Comparative Biochemistry and Physiology Part C 134 (2003) 199–206
Mitochondrial, sarcoplasmic membrane integrity and protein degradation in heart and skeletal muscle in exercised rats Andrea C. Perez, Antonio C. Cabral de Oliveira, Emma Estevez, Antonio J. Molina, Julio G. Prieto, Ana I. Alvarez* Department of Physiology, University of Leon, Leon 24071, Spain Received 14 July 2002; received in revised form 15 November 2002; accepted 19 November 2002
Abstract Several different exercise regimens varied in the severity of tissue damage induced. Therefore, this study investigated the effects of a single bout of exercise versus endurance training in heart and skeletal muscles with different predominant fiber types on indices of mitochondrial, endoplasmic reticulum (ER) integrity and protein degradation. Male Wistar rats performed different treadmill exercise protocols: exhaustive, maximal exhaustive, eccentric, training and exhaustive exercise after training. The maximal and eccentric exercises resulted in a significant loss of integrity of the sarcoplasmic and ER muscle, while no changes were observed in cardiac muscle. Mitochondrial membrane fluidity measured by the fluorescence polarization method was significantly increased post-acute exercises in heart and oxidative muscles. Regular exercise can stabilize and preserve the viscoelastic nature of mitochondrial membranes in both tissues. The highest increase in carbonyl content was obtained in heart after exhaustive exercise protocol, from 1"0.1 to 3.6"0.14 nmol mg proteiny1, such increase were not found after regular exercise with values significantly decreased. Nitrate heart levels showed attenuated generation of nitric oxide after training. Muscle protein oxidation was produced in all exhaustive exercises including eccentric exercise. 䊚 2002 Elsevier Science Inc. All rights reserved. Keywords: Exhaustive exercise; Cardiac muscle; Carbonyl; Exercise training; Fluorescence polarization; Membrane fluidity; Skeletal musclerat
1. Introduction Regular exercise is cardioprotective and many authors have reported that the survival rate of myocardial infarcts is greater in active individuals as compared with sedentary ones. Training improves myocardial responses to both ischaemia and reperfusion, promotes lower levels of lipid peroxidation, an increase in non-enzymatic antioxidants, and in general decreases muscle damage and inflammation, including the generation of fewer oxygen free radicals (Ji, 1999; Liu et al., 2000). *Corresponding author. Fax: q34-987-291267. E-mail address:
[email protected] (A.I. Alvarez).
It is well known that exercise has salutary effects. However, strenuous or unaccustomed physical exercise can induce morphological insult to the skeletal and myocardial muscles involved in the activity (Armstrong et al., 1991). Oxidative stress due to acute or chronic exercise elicits different responses, depending on the type of organ tissue and its endogenous antioxidant levels (Liu et al., 2000) and such damage ranges from considerable fibre disruption to subcellular damage. Exposure to reactive oxygen and nitrogen species (RONS) may cause lipid peroxidation in cell membranes, which in turn may generate species that damage cell proteins and promote their degradation (Davies and Goldberg, 1987). If exercise
1532-0456/03/$ - see front matter 䊚 2002 Elsevier Science Inc. All rights reserved. PII: S 1 5 3 2 - 0 4 5 6 Ž 0 2 . 0 0 2 4 7 - 8
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Fig. 1. Carbonyl content (nmol mg proteiny1) in vastus intermedius, EDL, tibialis and gastrocnemius muscles of control rats (C), acutely exercised rats: Maximal Exhaustive (Max-Ex), Eccentric (Ec), Exhaustive (Ex); and chronically exercised rats: Training (Tr) and Training Exhaustive (Tr–Ex). Values are means"S.E., *P-0.05 exercised vs. control, ns6.
training provides protection against injury it is probably due to exercise-induced changes in antioxidant capacity (Powers et al., 1999). Venditti and Di Meo (1996) have reported that exhaustive exercise gave rise to tissue damage irrespective of the trained state. Thus, endurance capacity was strongly increased by training. Most of the above degenerative processes have been quantified after exhaustive exercise (Alessio, 1993). However, it is also important to quantify the extent of damage, both within the different muscles involved in the exercise and as regards the different types of exercise performed. A number of indices have been used to assess the effects of damage due to strenuous exercise. Among them, carbonyl assays are useful for determining damage to proteins (Liu et al., 2000). In erythrocytes, proteolysis seems to occur independently of membrane damage and appears to be a more sensitive indicator of cell exposure to oxygen radicals than the degree of lipid peroxidation (Davies and Goldberg, 1987). Measurements of membrane fluidity and latency are important for analysing membrane integrity, since disruption of membranes increases the leakage of important cellular constituents, thereby compromising muscular function (Davies et al., 1982).
Nitric oxide (NO) can also be generated in muscle in response to exercise (Reid, 1996), and an excess of nitrogen free radicals damages muscle membranes and increases protein degradation both of which could impair the performance, integrity and metabolism of muscle cells. In addition, possible nitrating effects can be considered since tyrosine nitration is frequently linked to altered protein function during inflammatory conditions (Eiserich et al., 1999). Differences among muscle types may be related to their physiological, mechanical and structural properties and their utilisation during functional activity. However, to date no studies have focused on the effects of different types of exercise, the intensity of such exercise and training on the effects of muscle injury. Since the biochemical mechanisms through which regular exercise exerts beneficial effects are not well understood, here we were prompted to study the mechanism of adaptation and protection after physical training. 2. Materials and methods 2.1. Animals and exercise protocols Thirty six male Wistar rats weighing 210"30 g obtained from IFFA Credo (Madrid, Spain) were
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used. Animals were housed in a heat- and lightcontrolled animal facility and were allowed free access to water and commercial rat chow (Panlab, Barcelona, Spain). The animals were housed according to the Principles of Council Directive 86y609yECC: ‘On the Approximation of Laws, Regulation and Administrative Provision of the Member States regarding the protections of animal used for experimental and other scientific purposes’. The rationale for the selection of these animals was based on our experience that these rats are good runner and get to training efficiently (Ferrando et al., 1999; Cabral de Oliveira et al., 2001). Exercise was performed on a rodent treadmill on the basis of the following protocols: Exhaustive exercise (Ex): Rats were run to exhaustion at 20 m miny1 and 0% grade. Average endurance time was 80"4 min. Maximal exhaustive exercise (Max-Ex): Exercise started at 10 m miny1, 0% grade followed by a gradual increase in treadmill speed and grade every 4 min up to 30 m miny1, 15% grade. Average run-time to exhaustion was 60"5 min. Eccentric exercise (Ec): The rats ran an intermittent protocol downhill (y168 incline) at 16 m miny1 for a total of 90 min; 5 min bout (18 bouts) separated by 2 min rests. Training protocol: Rats were accustomed to treadmill running in a 4-week period, during which the intensity of the exercise was gradually increased to 27 m miny1, 15% grade for 1 h dayy1, 5 d weeky1. This intensity was maintained for a further 8 weeks. This group was then subdivided into 2 groups: in the first group, rats were killed at 24 h after exercise to minimize the residual effects of the last bout of exercise (Tr). The other rats were killed immediately after an exhaustive session of exercise lasting approximately 80"4 min to compare the effect of acute exercise after training (Tr– Ex). The control group (C) comprised sedentary animals. At the time of death, immediately after exercise protocols, the rats were anaesthetized with pentobarbital sodium (5 mg 100 g body wty1, ip), and the following muscles were removed: vastus intermedius, as an oxidative muscle (rich in I and IIa fibre types); mixed gastrocnemius (gastro), with an equal composition of oxidative and glycolytic fibres, and tibialis anterior and extensor digitorum longus (EDL) as glycolytic muscles rich in type IIb fibres. The muscle were rapidly excised, trimmed of extraneous fat and connective tissue,
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immersed in isopentane, dropped into liquid nitrogen and stored at y708 until further analysis. 2.2. Muscle protein oxidation This was evaluated by measuring carbonyl formation, using 2,4-dinitrophenylhydrazine (DNPH) as a reagent, according to Levine et al. (1990), with some modifications. Briefly, 200–400 mg muscle were homogenized at 0–4 8C in 5 mM potassium phosphate buffer (pH 7.4; wyv 1:10), including 0.1% Triton X and protease inhibitors. The homogenates were centrifuged at 500=g for 3 min, and an aliquot of 900 ml of supernatant was incubated with 100 ml of 10% streptomycin sulphate (in 50 mM HEPES). Samples were then vortexed vigorously and kept at room temperature for 15 min, after which they were centrifuged at 6000=g for 10 min at 4 8C and the supernatant used. After reacting with 10 mM DNPH–2 N HCl in the dark, protein was precipitated with 20% trichloroacetic acid, followed by centrifugation at 14 000=g for 10 min. The pellets were washed three times to remove excess DNPH, suspended in 6 M guanidine HCl (in 20 mM KH2PO4, pH 2.3), vortexed, and allowed to dissolve for 2 h at 37 8C. The absorbance of the samples was measured at 366 nm. Carbonyl contents were calculated using a molar absorption coefficient of 22 000 My1 cmy1. 2.3. Isolation of mitochondria Muscle samples were homogenized in 1y10 buffer A (containing 70=10y3 mol ly1 sucrose, 220=10y3 mol ly1 mannitol, 2.0=10y3 mol ly1 ethylene-diaminetetra-acetic acid, 5.0=10y3 y1 mol l 4-morpholinepropanesulphonic acid, and 0.5% bovine serum albumin, BSA) at pH 7.4 and at 0–4 8C. Mitochondria were isolated using a density gradient procedure according to the methods of Howard (1992), Schnaitman and Greenwalt (1968). The homogenates were centrifuged once for 10 min at 1000=g. The supernatant thus obtained was centrifuged once again at 1000=g for 10 min, after which the pooled supernatant was centrifuged for a further 20 min with buffer B (obtained by diluting buffer A 7.5-fold without BSA, pH 7.4) at 8000=g. The resulting intact mitochondrial pellets were suspended in buffer B. Mitochondrial protein contents were measured using the method of Lowry et al. (1951), with
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BSA as standard. Mitochondrial purity was evaluated by means of the inhibition percentage of adenosine triphosphatase activity (Drahota and Houstek, 1977) with sodium azide 10 mM, the inhibition obtained was higher than 70%. 2.4. Measurement of fluorescence anisotropy The lipid probe 1,6-diphenyl-1,3,5-hexatriene (DPH) in combination with fluorescence polarization techniques (Shinitzky and Barenholz, 1978; Li et al., 1999) has been used to investigate the dynamic properties of mitochondrial membranes. A high fluorescence polarization value correlates with low membrane fluidity and vice versa. Samples of mitochondria with 0.5 mg protein were incubated in 5 ml DPH solution 2=10y6 mol ly1, pH 7.4, at 25 8C for 30 min. Fluorescence polarization of the suspended solution was measured by a spectrofluorometer equipped with a polarization attachment at an excitation of 362 nm and an emission of 432 nm at 25 8C. From the intensity of the fluorescence measured successively with the polarizer parallel (I) and perpendicular (I9) to the excitation beam, the polarization was calculated according Ps(IyI9)y(IqI9). 2.5. Nitrate measurements Nitrate concentrations in the muscle cytosolic fraction were measured by an enzymatic method using nitrate reductase from Aspergillus species, as previously described by Granger et al. (1999). 2.6. Latency measurements of alkaline phosphatase activity Sarcoplasmic reticulum (SR) membrane integrity was assessed with latency measurements of alkaline phosphatase activity, as previously described by Forte et al. (1967) and according to Venditti and Di Meo (1996), using both initial and total (solubilized with Triton X 100-1%) activities. Briefly, the reaction kinetics was followed spectrophotometrically at 400 nm from 5 to 10 min. Reactions was carried out at 37 8C in a final volume of 1 ml containing 0.1 M Tris–acetate, pH 7.4, 4.8=10y3 M of p-nitrophenylphosphate, 4.8=10y3 M MgCl2, muscle homogenate (1y10 wyv in 125 mM KCl and 10 mM Tris pH 7.4) with or without Triton. The latency percentage of the activity was obtained from the ratio: (absorb-
Fig. 2. Carbonyl content (nmol mg proteiny1 ) in heart of control rats (C), acutely exercised rats: Maximal Exhaustive (Max-Ex), Eccentric (Ec), Exhaustive (Ex), and chronically exercised rats: Training (Tr) and Training Exhaustive (Tr–Ex). Values are means"S.E., *P-0.05 exercised vs. control, ns6.
ance changeymin (homogenateqTriton)yabsorbance changeymin (homogenate))y(absorbance changeymin (homogenateqTriton))=100. 2.7. Statistical analysis All data are expressed as means"S.E. The data were analysed by means of analysis of variance (ANOVAyMANOVA), using the Newman–Keuls test. Differences with a P-value of -0.05 were considered to be statistically significant. 3. Results Figs. 1 and 2 show the carbonyl contents in muscle and heart. A single bout of exercise dramatically increased the carbonyl contents of skeletal and cardiac muscle. The extent of such increases was clearly dependent upon the type and muscle exercised. Thus, the increase was higher in the vastus subjected to eccentric contractions (4.3"0.33 nmol mg proteiny1), (Fig. 1). The carbonyl contents in the trained group did not increase significantly in comparison with the controls (sedentary). Carbonyl contents in heart (Fig. 2) were significantly elevated by an acute bout of exercise (Max-Ex, Ec and Ex). In comparison with the controls, the carbonyl contents of both groups of animals from the trained groups were unchanged. Interestingly, basal carbonyl contents in cardiac muscle (1.0"0.1 nmol mg proteiny1) were lower than in skeletal muscle (1.8"0.2 nmol mg proteiny1 in the vastus intermedius), whereas after
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Table 2 Effect of different types of exercise on membrane integrity of heart and gastrocnemius muscle
Control Maximal-exhaustive Eccentric Exhaustive Training Training–exhaustive
Fig. 3. Nitrate concentration in rat myocardial muscle (nmol gy1) of control rats (C), acutely exercised rats: Maximal Exhaustive (Max-Ex), Eccentric (Ec), Exhaustive (Ex), and chronically exercised rats: Training (Tr) and Training Exhaustive (Tr–Ex). Values are means"S.E., *P-0.05 exercised vs. control, ns6.
acute exercise the values in the heart almost reached the highest value seen for skeletal muscles. Nitrate concentrations in cardiac muscle (Fig. 3) reflected the NO levels in the exercise experimental conditions. Thus, nitrate concentrations (nmol gy1) in the heart of rats (ns6) subjected to exhaustive and eccentric exercises were significantly (P-0.05) increased as compared with the controls (23.25"1.03) and clearly elicited oxidative stress. Training did not change the nitrate concentrations measured in heart (Tr 23.25"0.47) even after the exhaustive exercise session (Tr–Ex 24.50"0.64). The levels of NO, which can indirectly interfere with most of muscular events, were reduced in cardiac muscle after training. Table 1 shows the fluorescence polarization of muscle mitochondrial membrane in heart and muscle. The maximal exhaustive exercise significantly affected (P-0.05) mitochondrial membrane fluidity in the vastus intermedius and gastrocnemius muscles, whereas eccentric exercise significantly affected (P-0.05) the vastus intermedius. Table 1
Muscle
Heart
13.0"0.3 18.1"1.9* 25.0"0.3* 17.3"5.6 16.7"3.4 16.0"3.0
20.9"5.3 25.8"3.7 23.9"3.4 30.7"7.2 25.4"4.4 16.7"3.0
Latency percentage of alkaline phosphatase activity is expressed as: (absorbance changeymin (homogenateqTriton)yabsorbance changeymin (homogenate))y(absorbance changeymin (homogenateqTriton))=100. Values are means"S.E. * P-0.05 exercised vs. control, ns6.
also shows that a single bout of exercise, in this case Max-Ex, Ex and also Ec significantly increased (P-0.05) mitochondrial membrane fluidity in cardiac mitochondrial membrane. In the trained groups of rats, heart fluorescence polarization (P) was not significantly different from control group. Ps0.132"0.001 in training group (Tr) and 0.131"0.001 in exhaustive exercise after training group (Tr–Ex). Table 2 shows the results deduced from latency measurements of the enzyme alkaline phosphatase in gastrocnemius muscle and in heart. Collecting these results involved the determination of initial and solubilized activities in the skeletal and cardiac muscles that were unaffected by training. In contrast, activities were changed (P-0.05) following maximal exhaustive and eccentric exercises in gastrocnemius muscle, due to the increased (homogenateqTriton) activities, indicating alterations in the integrity of the sarcoplasmic and endoplasmic reticulum (ER) and membrane damage in this muscle. The values for heart were not statistically significant.
Table 1 Fluorescence polarization of muscles
Heart Vastus EDL Tibialis Gastro
C
Max-Ex
Ec
Ex
Tr
Tr–Ex
0.131"0.003 0.167"0.004 0.151"0.002 0.150"0.002 0.162"0.001
0.156"0.002* 0.202"0.005* 0.153"0.004 0.144"0.001 0.173"0.002*
0.145"0.002* 0.202"0.003* 0.150"0.004 0.154"0.002 0.151"0.002
0.148"0.001* 0.169"0.007 0.145"0.003 0.144"0.003 0.170"0.006*
0.132"0.001 0.164"0.002 0.154"0.003 0.147"0.002 0.167"0.002
0.131"0.001 0.165"0.003 0.153"0.002 0.142"0.008 0.163"0.001
Fluorescence polarization (P) of skeletal and cardiac muscles mitochondria in control (C), Maximal Exhaustive (Max-Ex), Eccentric (Ec), Exhaustive (Ex), Training (Tr) and Training Exhaustive (Tr–Ex) groups. Values are means"S.E. * P-0.05 vs. C, ns6.
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4. Discussion After exhaustive exercise increased rates of RONS in tissues mediate the oxidation and nitration of lipids, nucleotides, and susceptible protein amino acid residues. These products also suggest an impairment of biomolecule structure and function. In fact, disruption of the lipid bilayer may cause structural disorganisation of biological membrane. This will alter membrane fluidity and permeability, compromising membrane function (Sen, 1995). We chose five different exercise protocols because it is well known that different forms of exercise result in different levels of oxidative stress. Treadmill exercise is a standard form of exercise used for rodent experiments. Our animals were made to perform acute exercise to exhaustion in order to increase the oxidative stress. Since the aim of our work was to conduct a comparative study of both cardiac and skeletal tissues, we included the protocol designed as maximal exhaustive exercise to provide maximal cardiac stress, whose intensity was higher than that produced in exhaustive exercise (Ji, 1993). The other protocol, eccentric exercise, involved a polymetric (eccentric) muscle component and the animals were therefore subject 2 types of stress: oxidative and polymetric, the latter imposing muscle trauma (Hunter and Faulkner, 1997). The protective role of training against the oxidative stress generated by exercise in heart and muscles was assessed with the performance of an acute exercise session after training. In this study membrane fluidity of muscle mitochondria was assessed by the method of fluorescence polarization. This is the most convenient and frequently used index for estimating membrane fluidity. To avoid the possible influence of thermotropic transition of fluorescence polarization the temperature was well controlled at 25 8C when conducting the experiments (Astier et al., 1996; Li et al., 1999). The results obtained here revealed that in the heart changes in mitochondrial membrane fluidity due to exhaustive exercise are not subtle, suggesting metabolic changes that could contribute to membrane dysfunction or phospholipid degradation (Buja et al., 1991). The greatest changes in mitochondrial membrane fluidity were observed in vastus (type IIa-rich fibre) after the Max-Ex and Ec exercises. From animal studies (Ji et al., 1992), it is known
that the vastus muscle is strongly effected by acute exercise despite its high antioxidant capacity. It has been reported that exhaustive exercise can also alter membrane fluidity in other tissues of the body (cortex, hippocampus, etc.) Hiramatsu et al. (1993). Mitochondrial fluidity was not modified in either cardiac or skeletal muscle after training, Kim et al. (1996) have shown that a combination of exercise and dietary restriction may be effective in preserving membrane fluidity. It is known that endurance capacity is largely determined by the functional mitochondrial content of muscle and this can be increased by chronic endurance exercise training (Davies et al., 1981). Walsh et al. (2001), showed that the susceptibility of mitochondrial respiration to ROS was unchanged after endurance training in skinned fibre preparation, but the authors establish the vulnerability of the mitochondria isolated from their structural environment and the elimination of cytosolic soluble antioxidants enzymes. Comparing the results of skeletal muscle and heart mitochondrial membrane fluidity after exhaustive exercises, the heart showed lower levels. The increase in oxygen consumption in the liver and heart during exercise is rather modest compared with the situation in skeletal muscle, and the heart has an antioxidant capacity higher than muscle (Ji, 1993). The basal carbonyl content in the heart was also lower than in skeletal muscle. Our results pointed to a significant increase in carbonyl levels in the heart after acute exercises. Liu et al. (2000) did not find any increase in protein carbonyl level in the heart or skeletal muscle after acute exercise whereas they observed that chronic exercise induced an increase in slow muscle, however, their exercise protocol differed from ours. Radak et al. (1999) showed decreased extent of carbonylation and reduced accumulation of 8-OHdG after regular exercise in rat skeletal muscle. NO can indirectly interfere with many muscular events and has been shown to reduce the contractile force of the diaphragm and ventricular myocytes (Joe et al., 1998). NO production after a single bout of exercise may reflect a systemic inflammatory response to heavy exercise (Niess et al., 2000). During inflammatory conditions, some cytoskeletal proteins and cytochrome c serve as critical targets for oxidation and nitration-induced functional impairment. (Hinshaw et al., 1988; Cas-
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sina et al., 2000). In our study the trained animals presented lower NO heart levels and the basal expression of iNOS mRNA is down regulated by moderate endurance training (Niess et al., 2002). After the performance of eccentric exercise (Ec), the vastus muscle showed the highest value in carbonyl content. In animal studies, it has been demonstrated that exercise-induced damage occurs primarily in deep extensor muscles. Presumably, this is because deep red muscle fibres are specific fibres that are primarily recruited to produce force during exercise. Thus, deep red extensor muscles (such as the vastus intermedius) may be more affected than their synergistics (Komulainen and Vihko, 1994). The malondialdehyde concentration in muscle and plasma was unaltered after eccentric contraction (Child et al., 1999) but protein oxidation, damage of SR and mitochondrial membrane integrity in our study showed a free radical damage in the first 24 h after eccentric exercise according to Best et al. (1999). The pattern of membrane damage following exhaustive exercise may also be deduced from latency measurements of the SR and ER enzyme alkaline phosphatase. The results obtained here suggest that exhaustive exercises decrease SR and ER membrane integrity in muscle. Similar results have been obtained by Davies et al. (1982), Venditti and Di Meo (1996) after acute exercise. Functional alterations in SR may also be consequent to free radicals attack, also suggested by the presence of higher amount of carbonyl derivatives after exhaustive exercise, such results have been showed in skeletal muscle lesion during Mgdeficiency (Astier et al., 1996). Endurance training consistently upregulates GSH dependent defenses and other antioxidants enzymes with the effects most marked in highly oxidative muscle (Leeuwerburgh et al., 1997). Moreover, training results in several changes that could improve myocardial capacity. In fact, training is associated with an increase in both the Mn and Cu–Zn isoforms of SOD in the left ventricle and increase in myocardial glutathione (Ji, 1993), although this issue remains controversial since some investigators (Powers et al., 1999) failed to observe a traininginduced improvement in myocardial antioxidant capacity. Our study showed that a single period of exhaustive exercise (Max-Ex, Ec and Ex) led to increased formation of carbonyl and decreased membrane fluidity in mitochondria and that the heart is as
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responsive to all types of exercise as skeletal muscle. Both intensive exercises (exhaustive and maximal exhaustive) and eccentric exercise (which promotes mechanical damage) elicit mitochondrial injury in the heart and in the vastus intermedius muscle. Regular exercise ameliorates this acute oxidative stress in both tissues. Acknowledgments AC Perez was supported by a fellowship from the Comision Interministerial de Ciencia y Tecno´ Spain. All the experiments performed in this logıa, work comply with the current laws of the European Community. References Alessio, H., 1993. Exercise-induced oxidative stress. Med. Sci. Sports Exercise 25, 218–224. Armstrong, R.B., Warren, G.L., Warren, J.A., 1991. Mechanisms of exercise-induced muscle fibre injury. Sports Med. 12, 184–207. Astier, C., Rock, E., Lab, C., Gueux, E., Mazur, A., Rayssiguier, Y., 1996. Functional alterations in sarcoplasmic reticulum membranes of magnesium-deficient rat skeletal muscle as consequences of free radical-mediated process. Free Radic. Biol. Med. 20, 667–674. Best, T.M., Fiebig, R., Corr, D.T., Brickson, S., Ji, L.L., 1999. Free radical activity, antioxidant enzyme, and glutathione changes with muscle stretch injury in rabbits. J. Appl. Physiol. 87, 74–82. Buja, L.M., Miller, J.C., Krueger, G.R., 1991. Altered membrane fluidity occurs during metabolic impairment of cardiac myocytes. In Vivo 5, 239–243. Cassina, A.M., Hodara, R., Souza, J.M., et al., 2000. Cytochrome c nitration by peroxynitrite. J. Biol. Chem. 275, 21409–21415. Cabral de Oliveira, C., Perez, A.C., Merino, G., Prieto, J.G., Alvarez, A.I., 2001. Protective effects of Panax ginseng on muscle injury and inflammation after eccentric exercise. Comp. Biochem. Physiol. Part C 130, 369–377. Child, R., Brown, S., Day, S., Donnelly, A., Roper, H., Saxton, J., 1999. Changes in indices of antioxidant status, lipid peroxidation and inflammation in human skeletal muscle after eccentric muscle actions. Clin. Sci. 96, 105–115. Davies, K.J.A., Goldberg, A.L., 1987. Oxygen radicals stimulate intracellular proteolysis and lipid peroxidation by independent mechanisms in erythrocytes. J. Biol. Chem. 262, 8220–8226. Davies, K.J.A., Packer, L., Brooks, G.A., 1981. Biochemical adaptation of mitochondria, muscle and whole-animal respiration to endurance training. Arch. Biochem. Biophys. 209, 539–554. Davies, K.J.A., Quintanilha, A.T., Brooks, G.A., Packer, L., 1982. Free radicals and tissue damage produced by exercise. Biochem. Biophys. Res. Commun. 107, 1198–1205.
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