sublethal muscle fibre injuries after high-tension anaerobic exercise

Eur J Appl Physiol (1988) 57:360--368 European Journal of

p/ plied hysiology

and Occupational Physiology �9 Springer-Verlag 1988

Sublethal muscle fibre injuries after high-tension anaerobic exercise*

Jan Frid~n, Jan Seger, and Bj6rn Ekblom

Departments of Anatomy and Hand and Reconstructive Surgery, University of Umea, Sweden and the Department of Physiology III, Karolinska Institute, Stockholm, Sweden

Summary. The vastus lateralis muscles of eleven male elite sprinters (17--28 years) were investigated in order to examine the impact of high tension anaerobic muscular work on muscle fibre fine structure. In an attempt to reproduce the training regimen six subjects ran 20 repetitions of 25 s on a treadmill with 2 rain 35 s in between, at a speed corresponding to 86% of their personal best 200 m time. PAS-stained sections of biopsies taken approximately 2 h after training generally indicated glycogen depletion in type 1 and type 2B fibres. At the light microscopic level, no signs of inflammation or fibre rupture were observed. However, at the ultrastructural level, frequent abnormalities of the contractile material and the cytoplasmic organelles were detected. Z-band streaming, autophagic vacuoles and abnormal mitochondria were the most conspicuous observa- tions. Control specimens from sprinters who did not perform the acute exercise routine also displayed structural deviations, although to a lesser degree. It is hypothesized that during sprint training the leg musculature is put under great mechanical and metabolic stress which causes the degenerative response reported here.

Key words: Humans -- Skeletal muscles -- An- aerobic exercise -- H i s t o c y t o c h e m i s t r y - Ultra- structure -- Muscle cell injury

* This study was supported by grants from the Swedish So- ciety of Medical Sciences, the Tore Nilsson Foundation for Medical Research, the Coca Cola Export Company, Sweden and the Swedish Sports Research Council

of/print requests to: J. Frid~n, Department of Anatomy, Uni- versity of Ume~, S-901 87 Ume&, Sweden

Introduction

Eccentric muscle force development is character- ized by elongation of the muscle during contraction (Asmussen 1952; Newham et al. 1982; Arm- strong et al. 1983; Frid6n et al. 1983). The tension developed can become much higher with eccentric than with concentric contractions (Abbott et al. 1952; Asmussen 1952; Abbott and Bigland 1953). Since eccentric exercise demonstrates lower motor unit activity than concentric exercise for the same force developed, the tension per active unit will, consequently, be greater (Abbott et al. 1952; Asmussen 1952; Abbott and Bigland 1953). Strenous eccentric muscular exercise is known to induce muscle fibre membrane damage (Armstrong et al. 1983; Newham et al. 1983) as well as injuries within the muscle fibres (Frid~n et al. 1981; Newham et al. 1982; Armstrong et al. 1983; Frid6n et al. 1983). The intracellular structural deviations are expressed as distorsions of the contractile and cytoskeletal material (Frid6n 1984; Frid~n et al. 1984). The most conspicuous changes relate to the Z-disc, which seems to be the myofibrillar component most susceptible to chemical and mechanical influences (Meltzer et al. 1976; Cullen and Mastaglia 1982). Z-disc alterations observed after eccentric exercise include Z- disc streaming (zig-zag or wavy appearance) and smearing (extension of Z-disc material into the I- band) or even total Z-disc disruption (Frid~n 1984). Streaming and disintegration is a non-specific reaction and has been observed in a number of different pathological conditions (Engel 1967). Although animal studies on the effects of eccentric exercise reveal fibre necrosis (Armstrong et al. 1983), this is not found in humans (Frid6n et al. 1981, 1983). Morphological changes are inter- preted in two ways: The first interpretation states

J. Frid~n: Muscle fibre damage after anaerobic exercise 361

that the high tension developed during eccentric contractions leads to a primary rupturing of myofibrillar material (Frid~n et al. 1981; Newham et al. 1983), or to an intracellular structural disruption that stimulates activation or release of hydro- lases inherent to the fibres per se (Armstrong et al. 1983). The second interpretation involves a primary or secondary inflammatory response with degenerating muscle fibres and invading macrophages (Armstrong et al. 1983; Hikida et al. 1983; Hagberg 1984).

Eccentric contraction exercise protocols used by previous investigators have been unusual from a physiological point of view, employing eccentric exercises with no or only a minor component of concentric contractions. Therefore, we wanted to elucidate the effects of sprint training on muscle fibre fine structure. This type of dynamic exercise involves alternating, high-intensity eccentric and concentric contractions of leg muscles. Apart from the high production of lactic acid during sprint running, a significant vertical ground reaction force is produced by the legs when the foot hits the track (Nilsson et al. 1985). Thus we assume that sprint training places the muscle fibres under significant metabolic and mechanical stress which may be detected by electron microscopy. Also, a recent study on rats has shown that even after uphill running (involving primarily concentric contractions, cf. Magaria 1972) the soleus muscle is mechanically stressed (Armstrong et al. 1983).

The purpose of the present study, was to in- vestigate the specific adaptation of the fibre types to intense sprint training as seen at light microscopic and ultrastructural level. Different fibre types were distinguished by using enzyme histochemistry and the average image of the myofibrillar M-band was revealed by a specific photographic technique (Sj~str6m et al. 1982).

Material and methods

Subjects. Eleven male elite sprinters volunteered for the study. Six individuals (mean age 22 years, range 17--27 years) parti- cipated in the sprint training tests (test group), while the other five (mean age 24 years, range 21--28 years) constituted the controls. The average of their personal best time for 200 m was 23.1 sec (test group 22.8 s, reference group 23.3 s). Informed consent was obtained for the study, and was reviewed and ap- proved by the local committee on human experimentation.

Sprint training. Sprint running was performed using a motor driven treadmill. The speed was set at 86% of the sprinter's personal best time over 200 m. The subject ran 20 repetitions of 25 s each, including the time required for acceleration of the treadmill (average 4.6 sec). The rest between each repetition

was 2 rain 35 sec (start every third minute). Before training the subjects warmed-up as they would before a 200 m race. This protocol was designed to imitate the type of training the sprinters regularly performed.

Chronic exercise level. The subjects had trained mainly by sprint running for 2--3 h per day, 5--6 times a week all the year round for several years. Although the routines and intensity of training varied considerably over the year, the pro- grammes usually consisted of a warm up for about 30 min, strength training exercises, and brief, high-intensity running. Both test and control subjects avoided strenous exercise for one week prior to the test. The exercise test protocol was con- sidered much more strenous than the regular training.

Determination of blood lactate. Blood samples for lactate determination (Str6m 1949) were taken from the finger tip before the training session, after the 3rd, 6th, 10th, 15th and 20th repetition and also 1, 2, 3, 4, 5, 8 and 45 rain after the training had finished.

Muscle biopsy. Using local skin anaesthesia (1% Xylocain | open surgical biopsies were obtained from the right vastus lateralis approximately 2 hours after the last repetition. Control biopsies were taken from those subjects who did not perform the acute exercise. The biopsies were taken from a well-defined portion just beneath the fascia and 15 cm proximal to the lateral femoral condyle. The biopsy sample, 8--10 mm in length, and 4--5 mm in diameter, was excised together with the fascia. Biopsies were divided into two pieces, one of which was frozen and prepared for enzyme histochemistry, the other being embedded in plastic for light and electron microscopy.

Enzyme histochemistry. Each sample was sectioned transversely and orientated in O.C.T. embedding medium (Lab-Tek Products, Naperville, Illinois, USA) on a piece of paper and frozen in melting isopentane ( -159~ The samples were stored in a freezer ( - 8 0 ~ until sectioning. Serial transverse sections (10 lxm) were cut using a cryostat microtome at - 2 0 ~ and mounted on glass slides. The sections were stained with haematoxylin-eosin, treated for alkaline (pH 9.4) and acid (pH 4.6 and 4.2) stable myofibrillar adenosine tri- phosphatase (mATPase) (Dubowitz and Brooke 1973), and for NADH-tetrazolium reductase. Glycogen was visualized using the periodic acid-Schiff (PAS) reaction (Pearse 1980). Based on the mATPase properties of the fibres they were classified into type 1 (lightly stained at pH 9.4) and type 2 (heavily stained). At pH 4.6, lightly stained type 2 fibres were termed type 2A. At pH 4.2, weakly stained type 2 fibres, which were dark at pH 4.6, were termed type 2B (Brooke and Kaiser 1970).

Electron microscopy. The specimens were longitudinally oriented at their approximate rest length with needles on cork plates. Fixation was carried out overnight in ice-chilled glu- taraldehyde (2.5%) in an isotonic Tyrode buffer solution (pH 7.35). During rinsing, the middle portion of the biopsy, which was not mechanically damaged, was cut transversely into slices about 1 mm thick. One of these slices was cut into 8--10 pieces and post-fixed for two hours in 1% osmium tetroxide, dehydrated in a graded series of acetones and embedded in Vestopal. The blocks (4 per subject) were trimmed, and semithin (1 ~xm) and ultrathin ( - 6 0 rim) longitudinal sections were cut. The 1 ~xm thick sections, containing 15-20 fibres, were stained with toluidine blue or processed for the periodic-acid Schiff reaction until developed (2 to 4 h). The semithin sections were viewed under a Leitz-Dialux-20 light microscope. The ultrathin sections were contrasted with uranyl-acetate and

362

lead citrate and examined in an electron microscope (Philips 300).

Processin9 of average images of myofibrils. Conventional enlargement of a photomicrograph was employed with two different variations. First, our film negative was converted into a positive image. Second, a section of the enlargement was se- lected for smearing. This procedure consisted of projecting the enlarged image through a slit aperture which was moved along the photographic paper at a constant speed. The direction of this movement was parallel to the transverse striations of the myofibril. By employing this technique, the M-band pattern, i.e. number and density of M-bridges, as well as the Z-band widths, could be clearly visualized. Ultrastructural typing of abnormal fibres was always performed on a non-affected portion of the fibre. Further details of this method are given else- where (Sj6str6m et al. 1982).

Statistical method. The ~2 test was used for comparison of the occurrence of Z-band streaming between test and control subjects.

Results

Blood lactate concentration. The average value for the maximal blood lactate concentration through- out the exercise was 15 + 2 (+ SE) mMol- 1-1.

J. Frid+n: Muscle fibre damage after anaerobic exercise

Light microscopy

Gross morphology of specimens from control and test subjects. Cross-sections processed for enzyme histochemistry showed well-preserved fascicles with tightly packed, polygonal fibres of essen- tially equal size (Fig. 1). The fibre nuclei were localized peripherally, although occasionally some central nuclei were observed in samples from both groups. No focal abnormalities in individual fibres were noted. No inflammatory cells were observed. The fibre type distribution in the two groups are shown in Table 1. Type 2 fibres pre- dominated in 10 of 11 subjects studied.

PAS-stained specimens. While the PAS-stained sections from the controls displayed a homogene- ous intensity, 20% of the fibres in the PAS-treated sections from the test biopsies were lightly stained. The lightly stained fibres were identified as either type 1 or type 2B by serial sections (Fig. 1). Optical density of type 1 and type 2B fibres stained with PAS varied along a continuum, while

Fig. 1. Enzyme histochemically stained frozen cross-section of biopsy taken 2 h after sprint running, mATPase pH 4.2 (A), mAT- Pase pH 4.6 (B), NADH-TR (C), PAS (D). Different fibre types are labelled in (B). Bar= 100 l-tin. (• 160)

J. Frid6n: Muscle fibre damage after anaerobic exercise

Table 1. Fibre type proportions (mean_+ SD) in biopsies from the vastus lateralis of 11 male sprinters

Type 1 Type 2A Type 2B

Reference group 39.2 + 5.9 30.9 + 8.4 28.8 ___ 5.4 (n=5)

Test group 39.1 +9.9 37.0+8.9 23.6_+8.3 ( n = 6 )

2A fibres were always heavily and homogeneously stained. A similar difference in glycogen content between the fibre types was apparent in semithin survey cross sections and longitudinal sections of plastic-embedded specimens (Fig. 2). Control samples were homogeneously PAS- stained.

Electron microscopy

Controls. The contractile material was organized into well-defined, densely-packed myofibrils. Z- band streaming occurred in 6 of 92 fibres (6.5%) and was always focal (longitudinal dispersal <3 sarcomeres). Z-band changes were confined to the type 2 fibres. Mitochondria were occasionally ir- regularly shaped, but never displayed crystalloids. Components of the sarcoplasmic reticulum and of the cytoplasm were regularly shaped and inter- spersed between the myofibrils. Structural deviations were observed in all 5 subjects.

Test 9roup. In the specimens from all test subjects, morphological abnormalities were detected in 42

363

out of 118 fibres analyzed (=36%, p<0.001). Neither invading phagocytes nor regenerative fibres were observed. Disrupted sarcolemma was never seen, although contraction bands were occasionally found. The most distinctive changes of the contractile material were focal and extensive Z-band streaming (Fig. 3). Focal streaming was localized to 1, 2 or sometimes 3 sarcomeres. Ex- tensive streaming, which was more rare than focal streaming, extended over several sarcomeres and across many myofibrils. Streaming generally occurred in areas where few or no mitochondria were seen. Numerous polyribosomal clusters were observed close to the cell membrane. Autophagic response or Z-band streaming were found in both type 1 and type 2 fibres, although the majority of abnormalities (4 out of 5) was noted in fibres with narrow Z-bands and only three central M-bridges clearly visible, i.e. type 2B fibres (Fig. 4).

Autophagic vacuoles containing unidentified degradation products and osmiophilic bodies were frequently observed beneath the sarcolemma. Other perceptible changes were numerous multilamellar myeloid bodies (Fig. 5), lipofuscin granules, and bizarrely shaped mitochondria with paracrystalline inclusions (Fig. 6a), which occurred exclusively in the subsarcolemmal space. Subsarcolemmal mitochondria were frequently elongated, sometimes as long as 6 ~tm (Fig. 6b). The cristae were closely packed and parallel to each other and to the long axis of the mitochondria. In general, the density of the subsarcolemmal mitochondria was much greater than that of the intermyofibrillar mitochondria. Elongated mitochondria were frequently divided into two or three sections separated by a bulge of less density. Subsarcolemmal mitochondria were often associated with lamellar bodies (Fig. 6b).

Discussion

In this study, we report changes in muscle fibre contractile constituents as well as the cytoplasmic noncontractile components in association with sprint training.

Fig. 2. Longitudinal PAS-stained section from biopsy taken 2 h after exercise. Fibres with varying glycogen content can be observed. Bar = 50 gm. ( • 600)

Changes of the contractile apparatus

The most prominent reaction of the myofibrillar material was the Z-band irregularity. In rare in- stances, extensive smearing involving several myofibrils and sarcomeres were found, although the more common irregularities comprised Z-

364 J. Frid~n: Muscle fibre damage after anaerobic exercise

Fig. 3. Electron micrograph showing focus of Z-disc streaming extending over 4 sarcomeres and across several myofibrils. Bar= 1 ~m. (x 20000)

Fig. 4. Average image of electron micrograph of myofibril from type 1 (A) and type 2B (B) fibre. The different M-band pattern and Z-band widths are demonstrated. Note the different sarcomere lengths, reflected by the different widths of the I-bands. A = A-band, I = I-band, M = M-band, Z = Z- band. Vertical a r r o w s indicate M- bridges. ( x 78600)

J. Frid6n: Muscle fibre damage after anaerobic exercise 365

Fig. 5. Electron micrograph revealing frequent membranous bodies under the sarcolemma in a biopsy from sprint trained muscle. Arrows indicate clusters of ribosomes. Bar= 1 ~tm. (x 20000)

band widening and streaming in a small area. The Z-disc is thought to be the myofibrillar component most prone to pathological alterations (Cullen and Mastaglia 1982). Z-band streaming has even been reported in young, healthy individuals without prior exercise (Meltzer et al. 1976). The Z-disc constitutes the link which transfers tension between successive sarcomeres. We have previously suggested that the Z-disc is the weakest link in the myofibrillar contractile chain (Frid~n et al. 1981). Thus repeated stretching during contraction may induce disruption of the array of ac- tin filaments connected to the Z-disc.

Several histochemical studies show that the lysosomal system is activated, not only by invading macrophages, but also in association with exercise-induced sublethal fibre injuries (Vihko et al. 1978; Vihko et al. 1979; Salminen and Vihko 1984). Disruptions of myofibrillar material may expose the contractile components to hydrolysis

by proteases, or, perhaps, high tension work itself may activate the lysosomal system. Also, it has been shown that Ca2+-activated protease in the sarcoplasm of skeletal muscle preferentially de- grades the myofibrillar Z-band (Busch et al. 1972).

Serum lactate concentration during the acute exercise reached high levels. Presently, we cannot rule out lactic acidosis as a causative factor for the changes observed, although evidence for lactate-induced structural disturbances has not been presented. However, a recent animal study has shown considerable morphological abnormalities in the muscle after non-exhaustive submaximal exercise without increased blood lactate levels (Kuipers et al. 1983). The effect of concentric, anaerobic exercise (e.g. bicycling) on the morphological response of muscle cells has not yet been investigated. We favour the hypothesis that the significant Z-band streaming found here is the re-

366 J. Frid6n: Muscle fibre damage after anaerobic exercise

Fig. 6. Electron micrograph displaying numerous abnormally shaped mitochondria with dense crystalloids under the sarcolemma (A). Membranous whorls and deformed mitochondria beneath the sarcolemma (B). Bar= 1 ~xm. ( x 20000)

sult of local high mechanical stress and tearing, and may be in combination with high muscle lactate concentration. However, the ultrastructural alterations are much more discrete in this material than those observed after pure eccentric exercise (Frid6n et al. 1981, 1983, 1984; Armstrong et al. 1983; Frid6n 1984a, b).

Changes in cytoplasmic components

Although mitochondrial changes and autophagic response resembling those reported in response to ischaemia (Salminen and Vihko 1984) were observed, it is far-fetched to assume that repetitive occasions of insufficient blood flow are a causative factor for the morphological changes found among these highly trained athletes. No necrotic or regenerating fibres were observed. Apart from the autophagic vacuoles, the numerous abnormal

mitochondria beneath the sarcolemma were conspicuous. These mitochondria showed increased density, abnormal shape and frequent paracrystalline inclusions. Crystalloid-containing mitochondria have been described as an isolated find- ing in a variety of pathological conditions (for review, see Carpenter and Karpati 1984). It is reasonable to assume that the structurally abnormal mitochondria do not function normally (Car- penter and Karpati 1984). However, their sub- strate utilizations cannot be determined from their morphology. Polyribosome complexes were lo- cated beneath the sarcolemma or close to the areas with extensive streaming (arrows Fig. 5). Clusters of ribosomes reflect the fibres striving to repair injured material. The increased amount of morphological abnormalities in the test muscles as compared with those of the controls indicates that acute strenous exercise induces further le- sions in an already affected muscle.

J. Fridrn: Muscle fibre damage after anaerobic exercise 367

Eccentric vs. concentric contractions

During the normal stride cycle in level locomotion, antigravity muscles such as the soleus and quadriceps are put under considerable mechanical stress when the active tension is used to decel- erate the center of mass following foot placement (Walmsley et al. 1978). Running, especially sprint- ing, accentuates this effect (Armstrong et al. 1983; Nilsson et al. 1985). This submaximal "pre-land- ing" contraction of the muscles involved guaran- tees an efficient braking of the movement by op- timizing the energy absorbed as the muscles lengthen (Dyhre-Poulsen and Mosfeldt Laursen 1984; Nilsson et al. 1985). The tension development when performing the eccentric contractions also increases if the level of running is changed to a degree of decline (eccentric biased) or if the running speed is increased. It is reasonable to ex- pect that muscle fine structure changes are more closely related to peak tension developed in the muscles during the stride cycle than to the average total force (Newham et al. 1982, 1983; Arm- strong et al. 1983). Higher forces per active cross- sectional area of the muscle are probably produced during the deceleration (braking) phase of running than during acceleration.

Structural deviations in relation to fibre type

The majority of the fine structural alterations observed occurred in the ultrastructurally-defined type 2B fibres. This observation is made with a high degree of certainty since the M- and Z-band images of each abnormal fibre were strongly enlarged and processed using a specific photographic technique (Sjrstrrm et al. 1982). Briefly, this technique enables the observer to obtain a de- tailed average image of the M-bridge pattern across myofibrils. This pattern has been shown to provide an efficient tool for discriminating between subtypes of type 2 fibres, although slight differences both between individuals and different muscles seem to occur.

The PAS-staining pattern indicates that the type 2B fibres have been utilized to a certain ex- tent, although the majority of the type 2B fibres were not glycogen depleted. Type 2B fibres are known to be involved in force generation when fast movements are needed (Astrand and Rodahl 1977). Several studies have demonstrated that fibres with comparatively few mitochondria and rich in sarcoplasmic reticulum, i.e. corresponding to the 2B fibres, show the narrowest Z-bands

(Payne et al. 1975; Prince et al. 1981; Eisenberg 1983). In mechanical terms, this could indicate that the Z-discs of the type 2B fibres are the weakest, which may explain the possible tearing during repetitive high tension exercise.

In conclusion, this study demonstrates that human muscle subjected to intense sprint training display several morphological changes. We hypo- thesize that the alterations observed here repre- sent both the acute stress-related disruptions of contractile proteins and the effects of repetitive high-tension exercise over a longer period of time (several weeks).

Acknowledgements. We wish to express our thanks to Dr Rich- ard L. Lieber, Department of Orthopedic Research, University of California, San Diego, USA, for valuable criticism of the manuscript.

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Accepted August 13, 1987

sublethal muscle fibre injuries after high-tension anaerobic exercise

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