Cell Tissue Res (1984) 236:365 372 c e n a n d Tissue R e s e a l v . h �9 Springer-Verlag 1984

Changes in human skeletal muscle induced by long-term eccentric exercise Jan Frid~n Department of Anatomy, University of Umefi, Ume~, Sweden

Summary . The fine structure of muscle fibres from m. vastus lateralis of nine healthy males (mean age 26 years) was in- vestigated. Four individuals constituted non-exercised con- trols while five subjects participated in a two-months ec- centric muscular training program. Specimens from the controls showed a well-preserved, regular myofibrillar band pattern while changes in the myofibrillar architecture were constantly found in specimens taken after the training pro- gram. These changes consisted of Z-band alterations, Z- bands being out of register, extra sarcomeres, Z-band exten- sions and bisected Z-bands. Between the separated Z-band halves, thin and thick myofilaments as well as abundant glycogen particles and/or ribosomes, were observed. Type-2 (fast-twitch) fibres were predominantly affected. Contrary to the controls the trained individuals constantly showed a greater variation in sarcomere lengths in Type-2 fibres than in Type-1 fibres.

It is concluded that muscular work of high tension can induce fine-structural alterations. When repeated over a long period of time, extreme tension demands seem to initi- ate reorganization in the muscle fibres, predominantly in the, ultrastructurally defined, Type-2 fibres. This adapta- tion probably results in a better stretchability of the muscle fibres, reduces the risk for mechanical damage and brings about an optimal overlap between actin and myosin fila- ments.

Key words: Skeletal muscles - Myofibrils - Ultrastructure - Exertion - Man

Tension is an important factor influencing the longitudinal growth of skeletal muscle fibres (Williams and Goldspink 1971; Tabary et al. 1972; Barnett et al. 1980; Holly et al. 1980). Both the length of individual sarcomeres and the number of sarcomeres are influenced (Tabary et al. 1972; Tardieu et al. 1977). Elongation of sarcomeres is probably only a transient effect (Williams and Goldspink 1978; Bar- nett et al. 1980), whilst the increase in the number of sarco-

Send offprint requests to: Dr. Jan Frid6n, Dept. Anatomy, Univ. of Umeh, S-901 87 Ume~t, Sweden

Acknowledgements. This work was supported by grants from the Research Council of the Swedish Sports Federation, the Swedish Work Environment Fund, and the Swedish Medical Research Council

meres appears to be a long term change (Williams and Goldspink 1971 ; Tabary et al. 1972). Thus far experiments have used animals. Several of these have required denerva- tion of muscles so that they could be stretched by the action of their antagonists (Gutman et al. 1971; Goldberg et al. 1975). Other models have employed cast-immobilization of muscle in a lengthened position (Tabary et al. 1972; Wil- liams and Goldspink 1973; Williams and Goldspink 1978; Spector et al. 1982) or synergistic tenotomy (Schiffino and Hanzlikova 1970; Goldberg etal. 1975; Hofmann 1980). However, results of passive stretch induced growth of non- immobilized, non-denervated chicken wing muscles have been presented (Holly et al. 1980; Barnett et al. 1980). In addition, passive stretch appears to have a different effect on twitch and tonic muscles (Holly et al. 1980).

By employing autoradiography and other techniques, some workers have found that the new sarcomeres are formed at the end of the muscle fibre (e.g. Williams and Goldspink 1971). However, there are reports showing sar- comere generation taking place within the muscle fibre itself (Schmalbruch 1968).

The mechanism and the anatomical location of the length growth process is still obscure although several stu- dies indicate that the Z-disc is the origin of the sarcomero- genesis (Kelly 1968; Bishop and Cole 1969; Legato 1970; Jakubiec-Puka et al. 1982). If the Z-disc does serve as origin for formation of new sarcomeres and if the crucial stimulus for protein synthesis under physiological conditions is the tension put on the fibres, one might expect sustained stretching during simultaneous contraction to induce growth, possibly by lengthening of the muscle fibres. In parallel to this report morphological and strength perfor- mance data after eccentric training have been evaluated (Frid+n etal. 1983b). However, it soon became obvious that several fine structural alterations occurred in the mus- cles. Therefore, in this report results of a more detailed analysis of these findings are described and discussed.

Materials and methods

Subjects. Muscle biopsy was obtained from 9 healthy male physical education students (mean age 26 years, range 21 to 31 years). Five of these participated in an eccentric mus- cle training program involving the thigh muscles (see be- low). The remaining four individuals constituted the control grouP.

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All individuals were informed about the significance of the experiment and gave consent. The study was approved by the Ethical Committee of Ume~ University.

Training. Exercise was performed on a bicycle ergometer modified for use in eccentric work (Bonde-Petersen 1969). The subjects cycled 2-3 times per week for 8 weeks at a progressively increasing, individually adjusted load (Frid6n et al. 1983b). On every occasion they cycled until they be- came severely fatigued, i.e. 12-30 min.

Muscle biopsy. Under local anesthesia open surgical biop- sies were taken from the distal portion of m. vastus lateralis of the right leg from controls and exercised individuals. The controls did not perform any regular exercise during the week prior to the biopsy while the biopsies from the exercised individuals were taken three days after the last bout of eccentric exercise. Care was taken not to damage the muscle fibres mechanically throughout the procedure. Bundles of fibres, 8 to 10 mm in length and 4 to 5 mm in diameter, were excised together with the fascia.

Preparation for electron microscopy. The biopsy specimen was mounted at its approximate rest length with pins on a cork plate. Fixation was carried out in ice chilled 2.5 per cent glutaraldehyde in an isotonic Tyrode's buffer solu- tion overnight. While suspended in the buffer the mechani- cally undamaged middle protion of the biopsy was trans- versely cut into slices about 5 mm in length. From one of these, eight to ten tissue pieces were post-fixed for 2 h in one per cent osmiumtetroxide, dehydrated in graded se- ries of acetone and infiltrated with Vestopal. Two to four tissue blocks per specimen were chosen at random. Each of these was semithin sectioned (1 lam) for light microscopy and the sections were stained with toluidine blue. A selected area containing 15-25 mechanically undamaged and longi- tudinally oriented muscle fibres was trimmed and ultrathin sections (60 nm) were cut for electron microscopy. The sec- tions were contrasted with uranyl acetate and lead citrate. Further details including sampling procedure are given else- where (Sj6str6m et al. 1982b).

Determination of the relative frequency of Z-band streaming. Randomly taken micrographs (30-40 per individual, origi- nal magnification x 4400) were used to determine the oc- currence of Z-band changes. The areas to be photographed were chosen by the raster pattern scanning rules (Weibel and Bolender 1973). Alignment between the muscle fibre axis and the copper bars resulted sometimes in " random" selection of several areas from the same fibre. In such cases only the first area was photographed. Each micrograph with one or many areas of extended Z-band material (ex- tending over at least one fourth of the I-band width) was counted. Micrographs with Z-band extensions involving at least an entire sarcomere were considered separately.

Classification of fibres. The fibres were classified into Type 1 and Type 2 according to criteria defined elsewhere (Sj6s- tr6m et al. 1982a). Thus, fibres with M-bands showing all five M-bridges with equal density, were classified as Type 1 fibres. All other fibres were termed Type 2.

Measurement ofsarcomere length. Micrographs of low mag- nification (final magnification x 3000) were used for this

purpose. The number of sarcomeres along a 50 micrometer stretch of fibre was determined. Measurements were made at three different points on each fibre, avoiding areas lo- cated near the sarcolemma (< 10 micrometer). Ten fibres, from the same block, of Type 1 and Type 2, respectively, were analysed per individual. The coefficient of variation of sarcomere lengths for each individual and fibre type was calculated.

Statistical methods. The mean sarcomere lengths of fibres from untrained and trained muscles were compared by Mann-Whitney's non-parametric test.

Results

Control subjects. Longitudinal survey sections showed a reg- ular myofibrillar band pattern. However, Z-band streaming sometimes occurred, i.e. in 6 out of 162 (4%) randomly obtained micrographs. One per cent of the micrographs contained one or more regions with extensions of Z-band material involving at least an entire sarcomere (cf Frid6n et al. 1983b). Difference in the incidence of Z streaming between Type 1 and Type 2 fibres could not be reliably determined in the control material. Average sarcomere length was 2.92 ~tm in Type 1 and 2.89 lam (n.s.) in Type 2 fibres, respectively. The range of sarcomere lengths in both fibre types is shown in Table 1.

Trained subjects. Longitudinal survey sections frequently showed myofibrillar disturbances (mainly involving the Z- bands) (Fig. 1). Fibres with anomalous Z-band configura- tions were found in 42 out of 152 (28%) of the randomly obtained micrographs. However, only 5 per cent of the mi- crographs contained myofibrils with at least one area of Z-band material extending over a whole sarcomere (cf Fri- d6n et al. 1983b). The remaining Z-band anomalies oc- curred in essentially two different forms: either as Z-band widening and Z-band density extensions over parts of the sarcomere (Figs. 2-4) or bisected Z-band (Fig. 5a-b). All these changes occurred both just beneath the sarcolemma and deep in the fibres. Between the separated Z-band halves, both thin and thick filaments could be detected al-

Table l. Coefficient of variation of sarcomere lengths (C.V.) and sarcomere lengths (mean_+ SD) in Type 1 and Type 2 fibres from untrained and trained individuals

Type 1 Type 2

C.V. X_+SD C.V. X+SD

Untrained Subject 1 2.5 3.23_+0.08 3.5 3.30+0.12 Subject 2 4.9 3.22_+0.16 4.8 3.59+0.17 Subject 3 7.4 2.87_+0.18 7.4 2.83+_0.21 Subject 4 7.8 3.54_+0.28 5.3 3.09+0.16

Trained Subject 5 1.6 2.69+0.04 2.7 2.69+0.07 Subject 6 5.3 2.66+0.14 6.2 2.57+0.16 Subject 7 3.0 3.07+__0.09 3.7 2.96+0.11 Subject 8 1.6 2.69+0.04 2.5 2.71 +0.07 Subject 9 7.0 2.85 + 0.20 10.0 2.75 + 0.27

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Fig. 1. Electron micrograph of longitudinally sectioned muscle fibre after training. Distorted sarcomere registration (arrows) and extension of Z-band material (encircled) are seen. Capillary (c); n centrally located nucleus. Magnification • 1350

though they were not always completely parallel (Fig. 5 a - b). Band formations were sometimes visible between sepa- rated Z-band halves. An abundance of particular structures i.e. glycogen granules and ribosomes was observed adjacent to incomplete sarcomeres.

Parallel myofibrils adjacent to myofibrils containing Z- bands with intermediate zones had one sarcomere less. Thus, disordered sarcomeric registration was frequently seen. Extra sarcomeres were inserted into thickened, disor- ganized or discontinuous Z-band material (Fig. 4). Inter- connecting filaments were often appearent in the regions where the Z-bands were out o f register (Fig. 4). These fila- ments interconnected the Z-band of one myofibril and that o f the laterally extended arms of the disjoined, original Z- band in the adjacent myofibril (Fig. 4). Z-band changes

occurred in both fibre types but the Type 2/Type 1 ratio of anomalities was 4: 1.

Average sarcomere length was almost the same in Type 1 (2.77 gin) and Type 2 fibres (2.69 gm) (n.s.). The mean sarcomere length o f Type 1 and Type 2 fibres was not significantly different between trained and untrained individuals. However, a larger range of sarcomere lengths were constantly seen in Type 2 fibres than in Type 1 fibres f rom individuals who had trained (Table 1).

Discussion

This study describes a peripheral adaptation process in mus- cle fibres exposed to repeated powerful tension stimuli. Structural reorganization seems to result from repeated oc-

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Fig. 2. Electron micrograph showing material extending from a wavy Z- band into the I-band in a myofibril out of phase. Z-band (Z); mitochondrion (m). Magnification • 28000

casions of eccentric contractions of high intensity. It prob- ably represents a regenerative activity which makes it possi- ble for muscle fibres to adapt to a change in functional demands. This adaptation probably also serves as a defense against the potentially damaging tension that muscle fibres can be exposed to during eccentric contractions (Frid6n et al. 1981; Frid6n et al. 1983a). Hypothetically, the effect could be accomplished through lengthening of the myofi- brils by generation of new sarcomeres. This would, in its turn, produce better stretchability and a reduced risk of mechanically induced injuries.

Several studies point out the importance of tension in length increase (Gutman et al. 1971; Williams and Gold- spink 1971, 1973, 1978; Tabary et al. 1972; Barnett etal. 1980; Ashmore and Summers 1981). Muscle length can also be reduced by immobilization at a shortened length (Hayat 1978). Passive stretching can prevent or at least delay atro- phy after denervation (Tabary et al. 1972; Goldspink et al. 1974). Furthermore, passive stretching in studies in vitro has been shown to increase amino acid incorporation in myosin heavy chains (Vandenburg and Kaufmann 1979) and to decrease protein degradation (Goldberg et al. 1975). Many of these experiments have, however, involved dener- vation.

Under physiological conditions, high tensions can be reached, for example, by eccentric contractions (Bigland and Lippold 1954; Komi 1973). Eccentric contractions are characterized by elongation of the muscle at the same time as contraction. Since relatively little energy is required dur- ing eccentric work (Abbott et al. 1952) a long duration of work is possible at every training occasion. Electromyo- graphic studies have shown that comparatively fewer motor units are recruited with eccentric work than with corre- sponding concentric work (Bigland and Lippold 1954). This means that the tension per active motor unit is much higher in eccentric work.

In earlier studies, we have shown that occasional eccent- ric contractions give rise to disorganization of myofibrillar

material, probably by mechanical means (Frid6n et al. 1981, 1983 a). When comparing muscles which had contracted ei- ther concentrically or eccentrically, fibre abnormalities were found solely in the muscles which had contracted excentri- cally (Newham et al. 1983). In our experiments (Frid~n et al. 1983a) the regenerative process was rapid and the contractile material was repaired within a week. Similar extensive myofibrillar lesions observed after the single spell of work were not seen in the present study. On the other hand, different Z-band changes, including widening of the Z-band, extension of Z-band material and sarcomeres ap- parently in process of development, were observed. There are considerable difficulties in assessing whether the distur- bances represent de- or regenerative activity, or both. How- ever, it is reasonable to assume that the Z-band streaming represents damages caused by the eccentric work. Due to the high tension developed during eccentric contractions it might be supposed that actin and myosin filaments have been pulled apart. When the muscle is relaxed the thin fila- ments do not necessarily go back to their correct positions and thus the sarcomeres will be disorganized. Furthermore, a mechanical tearing of the intermediate filaments responsi- ble for the transverse alignment of the Z-disks could affect the structural integrity of the sarcomere. It is also possible that a primary shredding of filaments exposes contractile proteins to proteolytic attack (Armstrong et al. 1983). An- other possible explanation is that changes in the SR leading to calcium ion flooding, have caused a calcium-induced weakening of the Z-band (Hattori and Takahashi 1982). Disruption of SR is unlikely since such damage should have caused an increased level of myofibrillar Ca 2 +. This in turn would have caused contraction clots which were not found in the present study. However, the fact that the extent of myofibrillar disturbances were moderate indicates that the myofibrillar material has adapted to the high tension de- mands. Furthermore, it seems reasonable that sarcomeres as that shown in Fig. 5 ~ b are the result of an adaptation to stimulus repeated for a long period of time i.e. regenera-

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Fig. 3. Electron micrograph of Z- band smearing close to sarcomeres out of register. Magnification x 19000

Fig. 4. Electron micrograph of myofibrillar phase displacement. Disturbed Z-band alignment, separated Z-band material and interconnecting (intermediate) filament (arrows) are visible. Magnification x 19000

rive activity. The large number of glycogen particles and ribosomes is interpreted as a sign of increased protein syn- thesis. By using immunocytological techniques we have been able to show increased synthesis of desmin and reor- ganization of the cytoskeletal system in m. vas tus lateralis after eccentric load (Frid6n et al. 1983c). The desmin fila- ments would thereby act as mechanical integrators for the repair of sarcomeres. Moreover, strongly autofluorescent granules were observed beneath the sarcolemma. This find- ing is likely to represent increased iysosomal activity.

Some sarcomeres had disorganized contractile material whilst others had clearly defined thin and thick filaments and visible M-bands. These observations can be interpreted to be a sign of sarcomerogenesis. The results suggest that sarcomerogenesis can take place in the whole muscle and

not necessarily at the ends alone. This is in agreement with the findings of Schmalbruch (1968). The mechanisms un- derlying sarcomerogenesis in heart and skeletal musculature have been discussed frequently. Most workers agree that the Z-band plays a central role in this process (Kelly 1968; Legato 1970; Saetersdal et al. 1976; Jakubiec-Puka et al. 1982). Z-band anomalies occur spontaneously in both heart and skeletal musculature (Bishop and Cole 1969; Rowe et al. 1971) and frequently in nemaline myopathy (Price et al. 1965). The changes mainly consist of Z-band stream- ing. The biological reason is still unknown in most cases. Widening of the Z-band and insertion of extra sarcomeres has been regarded to be indirect evidence for ability of Z-bands to transform to contractile material (Thornell 1973; Jacubiec-Puka et al. 1982).

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Fig. 5. a Electron micrograph of sarcomere apparently developing after eccentric exercise. Bisected Z- band and myofilamentous material between the Z-band halves are observed. Magnification x 10500. b Detail of Fig. 5a. Thin and thick filaments as well as myofibrillar M- band (M) are apparent in the sarcomere. Magnifcation x 28000

In the present study, a range of Z-band anomalies were observed; these may be signs of a proliferative process in progress. In some sarcomeres the Z-band material was lying strictly l imited to the I -band, whilst in other sarcomeres it was located more laterally. In many sarcomeres, the Z- band appeared to be split into two halves. In parallel with separat ion of Z-band halves, synthesis of thin and thick fi laments was evident. M-band with distinct M-bridges could, thus, be seen in certain " i m m a t u r e " sarcomeres. This results in generat ion of new sarcomeres as seen in certain myofibri ls which had extra sarcomeres compared to the surrounding myofibrils. This entailed that the Z- bands were often seen to be out of register. However, one has to be caut ious when assessing dis turbed band al ignment because the str iat ions of the myofibri ls have been found

to have a helicoidal disposit ion (Peachey and Eisenberg 1978). The new sarcomeres were often seen to be inserted in thickened or extended Z-band material. It was remark- able that the proliferated Z-band appeared symmetrical and that the Z-band halves were mirror images of one another. This suggests that the proliferat ion starts centrally in the Z-band; these observations being well in line with the model for sarcomerogenesis that Legato (1970) described in heart musculature. The normal mechanism for breakdown and synthesis of myofibri l lar material is not yet clearly eluci- dated. Since one has not seen similar growth-zones in nor- mal, untrained musculature, it is improbable that normal breakdown and synthesis of myofibri l lar material takes place in the manner described above in trained musculature. The fact that the lateral mechanical integrator of myofibrils,

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i.e. the intermediate filament (Lazarides 1980), was often apparent can also be evidence for phase displacement of myofibrils. This in its turn can depend on the generation of new sarcomeres.

A crucial question is also whether different fibre types are engaged differently in this process. Although the validity of sarcomere length measurements is not as good as when measuring after in situ fixation, the large distribution in sarcomere length in Type 2 fibres of the trained individuals suggests that it is preferably the Type 2 fibres that undergo adaptation. This seems reasonable especially when one takes into consideration the high tension demands that are put on the musculature during eccentric work. When the muscle fibres are exposed to high tension the sarcomeres are initially too long for maximum tension to be developed. Subsequently an addition of sarcomeres and a reduction of sarcomere lengths occur. This is reflected by signs of sarcomerogenesis and a structural disturbance in the fibres predominant ly responsible for force generation under these circumstances, i.e. the Type 2 fibres. In eccentric work, the muscle becomes repeatedly stretched during its contractile phase. Even if all the muscle fibres became approximately evenly stretched, it is reasonable to assume that the tension stimuli become strongest in the fibres that contracted simul- taneously. These are probably Type 2 fibres according to an earlier study on comparable muscular work (Fr idrn et al. 1983 a). One should, however, keep in mind that ultrastruc- tural fibre typing is associated with many difficulties. In our experience we find that, for example, m. vas tus lateralis

is more difficult to type than m. tibialis anter ior (Sj6str6m et al. 1982b), that is to say that the differences between Type 1 and Type 2 fibres is less distinct in m. vas tus lateralis.

It can also be that training gives rise to a structural change in the myofibrillar M-band that in the present study was used as a criterion for discriminating fibre types. A succes- sive displacement in synthesis of M-band components is, in this case, another reason to consider strict fibre type limits with a certain scepticism. Muscle fibres continuously adapt both with regard to enzyme content and contractile protein structure.

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Accepted December 23, 1983