Enhanced Glycogen Storage of a Subcellular Hot Spot in Human Skeletal Muscle during Early Recovery from Eccentric Contractions

Unaccustomed eccentric exercise is accompanied by muscle damage and impaired glucose uptake and glycogen synthesis during subsequent recovery. Recently, it was shown that the role and regulation of glycogen in skeletal muscle are dependent on its subcellular localization, and that glycogen synthesis, as described by the product of glycogen particle size and number, is dependent on the time course of recovery after exercise and carbohydrate availability. In the present study, we investigated the subcellular distribution of glycogen in fibers with high (type I) and low (type II) mitochondrial content during post-exercise recovery from eccentric contractions. Analysis was completed on five male subjects performing an exercise bout consisting of 15 x 10 maximal eccentric contractions. Carbohydrate-rich drinks were subsequently ingested throughout a 48 h recovery period and muscle biopsies for analysis included time points 3, 24 and 48 h post exercise from the exercising leg, whereas biopsies corresponding to prior to and at 48 h after the exercise bout were collected from the non-exercising, control leg. Quantitative imaging by transmission electron microscopy revealed an early (post 3 and 24 h) enhanced storage of intramyofibrillar glycogen (defined as glycogen particles located within the myofibrils) of type I fibers, which was associated with an increase in the number of particles. In contrast, late in recovery (post 48 h), intermyofibrillar, intramyofibrillar and subsarcolemmal glycogen in both type I and II fibers were lower in the exercise leg compared with the control leg, and this was associated with a smaller size of the glycogen particles. We conclude that in the carbohydrate-supplemented state, the effect of eccentric contractions on glycogen metabolism depends on the subcellular localization, muscle fiber’s oxidative capacity, and the time course of recovery. The early enhanced storage of intramyofibrillar glycogen after the eccentric contractions may entail important implications for muscle function and fatigue resistance.     

Effects of exercise on plasma myosin heavy chain fragments and MRI of skeletal muscle.

The effects of a single series of high-force eccentric contractions involving the quadriceps muscle group (single leg) on plasma concentrations of muscle proteins were examined as a function of time, in the context of measurements of torque production and magnetic resonance imaging (MRI) of the involved muscle groups. Plasma concentrations of slow-twitch skeletal (cardiac beta-type) myosin heavy chain (MHC) fragments, myoglobin, creatine kinase (CK), and cardiac troponin T were measured in blood samples of six healthy male volunteers before and 2 h after 70 eccentric contractions of the quadriceps femoris muscle. Screenings were conducted 1, 2, 3, 6, 9, and 13 days later. To visualize muscle injury, MRI of the loaded and unloaded thighs was performed 3, 6, and 9 days after the eccentric exercise bout. Force generation of the knee extensors was monitored on a dynamometer (Cybex II+) parallel to blood sampling. Exercise resulted in a biphasic myoglobin release profile, delayed CK and MHC peaks. Increased MHC fragment concentrations of slow skeletal muscle myosin occurred in late samples of all participants, which indicated a degradation of slow skeletal muscle myosin. Because cardiac troponin T was within the normal range in all samples, which excluded a protein release from the heart (cardiac beta-type MHC), this finding provides evidence for an injury of slow-twitch skeletal muscle fibers in response to eccentric contractions. Muscle action revealed delayed reversible increases in MRI signal intensities on T2-weighted images of the loaded vastus intermedius and deep parts of the vastus lateralis. We attributed MRI signal changes due to edema in part to slow skeletal muscle fiber injury.(ABSTRACT TRUNCATED AT 250 WORDS)     

大负荷运动诱导大鼠骨骼肌损伤对其自噬超微结构及Beclin1和LC3-II/I的影响*

目的: 观察大负荷离心运动对大鼠骨骼肌自噬超微结构及自噬相关蛋白Beclin1和LC3II/I的影响。方法: 48只SD雄性大鼠适应性训练后随机分成对照组(C,n=8)和大负荷离心运动组(E,n=40)。E组于跑台进行90 min下坡跑,运动后0 h、12 h、24 h、48 h和72 h取比目鱼肌,透射电镜观察其自噬体超微结构变化;Western blot检测Beclin1和LC3II/I蛋白表达;免疫荧光观测LC3的定位及含量变化。结果: E组比目鱼肌自噬体数量在运动后0 h、12 h和24 h均有增加,并伴LC3自噬荧光明显增强(P＜0.01),同时运动后48 h自噬荧光仍有显著性升高(P＜0.05);Beclin1和LC3II/I在大负荷离心干预后表达升高(P＜0.05),运动后12 h～24 h达到峰值(P＜0.01),直至运动后72 h完全恢复。结论: 大负荷离心运动可诱导骨骼肌自噬超微结构变化,自噬蛋白表达增强,以上可能是运动损伤的骨骼肌功能下降的原因之一。     

Enhanced procollagen processing in skeletal muscle after a single bout of eccentric loading in humans.

Increases in procollagen processing within skeletal muscle have previously been reported in small animal models only. While indirect measurements in humans have suggested an increase procollagen processing, no intra-skeletal muscle measurements have confirmed these findings. In this study, eight young healthy male subjects performed a single bout of unaccustomed high intensity eccentric exercise on one leg, with the contralateral leg being the control. A significant increase in the muscle interstitial concentration of the N-terminal propeptide of procollagen type I (PINP) was observed (day 0: 1.96 +/- 0.44 ng ml(-1), day 2: 1.94 +/- 0.32 ng ml(-1), day 4: 3.90 +/- 1.03 ng ml(-1), day 8: 7.23 +/- 2.34 ng ml(-1)*, *P < 0.05 vs. basal and control) with no change being noted in the control leg. By day 2 post-exercise, an increase in the histological immunoreactivity of PINP and the N-terminal propeptide of procollagen type III (PIIINP) was also shown in the exercising leg only. Further, from day 2 post-exercise, immunoreactivity for tenascin C and reactive macrophages (CD68+ cells) was seen within the perimysial and endomysial connective tissue of the exercising leg only, indicating a high mechanical load and inflammation. This study shows that following a single bout of high intensity eccentric exercise there is an increase in procollagen processing within skeletal muscle in humans.     

Maximal lengthening contractions increase p70 S6 kinase phosphorylation in human skeletal muscle in the absence of nutritional supply

The aim of this study was to compare the training stimuli of eccentric (lengthening) and concentric (shortening) contractions regarding the effect on signaling enzymes involved in protein synthesis. Ten male subjects performed 4 x 6 maximal eccentric contractions on one leg followed by 4 x 6 maximal concentric contractions on the other. Six additional subjects performed the same protocol, but with maximal concentric and submaximal eccentric exercise of equal force to that of the maximal concentric contractions. Muscle biopsy samples were taken from the vastus lateralis before, immediately after, and 1 and 2 h after exercise in both legs. The average peak force produced during the maximal eccentric exercise was 31% higher than during the maximal concentric exercise, 2,490 (+/-100) vs. 1,894 (+/-108) N (P < 0.05). The maximal eccentric contractions led to two- to eightfold increases in the phosphorylation of p70 S6 kinase (p70(S6k)) and the ribosomal protein S6 that persisted for 2 h into recovery but no significant changes in phosphorylation of Akt or mammalian target of rapamycin (mTOR). Maximal concentric and submaximal eccentric contractions did not induce any significant changes in Akt, mTOR, p70(S6k), or S6 phosphorylation up to 2 h after the exercise. The results indicate that one session of maximal eccentric contractions activates p70(S6k) in human muscle via an Akt-independent pathway and suggest that maximal eccentric contractions are more effective than maximal concentric contractions in stimulating protein synthesis in the absence of a nutritional intake, an effect that may be mediated through a combination of greater tension and stretching of the muscle.     

Effects of step exercise on muscle damage and muscle Ca2+ content in men and women

Eccentric exercise often produces severe muscle damage, whereas concentric exercise of a similar load elicits a minor degree of muscle damage. The cellular events initiating muscle damage are thought to include an increase in cytosolic Ca. It was hypothesized that eccentric muscle activity in humans would lead to a larger degree of cell damage and increased intracellular Ca accumulation in skeletal muscle than concentric activity would. Furthermore, possible differences between men and women in muscle damage were investigated following step exercise. Thirty-three healthy subjects (18 men and 15 women) participated in a 30-minute step exercise protocol involving concentric contractions with 1 leg and eccentric contractions with the other leg. Muscle Ca content, maximal voluntary contraction (MVC), and muscle enzymes in the plasma were measured. In a subgroup of the subjects, T2 relaxation time was measured by magnetic resonance imaging. No significant changes were found in muscle Ca content in vastus lateralis biopsy specimens in women or in men. Following step exercise, MVC decreased in both legs of both genders. The women had a significantly larger strength decrease in the eccentric leg than the men had on postexercise day 2 (p < 0.01). Plasma creatine kinase increased following step exercise, with a sevenfold higher response in women than in men on day 3 (p < 0.001). The women, but not the men, had an increase in T2 relaxation time in the eccentrically working adductor magnus muscle, peaking on day 3 (75%) (p < 0.001). In conclusion, step exercise does not lead to Ca accumulation in the vastus lateralis but does induce muscle damage preferentially in the eccentrically working muscles, considerably more in women than in men. This indicates that gender-specific step training programs may be warranted to avoid excessive muscle damage.     

Whey protein supplementation accelerates satellite cell proliferation during recovery from eccentric exercise

Human skeletal muscle satellite cells (SCs) are essential for muscle regeneration and remodeling processes in healthy and clinical conditions involving muscle breakdown. However, the potential influence of protein supplementation on post-exercise SC regulation in human skeletal muscle has not been well investigated. In a comparative human study, we investigated the effect of hydrolyzed whey protein supplementation following eccentric exercise on fiber type-specific SC accumulation. Twenty-four young healthy subjects received either hydrolyzed whey protein + carbohydrate (whey, n = 12) or iso-caloric carbohydrate (placebo, n = 12) during post-exercise recovery from 150 maximal unilateral eccentric contractions. Prior to and 24, 48 and 168 h post-exercise, muscle biopsies were obtained from the exercise leg and analyzed for fiber type-specific SC content. Maximal voluntary contraction (MVC) and serum creatine kinase (CK) were evaluated as indices of recovery from muscle damage. In type II fiber-associated SCs, the whey group increased SCs/fiber from 0.05 [0.02; 0.07] to 0.09 [0.06; 0.12] (p < 0.05) and 0.11 [0.06; 0.16] (p < 0.001) at 24 and 48 h, respectively, and exhibited a difference from the placebo group (p < 0.05) at 48 h. The whey group increased SCs/myonuclei from 4?% [2; 5] to 10?% [4; 16] (p?p < 0.001) and muscle soreness and CK increased (p < 0.001), irrespective of supplementation. In conclusion, whey protein supplementation may accelerate SC proliferation as part of the regeneration or remodeling process after high-intensity eccentric exercise.     

Indices of free-radical-mediated damage following maximum voluntary eccentric and concentric muscular work

This study monitored plasma and skeletal muscle markers of free-radical-mediated damage following maximum eccentric and concentric exercise, to examine the potential role of free radicals in exercise-induced muscle damage. Fourteen male volunteers performed either (1) a bout of 70 maximum eccentric and a bout of 70 maximum concentric muscle actions of the forearm flexors (the bouts being separated by 4 weeks; n = 8) or (2) a bout of 80 maximum eccentric and a bout of 80 maximum concentric muscle actions of the knee extensors (the bouts being separated by 1 week; n=6). Plasma markers of lipid peroxidation, thiobarbituric acid-reactive substances (TBARS) and diene-conjugated compounds (DCC) were monitored in the arm protocol and skeletal muscle markers of oxidative lipid and protein damage, malondialdehyde (MDA) and protein carbonyl derivatives (PCD) respectively, were monitored in the leg protocol. In both protocols, the contralateral limb was used for the second bout and the order of the bouts was randomised between limbs. Repeated measures ANOVA indicated significant changes from baseline following eccentric arm work on the measures of serum creatine kinase activity (P < 0.05), maximum voluntary torque production (P < 0.01) and relaxed arm angle (P < 0.01). Subjective muscle soreness peaked 2 days after eccentric arm work (P < 0.05, Wilcoxon test). However, there were no changes in the plasma levels of TBARS or DCC following the eccentric or concentric arm exercise. Immediately after concentric leg exercise, skeletal muscle PCD concentrations was significantly higher than that observed immediately after eccentric work (P < 0.05). However, no significant difference between the eccentric and concentric knee extensor bouts was observed on the measure of skeletal muscle MDA concentration. The results of this study offer no support for the involvement of oxygen free radicals in exercise-induced muscle damage.     

Eccentric contractions leading to DOMS do not cause loss of desmin nor fibre necrosis in human muscle

High force eccentric muscle contractions can result in delayed onset muscle soreness (DOMS), prolonged loss of muscle strength, decreased range of motion, muscle swelling and an increase of muscle proteins in the blood. At the ultrastructural level Z-line streaming and myofibrillar disruptions have been taken as evidence for muscle damage. In animal models of eccentric exercise-induced injury, disruption of the cytoskeleton and the sarcolemma of muscle fibres occurs within the first hour after the exercise, since a rapid loss of staining of desmin, a cytoskeletal protein, and the presence of fibronectin, a plasma and extracellular protein, are observed within the muscle fibres. In the present study, biopsies from subjects who had performed different eccentric exercises and had developed DOMS were examined. Our aim was to determine whether eccentric exercise leading to DOMS causes sarcolemmal disruption and loss of desmin in humans. Our study shows that even though the subjects had DOMS, muscle fibres had neither lost staining for desmin nor contained plasma fibronectin. This study therefore does not support previous conclusions that there is muscle fibre degeneration and necrosis in human skeletal muscle after eccentric exercise leading to DOMS. Our data are in agreement with the recent findings that there is no inflammatory response in skeletal muscle following eccentric exercise in humans. In combination, these findings should stimulate the search for other mechanisms explaining the functional and structural alterations in human skeletal muscle after eccentric exercise.     

Utility of 17-(allylamino)-17-demethoxygeldanamycin treatment for skeletal muscle injury

Repeated eccentric contractions can injure skeletal muscle and result in functional deficits that take several weeks to fully recover. The 70-kDa heat shock protein (Hsp70) is a stress-inducible molecular chaperone that maintains protein quality and plays an integral role in the muscle’s repair processes following injury. Here, we attempted to hasten this recovery by pharmacologically inducing Hsp70 expression in mouse skeletal muscle with 17-(allylamino)-17-demethoxygeldanamycin (17-AAG) (40 mg/kg) both prior to and throughout the first 7 days after an injurious bout of 150 maximal eccentric contractions. Hsp70 content in the injured skeletal muscle was strongly induced following the eccentric contractions and remained elevated over the next 7 days as the muscle underwent repair. Treatment with 17-AAG increased Hsp70 content ～fivefold; however, this was significantly less than that induced by the injury. Moreover, 17-AAG treatment did not recover the decrements to in vivo isometric torque production following the bout of eccentric contractions. Together, these findings demonstrate that although Hsp70 content was induced in the uninjured skeletal muscle, treatment of 17-AAG (40 mg/kg) was not a preventive measure to either reduce the severity of skeletal muscle damage or enhance functional recovery following a bout of maximal eccentric contractions.     

Eccentric ergometry: increases in locomotor muscle size and strength at low training intensities

Lengthening (eccentric) muscle contractions are characterized by several unusual properties that may result in unique skeletal muscle adaptations. In particular, high forces are produced with very little energy demand. Eccentrically trained muscles gain strength, but the specific nature of fiber size and composition is poorly known. This study assesses the structural and functional changes that occur to normal locomotor muscle after chronic eccentric ergometry at training intensities, measured as oxygen uptake, that do not influence the muscle when exercised concentrically. Male subjects trained on either eccentric or concentric cycle ergometers for 8 wk at a training intensity starting at 54% and ending at 65% of their peak heart rates. The isometric leg strength increased significantly in the eccentrically trained group by 36%, as did the cross-sectional area of the muscle fiber by 52%, but the muscle ultrastructure remained unchanged. There were no changes in either fiber size, composition, or isometric strength in the concentrically trained group. The responses of muscle to eccentric training appear to be similar to resistance training.     

Low-frequency depression of tension in the cat gastrocnemius muscle after eccentric exercise.

Subjecting a muscle to a series of eccentric contractions in which the contracting muscle is lengthened results in a number of changes in its mechanical properties. These include a fall in isometric tension that is particularly pronounced during low-frequency stimulation, a phenomenon known as low-frequency depression (LFD). Reports of LFD have not taken into account the shift in optimum length for active tension generation to longer muscle lengths that takes place after eccentric contractions. Given the length dependence of the stimulation frequency-tension curve, we tested the hypothesis that the change in this relationship after eccentric exercise is due to the shift in optimum length. We measured LFD by recording tension in response to a linearly increasing rate of stimulation of the nerve to medial gastrocnemius of anesthetized cats, over the range 0-100 pulses per second. Tension responses were measured before and after 50 eccentric contractions consisting of 6-mm stretches starting at 3 mm below optimum length and finishing at 3 mm above it. An index of LFD was derived from the tension responses to ramp stimulation. It was found that LFD after the eccentric contractions was partly, but not entirely, due to changes in the muscle's optimum length. An additional factor was the effect of fatigue. These observations led to the conclusion that the muscle length dependence of LFD was reduced by eccentric contractions. All of this means that after eccentric exercise the tension deficit at low rates of muscle activation is likely to be less severe than first thought.     

Effects of muscle damage on stretch-shortening cycle function and muscle stiffness control

This experiment examined the effect of eccentric contraction-induced muscle damage on the stretch-shortening cycle and vertical leg spring stiffness during jumping activities. Ten moderately active male and female adult volunteers participated in this study (aged 23 +/- 2.3 years). Temporary muscle damage to the knee extensors was administered by a bout of eccentric contractions on an isokinetic dynamometer. Measurements were obtained of maximum voluntary force and of take-off velocities for single-leg countermovement jumps (CMJs), squat jumps (SJs), and drop jumps (DJs), performed on a specially constructed sledge and force plate apparatus. These measurements were obtained before and after the damage intervention, and the undamaged leg was used as a control. The results indicated that eccentric muscle damage significantly affected stretch-shortening cycle performance by causing relatively greater reductions in SJ performance than CMJ or DJ. The muscle damage intervention also significantly increased leg-spring stiffness, which indicates that the changes in leg stiffness may be an important adaptation resulting from eccentric exercise.     

Expression of lipocortins in human bronchial epithelial cells: effects of IL-1beta , TNF-alpha, LPS and dexamethasone

In this study, we investigated the expression of lipocortin I and II (annexin I and I in the human bronchial epithelium, both in vivo and in vitro. A clear expression of lipocortin I and II protein was found in the epithelium in sections of bronchial tissue. In cultured human bronchial epithelial cells we demonstrated the expression of lipocortin I and II mRNA and protein using Northern blotting, FACScan analysis and ELISA. No induction of lipocortin I or II mRNA or protein was observed after incubation with dexamethasone. Stimulation of bronchial epithelial cells with IL-1beta, TNF-alpha or LPS for 24 h did not affect the lipocortin I or II mRNA or protein expression, although PGE(2) and 6-keto-PGF(1alpha) production was significantly increased. This IL-1beta- and LPS-mediated increase in eicosanoids could be reduced by dexamethasone, but was not accompanied by an increase in lipocortin I or II expression. In human bronchial epithelial cells this particular glucocorticoid action is not mediated through lipocortin I or II induction.     

Human muscle function following prolonged eccentric exercise

4 subjects performed repeated eccentric contractions with leg extensors during prolonged downhill walking (-25% gradient) at 6.44 km.h-1 until collapse due to muscle weakness (range of exercise duration 29 to 40 min). During the exercise oxygen uptake rose progressively from approximately 45% of the previously determined VO2max at 10 min to approximately 65% at the end of the exercise. Following the exercise there was an immediate, significant, and sustained reduction in maximal voluntary isometric contraction, and short term (anaerobic) power output measured concentrically on an isokinetic ergometer. These reductions in muscle function persisted for 96 hours post exercise, and were reflected by significant reductions in the tension generated at low frequency (20 Hz) relative to higher frequency (50 Hz) percutaneous stimulation of the quadriceps. All four subjects showed an increase in plasma levels of creatine kinase post eccentric exercise. Performing concentric contractions by walking uphill for one hour at a significantly greater metabolic cost failed to induce comparable reductions in muscle function. These results provide evidence for the consequences of prolonged eccentric work upon dynamic function which complements earlier reports of structural, enzymatic, and static function changes.     

Lesions in the rat soleus muscle following eccentrically biased exercise

Morphological changes in skeletal muscle related to lengthening (eccentric) contractions have been noted by several laboratories. However, a systematic examination of skeletal muscle following repetitive eccentric contractions is lacking. This study was undertaken to study lesions and determine their relative densities in rat soleus muscle 30 min following level or downhill treadmill exercise. Following fixation in situ by vascular perfusion, toluidine-blue-stained 1-micron sections of the muscle samples selected at 73-micron intervals were scanned with a light microscope. Three types of lesions were noted: focal disruptions in the A-band, localized dissolution of Z-lines, and clotting of muscle fibers. Soleus muscle from the caged controls and the tibialis anterior muscle from downhill-exercised rats were essentially free of lesions. Eighty-nine percent of the soleus m. lesions in the downhill runner group and 97% of those in the level runner group were A-band disruptions. A-band lesion density was significantly higher in the soleus muscle of the downhill runners compared to level runners with the highest incidence in the distal half. A-band lesion density was lower in soleus muscles from level runners; however, the highest intramuscular incidence was in the proximal rather than the distal end. The results indicate that a disruption of the A-band is a principal change within some skeletal muscle fibers 30 min following repetitive eccentric contractions.     

Cellular adaptation to repeated eccentric exercise-induced muscle damage.

Eccentrically biased exercise results in skeletal muscle damage and stimulates adaptations in muscle, whereby indexes of damage are attenuated when the exercise is repeated. We hypothesized that changes in ultrastructural damage, inflammatory cell infiltration, and markers of proteolysis in skeletal muscle would come about as a result of repeated eccentric exercise and that gender may affect this adaptive response. Untrained male (n = 8) and female (n = 8) subjects performed two bouts (bout 1 and bout 2), separated by 5.5 wk, of 36 repetitions of unilateral, eccentric leg press and 100 repetitions of unilateral, eccentric knee extension exercises (at 120% of their concentric single repetition maximum), the subjects' contralateral nonexercised leg served as a control (rest). Biopsies were taken from the vastus lateralis from each leg 24 h postexercise. After bout 2, the postexercise force deficit and the rise in serum creatine kinase (CK) activity were attenuated. Women had lower serum CK activity compared with men at all times (P < 0.05), but there were no gender differences in the relative magnitude of the force deficit. Muscle Z-disk streaming, quantified by using light microscopy, was elevated vs. rest only after bout 1 (P < 0.05), with no gender difference. Muscle neutrophil counts were significantly greater in women 24 h after bout 2 vs. rest and bout 1 (P < 0.05) but were unchanged in men. Muscle macrophages were elevated in men and women after bout 1 and bout 2 (P < 0.05). Muscle protein content of the regulatory calpain subunit remained unchanged whereas ubiquitin-conjugated protein content was increased after both bouts (P < 0.05), with a greater increase after bout 2. We conclude that adaptations to eccentric exercise are associated with attenuated serum CK activity and, potentially, an increase in the activity of the ubiquitin proteosome proteolytic pathway.