Intracellular sodium in mammalian muscle fibers after eccentric contractions.

The effect of eccentric contractions on intracellular Na(+) concentration ([Na(+)](i)) and its distribution were examined in isolated rat and mouse muscle fiber bundles. [Na(+)](i) was measured with either Na(+)-binding benzofuran isophthalate or sodium green. Ten isometric contractions had no significant effect on force (measured after 5 min of recovery) and caused no significant change in the resting [Na(+)](i) (7.2 +/- 0.5 mM). In contrast 10 eccentric contractions (40% stretch at 4 muscle lengths/s) reduced developed force at 100 Hz to 45 +/- 3% of control and increased [Na(+)](i) to 16.3 +/- 1.6 mM (n = 6; P < 0.001). The rise of [Na(+)](i) occurred over 1-2 min and showed only minimal recovery after 30 min. Confocal images of the distribution of [Na(+)](i) showed a spatially uniform distribution both at rest and after eccentric contractions. Gd(3+) (20 microM) had no effect on resting [Na(+)](i) or control tetanic force but prevented the rise of [Na(+)](i) and reduced the force deficit after eccentric damage. These data suggest that Na(+) entry after eccentric contractions may occur principally through stretch-sensitive channels.     

Skeletal muscle collagen content in humans after high-force eccentric contractions.

The purpose of this study was to investigate the effects of high-force eccentric muscle contractions on collagen remodeling and on circulating levels of matrix metalloproteinases (MMP) and tissue inhibitors of metalloproteinases (TIMP) in humans. Nine volunteers [5 men and 4 women, mean age 23 (SD 4) yr] each performed a bout of 100 maximum voluntary eccentric contractions of the knee extensors. Muscle biopsies were taken before exercise and on days 4 and 22 afterward. Image analysis of stained tissue sections was used to quantify endomysial collagen staining intensity. Maximum voluntary contractile isometric force was recorded preexercise and on days 1, 2, 3, 4, 8, 11, and 14 postexercise. Venipuncture blood samples were also drawn on these days for measurement of serum creatine kinase activity and concentrations of MMP-9, TIMP-1, TIMP-2, and the MMP-2/TIMP-2 complex. Maximum voluntary contractile force declined by 39 +/- 23% (mean +/- SD) on day 2 postexercise and recovered thereafter. Serum creatine kinase activity peaked on day 4 postexercise (P < 0.01). Collagen type IV staining intensity increased significantly on day 22 postexercise to 126 +/- 29% (mean +/- SD) of preexercise values (P < 0.05). Serum MMP-9 levels increased on day 8 postexercise (P < 0.01), and serum TIMP-1 was also significantly elevated on days 1, 2, 3, 4, and 14 postexercise (P < 0.05). These results suggest that a single bout of eccentric muscle contractions results in remodeling of endomysial type IV collagen, possibly via the MMP pathway.     

Segmental muscle fiber lesions after repetitive eccentric contractions

Immunohistochemical and electron-microscopic techniques were used to analyze the extensor digitorum longus muscles of New Zealand White rabbits 1 h, 1 day, 3, 7, and 28 days after repetitive eccentric contractions. Loss of the cytoskeletal protein desmin was the earliest manifestation of injury. Apart from 1 h post-exercise, all desmin-negative fibers stained positively with antibody to plasma fibronectin, indicating loss of cellular integrity accompanying cytoskeletal disruption. Fiber sizes were significantly increased from 1–7 days after exercise. The large (hyaline) fibers found in histological sections after repetitive eccentric contractions resulted from segmental hypercontraction of the fiber. This phenomenon occurred proximally and distally to plasma membrane lesions of the muscle fiber and necrosis and manifested itself as very short sarcomere lengths. Thus, in serial sections, staining characteristics, sizes and shapes of one and the same fiber often varied dramatically. We conclude that the following sequence of events occurs: cytoskeletal disruptions, loss of myofibrillar registry, i.e., Z-disk streaming and A-band disorganization, and loss of cell integrity as manifested by intracellular plasma fibronectin stain, hypercontracted regions, and invasion of cells. When a fiber is disrupted, the remaining intact fibers apparently take up the tension put on the muscle and later fewer fibers are subjected to eccentric contractions.     

Role of the calcium-calpain pathway in cytoskeletal damage after eccentric contractions.

The mechanism(s) underlying eccentric damage to skeletal muscle cytoskeleton remain unclear. We examined the role of Ca(2+) influx and subsequent calpain activation in eccentric damage to cytoskeletal proteins. Eccentric muscle damage was induced by stretching isolated mouse muscles by 20% of the optimal length in a series of 10 tetani. Muscle force and immunostaining of the cytoskeletal proteins desmin, dystrophin, and titin were measured at 5, 15, 30, and 60 min after eccentric contractions and compared with the control group that was subjected to 10 isometric contractions. A Ca(2+)-free solution and leupeptin (100 microM), a calpain inhibitor, were applied to explore the role of Ca(2+) and calpain, respectively, in eccentric muscle damage. After eccentric contractions, decreases in desmin and dystrophin immunostaining were apparent after 5 min that accelerated over the next 60 min. Increased titin immunostaining, thought to indicate damage to titin, was evident 10 min after stretch, and fibronectin entry, indicating membrane disruption, was evident 20 min after stretch. These markers of damage also increased in a time-dependent manner. Muscle force was reduced immediately after stretch and continued to fall, reaching 56 +/- 2% after 60 min. Reducing extracellular calcium to zero or applying leupeptin minimized the changes in immunostaining of cytoskeletal proteins, reduced membrane disruption, and improved the tetanic force. These results suggest that the cytoskeletal damage and membrane disruption were mediated primarily by increased Ca(2+) influx into muscle cells and subsequent activation of calpain.     

Dihydropyridine and ryanodine receptor binding after eccentric contractions in mouse skeletal muscle.

The purpose of this study was to determine whether there are alterations in the dihydropyridine and/or ryanodine receptors that might explain the excitation-contraction uncoupling associated with eccentric contraction-induced skeletal muscle injury. The left anterior crural muscles (i.e., tibialis anterior, extensor digitorum longus, and extensor hallucis longus) of mice were injured in vivo by 150 eccentric contractions. Peak isometric tetanic torque of the anterior crural muscles was reduced approximately 45% immediately and 3 days after the eccentric contractions. Partial restoration of peak isometric tetanic and subtetanic forces of injured extensor digitorum longus muscles by 10 mM caffeine indicated the presence of excitation-contraction uncoupling. Scatchard analysis of [3H]ryanodine binding indicated that the number of ryanodine receptor binding sites was not altered immediately postinjury but decreased 16% 3 days later. Dihydropyridine receptor binding sites increased approximately 20% immediately after and were elevated to the same extent 3 days after the injury protocol. Muscle injury did not alter the sensitivity of either receptor. These data suggest that a loss or altered sensitivity of the dihydropyridine and ryanodine receptors does not contribute to the excitation-contraction uncoupling immediately after contraction-induced muscle injury. We also concluded that the loss in ryanodine receptors 3 days after injury is not the primary cause of excitation-contraction uncoupling at that time.     

Stretch-activated ion channels contribute to membrane depolarization after eccentric contractions

We tested the hypothesis that eccentric contractionsactivate mechanosensitive or stretch-activated ion channels (SAC) in skeletal muscles, producing increased cation conductance.Resting membrane potentials and contractile function were measured in rat tibialis anterior muscles after single or multiple exposures to aseries of eccentric contractions. Each exposure produced a significantand prolonged (>24 h) membrane depolarization in exercised musclefibers. The magnitude and duration of the depolarization were relatedto the number of contractions. Membrane depolarization was dueprimarily to an increase in Na⁺ influx, because theestimated Na⁺-to-K⁺ permeability ratio wasincreased in exercised muscles and resting membrane potentials could bepartially repolarized by substituting an impermeant cation forextracellular Na⁺ concentration. Neither theNa⁺/H⁺ antiport inhibitor amiloride nor thefast Na⁺ channel blocker TTX had a significant effect onthe depolarization. In contrast, addition of either of two nonselectiveSAC inhibitors, streptomycin or Gd³⁺, produced significantmembrane repolarization. The results suggest that muscle fibersexperience prolonged depolarization after eccentric contractions due,principally, to the activation of Na⁺-selective SAC.

     

Temperature dependency of force loss and Ca(2+) homeostasis in mouse EDL muscle after eccentric contractions

The goals of this study were first to determine the effect of temperature on the force loss that results from eccentric contractions in mouse extensor digitorum longus (EDL) muscles and then to evaluate a potential role for altered Ca(2+) homeostasis explaining the greater isometric force loss observed at the higher temperatures. Isolated muscles performed five eccentric or five isometric contractions at either 15, 20, 25, 30, 33.5, or 37 degrees C. Isometric force loss, caffeine-induced force, lactate dehydrogenase (LDH) release, muscle accumulation of (45)Ca(2+) from the bathing medium, sarcoplasmic reticulum (SR) Ca(2+) uptake, and resting muscle fiber free cytosolic Ca(2+) concentration ([Ca(2+)](i)) were measured. The isometric force loss after eccentric contractions increased progressively as temperature rose; at 15 degrees C, there was no significant loss of force, but at 37 degrees C, there was a 30-39% loss of force. After eccentric contractions, caffeine-induced force was not affected by temperature nor was it different from that of control muscles at any temperature. Loss of cell membrane integrity and subsequent influx of extracellular Ca(2+) as indicated by LDH release and muscle (45)Ca(2+) accumulation, respectively, were minimal over the 15-25 degrees C range, but both increased as an exponential function of temperature between 30 and 37 degrees C. SR Ca(2+) uptake showed no impairment as temperature increased, and the eccentric contraction-induced rise in resting fiber [Ca(2+)](i) was unaffected by temperature over the 15-25 degrees C range. In conclusion, the isometric force loss after eccentric contractions is temperature dependent, but the temperature dependency does not appear to be readily explainable by alterations in Ca(2+) homeostasis.     

The relationship of creatine kinase variability with body composition and muscle damage markers following eccentric muscle contractions

[Purpose]

The purpose of the study was to investigate the relationship between CK variability and body composition and muscle damage markers following eccentric exercise.

[Methods]

Total 119 healthy male subjects were recruited to perform 50 eccentric contractions consisted of 2 sets of 25 contractions. Then, blood creatine kinase (CK) activity was analyzed to divide into three groups based on their CK activity levels. Maximum isometric strength (MIS), muscle soreness (SOR) and body composition data were obtained before and after exercise.

[Results]

The results showed that high CK responders had a significant decrease in MIS (p<0.001) and greater SOR (p<0.01) following eccentric exercise compared to low CK responders. Percent body fat was also higher in high responders compared to low responders (p=0.014). Peak CK activity was significantly correlated with MIS and SOR but no correlation with % body fat, muscle mass, and body mass index.

[Conclusion]

CK variability following eccentric exercise is closely related to MIS and SOR and % body fat may be a potent factor for CK variability.     

Muscle creatine uptake and creatine transporter expression in response to creatine supplementation and depletion.

The total creatine pool size [Cr(total); creatine (Cr) + phosphocreatine (PCr)] is crucial for optimal energy utilization in skeletal muscle, especially at the onset of exercise and during intense contractions. The Cr(total) likely is controlled by long-term modulation of Cr uptake via the sodium-dependent Cr transporter (CrT). To test this hypothesis, adult male Sprague-Dawley rats were fed 1% Cr, their muscle Cr(total) was reduced by approximately 85% [1% beta-guanidinoproprionic acid (beta-GPA)], or their muscle Cr(total) was repleted (1% Cr after beta-GPA depletion). Cr uptake was assessed by skeletal muscle (14)C-Cr accumulation to Cr and PCr by using hindlimb perfusion, and CrT protein content was assessed by Western blot. Cr uptake rate decreased with dietary Cr supplementation in the white gastrocnemius (WG; 45%) only. Depletion of muscle Cr(total) to approximately 15% of normal increased Cr uptake in the soleus (21%) and red gastrocnemius (22%), corresponding to 70-150% increases in muscle CrT content. In contrast, the inherently lower Cr uptake rate in the WG was unchanged with depletion of muscle Cr(total) even though CrT band density was increased by 230%. Thus there was no direct relationship between apparent muscle CrT abundance and Cr uptake rates. However, Cr uptake rates scaled inversely with decreases in muscle Cr(total) in the high-oxidative muscle types but not in the WG. This implies that factors controlling Cr uptake are different among fiber types. These observations may help explain the influence of initial muscle Cr(total), time dependency, and variations in muscle Cr(total) accumulation during Cr supplementation.     

Decreased insulin action on muscle glucose transport after eccentric contractions in rats

Asp, Sven, and Erik A. Richter. Decreased insulinaction on muscle glucose transport after eccentric contractions in rats. J. Appl. Physiol. 81(5):1924-1928, 1996.We have recently shown that eccentriccontractions (Ecc) of rat calf muscles cause muscle damage anddecreased glycogen and glucose transporter GLUT-4 protein content inthe white (WG) and red gastrocnemius (RG) but not in the soleus (S) (S. Asp, S. Kristiansen, and E. A. Richter. J. Appl.Physiol. 79: 1338-1345, 1995). To study whetherthese changes affect insulin action, hindlimbs were perfused at three different insulin concentrations (0, 200, and 20,000 µU/ml) 2 daysafter one-legged eccentric contractions of the calf muscles. Comparedwith control, basal glucose transport was slightly higher (P < 0.05) in Ecc-WG and -RG,whereas it was lower (P < 0.05) atboth submaximal and maximal insulin concentrations in the Ecc-WG and atmaximal concentrations in the Ecc-RG. In the Ecc-S, the glucosetransport was unchanged in hindquarters perfused in the absence orpresence of a submaximal stimulating concentration of insulin, whereasit was slightly (P < 0.05) higherduring maximal insulin stimulation compared with control S. At the endof perfusion the glycogen concentrations were lower in bothEcc-gastrocnemius muscles compared with control muscles at all insulinconcentrations. Fractional velocity of glycogen synthase increasedsimilarly with increasing insulin concentrations in Ecc- and control WGand RG. We conclude that insulin action on glucose transport but notglycogen synthase activity is impaired in perfused muscle exposed toprior eccentric contractions.

     

E-C coupling failure in mouse EDL muscle after in vivo eccentric contractions

The objectives of this research were to determine thecontribution of excitation-contraction (E-C) coupling failure to the decrement in maximal isometric tetanic force(P_o) in mouse extensor digitorumlongus (EDL) muscles after eccentric contractions and to elucidatepossible mechanisms. The left anterior crural muscles of femaleICR mice (n = 164) wereinjured in vivo with 150 eccentric contractions.P_o, caffeine-,4-chloro-m-cresol-, andK⁺-induced contracture forces,sarcoplasmic reticulum (SR) Ca²⁺release and uptake rates, and intracellularCa²⁺ concentration([Ca²⁺]_i)were then measured in vitro in injured and contralateral control EDLmuscles at various times after injury up to 14 days. On the basis ofthe disproportional reduction inP_o (~51%) compared with caffeine-induced force (~11-21%), we estimate that E-C coupling failure can explain 57-75% of theP_o decrement from 0 to 5 days postinjury. Comparable reductions inP_o andK⁺-induced force (51%), and minorreductions (0-6%) in the maximal SRCa²⁺ release rate, suggest thatthe E-C coupling defect site is located at the t tubule-SR interfaceimmediately after injury. Confocal laser scanning microscopy indicatedthat resting[Ca²⁺]_iwas elevated and peak tetanic[Ca²⁺]_iwas reduced, whereas peak4-chloro-m-cresol-induced[Ca²⁺]_iwas unchanged immediately after injury. By 3 days postinjury, 4-chloro-m-cresol-induced[Ca²⁺]_ibecame depressed, probably because of decreased SRCa²⁺ release and uptake rates(17-31%). These data indicate that the decrease inP_o during the first several daysafter injury primarily stems from a failure in the E-C couplingprocess.

     

Muscle damage induced by eccentric contractions of 25% strain

Contractile and morphological properties were measured in the rabbit tibialis anterior muscle 1 h after isometric contraction (IC), passive stretch (PS), or eccentric contraction (EC). Maximal tetanic tension (Po) was reduced after 30 min of PS (P less than 0.001), IC (P less than 0.001), or EC (P less than 0.0001). However, the magnitude of the force deficit was a function of the treatment method. After 30 min of cyclic PS, Po decreased by 13%, whereas after IC or EC, Po decreased by 31 and 69%, respectively. The time course of tension decline in the various groups suggested that the EC-induced injury occurred during the first few minutes of treatment. Although the morphology of samples from the PS and IC groups appeared normal, eccentrically exercised muscles exhibited portions of abnormally large fibers (diam greater than or equal to 110 microns) when viewed in cross section. Examination of 231 such fibers from 6 muscles revealed that all enlarged fibers were exclusively of the fast-twitch glycolytic fiber type. Although no ultrastructural abnormalities were observed in any of the muscles from the IC or PS groups, a significant portion of the fibers in the EC group displayed various degrees of disorganization of the sarcomeric band pattern. Taken together, these studies highlight the importance of fiber oxidative capacity in EC-induced injury, which may be related to the damage mechanism.     

Rises in whole muscle passive tension of mammalian muscle after eccentric contractions at different lengths.

This is a report of experiments carried out on the medial gastrocnemius muscle of the anesthetized cat, investigating the effects of eccentric contractions carried out at different muscle lengths on the passive and active length-tension relationships. In one series of experiments, the motor supply to the muscle was divided into three approximately equal parts; in the other, whole muscles were used. Fifty eccentric contractions were carried out over different regions of the active length-tension curve for each partial or whole muscle. Active and passive length-tension curves were measured before and after the eccentric contractions. When eccentric contractions were carried out at longer lengths, there was a larger shift of the optimum length for active tension in the direction of longer muscle lengths and a larger fall in peak isometric tension. Passive tension was higher immediately after the eccentric contractions, and if the muscle was left undisturbed for 40 min, it increased further to higher values, particularly after contractions at longer lengths. A series of 20 passive stretches of the same speed and amplitude and covering the same length range as the active stretches, reduced the passive tension which redeveloped over a subsequent 40-min period. It is hypothesized that there are two factors influencing the level of passive tension in a muscle after a series of eccentric contractions. One is injury contractures in damaged muscle fibers tending to raise passive tension; the other is the presence of disrupted sarcomeres in series with still-functioning sarcomeres tending to reduce it.     

Effect of antioxidant vitamin supplementation on muscle function after eccentric exercise

This study investigated the effects of antioxidant vitamin supplementation upon muscle contractile function following eccentric exercise and was performed double blind. Twenty-four physically active young subjects ingested either placebo (400 mg; n = 8), vitamin E (400 mg; n=8) or vitamin C (400 mg; n = 8) for 21 days prior to and for 7 days after performing 60 min of box-stepping exercise. Contractile function of the triceps surae was assessed by the measurement of maximal voluntary contraction (MVC) and the ratio of the force generated at 20 Hz and 50 Hz tetanic stimulation before and after eccentric exercise and for 7 days during recovery. Following eccentric exercise, MVC decreased to 75 (4) % [mean (SE); n = 24; P < 0.05] of the preexercise values and the 20/50 Hz ratio of tetanic tension from 0.76 (0.01) to 0.49 (0.03) [mean (SE); n = 24; P<0.05). Compared to the placebo group no significant changes in MVC were observed immediately post-exercise, though recovery of MVC in the first 24 h post-exercise was greater in the group supplemented with vitamin C. The decrease in 20/50 Hz ratio of tetanic tension was significantly less (P < 0.05) post-exercise and in the initial phase of recovery in subjects supplemented with vitamin C but not with vitamin E. These data suggest that prior vitamin C supplementation may exert a protective effect against eccentric exercise-induced muscle damage.     

Taurine supplementation decreases oxidative stress in skeletal muscle after eccentric exercise

Infrequent exercise, typically involving eccentric actions, has been shown to cause oxidative stress and to damage muscle tissue. High taurine levels are present in skeletal muscle and may play a role in cellular defences against free radical‐mediated damage. This study investigates the effects of taurine supplementation on oxidative stress biomarkers after eccentric exercise (EE). Twenty‐four male rats were divided into the following groups (n = 6): control; EE; EE plus taurine (EE + Taurine); EE plus saline (EE + Saline). Taurine was administered as a 1‐ml 300 mg kg^?1 per body weight (BW) day^?1 solution in water by gavage, for 15 consecutive days. Starting on the 14th day of supplementation, the animals were submitted to one 90‐min downhill run session and constant velocity of 1·0 km h^?1. Forty‐eight hours after the exercise session, the animals were killed and the quadriceps muscles were surgically removed. Production of superoxide anion, creatine kinase (CK) levels, lipoperoxidation, carbonylation, total thiol content and antioxidant enzyme were analysed. Taurine supplementation was found to decrease superoxide radical production, CK, lipoperoxidation and carbonylation levels and increased total thiol content in skeletal muscle, but it did not affect antioxidant enzyme activity after EE. The present study suggests that taurine affects skeletal muscle contraction by decreasing oxidative stress, in association with decreased superoxide radical production. Copyright © 2010 John Wiley & Sons, Ltd.     

Rat hypothalamic proopiomelanocortin messenger RNA is unaffected by adrenalectomy.

The negative feedback control of hypothalamic cortocotrophin releasing factor (CRF) and anterior pituitary proopiomelanocortin (POMC) by corticosteroids is well understood. However, less is known about the mechanisms that regulate POMC gene expression in the arcuate nuclei in the medial basal hypothalamus (MBH). Using a sensitive and specific S1 endonuclease protection assay, we have examined the effect of adrenalectomy on POMC mRNA in the rat MBH and pituitary. Our results show that adrenalectomy does not change POMC mRNA levels in the MBH at 7 or 14 days post surgery. The neurointermediate lobe of the pituitary was similarly unaffected by adrenalectomy, while in the anterior lobe, POMC mRNA increased 7-10 fold at both time points, effects that were prevented by dexamethasone treatment. We conclude that while POMC mRNA in the anterior lobe of the pituitary is regulated by plasma glucocorticoids, in the MBH and neurointermediate lobe, it is not.     

Capillary and venous samples of total creatine kinase are similar after eccentric exercise

Circulating creatine kinase (CK) levels are often monitored as an indirect biomarker of muscle damage after resistive exercise. The purpose of the present investigation was to evaluate whether capillary whole-blood sampling, a simpler and less invasive method for obtaining a venous blood sample, would allow for a reliable measurement of total CK compared to venipuncture. Fifteen untrained subjects performed 50 maximal eccentric elbow extensions to induce muscle damage of the biceps brachii. Capillary (fingerstick) and venous whole-blood samples were collected contemporaneously at baseline and again at 24, 48, 72, and 96 hours post-exercise. Using a commercial CK analysis kit with a protocol modification to account for a reduced sample size, total CK activity of the capillary and venous samples was analyzed concurrently via spectrophotometry. Results indicated a 0.997 correlation between sampling sites for total CK, with disagreement between the venous and capillary samples estimated at <12% across the range of CK values. These findings indicate capillary sampling for total CK activity provides a valid alternative to venipuncture and should be considered by researchers, clinicians, and strength and conditioning specialists as an alternate sampling technique when indirectly evaluating muscle damage after exercise.     

Decreased contraction economy in mouse EDL muscle injured by eccentric contractions

Warren III, Gordon L., Jay H. Williams, Christopher W. Ward,Hideki Matoba, Christopher P. Ingalls, Karl M. Hermann, and R. B. Armstrong. Decreased contraction economy in mouse EDL muscleinjured by eccentric contractions. J. Appl.Physiol. 81(6): 2555-2564, 1996.The objective ofthis study was to find out whether basal and/or active energymetabolism are altered in isolated mouse extensor digitorum longusmuscle injured by eccentric (Ecc) contractions. Measurements of basalO₂ consumption and isometric tetanus O₂ recovery cost were madeat 25°C on muscles that had done either 10 Ecc, 10 isometric (Iso),or no contractions (No). In parallel experiments, rates of lactate andpyruvate production were measured to estimate the anaerobiccontribution. Basal O₂ consumptionwas unaffected by the type of protocol performed(P = 0.07). However, the tetanusO₂ cost per force-time integral was elevated by 30-36% for the Ecc protocol muscles over that forthe Iso and No protocol muscles. When including the increased lactateproduction by the Ecc protocol muscles, the total energetic cost perforce-time integral was 53% higher than that for the Iso protocolmuscles [2.35 ± 0.17 vs. 1.54 ± 0.18 µmolO₂/(N ^· m ^· s)].The decreased economy was attributed to two factors. First, in skinnedfibers isolated from the injured muscles, the ratio of maximalactomyosin adenosinetriphosphatase activity to force production was upby 37.5%, suggesting uncoupling of ATP hydrolysis from forceproduction. Second, increased reliance on anaerobic metabolism alongwith the fluorescent microscopic study of mitochondrial membranepotential and histochemical study of ATP synthase suggested anuncoupling of oxidative phosphorylation in the injured muscles.