Eccentric exercise-induced injuries to contractile and cytoskeletal muscle bre components

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1 Acta Physiol Scand 2001, 171, 321±326 Eccentric exercise-induced injuries to contractile and cytoskeletal muscle bre components J. FRIDEÂ N 1 and R. L. LIEBER 2 1 Department of Hand Surgery, Sahlgrenska University Hospital, GoÈteborg, Sweden 2 Departments of Orthopaedics and Bioengineering, University of California and Veterans Affairs, San Diego, USA ABSTRACT Exercise involving lengthening of an activated muscle can cause injury. Recent reports documented the mechanics of exercise-induced muscle injury as well as physiological and cellular events and manifestations of injury. Loss of the cytoskeletal protein desmin and loss of cellular integrity as evidenced by sarcolemmal damage occur early during heavy eccentric exercise. These studies indicate that the earliest events in muscle injury are mechanical in nature, while later events indicate that it may be more appropriate to conclude that intense exercise initiates a muscle remodeling process. We conclude that muscle injury after eccentric exercise is differently severe in muscles with different architecture, is fibre type-specific, primarily because of fibre strain in the acute phase, and is exacerbated by inflammation after the initial injury. Keywords cytoskeleton, delayed-onset muscle soreness, muscle injury, muscle morphology, muscle strength. Received 12 December 2000, accepted 24 January 2001 Delayed-onset muscle soreness (DOMS) is the sensation of muscular discomfort and pain during active contractions that peaks 24±48 h after strenuous exercise. The initial symptoms are most evident at the proximal and distal muscle tendon junctions and spreading throughout the entire muscle. Skeletal muscle soreness and injury are associated with intense exercise but are more pronounced if the individual is unaccustomed to the exercise performed. Thus, even individuals in excellent athletic condition may experience muscle soreness and damage when performing exercise which is new to them. Although loss of strength has been observed very soon (within minutes) after exercise, the underlying causes of the relation between DOMS and the loss of muscle strength has yet to be explicitly proven. ECCENTRIC MUSCULAR ACTIVITY Erling Asmussen established in the 1950s that DOMS is directly associated with the eccentric component of exercise (Asmussen 1952, 1956). During an eccentric action, a contracting muscle is forced to lengthen while producing tension. During concentric action the muscle produces tension while shortening. The intermediate, isometric contraction, produces tension while the muscle remains essentially at the same length. All three actions are common components of daily movement. The tension generated during eccentric action is higher than that for either of the other actions (Fig. 1) (Katz 1939, Singh & Karpovich 1966, Komi & Rusko 1974, Stauber 1989) although fewer motor units are recruited (Bigland-Ritchie & Woods 1976). In addition to relative differences in tension between actions, Fig. 1 also demonstrates variations in tension as a function of contraction velocity. During concentric activity the tension/velocity relationship is explicitly formulated as described by the Hill equation (Hill 1938) and is attributed to the alignment of actin and myosin optimizing cross-bridge formation (Huxley 1969). Because of the nature of an eccentric action, the tension generating mechanism can be expressed in two phases. First, the muscle contracts to generate tension and then the muscle is passively stretched by an external force while generating additional tension (Katz 1939, Stauber 1989). The passive stretching has led investigators to suspect elastic components of the intact muscle-tendon system as possible contributors to the additional tension (reviewed by Chapman 1985). Recent studies imply that muscle passive tension is determined by the particular titin isoform expressed and that it is variable between different striated muscles, even within the Correspondence: Jan FrideÂn, Department of Hand Surgery, Sahlgrenska University Hospital, SE GoÈteborg, Sweden. Ó 2001 Scandinavian Physiological Society 321

2 Exercise-induced muscle injury J FrideÂn and R L Lieber Acta Physiol Scand 2001, 171, 321±326 Figure 1 Generalized relationship between muscle length, force and velocity. At a given force `plane', a variety of sarcomere lengths velocity combinations are possible. same species (Wang et al. 1993, Labeit & Kolmerer 1995). Other investigators attribute, in theory, the energetics of cross-bridge formation during the contraction/stretching phase as the reason for the higher tension for eccentric activity (Stauber 1989). Recently, Linari and coworkers demonstrated that during lengthening the cross-bridge number increases while the average degree of cross-bridge strain is similar to that induced by the force-generating process in isometric conditions (Linari et al. 2000). OBJECTIVE FINDINGS ASSOCIATED WITH DELAYED ONSET MUSCLE SORENESS The general ndings when examining an individual with DOMS symptoms include prolonged strength loss, a reduced range of motion, and often elevated levels of serum creatine kinase (CK). A multitude of studies have demonstrated that eccentric exercise results in increase in serum CK levels 24±48 h after the exercise bout and may persist for 3±6 days. Creatine kinase is an intramuscular enzyme responsible for maintaining adequate ATP levels during muscle contraction. Its appearance in blood is interpreted as an increased permeability or breakdown of the membrane surrounding the muscle cell. The perception of soreness after eccentric exercise is out of phase with serum CK levels (Newham et al. 1988). It is also important to note that the substantial muscle injuries and necrosis demonstrated in animal exercise experimentation cannot be reproduced with eccentric exercise protocols in humans. Investigators have demonstrated a protective effect of previous training, comparing CK levels to untrained control values (Evans et al. 1985). It is often assumed that the level of CK activity is somehow related to the magnitude of muscle injury, although this potential correlation has not been explicitly proven. In a preliminary study of 26 rabbits performing eccentric exercise with their ankle dorsi exors we found a poor correlation between serum CK levels and depressed skeletal muscle force generating capacity after eccentric exercise (Fig. 2) (FrideÂn & Lieber 2000). It may not be surprising that this relationship is relatively poor because a muscle bre's permeability to intramuscular enzymes may or may not be correlated with cellular contractile function. For example, we previously demonstrated that numerous muscle bres subjected to eccentric exercise that retained their ability to exclude plasma bronectin demonstrated signi cant structural abnormalities such as loss of intracellular desmin, myo brillar disruption, and Z-disk disintegration. MUSCLE INJURY The immediate loss of strength after eccentric exercise has been referred to as overstretch of sarcomeres, resulting in a non-optimal overlap between actin and myosin laments length (Faulkner et al. 1993) as well as changes in excitation-contraction coupling (Balnave & Allen 1995, Morgan & Allen 1999). Faulkner and coworkers suggested that during eccentric exercise some sarcomeres are stretched beyond overlap and thereby injured while others maintain their length. It is well known that eccentric exercise induces greater changes than comparable contractions of either isometric or concentric activity (Warren et al. 1993). Despite the greater force loss after eccentric contractions, the resting membrane potential of isolated muscles fatigued by either eccentric or isometric contractions were similar (Warren et al. 1993). Because Figure 2 Relationship between serum creatine kinase (CK) and maximum dorsi exion torque. Data are presented for torque measured 1, 2, 7 or 14/28 days after a single eccentric exercise bout. 322 Ó 2001 Scandinavian Physiological Society

3 Acta Physiol Scand 2001, 171, 321±326 Figure 3 Schematic drawing of longitudinal section of muscle bre with segmental damage surrounded by two normal bres. Bilateral to the central necrotic zone are hypercontraction zones noted. In the region of necrosis, phagocytes are observed both inside and outside the partially damaged muscle bre membrane. The hypercontraction zones displace adjacent bres at the level of lesion. there is no evidence of excitation failure the ability to produce action potentials is expected to be unaffected. Regardless of exact mechanism, it is generally agreed that initial force decline is caused by mechanical injury. A number of mechanical factors such as muscle length, force and velocity seem to play a role in the consequences of eccentric contractions. Newham et al. (1988) found more pronounced strength loss after eccentric exercise at a long muscle length compared with a short muscle length. Although the nature of morphological injury to the muscle has been well documented the mechanism of the injury is not fully understood. Although muscle tissue is extremely plastic (Pette 1990) destructive changes of muscle ultrastructure may occur in response to unusual demands (Hoppeler 1986). Morphological abnormalities after various types of exercise have been reported in both animal (Armstrong et al. 1983, Kuipers et al. 1983, McCully & Faulkner 1985, Lieber & FrideÂn 1988) and human models (FrideÂn et al. 1981, 1983, 1984, Newham et al. 1983). Different types and locations of cellular lesions after heavy exercise are shown in Table 1. The vast majority of morphological studies indicate that the Z disk is the most vulnerable structure to eccentric exercise induced injury. Damage has also been found in the sarcolemma, T tubules, myo brils and the J FrideÂn and R L Lieber Exercise-induced muscle injury cytoskeletal system. In the mild type of damage, immediately after exercise, single or very few sarcomeres throughout the muscle may be affected, usually showing Z-disk streaming (wavy appearance) with myo brillar disarray. In more severe cases, extensive streaming (wavy appearance and widening of Z-disk stain) and smearing (dispersion of Z-disk material into neighbouring sarcomeres) of the Z disk, focal loss of the Z disk and displacement of Z-disk material may occur. Both type 1 and type 2 bres seem to be affected although the majority of studies demonstrate type 2 biased damage (Lieber & FrideÂn 1988). It has been shown that sarcoplasmic reticulum channel proteins may play a role in the aetiology of muscle injury (Gilchrist et al. 1992). Several studies indicate that disturbances in calcium metabolism may be associated with muscle changes and weakness after heavy exercise (Reid et al. 1994). More recent studies have suggested that the early cytoskeletal changes (Lieber et al. 1996) may be attributed to cytoskeletal proteins located at the level of the Z disk (a -actinin, vimentin and desmin). The most striking change observed after eccentric rabbit dorsi exion exercise was the loss of desmin staining in a signi cant portion of muscle bres across the muscle (Lieber et al. 1996). Three days following eccentric exercise, over 30% of the EDL bres had lost desmin staining. This percentage rapidly decreased, so that by 7 days post-injury the percentage was about 10% for the EDL. By 28 days post-injury, less than a fraction of a percentage of desmin negative bres could be found. Interestingly, most but not all desmin negative bres were also bronectin positive, indicating loss of cellular integrity accompanying cytoskeletal disruption. It should be noted that desmin staining was lost although the muscle bre still maintained contractile and oxidative enzymes. Belcastro (1993) reported increased calpain activity in hindlimb muscles after treadmill running. He postulated that early mechanical events which initiate injury and which result in later in ammation require an intermediate event. A most likely intermediate event is an increase in intracellular calcium level (Balnave & Allen 1995). While cyclic calcium concentration changes are normal in the muscle contraction cycle, Table 1 Evidence of exercise-induced structural changes in muscle bres Site of bre lesion Reference (1) Primary or secondary sarcolemmal disruption (Jenkins 1988, Armstrong 1990, Duan et al. 1990) (2) Swelling or disruption of the sarcotubular system (FrideÂn & Lieber 1996, Armstrong 1990, Willems et al. 1999) (3) Distortion of the myo brils' contractile components (Armstrong et al. 1983, Newham et al. 1983, FrideÂn et al. 1984, FrideÂn et al. 1988) (4) Cytoskeletal damage (FrideÂn et al. 1984, Lieber et al. 1996, FrideÂn & Lieber 1998) (5) Extracellular myo bre matrix abnormalities (Stauber 1989) Ó 2001 Scandinavian Physiological Society 323

4 Exercise-induced muscle injury J FrideÂn and R L Lieber Acta Physiol Scand 2001, 171, 321±326 Figure 4 Schematic diagram of a hypothetical mechanism whereby Z disk is disrupted. Myosin laments which are displaced based on force inequalities across sarcomeres may be pulled to one side of the sarcomere. (a) Normal myo brillar array (b) Myo brillar displacement. The wider Z disks compared with gure a represent abnormal Z disks as evidenced by Z-disk smearing in the electron microscope. chronically elevated intracellular calcium can activate endogenous proteases (e.g. calpain), causing muscle deterioration. As a result, increased intracellular calcium remains an attractive feature for most muscle injury models. Belcastro also demonstrated an increased af nity of calpain for calcium after exercise, suggesting that exercise itself increased the total amount of calpain available to destroy muscle tissue as well as its af nity for calcium. A given amount of damage could thus occur after exercise at a lower calcium concentration. Interestingly, the calpain is speci c for cytoskeletal proteins, whereas actin and myosin do not appear to be affected. Two proteins associated with the intermediate lament system (a-actinin and desmin) are highly affected. MORPHOLOGICAL MANIFESTATIONS In a number of previous animal muscle injury studies, bres with abnormal shapes and sizes have been used as morphological indices of acute exercise-induced muscle injury (Armstrong et al. 1983, McCully & Faulkner 1985, Lieber & FrideÂn 1988, FrideÂn et al. 1991, Lieber et al. 1991). Particularly, very large bres were demonstrated as a result of eccentric exercise. These bres were always identi ed as the fast-twitch glycolytic (FG) bre type. Furthermore, these large bres frequently displayed intracellular deposits of plasma bronectin indicating membrane lesion (FrideÂn et al. 1991). The large bres that were found in histological sections following repetitive eccentric contractions represent segmental hypercontraction of the bre (FrideÂn & Lieber 1998). This phenomenon occurs adjacent to muscle bre plasma membrane lesions and necrosis and manifest itself as very short sarcomere lengths (Fig. 3). On both sides of a lesion, the bre regained its contractile elements and myo brils became increasingly densely packed. The large bres appeared at variable distances from the bulging zones as bres with normal thickness or again signi cantly wider than neighbouring `normal' bres. At the border region between wide and thin bre portions, invading cells were found although consistently to a much lesser extent than in the thin region. In the thin region, the plasma membrane was only infrequently intact and numerous macrophages were always present at 3 days post-exercise. It is important to remember that these `extreme' exercise models may not directly relate to the human DOMS models where much less damage have been demonstrated. After an eccentric exercise protocol in the rabbit, ultrastructural analyses revealed that the typical A-band lattice was consistently lost, and Z-disk lattices and even Z-disk material were absent in the biopsies taken 1 h post-exercise (FrideÂn & Lieber 1996). Thin and thick laments were severely disorganized and the remnants of the band pattern were wavy and consistently broken in multiple places. Sarcoplasmic reticulum and T tubules were either normal or dilated in the 1-h post-exercise biopsies while they appeared rounded and with an increased and often granular density in biopsies taken later after eccentric exercise. The bres of lower antidesmin density but negative anti bronectin staining presumably correspond with the previously reported immediate ultrastructural consequence of eccentric load, i.e. distortion of the alignment of the A and I bands, irregular Z disks and slippage of the thick laments out of the thin lament lattice (Dix & Russell 1991, Lieber et al. 1991). It is reasonable to assume that the bre as an entity is essentially in good structural condition unless the focal injuries appear multiple times in the same bre. This is, however, not possible because once a bre is disrupted the remaining intact bres supposedly take up the tension put on the muscle. This would mean that eventually fewer bres will be subjected to forced lengthening as has been proposed earlier (FrideÂn et al. 1983, Armstrong 1990). On the other hand, a focally damaged bre may function normally, especially 324 Ó 2001 Scandinavian Physiological Society

5 Acta Physiol Scand 2001, 171, 321±326 considering the fact that longitudinal intermediate laments may span the injured segments and transmit tension (Wang et al. 1993). To test the role of desmin in normal and injured muscle, EDL muscles were obtained from young wildtype mice, and desmin homozygous knockout mice in which the desmin gene had been deleted (Milner et al. 1996, Sam et al. 2000). The mice ankle dorsi exors were exercised eccentrically using a dual mode ergometer that could impose rapid length changes. A highly signi cant difference in percentage Z-disk damage was demonstrated between exercised and control muscles but no signi cant difference between knockouts and wildtype. On the other hand, a signi cantly higher degree of lateral myo brillar displacement was found in the desmin knockouts compared with wildtype. These data demonstrate a dissociation between Z-disk disruption and Z-disk misalignment and that the increased longitudinal translation of Z disks along the myo brillar axis in knockout muscles may indicate that the Z disks are less well anchored to one another (Fig. 4). ADAPTATION It may be a misnomer to propose that exercise result in muscle injury. Rather, it may be more appropriate to conclude that intense exercise initiates a muscle remodelling process that enables skeletal muscle to hypertrophy. When in ammation occurs post-exercise it appears to be uniquely associated with the eccentric component in that the same force decrement is not observed after isometric exercise of the same duration. Thus, we hypothesize that eccentric exercise initiates events which result in extravasation of leucocytes and monocytes from the bloodstream and in ltration by these cells into the tissue. This cellular in ltration results in further tissue degradation, probably by deposition of proteolytic enzymes designed to enable tissue remodelling. A study of the effect of non-steroidal anti-in ammatory drugs (NSAIDs) after eccentric exercise, demonstrated that systemic administration of urbiprofen following muscle injury can improve contractile function and reduce circulating levels of in ammatory cells and muscle enzymes (Mishra et al. 1995). These data suggest that the in ammation process itself, which may occur secondary to the initial muscle injury, also results in an additional delayed injury to the muscle, i.e. a short-term bene cial effect but long-term negative effect of NSAIDs after exerciseinduced muscle injury. A morphological mechanism for rapid adaptation has been hypothesized in that repetition of eccentric exercise could make myo brils, cytoskeleton and cell membranes more resistant to stress and strain (Gibala et al. 1995). Rapid recovery has been demonstrated after eccentric quadriceps exercise. J FrideÂn and R L Lieber Exercise-induced muscle injury HortobaÂgyi et al. (1998) found that this recovery occurred independently of cell disruption and concluded that it was mediated by neural factors. HortobaÂgyi's results were based on the fact that after a second bout of eccentric exercise EMG, patellar tendon re ex amplitude and CK level were normalized although 23% of the muscle tissue was damaged. In summary, we believe that the following series of muscle bre events are taking place after DOMScausing exercise: cytoskeletal disruptions, loss of myo brillar registry, i.e. Z-disk streaming and A-band disorganization, loss of cell integrity as manifested by intracellular plasma bronectin stain, hypercontraction of injured bre regions and invasion of in ammatory cells. 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