376 J.A. Faulkner et al. With maximum activation, the forces developed are greatest during lengthening contractions, intermediate during isometric contractions, and least during shortening contractions. The explanation for the greater force during lengthening contractions than during isometric contractions is that during isometric contraction only the cross-bridges that are in their driving stroke generate tension, but when a maximally activated muscle is stretched, additional strongly-bound cross-bridges that have not progressed into their ‘driving stroke’ resist the ‘lengthening’ of the skeletal muscle, are strained and generate force. Consequently, the force developed during a lengthening contraction can exceed that developed during an isometric contraction by as much as twofold. The high forces developed during lengthening contractions are partially responsible for the high susceptibility of muscles to contraction-induced injury during this type of contraction. In fact, only the lengthening contractions are capable of producing a contraction-induced injury. 3 Age-Related Muscle Wasting and Muscle Weakness The ‘wasting’ or ‘atrophy’ of a skeletal muscle refers to a loss in the mass of the skeletal muscle, a condition that arises from a reduced usage of skeletal muscles at any age. The reduction in the daily usage may arise from: (a) sickness and imposed bed-rest, (b) disuse of a specific muscle due to immobilization by casting, or to the placement of an injured arm in a sling. In addition, by 70–80 years of age an outright loss of skeletal muscle fibers occurs that is estimated, based on data from vastus lateralis muscles, to be as high as 50% of the fibers (Lexell et al. 1988). The loss in the number of muscle fibers contributes significantly to the concurrent loss of muscle mass and myofibrillar protein. In contrast to atrophy, ‘weakness’ of a muscle reflects an inability of a muscle to generate the normal or expected force when activated. As people age, particularly into advanced old age, the vast majority of humans, both men and women, become less physically active and invariably show signs of both muscle wasting and muscle weakness. Particularly in old age, the combined impact of decreased physical activity and muscle wasting and weakness lead to the debilitating condition of frailty (Hadley et al. 1993). The increase in physical frailty with old age has serious consequences in terms of the health and longevity of the elderly. Physical frailty invariably leads to a further decrease in physical activity as well contributing to respiratory and cardiovascular problems (Hadley et al. 1993). Despite the magnitude of the problem, even in the elderly, these conditions are at least partially reversible by re-establishing an increased level of physical activity, but such programs must be carefully designed with a slow progression and close supervision by highly trained exercise leaders. Although some amelioration of muscle atrophy is achievable through exercise, the component of muscle atrophy that is due to the loss of muscle fibers appears inevitable and irreversible. Consequently, the magnitude of the improvements attainable with physical training of the frail elderly must be realistic and kept in perspective with the limitations of the participants. 377Role of Contraction-Induced Injury in Age-Related Muscle Wasting and Weakness 4 Late-Onset Muscle Soreness The phenomenon of a contraction-induced injury to skeletal muscle fibers was first recognized inadvertently by Theodore Hough, during experiments on the fatigue of finger flexor muscles (Hough 1901, 1902). Hough’s subjects performed a highly fatiguing muscle contraction protocol using a pulley-system that enabled lifting and lowering a weight with flexion and extension of the middle finger. Some of the participants complained of pain in the forearm between 8 and 12 h after the comple- tion of the protocol, with the soreness increasing and reaching its highest level 48 or even 60 h afterward. In these experiments, it was not recognized that the sore- ness was initiated by the lowering of the weight. The phenomenon of muscle sore- ness encountered in the Hough studies was ignored for almost 80 years, and then re-surfaced as ‘delayed onset muscle soreness’ in the early 1980s. Late onset muscle soreness has been observed after a number of different protocols that involved the lowering of a weight or the ‘stretching’ of the activated skeletal mus- cle fibers and a number of inventive protocols were developed to investigate the factors involved in the lengthening contractions that initiated the delayed soreness of the muscle. These early protocols involved repeatedly stepping up with one leg and down with the other leg on and off a fairly high stool (Newham et al. 1983a, b), raising and lowering a weight with forearm flexion and extension (Newham et al. 1987), and resisting the reverse-rotation of the pedals of a bicycle ergometer (Friden et al. 1983). Needle biopsy samples of both arms and legs indicated that these protocols of lengthening contractions invariably caused morphological evi- dence of injury to skeletal muscle fibers (Fig. 2a–c). Lengthening contractions produce a decrease in maximum strength and assays of blood samples indicate a peak in plasma creatine kinase several days after the initial injury (Fig. 3a). Subjective assessments of pain indicate that the exact timing of the onset of muscle soreness varies somewhat with the individual and with the type of exercise, but typically peaks after ~2 days and is resolved within 5 days. The recovery of strength and reestablishment of pre-injury levels of circulating creatine kinase take anywhere from 1 to 2 weeks depending on the severity of the injury and repeated bouts of training with lengthening contractions reduce the occurrence of late onset muscle soreness (Newham et al. 1983a, b). The experi- ments on volitional lengthening contractions performed by human subjects were soon followed up with more definitive experiments on mice and rats (Armstrong et al. 1983; McCully and Faulkner 1985). The experiments on small mammals substantiated the time course of the injury to muscle fibers and that the magnitude of the injury was greatest approximately 3 days after the lengthening contraction protocol with complete recovery requiring 3–4 weeks (Fig. 3b). A number of fac- tors have been cited as the likely causes of the late-onset muscle soreness. The most plausible of these factors are the actual damage to muscle fibers and connective tissue and inflammation (Cheung et al. 2003; Friden et al. 1983, 1986; Newham et al. 1983a, b; Jones et al. 1986; Schwane and Armstrong 1983). From the begin- ning, Hough (1902) cited the ruptures within the muscles as the cause of the Fig. 2 Electron micrographs from an EDL muscle of a young mouse after a protocol of 75 lengthening contractions. (a) A longitudinal section of a single fiber at high magnification taken immediately after a severe lengthening contraction protocol. Note that some sarcomeres have actually shortened down to a 1.40 mm length, whereas the weaker sarcomeres have been damaged severely through a stretch out to a 3.80 mm that has displaced the thick filament to one end of the sarcomere or the other. This segment of this fiber will undergo the degenerative and regenerative stages shown in Fig. 6. This photomicrograph depicts a part of a single fiber in Stage 2. (b) A longitudinal section of a myofiber 10 min after a lengthening contraction protocol showing areas of focal damage (*) within single or small groups of sarcomeres. In some sarcomeres, the damage appears to be in the A-band region, with Z-lines remaining intact, whereas in other sarcomeres the damage involves the Z-lines. (c) Transverse sections of a muscle 3 days after the protocol. Muscle fibers range from those with intact myofibrils (M3 and M4) to those with degenerating myofibrils (M1) or devoid of cytosolic constituents (M2). Fiber M2 has phagocytes (P) within the basement membrane (arrows). C is a capillary (Figure 2b and c reproduced from Faulkner et al. 1995 with permission of Oxford University Press) Hours 01 2310614 Maximum Value (%) 0 20 40 60 80 100 Muscle pain Plasma creatine kinase Maximum isometric strength Days Time After Initial Injury Hours 01 23610 14 Maximum Value (%) 0 20 40 60 80 100 EDL muscles TBA muscles Days Time After Initial Injury Maximum isometric force of a b Fig. 3 Data are given for several indices of contraction-induced injury measured prior to and at selected time periods following a protocol of lengthening contractions administered to (a) the elbow flexor muscles of human beings and (b) the ankle dorsiflexor muscles of mice. The values indicated on the abscissa are the times in “hours” and “days” after the initiation of the contraction protocols. (a) Eight human subjects (age 24–43 years) performed maximal lengthening contractions of the elbow flexor muscles once every 15 s for 20 min. (b) The dorsiflexor muscle group of mice was exposed to a maximal lengthening contraction every 5 s for 30 min during plantar flexion of the ankle with the foot in a “shoe” apparatus. Data are shown for the maximum isometric forces developed by the tibialis anterior (TBA) and extensor digitorum longus (EDL) muscles measured in vitro following the injury protocol (n = 4−9 for each data point). All values are expressed as percentages of the maximum value for each variable. For isometric strength and maximum isometric force, the maximum values were achieved by all subjects prior to the exercise and are taken as 100%. For muscle pain and plasma creatine kinase, each subject did not reach his or her maximum values on the same day. Therefore, the peak values for these variables do not correspond to 100%. Values are given as means ± standard errors. When no error bars are shown, they are contained within the symbol (Modified from data in Newham et al. 1987; Faulkner et al. 1989; with permission. Reprinted from Faulkner et al. 1993; with permission of the American Physical Therapy Association. This material is copyrighted, and any further reproduction or distribution is prohibited) 380 J.A. Faulkner et al. soreness, although he had no direct evidence for this. Later needle biopsy studies of humans definitively demonstrated ultrastructual disruptions within muscle fibers associated with late-onset muscle soreness. 5 The Cause of the Contraction-Induced Injury The concept of a contraction-induced injury that occurred only when skeletal muscle fibers were activated to produce high forces and then stretched was slow to evolve. Early investigations of lengthening contractions focused primarily on the absorption of the work done on the muscle and the ‘heat of lengthening’ (Abbott et al. 1951). A major advance occurred in the understanding of the physiological cost of positive and negative work with the Abbott et al. (1952) study utilizing the modified bicycle-ergometer that enabled both positive and negative work to be performed. Knuttgen and his colleagues (Knuttgen and Saltin 1972; Knuttgen et al. 1982) also modified a bicycle ergometer to enable subjects to pedal against the load and perform lengthening contractions with either the arms or the legs. The fourfold difference observed between the energy cost during the shortening compared with the lengthening contractions is rather amazing (Fig. 4) and the complex physiologi- cal implications of this difference in energy cost are still not understood. The focus of the research on lengthening contractions gradually shifted to the effects of the lengthening contraction protocols on muscle pain and damage. The prevailing view initially was that as long as a given protocol of contractions was sufficiently intense, select populations of fibers would be injured. Armstrong (1990) expressed this view at a Symposium on Muscle Injuries, when he wrote that “muscular Work (kg m/min) 1500 1000 500 0 500 1000 1500 Oxygen consumption (l./min) 0.5 1.0 1.5 2.0 2.5 3.0 3.5 Free-wheeling (mean) Resting (mean) Fig. 4 Variation in the rate of oxygen consumption with the rate of work in pedaling for both positive and negative work (Reproduced from Abbott et al. 1952 with permission of Wiley) 381Role of Contraction-Induced Injury in Age-Related Muscle Wasting and Weakness exercise commonly results in injury to fibers in active muscles, particularly when the exercise is relatively intense, is of long duration, and/or includes lengthening contractions”. The hypothesis that “eccentric” exercise (exercise that involves lengthening contractions of muscles) preferentially damages fibers (Newham et al. 1987) was explored using comparable protocols of lengthening, shortening and isometric contractions of isolated muscles of mice (McCully and Faulkner 1985). With experiments on in situ single muscles of mice or rats (McCully and Faulkner 1985; Brooks and Faulkner 1990; Brooks et al. 1995) or single permeabilized fibers obtained from muscles of mice or rats (Macpherson et al. 1996; Brooks and Faulkner 1996; Lynch et al. 2008), precise protocols of lengthening contractions were designed to investigate the underlying mechanisms responsible for the injury associated with lengthening contractions. Such experiments demonstrated conclusively that injury was only observed following lengthening contractions regardless of the intensity of the shortening or isometric contraction protocol (McCully and Faulkner 1985). Furthermore, the magnitude of the injury induced by a given protocol of lengthening contractions was found to be a function of the force developed during the lengthening contraction, the magnitude of the stretches imposed, and the number of repetitions of the lengthening contractions in a given protocol (Brooks et al. 1995; Lynch et al. 2008; McCully and Faulkner 1986). Contraction-induced injury is thus most likely to occur during activities that involve a severe lengthening of a maximally activated muscle, such as lowering a very heavy object, or with multiple lengthening contractions of smaller groups of motor units as in distance running (Komi 2000). Running at relatively high speed, even on the level, involves stretching of the quadriceps muscles on the landing (Komi 2000), and running faster or longer distances than a runner is accustomed to may result in contraction-induced injury to fibers in the muscles involved. In any given activity, untrained participants are much more likely to experience a contraction- induced injury than trained subjects. Despite the protection provided by training, even trained athletes may sustain a contraction-induced injury during transition periods when training loads or work-outs are increased or modified. After single lengthening contractions (Brooks and Faulkner 1990; Li et al. 2006) or a protocol of many lengthening contractions (McCully and Faulkner 1986), the severity of a contraction-induced injury is most accurately assessed by the deficit in force generation (Fig. 5). An immediate force deficit occurs when a maximally activated fast skeletal muscle fiber of a rat is stretched through a single 20% strain (Macpherson et al. 1996; Lynch and Faulkner 1998; Panchangam et al. 2008) or an in situ skeletal muscle is stimulated maximally and stretched through a 20% strain for three 5-min contraction periods separated by 5 min (McCully and Faulkner 1985). The single 20% lengthening contraction of the single fiber produced a 17% force deficit in fast fibers of rats (Macpherson et al. 1996; Panchangam et al. 2008), whereas the 450 lengthening contractions of extensor digitorum longus muscles of the mice produced a 60% force deficit immediately afterward (McCully and Faulkner 1985). Force deficits invariably cause a more severe initial injury in muscles of old compared with young or adult animals. When activated maximally and exposed to 382 J.A. Faulkner et al. a single stretch through 30% of fiber length, a small 8–10% force deficit was observed for in situ extensor digitorum longus (EDL) muscles of young and old mice, but 40% and 50% strains produced large force deficits with the muscles of the old experiencing twofold greater force deficits than those of the young and adult mice (Fig. 5a and b). For single permeabilized fibers from fast muscles of rats, Strain (% L f ) 01020304050 Force Deficit (%) 0 20 40 60 80 100 a b Work (J/kg) 050 100 150 200 250 300 Force Deficit (%) 0 20 40 60 80 100 Young Mice (Brooks et al., 1995) Adult Mice Old Mice Fig. 5 The force deficits following single stretches of maximally activated muscles.Data are presented for single stretches varying in magnitude but not velocity(V = 2 L f s −1 ) for pooled young and adult mice ( • ) and old mice ( • ) in (a) and in situ EDL muscles of young (Ñ), adult ( ° ) and old ( ° ) mice in (b). The work input during the stretch is normalized by muscle wet mass (J kg −1 ), strain is expressed as a percentage of optimum fiber length (L f ), and the force deficit observed 1 min after the stretch is expressed as a percentage of the isometric force developed just prior to the stretch. Each symbol in (b) indicates a data point from a single stretch. The coefficients of deter- mination for the regression relationships for data from adult mice (continuous line) and old mice (dashed line) are 0.59 and 0.77, respectively. The slopes of the relationships, 0.20 for muscles in adult mice and 0.39 for muscles in old mice, are significantly different. Data for young mice (r 2 = 0.73; slope − 0.13) are reproduced from Brooks et al. 1995. Data in (a) are presented as means ± S.E.M. Sample size is from 3 to 12 for each point. *Significant difference (P <− .05) in the mean force deficits between the two groups (Reprinted from Brooks and Faulkner 1995) 383Role of Contraction-Induced Injury in Age-Related Muscle Wasting and Weakness force deficits immediately after single strains of 10% or greater were approximately twofold larger for single fibers from muscles of old compared with those from adult animals (Brooks and Faulkner 1996; Lynch et al. 2008). In combination, the whole muscle and single fiber experiments indicate a greater susceptibility of muscles in old animals to injury that is due at least in part to a mechanically compromised sarcomeric structure that is less able to withstand stretch. 6 Progression of the Injury The severity of the contraction-induced injury is a direct function of how severely single fibers are injured and how many fibers are injured sufficiently to initiate the cascade of events associated with a secondary injury. This cascade of events involves phases of contraction-induced injury to skeletal muscles that can be broadly categorized as: (1) the initial lengthening contraction that triggers the injury; (2) an autogenic stage that includes degradation by proteolytic and lipolytic systems indigenous to the fibers, (3) a phagocytic stage from 4 to 6 h through 2–4 days including an inflammatory response, and (4) a regenerative stage beginning at 4–6 days and extending to 10–14 days depending on the severity of the injury (for review see Tidball 1995). These four phases match well with the seven phases depicted in Fig. 6, with Phases (c) and (d) the phagocytic stage and (e) and (f) depicting the regenerative phase. During lengthening contractions, the actual injury to sarcomeres in a myofibril appears to occur when thick filaments of single sarcomeres are displaced to one end of the sarcomere and some or all of the filaments fail to interdigitate properly within the myofibril when the sarcomere attempts to return to its resting length (Fig. 2a). Usually the injury occurs to a highly localized cluster of sarcomeres within a single fiber. Damage to the muscle fiber compromises the fiber’s ability to maintain proper calcium homeostasis. The prolonged increase in intracellular calcium levels in damaged muscle fibers activates the m-calpain protease system. M-calpain and related proteases perform the initial disassembly of damaged myofibrils (Jackman and Kandarian 2004). Once the sarcomere has been disassembled, the damaged proteins are broken down into their constitutive amino acids by the ubiquitin-proteasome system. Within a few days following injury, protein synthesis pathways are activated and new sarcomeres are synthesized. Following severe protocols of lengthening contractions, the large force deficits displayed by muscles from both young and old mice indicate that throughout the cross-sections of individual fibers a substantial number of sarcomeres have been injured and that portions of these fibers will undergo additional degeneration of the total cross-section of the injured fibers (Rader et al. 2006). The additional steps include: a sealing off of the damaged area accompanied by the infiltration of inflammatory cells, phagocytosis of the damaged tissues, and subsequent activation of satellite cells and regeneration of entirely new segment of fiber (Fig. 6). Satellite cells are muscle precursor cells that reside between the sarcolemma and the basal lamina in skeletal muscle fibers. Satellite cells normally exist in a quiescent state, but upon injury the satellite cells are activated, migrate to the site of injury, proliferate, 384 J.A. Faulkner et al. and fuse with the damaged fiber to replace the nuclei lost as a result of the injury. Mechanical disruption of the endomysium causes the release of inactive hepatocyte growth factor (HGF) (Tatsumi and Allen 2004). The HGF is activated within the injured tissue (Tatsumi et al. 2006) and binds to the c-met receptor on the plasma membrane of the resident satellite cells, which are thus activated from their quiescent state and migrate to the site of injury. As satellite cells migrate to the site of injury, they also undergo several rounds of proliferation. The initial proliferation of satellite cells is brought about by an increase in the expression of the basic helix-loop-helix (bHLH) transcription factor MyoD. MyoD is one of four members of myogenic regulatory factor (MRF) family that also a b c d e f g Fig. 6 Schematic diagram of the sequence of events for a typical muscle fiber following a severe LCP. Within several hours following focal injury, the plasma membrane is damaged, an influx of calcium activates proteases intrinsic to the muscle fiber, and myofibrils hypercontract, resulting in a zone of necrosis. The freely permeable basement membrane remains intact. By 1 day, the hypercontracted myofibrils degenerate while vesicles accumulate to seal off the viable portions from the necrotic segments of the fiber. Neutrophils infiltrate at this time. Between 2 and 5 days, macrophages infiltrate, releasing more cytotoxic substances such as ROS that break down damaged tissue further, as well as previously uninjured tissue, resulting in a secondary injury. Satellite cells migrate to the site of injury. At 5–30 days, satellite cells proliferate and fuse across the necrotic segment so that recovery takes place (Reproduced with modifications based on a previously published figure (Bischoff 1994) with permission of the McGraw-Hill Companies. Figure also published in Rader et al. 2006 with permission Wiley) 385Role of Contraction-Induced Injury in Age-Related Muscle Wasting and Weakness include Myf-5, myogenin and MRF-4. The MRFs induce the “myogenic program” in these proliferating satellite cells, causing the cells to begin to express skeletal muscle contractile proteins. Once in proximity of the damaged region of the muscle fiber, satellite cells fuse with each other to form multinucleated structures called myotubes. Myotubes fuse with the damaged muscle fiber and restore the nuclei lost after the initial injury. Some proportion of the satellite cells that underwent prolifera- tion do not form myotubes, but instead resume a sub-basal lamina position, return to the quiescent state, and repopulate the satellite cell pool. In addition to satellite cells, fibroblasts and inflammatory cells are attracted to the site of injury within the muscle. These cells assist in the removal of cellular debris and in the repair of the extracellular matrix (ECM). If there is a severe disruption of the ECM, fibroblasts respond with an overproduction of ECM resulting in the clinical condition of fibrosis, or scar tissue accumulation (Huard et al. 2002). The prevention of scar tissue accumulation is an important goal in the initial treatment of muscle injuries, as this scar tissue is disruptive to the normal function of muscle tissue and, once formed, is relatively permanent (Järvinen et al. 2005). Clear evidence shows that recovery from contraction-induced injury is impaired in muscles of old compared with adult animals (Brooks and Faulkner 1990; McArdle et al. 2004), but the basis for the regeneration defects remain an active area of investigation (Carlson et al. 2009; Conboy et al. 2003). Moreover, the impaired regenerative potential of skeletal muscle in old animals is associated with an increase in tissue fibrosis (Brack et al. 2007). 7 Contribution of Lateral Transmission of Force to Contraction-Induced Injury A contraction-induced injury to a muscle fiber occurs when a segment, or segments, within the fiber contains groups of sarcomeres that are weaker than the sarcomeres in series with them (Fig. 6). The weaker sarcomeres normally receive lateral sup- port from the adjacent sarcomeres in the myofibrils surrounding them through intermediate filament proteins, including desmin, located at the z-discs (Fig. 7a). The desmin anchors each of the z-discs of a myofibril to the z-lines of each of the surrounding myofibrils so that the force generated by each myofibril is transmitted laterally, providing stability for all of the myofibrils within a fiber. For the myofi- brils that are immediately adjacent to the sarcolemma of a fiber, the z-discs are anchored into the sarcolemma by costameres (Fig. 7a). The costameres (Fig. 7) include the dystrophin-associated glycoprotein (DAG) complex, a portion of which extends into the ECM. The DAG appears to be situated in a position suitable for the transmission of the force laterally through the sarcolemma into the ECM. The lat- eral transmission of force continues without decrement through the intermediate filaments at each z-disc from myofibril to myofibril throughout the muscle fiber (Fig. 7b) and then through costameres from fiber to fiber throughout the muscle. This concept is supported by the successful demonstration of the lateral transmis- sion of force from a maximally activated single fiber partially dissected free in a . Muscle Wasting and Muscle Weakness The wasting or ‘atrophy’ of a skeletal muscle refers to a loss in the mass of the skeletal muscle, a condition that arises from a reduced usage of skeletal muscles. men and women, become less physically active and invariably show signs of both muscle wasting and muscle weakness. Particularly in old age, the combined impact of decreased physical activity and. of Contraction-Induced Injury in Age-Related Muscle Wasting and Weakness 4 Late-Onset Muscle Soreness The phenomenon of a contraction-induced injury to skeletal muscle fibers was first recognized