two different but linked mechanisms that mediate protein efflux and the destruction of the contractile apparatus (6). Therefore an increase of MHC in plasma, which requires enzymatic degradation of myosin and a leaky plasma membrane, indicates a larger damage to muscle fibers than the release of cytoplasmic proteins, where only membrane leakage is required. Thus the early rise in plasma myoglobin and CK during the first 48 h after the eccentric exercise bout may be due to membrane leakage of still viable muscle fibers, whereas the later MHC peaks reflect damage and subsequent degradation of the subcellular contractile apparatus. These considerations and the morphological results obtained in previous studies of human muscle biopsies after eccentric exercise (17,32) suggest that the increase in MHC fragments observed in plasma probably indicates irreversible cell damage of some slow-twitch skeletal muscle fibers of the quadriceps femoris muscle in response to eccentric exercise. N~~comment is possible on the extent of injury in fast-twitch fibers, which has been
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662
PLASMA
MHC AND MRI OF MUSCLE
described in previous reports (10, 11, 17). It cannot be excluded that the contractile appartus and the plasma membrane after muscle fiber injury are repaired without cell death. However, the time course of plasma MHC fragment concentrations resembles that of histological signs of fiber degeneration described by Jones et al. (17) in muscle biopsies obtained after eccentric exercise. Skeletal muscle damage after eccentric muscle action results from mechanical rather than metabolic factors (28). Eccentric muscle action requires less energy than comparable concentric muscle action (31). The muscle, however, develops more tension than during concentric exercise. Presumably the mechanical strain is greater than the ability of the muscle to resist, which probably results in disruption of the contractile apparatus, some myofibers, and mechanical damage of the plasma membrane and sarcoplasmic reticulum (31). Calcium leakage from the latter could induce a cascade of events leading to damage to the myofilament apparatus and degeneration of muscle fibers (6). Muscle soreness and the decrements in mu .scle torque appear to occur before muscle protein peak concentrations and maximum MRI signal intensity differences between the exercised leg and the control. It is unclear what the large strength loss 2 h after the exercise exactly reflects. It somehow has to be a combination of injury and fatigue. On the other hand, muscle soreness is also out of phase with peak plasma concentrations of proteins indicative of muscle injury. This supports the hypothesis that pain, soreness, and muscle injury occur by different mechanisms. Pain and soreness in injured muscles after eccentric exercise may result from overuse of the connective tissue around the active muscle rather than from injury to the myofiber itself (31). The time course of increases in muscular MRI signal intensity after exercise appears similar to that of MHC and CK release into blood, which means that muscle protein release and MRI signal intensity changes are related to the same aspect of muscle damage and that MRI can be used to identify injured muscles from which proteins are being released into the circulation. Moreover, the uptake of the radioisotope technetium by muscle, which is indicative for increased permeability of the cell membrane, has also been reported to be highest -1 wk after eccentric exercise (17). Delayed reversible MRI signal intensity increases were strongest on or about the 6th9th day after the eccentric exercise bout. They were most apparent in the vastus intermedius and in the deep parts of the vastus lateralis on T,-weighted images. A few days after eccentric exercise, active and inactive muscles could be clearly distinguished in all but one subject. These findings confirm prior observations (9, 20). The large signal intensity difference between T,- and T,weighted images reveals changes in the amount and distribution of free water protons (edema) as the major underlying causes of increases in MRI signal intensities. MRI studies, however, detect total tissue water change and cannot distinguish intracellular from interstitial water. The proportion of slow-twitch fibers in the whole vastus muscle is ~50% (13, 25). The distribution of different fiber types varies with .in the vastus lateral .is, mainly as a function of depth, with a predominance of
AFTER
EXERCISE
fast-twitch fibers at the surface and slow-twitch fibers in the deeper regions of the muscle (25). The location of MRI signal intensity changes and the increases in plasma MHC fragments of slow-twitch skeletal myosin indicate that MRI findings in our subjects are at least partly due to edema of slow-twitch fiber injury. In addition, interstitial edema from extracellular matrix disruption (32) may also contribute to the increases in MRI signal intensity on T,-weighted images. Thus, muscle fiber injury, breakdown of connective tissue, release of muscle-specific proteins, and their slow removal from the extracellular matrix via the lymph probably provide the oncotic substances necessary for the localized edema observed. From the data presented, MRI is a useful tool to visualize exercise-induced skeletal muscle injury. MRI could help avoid sampling errors in obtaining muscle biopsies after exercise. The observed increases in MRI signal intensity of the loaded thigh were due to edema, resulting in part from slow-twitch muscle fiber injury. In all participants, however, there was no residual functional impairment of the loaded quadriceps. Whether such muscle fiber injury after exercise should be regarded merely as mechanical damage or whether it might be stimulus or part of an adaptational degenerating-regenerating repair mechanism of the kind observed in higher animals (27, 34) remains to be investigated. Address for reprint requests: B. Puschendorf, Institut fur Medizinische Chemie und Biochemie, Fritz-Preglstrasse 3, A-6020 Innsbruck, Austria. Received 11 February 1991; accepted in final form 4 September 1991. REFERENCES R. B., R. W. OGILVIE, AND J. A. SCHWANIZ. Eccentric exercise-induced injury to rat skeletal muscle. J. Appl. Physiol. 54:
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24. LEGER, J. 0. C., C. LARUE, TAO MING, C. CALZARONI, P. GAUTIER, C. MOUTON, R. GROLLEAU, P. LOUISOT, P. PUECH, B. PEPERSTRAETE, M. STAROUKINE, M. TELERMAN, B. PAU, AND J. J. LEGER. Assay of serum cardiac myosin heavy chain fragments in patients with acute myocardial infarction: determination of infarct size and long-term follow-up. Am. Heart J. 120: 781-790, 1990. 25. LEXELL, J., K. HENRIKSSON-LARSEN, AND M. SJOSTROM. Distribution of different fibre types in human skeletal muscle. Actu PhysioZ. Scud. 117: 115-122, 1983. 26. LINDENA, J., W. K~PPNER, AND I. TRAUTSCHOLD. Enzyme activities in thoracic duct lymph and plasma of anaesthetized and conscious resting and exercising dogs. Eur. J. AppZ. Physiol. Occup. Physiol. 52: 188-195, 1984. 27. MAIER, A., B. GAMBKE, AND D. PETTE. Degeneration-regeneration as a mechanism contributing to the fast to slow conversion of chronically stimulated fast-twitch rabbit muscle. Cell Tissue Res. 244: 635-643,1986. 28. NEWHAM, D. J. The consequences of eccentric contractions and their relationship to delayed onset of muscle pain. Eur. J. AppZ. Physiol. Occup. Physiol. 57: 353-359, 1988. 29. NEWHAM, D. J., G. MCPHAIL, K. R. MILLS, AND R. H. T. EDWARDS. Ultrastructural changes after concentric and eccentric contractions of human muscle. J. Neural. Sci. 61: 109-122, 1983. 30. SJOGAARD, G., AND B. SALTIN. Extraand intracellular water spaces in muscles of man at rest and with dynamic exercise. Am. J. PhysioZ. 243 (Regulatory Integrative Camp. Physiol. 12): R271R280,1982. 31. STAUBER, W. T. Eccentric action of muscles: physiology, injury and adaptation. Exercise Sport Sci. Rev. 17: 157-185, 1989. 32. STAUBER, W. T., P. M. CLARKSON, V. K. FRITZ, AND W. J. EVANS. Extracellular matrix disruption and pain after eccentric muscle action. J. AppZ. Physiol. 69: 868-874, 1990. 33. WARRICK, H. M., AND J. A. SPUDICH. Myosin structure and function in cell motility. Annu. Rev. Cell BioZ. 3: 379-421, 1987. 34. WERNIG, A., A. IRINTCHEV, AND P. WEISSHAUPT. Muscle injury, cross-sectional area and fibre type distribution in mouse soleus after intermittent running. J. Physiol. Lord. 428: 639-652, 1990. 35. YAMAUCHI-TAKIHARA, K., M. J. SOLE, J. LIEW, D. ING, AND C.-C. LIEW. Characterization of human cardiac myosin heavy chain genes. Proc. NatZ. Acad. Sci. USA 86: 3504-3508, 1989.
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