Mechanisms of exercise-induced muscle fibre injury.

REVIEW ARTICLE

Sports Medicine 12 (3): 184-207, 1991 0112-1642/91/0009-0184/$05.00/0 © Adis International Limited. All rights reserved. SP0146A

Mechanisms of Exercise-Induced Muscle Fibre Injury R.B. Armstrong, GL Warren and l.A. Warren Exercise Biochemistry Laboratory, University of Georgia, Athens, Georgia, USA

Contents 184 186 186 186 190 190 190 190 193 197 197

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198 199 200 202

Summary

Summary 1. Initial Event in Muscle Fibre Injury 1.1 Physical Hypotheses 1.1.1 Mechanically Induced Mechanisms 1.1.2 Temperature-Induced Mechanisms 1.2 Metabolic Hypotheses 1.2.1 Insufficient Mitochondrial Respiration 1.2.2 Free Radical Production 2. Loss of Ca++ Homeostasis 3. Autogenetic Mechanisms During Muscle Injury 3.1 Contracture 3.2 Mitochondrial Ca++ Overload 3.3 Ca++-Activated Neutral Proteases

3.4 Lysosomal Proteases 3.5 The Phospholipase A2 (PLA2) Pathway 4. Conclusions

Exercise for which a skeletal muscle is not adequately conditioned results in focal sites of injury distributed within and among the fibres. Exercise with eccentric contractions is particularly damaging. The injury process can be hypothesised to occur in several stages. First, an initial phase serves to inaugurate the sequence. Hypotheses for the initial event can be categorised as either physical or metabolic in nature. We argue that the initial event is physical, that stresses imposed on sarcolemma by sarcomere length inhomogeneities occurring during eccentric contractions cause disruption of the normal permeability barrier provided by the cell membrane and basal lamina. This structural disturbance allows Ca++ to enter the fibre down its electrochemical gradient, precipitating the Ca++ overload phase. If the breaks in the sarcolemma are relatively minor, the entering Ca++ may be adequately handled by ATPase pumps that sequester and extrude Ca++ from the cytoplasm ('reversible' injury). However, if the Ca++ influx overwhelms the Ca++ pumps and free cytosolic Ca++ concentration rises, the injury becomes 'irreversible'. Elevations in intracellular Ca++ levels activate a number of Ca++-dependent proteolytic and phospholipolytic pathways that are indigenous to the muscle fibres, which respectively degrade structual and contractile proteins and membrane phospholipids; for instance, it has been demonstrated that elevation of intracellular Ca++ levels with Ca++ ionophores results in loss of creatine kinase activity from the fibres through activation of phospholipase A2 and subsequent production of leukotrienes. This autogenetic phase occurs prior to arrival of phagocytic cells, and continues

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during the inflammatory period when macrophages and other phagocytic cells are active at the damage site. The phagocytic phase is in evidence by 2 to 6 hours after the injury, and proceeds for several days. The regenerative phase then restores the muscle fibre to its normal condition. Repair of the muscle fibres appears to be complete; the fibres adapt during this process so that future bouts of exercise of similar type, intensity, and duration cause less injury to the muscle.

The topic of exercise-induced muscle fibre injury has been discussed in several recent reviews (Armstrong 1984, 1986, 1990; Ebbeling & Clarkson 1989; Stauber 1989). It is clear from the literature that exercise damages fibres; conditioning of the muscle reduces the amount of injury (Armstrong 1984; Ebbeling & Clarkson 1989; Schwane & Armstrong 1983; Stauber 1989). The damage is associated with eccentric contractions, in which the muscle is lengthened while it is active (Armstrong et al. 1983b; Lieber & Friden 1988; McCully & Faulkner 1985) and varies with the intensity (McCully & Faulkner 1986; Tiidus & Ianuzzo 1983), and the duration (McCully & Faulkner 1986; Tiidus & Ianuzzo 1983) of the exercise. The injury is accompanied by loss of contractile force (Davies & White 1981; Friden et al. 1983; Hough 1901; Newham et al. 1983; Ogilvie et al. 1985; Warren et al. 1990) and the sensation of soreness (Armstrong 1984; Ebbeling & Clarkson 1989), so the pathology has pr~ctical consequences. There is no evidence that the muscle is permanently impaired from this type of injury; in fact, it has been suggested that this damage is a normal precursor to muscle adaptation to increased use (e.g. see Armstrong 1984, 1990). In spite of the frequent occurrence of the injury, and its practical consequences, surprisingly little is known about the causative factors or the cellular mechanisms involved. The purpose of this review is to consider hypotheses that explain why the injury occurs and the immediate response of the muscle cells to the insult. The review focuses on intrafibre mechanisms, although it seems clear from the literature that the interfibre damage is also involved in the aetiology of exercise-induced injury (e.g. Stauber 1989). It was previously suggested that the muscle injury sequence can be divided into the following hypothetical events (Armstrong 1990).

First, some initial event occurs in the muscle fibre that serves to inaugurate the injury process. Most of the hypotheses about the initiation of the injury can be categorised as either physical or metabolic in nature. Second, the initiating event leads to a focal loss ofCa++ homeostasis in the muscle fibre, which will be referred to as the Ca++ overload phase. Cells have a number of mechanisms for regulating free cytosolic Ca++ levels (Carafoli 1985; Klug & Tibbits 1988). When these buffering and translocation mechanisms are overwhelmed in the face of elevated intracellular Ca++ levels, several intrinsic degradative pathways are activated in the fibres. Thus, the third step in the hypothetical injury sequence is Ca++ activation of degradative autogenetic mechanisms at the local site of the injury. These include the phospholipase A2 (PLA2) cascade (which produces arachidonic acid, prostaglandins, and leukotrienes), Ca++-activated proteases and lysosomal proteases. In addition, elevated intracellular Ca++ levels can disrupt normal mitochondrial respiration and cause sarcomere contracture. These various autogenetic factors in the muscle fibres occur prior to, and continue after, invasion of phagocytic and inflammatory cells into the sites of injury. By 2 to 6 hours after the initiation of the injury, the phagocytic phase is in evidence in the muscles. These inflammatory processes are important in the removal of injured tissue and for stimulating regeneration of the damaged fibres. Four to 5 days after the injury, there is clear evidence the injured fibres are in the regenerative phase, and it appears that the muscle completely heals during the regeneration (Armstrong et al. 1983b). Like the preceding phases, the regenerative processes are restricted to the focal site of injury, except for the apparent migration of satellite cells

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to the damaged area from uninjured parts of the fibre (Schultz 1989); thus, the degenerative and regenerative processes do not affect the entire fibre (Armstrong et al. 1983b; Kuipers et al. 1983). The phagocytic and regenerative steps in the injury sequence seem to follow the same general order as described in the literature for other muscle injury and regeneration models (e.g. see Carlson & Faulkner 1983; Mauro 1979). However, very little is known about the mechanisms underlying the initial, Ca++ overload, and autogenetic phases of skeletal muscle fibre injury resulting from exercise. This review focuses on these early events in the aetiology. It should again be emphasised that the presentation is hypothetical; it is hoped that it will stimulate experimental testing of the hypotheses.

1. Initial Event in Muscle Fibre Injury The specific event that serves to initiate exercise-induced muscle fibre injury is not known. The following discussion presents some of the hypotheses proposed to explain the initial event. The premises are categorised as physical or metabolic in nature, although in several cases this distinction is equivocal. 1.1 Physical Hypotheses

The possible physical mechanisms for the initiation of muscle fibre injury may be divided into 2 categories; mechanically induced and temperature-induced. It is generally recognised that exercise-induced muscle fibre injury is particularly associated with eccentric contractions (Armstrong 1984; Ebbeling & Clarkson 1989; Stauber 1989). Therefore, the proposed mechanically induced mechanisms focus on aspects of the eccentric contraction that distinguish it from isometric or concentric contractions (e.g. the greater force production or longer muscle length attained during an eccentric contraction). The proposed temperatureinduced mechanisms focus on the idea that local muscle temperature is higher during eccentric contractions, thus predisposing the muscle fibre to deleterious structural and/or metabolic changes.

1.1.1 Mechanically Induced Mechanisms Analysis of muscle fibre injury from a materials science standpoint provides insight into the unique mechanical role of the eccentric contraction in the injury process. Skeletal muscle meets the criterion for a ductile material (i.e. a material that may elongate by 5% or more), for which there is a wealth of knowledge regarding materials failure (e.g. Popov 1990). However, muscle also is a complex composite material, which makes the identification and study of the 'failed' component extremely difficult. In this discussion, the term 'failed muscle fibre' is not meant to imply that it is not capable of force generation, but rather that a structural component of the fibre has ruptured or been weakened so that a reduction in muscle fibre performance results. In other words, the muscle fibre has been injured irreversibly. Skeletal muscle fibre failure may result from either a single contraction or from the cumulative effects of many contractions. During any given contraction, a muscle fibre will fail if the tensile stress of a structural component in the fibre exceeds the yield strength of the component (maximum normal stress theory). If the tensile stress exceeds the tensile strength, then the structural component ruptures (fig. 1). Alternatively, failure of a structural component will occur if the shear

Tensile strength VielO strength

+

Strain

1

Fig. 1. Stress-strain curve typical of a ductile material in tension. The material behaves elastically until stress exceeds the yield strength. As stress exceeds the yield strength, the material undergoes a permanent change of shape (i.e. plastic deformation). When stress equals the tensile strength, the material fractures.


187

,, \ \

10

102

,,

,,

" ... ...

... ...

... ......

104

Number of cycles to failure

Fig. 2. Materials fatigue curves typical of ductile materials. As the mean stress applied during a tension-compression cycle is increased, the number of cycles to failure is reduced. The horizontal dotted line is the endurance limit of the material and represents the stress below which an infinite number of tension-compression cycles can be performed without failure. The dashed line represents a material that is more fatigueresistant than that represented by the solid line.

stress within a fibre (maximum shear-stress theory) or the weighted average of stresses in the 3 dimensions (maximum distortion-energy theory) exceeds some critical value (Popov 1990). The study of muscle fibre injury resulting from the cumulative effects of many contractions relies on knowledge from the field of materials fatigue. In this field, a material is subjected to alternating tension and compression (or relaxation) until the material fails. For most ductile materials, the relation between stress and the number of cycles to failure is exponential (fig. 2). As stress is increased, the number of cycles to failure decreases (Ashby & Jones 1988). From materials fatigue theory, the energy absorbed by a muscle fibre being forcibly lengthened must be dissipated either in the form of heat or as plastic deformations (i.e. permanent changes in the size or shape of structural components). Plastic deformations in a muscle fibre could initiate 'cracks' in one or more structural components, which would grow with ensuing contractions until the component ruptures. In addition, increasing the rate of stress development tends to reduce . the number of cycles to failure, suggesting that the velocity of muscle fibre lengthening could have an effect on the injury process.

Analysis of the muscle injury literature from the materials science perspective is difficult. First, there have been no studies of the relationship between tensile or shear stress and the degree of injury. Few studies have directly measured muscle force, and even when the measurement is made, the force determined at the tendon is the sum of the stresses on the individual structural components multiplied by their respective cross-sectional areas. It is difficult to analyse the potential for component failure from these relatively gross measurements. Second, the yield and tensile strengths of the individual force-bearing elements in the muscle fibre are unknown. Finally, there are few data on the amount of work done on muscle during eccentric contractions and its relation to the degree of injury, and there are no data on the proportion of absorbed energy that is dissipated as plastic deformations. Despite these shortcomings, the literature does suggest that muscle fibres may fail as a result of excessive tensile stress during a single eccentric contraction. First of all, force production during an eccentric contraction may exceed maximal isometric force (Po) by 50 to 100% (Woledge et al. 1985). Second, in order to meet the requirement for an isovolumic contraction, fibre cross-sectional area must be reduced as a fibre is lengthened. Therefore, the average tensile stress across a fibre actively lengthened to 130% of its resting length (1.0) may be 100 to 160% greater than for a maximal isometric contraction performed at 1.0. However, it is likely that it is the cross-sectional area of cellular matrix that is reduced upon lengthening, and not that of the main structural components. Third, the number of attached crossbridges may decrease with increasing lengthening velocity (McMahon 1984). This would result in an increased force per crossbridge, possibly disposing the contractile proteins to failure. Experimental evidence relating injury to the muscle force generated during eccentric contractions supports this line of thought. Katz (1939) may have been the first to observe the relation of muscle force produced during an eccentric contraction to injury. After 3 eccentric contractions of an isolated

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frog sartorius muscle, the rate of force development became depressed and there appeared to be a shift in the length-tension curve towards longer muscle lengths. In most instances, this injury appeared only after contractions in which force exceeded 180% Po. Data from the studies of McCully and Faulkner (1985, 1986) also suggest that muscle fibre injury results from high tensile stresses occurring during eccentric contractions. Using an in situ mouse extensor digitorum longus (EDL) muscle preparation, the peak force generated on the first of a series of eccentric contractions was found to be related to the Po developed 3 days after the injury protocol (r = -0.70) [McCully & Faulkner 1986]. The degree of reduction in Po was correlated with the histological evidence of injury (r =0.79). The greatest amount of injury occurred during the faster stretches, but it was shown that the degree of injury was primarily related to the peak force and not to the lengthening velocity. McCully and Faulkner (1986) found that injury resulted from eccentric contractions eliciting 85% Po, but not from isometric or concentric contractions eliciting 85% Po. They interpreted this to mean that a characteristic of the eccentric contraction other than peak force was also involved in the injury process. This characteristic may have been the longer muscle lengths at which peak forces were produced during the eccentric contractions. Peak force during the eccentric contractions occurred at a longer muscle length than during either the isometric or concentric contractions (110 vs ~ 100% 1.0) [McCully & Faulkner 1985]. Both Katz (1939) and Newham et al. (1988) have observed length-dependent components in the injury process. In these studies, it was found that greater injury occurred when eccentric contractions were initiated at longer muscle lengths. Eccentric contractions performed at muscle lengths greater than 1.0 may result in excessive tensile stresses in the active and/or passive elements. Regarding the former, in the studies of McCully and Faulkner (1985, 1986), the number of attached crossbridges at the time of peak force production was probably less for the eccentric contractions than


for the isometric or concentric contractions. It follows that at their 85% Po peak force level, the stress per crossbridge would have been greater for the eccentric contractions, and rupture of the heavy meromyosin subunits might have resulted. This logic is correct only if the length-tension curve for the mouse EDL muscle exhibits a descending limb for total tension. If the mouse EDL does not exhibit a descending limb for total tension, it is possible that the passive elements could have experienced excessive tensile stresses. Active tension declines as muscle lengths exceed 1.0; passive tension rises, making a greater contribution to total force production. For a given level of force production, it follows that the stresses borne by the passive elements could have been greater during the eccentric contractions. However, McCully and Faulkner (1986) and Faulkner et al. (1989) reported that injury did not occur during passive stretches performed at the same velocities as the eccentric contractions. This could be explained if the loading of the passive elements is not the same for active and passive stretches. There are few data to suggest which one of the passive elements might fail during eccentric contractions. Most of the passive tension is borne by the sarcolemma at sarcomere lengths greater than 140 to 150% of resting length (Casella 1951; Higuchi & Umazume 1986; Rapoport 1972). Because sarcomere length inhomogeneities occur during eccentric contractions (Colomo et al. 1988; Julian & Morgan 1979), some sarcomeres may undergo extreme lengthening even when whole muscle length changes are relatively small. Damage to the sarcolemma adjacent to these overlengthened sarcomeres could occur. The importance of sarcolemmal integrity is illustrated by the pathology associated with Duchenne muscular dystrophy. This disease is believed to result from development of structural defects in the sarcolemma (Bhattacharya et al. 1989). Recent work has shown that a defective gene in Duchenne muscular dystrophy results in a lack of the protein dystrophin, which is associated with the sarcolemma (Hoffman et al. 1987; Zubrzycka-Gaam et al. 1988). Karpati and Carpenter (1989) suggest that dystrophin may be essential for


'mechanical stability of the plasma membrane so it can withstand the normal contraction-induced strains', and that the protein may be important in maintaining apposition between the basal lamina and plasma membrane. Alternatively, intermediate filaments in the cytoskeletal network may fail when inhomogeneous lengthening of sarcomeres occurs. The interfibrillar intermediate filaments are believed to be responsible for maintaining the sarcomeres in register (Wang & Ramirez-Mitchell 1983). Two adjacent sarcomeres in parallel undergoing differing degrees of lengthening could produce excessive tensile stresses in these filaments. In addition, Higuchi and U mazume (1986) have reported that a radial compressive force exists in muscle fibres, which increases with sarcomere lengths greater than 140% of resting length. This radial stress coupled with what may be a 'normal' tensile stress may exceed the minimum criterion for failure under the maximum distortion-energy theory. Because of the 3dimensional nature of the cytoskeletal network, it is a likely site for this type of failure. There are data to suggest that the basement membrane may be the failure site (Tidball 1986; Tidball & Daniel 1986). When rigoured frog semitendinosus muscle fibres performed sinusoidal length changes, it was found that there was about a 2-fold increase in energy loss as sarcomere length increased from 2.4 to 3.1~m (Tidball & Daniel 1986). Because there was a decrease in actomyosin overlap as sarcomere length increased, they concluded that the energy must have been dissipated as heat or plastic deformations in the sarcolemma, sarcoplasmic reticulum (SR), basal lamina or cyto skeletal network. Tidball (1986) performed similar experiments on fibres whose basement membranes had been removed. His data show that approximately 77% of the total energy lost during a stretch-shortening cycle was dissipated in the basement membrane. Stauber and coworkers (see Stauber 1989) have reported histochemical and immunohistochemical evidence for injury to the basal lamina/endomysium in muscles that have performed lengthening contractions. A novel explanation for eccentric contraction-

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induced muscle fibre injury has been proposed by Morgan (1990). His idea incorporates the fact that sarcomere length inhomogeneities exist during eccentric contractions. He proposes that at the beginning of a contraction, there is a distribution of sarcomere lengths within a given fibre or myofibril, but the distribution is confined to the plateau of the sarcomere length-tension curve. Lengthening of the sarcomeres does not affect their strength until they are stretched to the descending limb of the curve. Once the longest sarcomere is on the descending limb, it becomes progressively weaker with increasing length until it is unable to maintain existing tension at any velocity. It then lengthens more rapidly until rising passive tension eventually halts further lengthening. This process is then repeated in the next weakest sarcomere. This sequence of events continues until the fibre ceases lengthening and/or there is a reduction in fibre tension. The sequence for injury as proposed by Morgan (1990) is: (a) eccentric contractions cause some sarcomeres in each myofibril to extend to very long lengths; (b) some of these sarcomeres do not interdigitate properly upon relaxation; (c) subsequent eccentric contractions quickly stretch these sarcomeres, placing greater tensile stresses on neighbouring myofibrils; and (d) consequently, adjacent sarcolemma and sarcoplasmic reticulum fail. Additionally, the intrafibrillar intermediate filaments are believed to be important in limiting extreme sarcomere length changes (Wang & RamirezMitchell 1983), so these filaments could be affected by the stretched sarcomeres. This model predicts that there would be a greater number of sarcomere failures for contractions performed at longer lengths. It also predicts that these failures would occur at random throughout the fibre. Such diffuse injury would be difficult to observe histologically and could possibly explain why McCully and Faulkner (1986) were unable to detect histological evidence of injury when lengthening velocities were low. The work of McCully and Faulkner (1986) also provides evidence that fibre structural components may undergo materials fatigue during repetitive eccentric contractions. As the number of eccentric

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contractions performed by the muscles rose from 15 to 150, a linear decrease in Po at 3 days and a linear increase in the injured area was observed. This suggests that the degree of injury was proportional to the amount of work done on the muscle. Muscles performing more than 150 contractions failed to exhibit greater injury. These findings may be interpreted in 2 ways. First, the weakest structural component of the muscle fibres could have completely ruptured after 150 contractions. The next weakest structural component could have a fatigue curve (fig. 2) that is displaced well to the right so that 'crack' propagation through this component proceeds slowly. Second, in this study, peak eccentric force after 150 contractions had dropped to 20 to 40% of that elicited during the first contraction of the injury protocol. Therefore, one may predict that the average cyclic stress experienced by the fibres would have been reduced by 60 to 80%. From figure 2, it can be seen that this would result in a decreased rate of 'crack' propagation and a large increase in the number of contractions until the structural component ruptures.

1.1.2 Temperature-Induced Mechanisms There are reports that intramuscular temperature is higher during negative work than during positive work when compared at equivalent metabolic or heat production rates (Nadel et al. 1972; Nielsen 1969; Pahud et al. 1980). At the same level of heat production (equal to metabolic rate minus work rate), eccentric contraction-biased exercise elicited a 1.2°C higher muscle temperature (Nadel et al. 1972). Under such conditions, the QIO effect could account for an 18% higher rate of structural lipid and protein degradation during the negative work. Also, a 1.2°C higher temperature would be expected to decrease sarcolemma viscosity by approximately 7% (Nagatomo et al. 1984). This decrease in viscosity might be sufficient to bring phospholipase A2 into apposition with its substrate, thus enhancing the rate of membrane degradation (Chang et al. 1987). In addition, preliminary evidence suggests that an increase in temperature from 25 to 35°C in an in vitro muscle


preparation performing eccentric contractions increases injury by approximately 50% (Zerba et al. 1990b). Caution must be used in analysing the possible role that temperature plays in muscle fibre injury. First of all, peak muscle temperature appears to be the same or only slightly higher during negative work than during positive work (Nadel et al. 1972). Second, comparison of muscle temperatures at equivalent absolute metabolicl'ates may not be appropriate. In the study of Nadel et al. (1972), peak metabolic rate during positive work appeared to be approximately 50% higher. Thus, muscle temperatures during the two modes of exercise would have been more similar if compared at equivalent relative metabolic rates. Finally, the Fenn effect predicts a lower (not higher) rate of heat production during eccentric contractions than during isometric or concentric contractions. This has been confirmed experimentally for eccentric contractions eliciting a force less than 140% Po (Abbot & Aubert 1951). For faster stretches where force exceeds 180% Po, heat production is greater than for an isometric contraction but much less than expected from the amount of work done on the muscle (Abbot et al. 1951). These observati~ns suggest that if a higher muscle temperature occurs during eccentric contraction-biased exercise, it is probably a consequence of a lower rate of heat removal and not due to a higher rate of heat production within the muscle. 1.2 Metabolic Hypotheses

1.2.1 Insufficient Mitochondrial Respiration During exercise, mitochondrial respiration is elevated to match ATP synthesis to ATP hydrolysis. This match is normally quite good during low to moderate intensity exercise, so that the active muscle fibres maintain ATP concentrations near resting levels (e.g. Krisanda et al. 1988). However, there always is some reduction in the concentrations of the high energy phosphates during muscular activity (Krisanda et al. 1988), and the possibility that the reductions occur within specific compartments in the fibres makes this a viable hy-


pothesis for the initiating event in muscle fibre injury. For example, if there is an attenuation in ATP levels in the vicinity of the Ca++-ATPase in the SR or sarcolemma, removal of Ca++ from the cytoplasm may be compromised, allowing an elevation in cytosolic Ca++ levels. The importance of maintaining functional Ca++ pumps to the health of the cell is indicated by experiments using ruthenium red, which inhibits Ca++-ATPase on sarcolemma, SR and mitochondria (Duncan 1987). In isolated intact skeletal muscles, ruthenium red treatment causes rapid and dramatic damage to the ultrastructure of the muscle (Duncan et al. 1980). Also, it has been demonstrated that reduction in energy supply in cells leads to Ca++ release from internal stores (Duchen et al. 1990). The fact that argues most persuasively that insufficient respiration in the fibres is not the initiating event in muscle injury is the common observation that a given force or power output produced with an eccentric contraction is metabolically 'cheaper' than that produced by concentric or isometric contractions (Bonde-Petersen et al. 1972; Curtin & Davies 1970; Infante et al. 1964), but the eccentric contractions cause more injury to the muscle (Armstrong et al. 1983b; Asmussen 1956; Newham et al. 1983). This dissociation between metabolic cost and injury in eccentric contractions indicates that the injury in the muscle fibres is not a result of insufficient ATP production. Aldridge et al. (1986) directly measured high energy phosphate levels in forearm muscles with 31 P-NMR immediately after eccentric exercise that induced postexercise soreness. There were no changes in ATP or CP levels or pH in the injured muscles, although 24 hours after exercise there was a significant elevation in inorganic phosphate levels. Similarly, it would be expected that exercise with concentric contractions would produce lower pH in the active muscles than exercise with negative contractions, indicating decreased pH is not the initiating mechanism for damage in the muscle. It has been demonstrated in chemically skinned muscle fibres that rapid myofibrillar damage can be induced by Ca++ at neutral pH (pH 7.3 to 7.6)

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with 3 mmolfL ATP in the medium (Duncan 1987). This does not prove that depleted ATP or low pH are not involved in the muscle injury process, but it demonstrates that muscle damage can occur without these metabolic complications. One note of caution, however, is that metabolic disparities in the fibres may be focal, so that in localised regions of the cells there may be energydepleted or acidic foci that are not resolvable in whole muscle assays. The lesions in the injured muscles are small (Kuipers et al. 1983; Ogilvie et al. 1988), and there is not final proof that they do not have a metabolic origin, which may well be insufficient respiration rate in the mitochondria in those focal areas. Lieber and Fridlm (1988) recently hypothesised that injury to fast glycolytic fibres in rabbit tibialis anterior muscle from eccentric contractions has a metabolic aetiology. Decreases in force production after exercise have been attributed in the literature to metabolic fatigue and, under many circumstances, this is the appropriate conclusion. However, it also is apparent that reductions of muscle force can result from injury to the muscles independent of metabolic deficits. Human studies clearly demonstrate that eccentric contractions cause attenuation of maximal force compared to concentric contractions, even though the concentric contractions are metabolically more demanding (Davies & White 1981; Friden et al. 1983; Newham et al. 1983). Following downhill walking in rats, which has a relatively low metabolic cost (Armstrong et al. 1983a), force production by soleus muscles is reduced in association with histological injury in the muscles (Ogilvie et al. 1985; Warren et al. 1990). Thus, from an experimental standpoint, clearly differentiating between the contribution of fatigue and injury mechanisms to decreased muscle force can be difficult.

1.2.2 Free Radical Production Another consequence of elevated metabolism during exercise is an increased production of free radicals (for reviews, see Jenkins 1988; Packer 1985, 1986). This results primarily from increased reducing equivalent flux through the mitochondrial electron transport system (Blake et al. 1987). Dur-

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ing the Q cycle of electron transport, the single electron reduction of ubiquinone produces ubisemiquinone, which contains only one electron in its outer valence shell. Ubisemiquinone is normally oxidised by the cytochrome bel complex, but it also can react with molecular oxygen, producing the superoxide anion (Boveris et al. 1976; Cadenas et al. 1977) and other potent free radical species (Nohl et al. 1986). Under most conditions, these activated oxygen species are controlled by an arsenal of antioxidant molecules and enzymes (Del Maestro 1980), but, in some circumstances these protective mechanisms can be overwhelmed (Demopoulos 1973b; Jenkins 1988). Uncontrolled free radical production can result in damage to the cell through the ensuing oxidation of phospholipids (Blake et al. 1987; Demopoulos 1973a), DNA (Cochrane et al. 1988), carbohydrates (Blake et al. 1987), and proteins (Tappel 1973; Wolff et al. 1986). Membrane lipid peroxidation may disrupt the normal permeability barrier provided by the sarcolemma (Quintanihla et al. 1982), permitting abnormal diffusion of molecules (e.g. Ca++ and intramuscular enzymes) down their respective concentration gradients (Braughler 1988; Malis & Boventre 1988). It has been suggested that Ca++ATPase may be a particularly susceptible target for membrane oxidation reactions (Braughler 1988; Kako 1985); inactivation of this enzyme would affect Ca++ homeostasis in active muscle fibres, which in turn could activate various degradative pathways in the cell (see below). Research supporting the role of free radicals in the initiation of injury during eccentric contractions is limited. The most convincing evidence for involvement of free radicals in the early phases of the injury process is the work ofZerba et al. (1990a), who reported that intraperitoneal injections of superoxide dismutase (SOD) in old mice attenuate the reductions in Po in the muscles immediately following eccentric contractions in situ. Interestingly, SOD treatment did not provide this protection for muscles from young or adult animals. SOD treatment reduced the loss of Po in the muscles from all age groups at 3 days after the eccentric contractions, emphasising the important role of free


oxygen radicals during the phagocytic phase of tissue necrosis. We (Warren et al. 1990) found that elevating a-tocopherol (a membrane-specific antioxidant) in the muscles through dietary manipulation did not attenuate muscle injury resulting from downhill walking in rats, either immediately or 2 days after the exercise. Thus, with the exception of the data for the old mice (Zerba et al. 1990a), evidence for a significant role for free radicals in the initiation of exercise-induced injury is lacking. In addition to lacking empirical support, the hypothesis that free radical production serves as the initiating mechanism in exercise-induced muscle fibre injury faces the same challenge as other metabolic hypotheses, i.e. why would one expect a greater free radical production during eccentric contractions? It has been proposed (Lehninger 1975) that during ischaemia in the ischaemia/reperfusion injury model in heart there is a disruption in the normally tight association between electron carrier elements. This would allow for the preferential donation of electrons to molecular oxygen rather than to the cytochrome complex (Demopoulos 1973a). Free radical production in this model is particularly evident during the reperfusion stage, when there are elevated oxygen concentrations in the tissue (Arkhipenko et al. 1983; Faust et al. 1988; Fisher 1988; Hess et al. 1982). It is possible that high specific tensions produced in muscle fibres during eccentric contractions (Bigland-Richie & Woods 1976) could alter the normal cytoskeletal framework, which functions to stabilise organelles (e.g. the mitochondria) in position in the cell. Disruption of the cytoskeleton could in turn cause physical disruption in the association of the components of the mitochondrial respiratory chain (Demopoulos 1973a; Packer 1985). This disturbance in the electron transport system could lead to production of excessive amounts of free radicals, which in turn could oxidise critical membrane components and enzymes. This explanation requires that sufficient molecular oxygen be present in the muscle to accept the misguided electrons from the respiratory chain. During downhill walking in rats, 02 delivery to the deep slow-twitch muscles (soleus and vastus intermedius) that show


fibre injury is equivalent to that during level walking at the same treadmill speed (unpublished observations). Presumably 02 consumption by muscles due to terminal 02 reduction in the electron transport system is lower during downhill walking, because whole animal V02 is less (Armstrong et al. 1983a). If muscle V02 is lower in the downhill walk, there is a relative 'overperfusion' of the muscle, which should result in an elevated intracellular P02. Thus, any disruption in respiratory chain components could result in abnormal production of free oxygen radicals and could serve as an initiating mechanism in exercise-induced muscle fibre injury. Data from other studies argue against an 'overperfusion' of muscles during exercise with eccentric contractions; e.g. Bonde-Petersen et al. (1970) and Stainsby (1976) reported that muscle blood flow is directly related to muscle V02 during eccentric exercise, similar to the relationship between these variables during exercise employing concentric contractions.

2. Loss 0/ Ca++ Homeostasis Whether the initiating event is mechanical or metabolic in nature, the next sequential step in the injury process is hypothesised to be an elevatio~ in intracellular Ca++ concentrations at the site of the lesion. A sizable toxicology literature (see Boobis et al. 1990; Murphy'& London 1988) and evidence from several muscle injury models (Baracos et al. 1984; Carpenter 1989; Kameyama & Etlinger 1979; Publicover et al. 1978; Statham et al. 1976) indicate that cellular necrosis is accompanied by loss of Ca++ homeostasis. Also, elevations in intracellular Ca++ levels are evident in muscles in patients with muscular dystrophy and other muscle diseases (Jackson et al. 1985; Turner et al. 1988; Wrogemann & Pena 1976). The importance of maintaining free cytosolic Ca++ concentrations within relatively narrow limits is indicated by the number of mechanisms the cell has for transporting Ca++ from the cytosolic compartment (Gillis 1985; Klug & Tibbits 1988; Tibbits & Thomas 1989). As pointed out by Carafoli (1985), there are at least 7 systems in the membranes for transporting Ca++

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including ATPases, Na+: Ca++ exchangers, channels, and electrophoretic uniporters. At the present time, there is no direct evidence that elevations in intracellular Ca++ levels are mechanistically involved in exercise-induced muscle fibre injury. However, preliminary work using the rat downhill walking model has demonstrated that total muscle Ca++ and mitochondrial Ca++ levels are increased in the injured muscles (Duan et aI. 1990a,b). How might the various initiating factors hypothesised above lead to increases in intracellular calcium concentrations? Disruption of the sarcolemma, which serves as a barrier for maintenance of concentration and electrical gradients between the extra- and intracellular spaces, could allow Ca++ to enter the cell down its electrochemical gradient. The extracellular free Ca++ concentration is 2 to 3 mmoljL, whereas the free cytosolic Ca++ level is about 0.1 ~moljL in the resting muscle fibre. Thus, there is a large gradient for this cation; any break in the normal permeability barrier provided by the sarcolemma would allow influx of Ca++ from the interstitium. The importance of extracellular Ca++ to the injury process in muscles is indicated in the studies in which removal of Ca++ from the incubation medium markedly reduces the evidence of damage (Jackson et al. 1984; Jackson & Edwards 1986) and attenuates the degradation of protein (Baracos et al. 1984), although interpretation of data from these experiments is complicated by the fact that the muscles lose significant amounts of Ca++ to the medium when incubated in Ca++-free solutions (Duan et al. 1990b). That there is disruption of the cell membrane in diseased and toxic conditions is supported by the changes in various cellular soluble constituents as predicted from their respective concentration gradients across the membrane. Thus, there is a loss of intracellular enzymes, myoglobin, adenine nucleotides, K+ and Mg++, and increases in cellular Na+ and Ca++ concentrations (Carpenter 1989; Ebbeling & Clarkson 1989; Jackson et al. 1985; Kagen 1972; Murphy & London 1988; Trump et al. 1971). In saponin-skinned fibres, incubation of the muscle in Ca++ concentrations of 0.5 to 8 ~moljL

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stimulates destruction of myofibrils and hypercontraction of sarcomeres (Duncan 1987, 1988). These Ca++ concentrations are in the physiological range ofthose in contracting muscles, indicating the free cytosolic Ca++ level is high enough during normal activity to initiate degradation of muscle structure. Several explanations for why this does not occur can be suggested. First, the elevations of cytosolic Ca++ concentrations during contraction in vivo are transient. When Ca++ is released by SR during excitation, the Ca++ is rapidly bound to regulatory proteins, including troponin C, so that the free cytosolic Ca++ level is only briefly elevated (Robertson et al. 1981). If the rate of binding in the Ca++activated degradative pathways in the fibres is lower than those for the contractile regulatory proteins, the events may occur too rapidly for significant activation of the proteolytic enzymes to occur. A second possibility is that the degradative enzymes are compartmentalised so they are not influenced by the Ca++ involved in excitation-contraction. On the other hand, damage to membranes, either sarcolemma or SR, could result in elevations in Ca++ concentrations in those compartments within the fibres, allowing Ca++ to contact the binding sites on degradative enzymes. Duncan (1987) concluded that it is not the absolute Ca++ level that is important in initiation of muscle damage, but the duration of magnitude of active movement ofCa++ across key membranes in the muscle fibres. Carpenter and Karpati (1989) used micropuncture of skeletal muscle fibres to produce segmental necrosis of the cells. This procedure appears to initiate lesions that are quite similar to those observed in muscle fibres following eccentric exercise (e.g. Armstrong et al. 1983b; Ogilvie et al. 1988). Following puncture of the sarcolemma (Carpenter & Karpati 1989), degeneration occurs in sarcomeres at the level of the wound, but the necrotic area is effectively 'walled off so that adjacent sarcomeres are protected from the degradative processes. The walling off was accomplished by hypercontraction of myofilaments; this region was heavily stained for precipitated Ca++. Also, adjacent to the hypercontracted filaments a demarcating membrane formed, which separated the necrotic area


from the healthy sarcomeres. Similar mechanisms probably are at play in exercise-induced muscle fibre injury, because the lesions normally only affect the fibres in segments of several sarcomeres (Armstrong et al. 1983b; Kuipers et al. 1983; Ogilvie et al. 1988). Several muscular disorders appear to result from elevated intracellular Ca++ concentrations caused by disruption of the normal permeability barrier of the sarcolemma to Ca++. In dystrophic mdx mice, which have a similar genetic deficiency to human Duchenne muscular dystrophy patients (Hoffman et al. 1987; Karpati & Carpenter 1989; ZubrzyckaGaarn et al. 1988), the intracellular Ca++ level is elevated in the diseased muscles (Turner et al. 1988), presumably because of disruption of sarcolemma. Protein degradation in the dystrophic muscles is directly related to the increases in intracellular Ca++ levels (Turner et al. 1988). Another genetically-predisposed condition that primarily affects skeletal muscle and is associated with elevated intracellular Ca++ concentrations is malignant hyperthermia, which is found in particular in humans and pigs (for review see Cheah & Cheah 1985). In this condition, anaesthetic agents cause a sustained increase in intracellular Ca++ levels in the involved muscle fibres, resulting in uncontrolled contraction and heat production (Nelson 1989). One of the hypotheses proposed to explain this condition is that membrane permeability of Ca++ is altered, allowing Ca++ to enter the cells down its concentration gradient (Cheah & Cheah 1985). A second mechanism for elevating free cytosolic Ca++ levels and promoting muscle damage is dysfunction of the SR. It appears that Ca++ influx from the extracellular space does occur in eccentric-exercise-induced muscle fibre injury (Duan et al. 1990b), but SR failure to resequester Ca++ could also contribute to the elevation in cytosolic Ca++ concentrations. It has been demonstrated that both exhaustive moderate- and high-intensity exercise reduce the ability of the SR to sequester Ca++ (Byrd et al. 1989a,b). There are no data on the possible differential effects of eccentric, concentric or isometric contractions on SR function, but as dis-


cussed above, sarcomere length inhomogeneities during eccentric contractions could adversely affect adjacent SR segments. When isolated muscles are incubated with caffeine, which promotes Ca++induced Ca++ release from SR, myofibrillar deterioration is observed (Duncan & Smith 1978). Also, as indicated above, incubation of muscles in ruthenium red, which inhibits Ca++-ATPase, results in significant damage to myofibrillar structures (Duncan et al. 1980). Thus, loss of Ca++ homeostasis in injured muscle fibres may be in part due to decreased function of the normal sequestering mechanisms. In this review, emphasis is placed on the hypothesis that mechanical disruption of membranes is primarily responsible for the increase in intracellular Ca++ concentrations in injured muscle fibres. However, there are other mechanisms by which Ca++ homeostasis could be disturbed in the cells. Altered SR uptake of Ca++ was mentioned above; alterations in one or more of the following mechanisms could also affect intracellular concentrations: (a) Ca++ influx through voltage- or stretchactivated slow Ca++ channels; (b) Ca++ influx or effiux via the Na+ : Ca++ exchanger; and (c) ligand binding to Ca++-mobilising receptors that induce Ca++ influx from the extracellular space. Skeletal muscles possess voltage-dependent Ca++ channels that are primarily located in t-tubular membranes (Godfraind et al. 1986). Dihydropyridine-sensitive Ca++ channels in the t-tubules of rabbit skeletal muscles contain a peptide that is phosphorylated by cyclic AMP-dependent protein kinase, suggesting that Ca++ influx through these channels may be regulated by receptors associated with adenyl cyclase (Cooper et al. 1989). Whereas Ca++ entry through these channels is not generally considered to be important in excitation-contraction coupling in skeletal muscle, high concentrations of calcium channel blocking drugs (e.g. verapamil, diltiazem and D 600) attenuate force production in isolated muscles (Duan et al. 1990b; Gallant & Goettl 1985; Varagic & Kentera 1978). Using an isolated rat muscle model, we (Delp et al. 1989) have observed that static stretch of muscles results in elevations in intracellular Ca++

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levels which can be prevented with verapamil, a slow calcium channel antagonist. Others have also shown that skeletal muscles have stretch-sensitive calcium channels (LOpez et al. 1985; Snowdowne & Lee 1980); because muscles are stretched while they are active during eccentric contractions, it is possible that these channels are involved in elevating intracellular Ca++ concentrations during this type of exercise. Verapamil administration attenuated somewhat the elevations in muscle mitochondrial Ca++ levels resulting from downhill walking in rats (Duan et al. 1990a), but the drug does not block the uptake of Ca++ in soleus muscles isolated from rats that have just completed downhill walking (Duan et al. I 990b ). These findings suggest that the primary mechanism of Ca++ entry in muscles injured during exercise is not through slow channels, but do not finally disprove this hypothesis. Verapamil does attenuate injury in skeletal muscle resulting from ischaemia-reperfusion (Paul et al. 1990). In this type of injury, Ca++ influx into myocytes results in its accumulation in mitochondria (Murphy et al. 1987). Paul and coworkers (1990) suggested that the calcium channel blockers function in this model to inhibit Ca++ entry into the mitochondria, thereby protecting the respiratory capability of the organelles. Uptake and extrusion ofCa++ from the cell can occur via the Na+ : Ca++ exchanger (for review see Allen et al. 1989). There is no evidence that this mechanism is involved in Ca++ overload in skeletal muscles injured by exercise. However, the exchanger is implicated in the loss of Ca++ homeostasis that occurs in ischaemia/reperfusion injury to the myocardium (Grinwald & Brosnahan 1987; Philipson & Bersohn 1986; Renlund et al. 1986). In this model, high intracellular Na+ results in large influx of Ca++ in exchange for the Na+, or otherwise disrupts normal exchange of the two cations. Similarly, when cardiac muscle is incubated or perfused with Ca++-free medium, and then reperfused with Ca++, there is an influx of Ca++ during the reperfusion period resulting in loss of contractile ability and intracellular constituents (Zimmermann & Hulsmann 1966). Chapman and colleagues (see Chapman 1990) have presented sup-

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port for the hypothesis that during the Ca++-free perfusion there is an influx of Na+ through Ca++ channels. During the reperfusion period, the intracellular Ca++ concentration then increases through Na+: Ca++ exchange. According to this hypothesis, the critical event is the rise in the intracellular Na+ concentration during the Ca++-free perfusion phase. Several types of receptors exist in the muscle cell membrane that when bound by their agonists act to release Ca++ from its internal stores or to allow Ca++ influx from the interstitium (Abdel-Latif 1986; Axelrod et al. 1988; Exton 1988; Minneman 1988; Rosenthal et al. 1988). Binding of agonist to these Ca++-mobilising receptors activates phospholipase C through G-proteins; the resulting inositol 1,4,5-triphosphate and inositol cyclic1:2,4,5-triphosphate act on SR to promote Ca++ release into the cytoplasm (Putney et at 1989). The mechanism by which receptor activation causes Ca++ influx from the extracellular space is less clear, although there is evidence of G protein mediation (Exton 1988; Rosenthal et al. 1988). Inositol 1,4,5triphosphate may be responsible for stimulating Ca++ entry into cells, but not by acting directly on the Ca++ channels in the sarcolemma (Putney et al. 1989). Incubation of rat skeletal muscle with a adrenoceptor agonists [adrenaline (epinephrine), noradrenaline (norepinephrine) or phenylephrine] stimulates marked increases in inositol monophosphate (Schadewaldt et al. 1987); the effect can be blocked with a adrenoceptor antagonists (e.g. prazosin). A second receptor that has been shown to stimulate polyphosphoinositide turnover in skeletal muscle is the nicotinic-cholinergic receptor (Abdel-Latif 1986). It has been demonstrated, e.g. that blocking cholinesterases at the neuromuscular junction produces Ca++ influx into the muscles and local necrosis and contracture of the fibres, and that the pathology can be inhibited with a-bungarotoxin (nicotinic-cholinergic receptor blocker) or by deleting Ca++ from the incubation medium (Leonard & Sal peter 1979). Other receptor types that potentially could be involved in mobilising Ca++ and increasing Ca++ influx from the extracellular space are those for which bradykinin and histamine serve


as agonists (Axelrod et at 1988; Hill 1990; Kaya & Hong 1989). One of the experimental approaches that supports the importance of Ca++ in muscle injury is exposure of muscles to Ca++ ionophore (e.g. A23187) in vitro. The ionophore allows Ca++ to enter the fibres down its concentration gradient and causes SR to release Ca++ (Publicover et at 1978); both of these processes produce increases in free cytosolic Ca++ levels (Goodman 1987). This results in activation of contraction (Etlinger et al. 1981) and in rapid « 30 minutes) destruction of myofibrillar structure and loss of intramuscular enzymes into the incubation medium (Duncan & Jackson 1987). When there is no Ca++ in the incubation medium, the effect of the ionophore is significantly reduced (Rodemann et al. 1982). Also, the effects of the ionophore are dose-dependent; incubation of rat soleus muscles in relatively low concentrations of A23187 result in elevations in protein degradation, but no loss of myofibrillar structure or reduction in maximum twitch tension (Kameyama & Etlinger 1979). Data from this experimental model are important in the development of hypothetical constructs for exercise-induced muscle fibre injury, because two of the primary criteria for exercise-induced injury, i.e. enzyme loss and histopathology, have been shown to result from experimentally elevating intracellular Ca++ levels with ionophore treatment. In apparent contradiction to the reports of Duncan and colleagues (e.g. Duncan & Jackson 1987; Publicover et al. 1978), Goodman (1987) found that A23187 caused degradation of nonmyofibrillar proteins, but not those of myofibrillar origin. He based this conclusion on the observations that A23187 stimulated _protein degradation in the muscles, but did not cause increased release of methylhistidine, a product of contractile protein breakdown. The explanation for this apparent disparity is not totally clear, although it may simply reflect differences in ionophore dose. One of the consequences of elevated intracellular Ca++ concentrations in cells is referred to as blebbing, which involves formation of cytoplasmic enlargements on the cell surface. It is believed that

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cytoplasmic blebbing results from alterations in the relationship between cytoskeletal proteins (e.g. actin and tubulin) and the cell membrane (Orren ius et al. 1989; Phelps et al. 1989). In hypoxic hepatocytes, bleb rupture appears to signal cell death (Lemasters et al. 1989). Phelps et al. (1989) found that inhibitors of calmodulin attenuate bleb formation, suggesting Ca++ binding to calmodulin may regulate the process. Alternatively, activation of neutral proteases may degrade cytoskeletal proteins, causing disruption of the filament network and formation of blebs (Orrenius et al. 1989). Although the phenomenon of blebbing has primarily been described for nonmuscular cells, it has also been observed in myocardial cells in the ischaemia/reperfusion model (Ganote & Humphrey 1985). Implicit in this discussion of the probable role of Ca++ in the muscle fibre injury is the reversibility of the process during the phase in which Ca++ influx is initiated. Theoretically, the initiating lesions, whatever their nature, could occur to some extent in all the muscle fibres participating in a bout of exercise. However, according to the hypothetical model of injury presented in this review, if the cell is able to handle the local Ca++ influx effectively and maintain relatively low intracellular Ca++ levels, the sequence may never proceed to the autogenetic phase. On the other hand, if the intracellular level is elevated to the point that autogenetic Ca++-activated pathways are activated, the injury becomes irreversible. This scheme is not unlike that proposed to explain irreversible damage in the ischaemia/reperfusion model in the heart (e.g. Parsons et al. 1989). The emphasis in this review is on the various roles Ca++ may play in the degradative processes associated with exercise-induced injury. It should also be mentioned that the ion could be important in the adaptive response to the fibre to the exercise. As an interesting example of this possibility, elevations in intracellular Ca++ concentrations have been suggested to serve to activate stress protein gene transcription in cells (Welch 1990). Stress proteins are produced by cells in response to heat or other forms of stress (for reviews see Morimoto et

al. 1990; Pelham 1990; Schlesinger 1990; Welch 1990). Their induction provides the cells with a tolerance for further exposures to the stress, although the tolerance is transient. The protective functions of these proteins is not well understood; some appear to associate specifically with cytostructural proteins in the cells. There is evidence that these proteins are induced in muscles by exercise (Locke et al. 1990), and this mechanism could provide muscle fibres with a means of responding to exercise-induced trauma to protect the cells from future exposures.

3. Autogenetic Mechanisms During Muscle Injury The thesis has been developed that during exercise there is an initiating failure in some structural component of the muscle fibre; in particular, physical forces cause a disruption of the normal permeability barrier to extracellular Ca++. This allows Ca++ to enter the cell at the site of membrane damage, overwhelming the Ca++ buffering systems in the fibre (i.e. Ca++-binding proteins, SR, mitochondria, and nuclei). Once free cytosolic Ca++ concentrations attain critical levels, are elevated for sufficient periods of time, or are elevated within specific compartments within the fibres, various Ca++-activated degradative mechanisms start to operate in the injured muscle fibres. In myocardial ischaemia, free cytosolic Ca++ levels increase, but are not immediately associated with lethal injury (Steenbergen et al. 1987b). These authors suggest, however, that if a high cytosolic Ca++ concentration is maintained, degradative enzymes would be activated in the cells. As described in the preceding section, this may represent the point in time when the injury becomes irreversible. The following discussion covers some of the autogenetic, or intrinsic, pathways in the muscle fibre that may be activated when Ca++ homeostasis is lost in the cell. 3.1 Contracture Although this phenomenon is not a 'degradative pathway' in the same sense as the enzyme-catalysed processes discussed below, loss of Ca++

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homeostasis in the muscle fibre can result in uncontrolled contraction of the sarcomeres in the affected area. This phenomenon has been observed in the muscle fibres of rats that have walked downhill (Ogilvie et al. 1988). Immediately following the exercise, some parts of some myofibrils in the injured muscles were 'hypercontracted' or 'clotted'. The incidence of this type of disruption was low « 5% of the lesions), but if rapid elevations of intracellular Ca++ levels occur in sarcomeres that have sufficient ATP to support contraction, it is reasonable to expect that these sarcomeres would go through a period of contracture. As described above, this band of hypercontracture may serve to wall otT the injury and protect adjacent sarcomeres from the degradative processes (Carpenter & Karpati 1989). Contracture of sarcomeres has also been described in other injury models in which there are elevations in intracellular Ca++ levels (Publicover et al. 1978). Also, when isolated muscles are exposed to the Ca++ ionophore A23187, resting tension increases (Edinger et al. 1981), suggesting the elevation in the Ca++ concentration activates contraction. Like skeletal muscle, when cardiac muscle has elevated intracellular Ca++ levels, hypercontraction is observed (Rudge & Duncan 1984). Uncontrolled contraction of the muscle fibre could have several detrimental consequences. First, there may be a local depletion of high energy phosphates as a result of the continuous contraction. This reduced ATP concentration would contribute to the positive feedback loop described above in which energy stores decrease as the intracellular Ca++ level increases. This suggestion is supported by the findings of Goodman (1987), who showed that exposure of isolated rat muscles to Ca++ ionophore (A23187) resulted in ATP depletion and elevated lactate production. Secondly, uncontrolled contraction could produce mechanical forces in the fibres that further damage structural components in the membranes or contractile elements. Thus, uncontrolled contraction of sarcomeres may simply be one etTect of elevated intracellular Ca++ concentrations, but it would contribute to the local progression of the pathology while simultaneously


protecting the surrounding tissue through walling otT of the lesion (Carpenter & Karpati 1989). In concluding this section, it should be pointed out that if depletion of ATP (e.g. from elevated Ca++-ATPase activity) precedes loss of Ca++ homeostasis, contracture of atTected sarcomeres would presumably be minimal. As pointed out above, the sequence of events is not known, and both intracellular Ca++ elevation and decreased ATP concentrations occur as part of the vicious cycle associated with the injury process. 3.2 Mitochondrial Ca++ Overload The mitochondria in muscle fibres serve to butTer elevations in cytosolic Ca++ levels. Although it is generally believed that the uptake of Ca++ by mitochondria is too slow for the organelle to contribute significantly to muscle fibre relaxation, the mitochondria can accumulate large quantities of the ion (up to 3 ~mol/mg mitochondrial protein) under pathological conditions (Gillis 1985). Slowtwitch oxidative fibres may be particularly prone to accumulating Ca++, since mitochondria from slow fibres can sequester Ca++ at a rate 2 to 3 times that in fast-twitch fibres (Sembrowich et al. 1985). Uptake of excessive amounts of Ca++ by the mitochondria is accompanied by uptake of phosphate, so that precipitates of calcium phosphate may deposit in the intramitochondrial space (Gillis 1985). Whereas increases in mitochondrial Ca++ levels in the nanomolar range serve to stimulate mitochondrial respiration (Hansford 1985; McMillin & Madden 1989), accumulation of Ca++ in the micromolar range depresses mitochondrial function (Wrogemann & Pena 1976). 3.3 Ca++-Activated Neutral Proteases The Ca++-dependent proteases, referred to as cal pains by Murachi et al. (1981), are identified as type I or type 2 depending on the Ca++ level required to activate them. The type 1 isoform is active in the presence of micromolar amounts of Ca++, the type 2 enzyme in the presence of millimolar quantities (Murachi et al. 1981). Unlike the


lysozomal proteases, these enzymes have neutral pH optima. Whereas they are not highly specific for any protein or peptide sequence (Mellgren 1987), their activation is associated with degradation of particular structures in the muscle cell. This suggests the enzymes are localised in the fibres. For example, Ca++-activated proteases may specifically degrade Z-discs (Busch et al. 1972; Cullen & Fulthorpe 1982; Dayton et al. 1976; Ishiura et al. 1980) or contractile filament components (Cullen & Fulthorpe 1982; Dayton et al. 1976, 1979). Ca++-activated proteases associated with these specific structures could explain the observed Z-line streaming (Friden et al. 1981, 1983; Ogilvie et al. 1988) or the disruption of the A-band (Friden et al. 1983; Newham et al. 1983; Ogilvie et al. 1988) that occur in muscle fibres injured by eccentric exercise. Calpain appears to degrade Z-discs by digesting the proteins zeelin 1 and zeelin 2, which are thought to anchor a-actinin in the disc; a-actinin is released by the action of the protease (Bullard et al. 1990). At low Ca++ levels, activity ofthe enzyme is increased by presence of phospholipids (Bullard et al. 1990). There also is evidence that the cytoskeletal proteins that anchor intracellular components to the sarcolemma are particularly good substrates for the Ca++-activated proteases (Pontremoli & Melloni 1986). It has been proposed that proteolysis of vinculin, a cytoskeletal protein that anchors the cell membrane to the cytoskeleton, by Ca++-activated proteases leads to increased sarcolemmal fragility in myocardial cells during ischaemia (Steenbergen et al. 1987a). Cells also contain a Ca++-dependent protease inhibitor, or calpastatin, which effectively inhibits both types of the Ca++-activated neutral proteases (Mellgren & Carr 1983; Murachi et al. 1981). Although the protein is assumed to be cytosolic, there is evidence that it is associated with the sarcolemma and possibly SR in heart cells (Lane et al. 1985; Mellgren et al. 1987). It has been suggested that the inhibitor protein functions to protect membrane proteins from proteolysis during brief exposures to Ca++ (Mellgren 1987). Studies on isolated muscles using Ca++ ionophore treatment do not support a significant role

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for the Ca++-activated proteases in muscle damage or protein degradation. Rudge and Duncan (1984) attempted to attenuate the structural injury in muscles induced by Ca++ ionophore (A23l87) by inhibiting Ca++-activated proteases with leupeptin. The drug was not effective in preventing the myofibrillar damage, suggesting the rapid ionophoreinduced injury is not caused by Ca++-activated proteases. These findings agree with those of Rodemann et al. (1982), who showed inhibition of Ca++-activated proteases with mersalyl does not alter the rate of protein degradation induced by A23187. Taken together, these data indicate that the muscle degradation accompanying rapid elevations in intracellular Ca++ concentrations induced by ionophore is not caused by the Ca++-activated proteases. It is possible that increases in intracellular Ca++ level of the magnitude induced by the Ca++ ionophore simply overwhelm the cell and mask more subtle proteolytic processes that occur with smaller changes in intracellular Ca++ levels. 3.4 Lysosomal Proteases Myofibrillar proteins can be degraded by proteolytic enzymes contained in lysosomes in the muscle fibres (Schwartz & Bird 1977; Matsumoto et al. 1983). It is reasonable to hypothesise that these proteases may play a role in the autogenic phase of exercise-induced muscle injury. Following exhaustive treadmill running in mice there are large increases in the activities of lysosomal acid hydrolases in the muscles (Vihko et al. 1978), but these elevations are not observed until the second day after exercise. The authors concluded that a significant proportion of the elevated protease activity was contributed by the muscle fibres per se, although infiltrating cells were responsible for part of the change. Nonetheless, there is no evidence that intrinsic lysosomal proteases are released or activated during, or immediately after, exercise that injures skeletal muscles. In some tissues it has been demonstrated that calmodulin is associated with lysosomal vesicles, indicating Ca++ may serve to activate the pro-

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teases, perhaps through exocytosis of the enzymes from the organelles (Nielsen et al. 1987). In leucocytes, Ca++ stimulates lysosomal enzyme discharge (Hoffstein & Weissmann 1978). Also, there is evidence that lysosomal enzymes are activated when muscles are exposed to Ca++ ionophore (A23187) [Rodemann et al. 1982). These authors interpreted their data to indicate that elevated intracellular Ca++ levels activate phospholipase A2 (PLA2), which increases production of prostaglandins. In particular, prostaglandin E2 (PGE2) produced by PLA2 activation appeared to stimulate lysosomal protease activity. They arrived at this conclusion because protein degradation could be inhibited similarly by blocking cyclo-oxygenase or PLA2 or by blocking lysosomal thiol proteases. However, from more recent studies from the same laboratory, it was concluded that lysosomal proteases are not involved in protein degradation by incubation in A23187 or injury by cutting (Furuno & Goldberg, 1986). Also, the rapid myofibrillar damage that occurs in isolated muscles exposed to the ionophore is not blocked in chemically-skinned fibres or isolated muscles by inhibitors of various lysosomal proteases (Duncan 1987; Duncan et al. 1979; Duncan & Rudge 1988), suggesting that these enzymes are not involved in this pathology. In conclusion, muscle fibres contain Ca++-dependent mechanisms for stimulation of lysosomal proteases; these pathways may play a role in the autogenetic phase of exercise-induced muscle fibre injury, although findings from studies using other models do not indicate they play an important role. This possibility awaits further clarification. 3.5 The Phospholipase A2 (PLA2) Pathway PLA2 is the first enzyme in the pathway that uses membrane phospholipids as substrate for production of arachidonic acid and, subsequently, prostaglandins, leukotrienes and thromboxanes. The enzyme is located in the sarcolemma, membranes of organelles, the cytosolic compartment and in lysosomes (van der Vusse et al. 1989). PLA2 associated with mitochondrial membranes has been hypothesised to play a role in the loss of Ca++


homeostasis that occurs in malignant hyperthermia (Cheah & Cheah 1985). There are Ca++independent forms of PLA2 in the cytosol or lysosomal compartments that have acid pH optima, but the primary form of the enzyme in most tissues is membrane-bound, Ca++-dependent and maximally active in neutral or basic pH (Chang et al. 1987; Scheuer 1989). There is some question about the physiological role of Ca++ in controlling these enzymes (Chang et al. 1987; Irvine 1988; van der Vusse et al. 1989). PLA2 activity may be regulated by receptors through G proteins (Axelrod et al. 1988; Irvine 1988), which are different than those associated with phospholipase C activation (Axelrod et al. 1988). The enzyme also appears to be regulated by endogenous stimulatory and inhibitory peptides. An example is lipocortin, an inhibitory peptide, whose synthesis is induced by glucocorticoids (Chang et al. 1987; Vane & Botting 1987). Although lipocortins are effective at blocking arachidonic acid production, there is some disagreement about their mode of action; they appear to be identical to the proteins referred to as calpactins, which bind Ca++ and phospholipids (Vane & Botting 1987). Thus, the lipocortins may attenuate PLA2 activity through means other than direct inhibition of the enzyme. Elevations in intracellular Ca++ concentrations would thus be expected to stimulate PLA2 activity, resulting in a variety of products that could play autogenetic roles in exercise-induced muscle fibre injury. Arachidonic acid and lysophospholipids, the immediate products of PLA2 activity, can have a detergent effect on cell membranes, affecting stability of the structures (Chang et al. 1987; Jackson & Edwards 1986). Some lysophospholipids are also stimulators or inhibitors of protein kinase C, thus serving a regulatory role as second messengers (Oishi et al. 1989). As indicated in the preceding section, PGE2 produced in the cyclo-oxygenase pathway may serve to activate lysosomal proteases, and hence, protein degradation (Rodemann et al. 1982) [although more recent work from the same laboratory indicates this is not the case (Furuno & Goldberg 1986»). Leukotrienes have also been implicated in the release of intramuscular enzymes from muscles exposed to


Ca++ ionophores (Duncan & Jackson 1987; Jackson et al. 1987), as described in more detail below. Finally, cis-epoxyeicosatrienoic acid products of the 'epoxygenase' pathway have been shown to elevate free cytosolic Ca++ in some tissues (Fitzpatrick & Murphy 1989). An appreciation for the potency of PLA2 and its products in muscle can be gained from research on venoms. The enzyme is one of the primary active ingredients in snake and bee venom; PLA2 purified from snake venom reproduces all of the symptoms of snake bite (Chang et al. 1987). Injection of coral snake venom into mouse muscle produces injury that is qualitatively similar to that resulting from eccentric exercise, i.e. the muscles lose creatine kinase and experience hypercontraction and disruption of ultrastructural components (Arroyo et al. 1987). However, the necrosis of the muscle fibres caused by the venom is rapid and drastic (Arroyo et al. 1987). Injection of 2~g of venom from the Australian tiger snake into the soleus muscle ofa rat destroys all of the fibres within 24 hours (Harris 1989). Thus, activation of the PLA2 pathway through elevations in intracellular Ca++ levels or by other means (e.g. van der Vusse et al. 1989) during and immediately after exercise could serve as an important autogenetic mechanism in exercise-induced muscle fibre injury. Although the focus in this review is on the potential involvement ofPLA2 in degradative processes in the muscle fibres, the enzyme also serves a 'protective' role in that its activity increases during peroxidation of membrane phospholipids; PLA2 excises oxidised fatty acids, thus protecting the membranes from oxidative injury (Van Kuijk et al. 1987). Jackson et al. (1984) found that electrical stimulation of isolated mouse muscles in the presence of dinitrophenol and Ca++ resulted in loss of lactate dehydrogenase (LDH) activity into the incubation medium. When Ca++ was left out of the incubation medium, LOR loss was markedly attenuated. Furthermore, inclusion of PLA2 inhibitors [chlorpromazine, dibucaine, or mepacrine (quinacrine») in the medium in the presence ofCa++ reduced the enzyme loss. These results implicated

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Ca++ in the pathology, and indicated that PLA2 activation is a contributing mechanism to the loss of intramuscular enzymes during muscle fibre injury. In further experiments, Jackson et aI. (1987) found that A23187 causes muscles to release prostaglandins, but that blocking prostaglandin synthesis with cyclo-oxygenase inhibitors did not prevent enzyme loss. On the other hand, lipoxygenase inhibitors effectively protected the muscles from enzyme loss during incubation with the ionophore. Duncan and Jackson (1987) reported that different Ca++-activated mechanisms are responsible for the loss of intramuscular enzymes and for myofibrillar damage when muscles are exposed to A23187. Chlorpromazine, an inhibitor of PLA2 activity, blocks release of creatine kinase into the medium when muscles are incubated with ionophore, but does not prevent the myofibrillar damage to the fibres. These authors, in agreement with the results of Jackson et al. (1987), showed that nordihydroquaiaretic acid, an inhibitor of lipoxygenase, effectively blocks the loss of enzymes, indicating that leukotrienes (or possibly free oxygen radicals produced in the lipoxygenase reaction) are responsible for the phenomenon. Thus, Ca++ appears to cause release of intramuscular enzymes through activation of the PLA2 pathway and, subsequently, lipoxygenase; the mechanism by which Ca++ causes destruction of myofibrils is undetermined. It is not known whether or not Ca++-activation of PLA2 is responsible for the enzyme loss from muscles that occurs during and after exercise. This possibility should be experimentally investigated. When muscles are incubated in the presence of arachidonic acid, protein degradation is increased with no change in protein synthesis (Rodemann & Goldberg 1982). This stimulation of proteolysis by arachidonic acid can be blocked with inhibitors of prostaglandin synthesis (aspirin, indomethacin and meclofenamate) [Rodemann & Goldberg 1982). Some investigators (e.g. Barnett & Ellis 1987) have not found that PGE2 plays a role in protein degradation in isolated muscles. PLA2 activation by elevations in intracellular Ca++ concentrations is one of the primary hypotheses proposed to explain the injury to myo-


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cardium from ischaemia (Buja & Willerson 1989); e.g. in the ischaemic heart, arachidonic acid accumulates in significant amounts when ATP level falls below 8 to 10 I'molfg dry weight (van der Vusse et al. 1989). According to this model, ischaemia leads to alterations in Ca++ transport from the myocytes and elevated release from SR and mitochondria, which in tum lead to activation of Ca++-dependent phospholipases and other proteases. This results in a vicious cycle leading to further loss of membrane integrity and Ca++ loading. This positive feedback system would eventually terminate in irreversible cell injury. Whereas most hypotheses concerning ischaemic injury in the heart attribute functional significance to elevations in intracellular Ca++ levels, increases in the ion per se may not be the cause of the damage (Lefurgey et al. 1989), but elevations in the face of decreased ATP concentrations may be what causes irreversible injury. Human studies, in which SUbjective sensations of soreness and/or measures of plasma levels of intramuscular enzymes were used as criteria of injury, have been used to assess the efficacy of antiinflammatory drugs (which block cyclo-oxygenase, and to a lesser extent, PLA2) in attenuating exercise-induced injury (Donnelly et al. 1988; Francis & Hoobler 1987; Kuipers et al. 1985). Whereas these studies do not provide a clear answer to whether or not anti-inflammatory drugs block the injury response to exercise, it would have to be assumed that from the injury criteria used, they would not provide much evidence concerning the role of autogenetic PLA2 activation within the muscle fibres during the immediate postexercise period. For example, PGE2 levels in the blood following eccentric exercise are not significantly elevated until 24 hours after the exercise (Smith LL, personal communication).

4. Conclusions The focus of this review has been quite narrow. It has dealt specifically with potential mechanisms responsible for the initiation of exercise-induced skeletal muscle fibre injury. Unfortunately, many

Phases

Mechanisms

Initial Eccentric contractions

+ +

Sarcomere inhomogeneities

sarcolemmi disruption Ca++ Overload Ca++ influx

t

[Ca++]j '" 0.1 "moi/L (Ca++-ATPase activity> Ca++ influx; t [ATP]; 'reversible')

+

[Ca++]j > > 0.1 "mol/L (Ca++-ATPase activity < Ca++ influx; ~ [ATP]; 'irreversible') Autogenetic

f

,

I

t

,

t Protease

t Phospholipase

activity

activity

Myofibrillar degradation

Membrane degradation

(PhagocytiC) (Regenerative)

Fig. 3. Simplified hypothetical model of the phases of exercise-induced muscle fibre injury. Refer to the text for descriptions of the phases, and for alternative hypotheses.

interesting questions peripheral to this focus could not be addressed, such as why does one fibre fail during exercise, while adjacent fibres remain intact? What cellular mechanisms explain the rapid adaptation in muscles that protect them from injury in subsequent bouts? With the advances in research technology and our understanding of cell biology, these questions, and those posed throughout this review, can be answered. However, the problems are complex, and almost certainly involve a variety of pathways and phenomena that are sequentially activated in the injury process. For this reason care must be observed in suggesting an ov-


ersimplified model of exercise-induced muscle fibre injury. Nonetheless, figure 3 presents a hypothetical pathway for the injury process that incorporates some of the information contained in this review. It seems reasonable in light ofthe literature, and the steps can be experimentally tested in the laboratory.

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Fibre type-specific hypertrophy mechanisms in human skeletal muscle: potential role of myonuclear addition.

Fibre number and fibre size in a surgically overloaded muscle.

Jitter in the muscle fibre.

Muscle fibre splitting--a reappraisal.

Muscle fibre type and aetiology of obesity.

Muscle injury, cross-sectional area and fibre type distribution in mouse soleus after intermittent wheel-running.

Muscle fibre number following hindlimb immobilization.

Muscle fibre type and habitual snoring.

Histochemical fibre types in human extraocular muscle.

Muscle fibre type changes in hypothyroid myopathy.

The influence of muscle length on muscle fibre conduction velocity and development of muscle fatigue.

Do muscle fibre size and fibre angulation correlate in pennated human muscles?

Histochemical and functional fibre typing of the rabbit masseter muscle.

Ultrastructure of the syndrome of continuous muscle fibre activity.

Fatiguability and fibre composition of human skeletal muscle.

Metabolic characteristics of fibre types in human skeletal muscle.

Metabolic substrates, muscle fibre composition and fibre size in late walking and normal children.

Rigorous analysis of light diffraction by a striated muscle fibre.

Postnatal centralization of muscle fibre nuclei in centronuclear myopathy.

Influence of muscle geometry on shortening speed of fibre, aponeurosis and muscle.

Isometric strength performance and muscle fibre type distribution in man.

Quadriceps muscle fibre dysfunction in patients with pulmonary arterial hypertension.

Muscle fibre type composition and body composition in hammer throwers.

Evidence for cross bridge slippage in a stretched muscle fibre.