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1 School of Exercise and Sport Science, Faculty of Health Sciences, The University of Sydney, Sydney, NSW 1825, Australia 2 Muscle Development Unit, Children's Medical Research Institute, Locked Bag 23, Wentworthville, NSW 2145, Australia
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(Received 8 November 2006;
accepted after revision 26 November 2006; first published online 30 November 2006)
Corresponding author M. W. Thompson: School of Exercise & Sport Science, The University of Sydney, Sydney, NSW 1825, Australia. Email: m.thompson{at}fhs.usyd.edu.au
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Introduction |
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Warren et al. (1993) suggested that impaired excitation–contraction (E–C) coupling occurred in eccentrically damaged mammalian muscle, which was subsequently confirmed by direct measurements of intracellular calcium (Balnave & Allen, 1995), and that most of the early strength loss can be attributed to a failure of E–C coupling (Warren et al. 2001). It has been proposed that the muscle damage process begins with overstretching of sarcomeres within myofibrils and that T-tubules and SR would be susceptible to damage in the overstretched regions, which may be the basis for the E–C coupling damage (Morgan & Allen, 1999; Yeung et al. 2002).
The investigation of SR calcium regulation in muscle damaged by eccentric contractions can yield information on the mechanism of E–C coupling failure. Such studies are few and yield conflicting results. One animal study (Yasuda et al. 1997) has demonstrated that SR membrane integrity is altered in rat skeletal muscles following eccentric contractions induced by percutaneous electrical stimulation with no changes in muscle SR Ca2+-ATPase pump function. However, in a human study that investigated changes in SR Ca2+ uptake and Ca2+-ATPase activity in exercised vastus lateralis with 60 min of eccentric cycling exercise (Enns et al. 1999), SR Ca2+ uptake was depressed only during the recovery period but not immediately following the exercise, whereas the SR Ca2+-ATPase activity showed a biphasic response, increasing during the first 2 days of recovery, returning to the pre-exercise level at 6 days of recovery, and then increasing again by the end of the recovery period. In contrast, no changes in SR function were found during the first 24 h following eccentric exercise in young men even though there were significant changes in muscle contractility (Nielsen et al. 2005). There are no studies that have examined the magnitude and time course of changes in SR function, including Ca2+ uptake, Ca2+ release and Ca2+-ATPase, together with examination of muscle ultrastructure over a longer time course. Thus, there is a clear need for further research in this area.
The objective of this study was to examine the effects of eccentric exercise on SR Ca2+ uptake, Ca2+ release and Ca2+-ATPase activity in rat skeletal muscle, in terms of both magnitude and time course. In addition, ionophore stimulation was determined to assess vesicle integrity by measuring the ratio of calcium-dependent ATPase activities in the presence and absence of the calcium ionophore A23187 [GenBank] (Byrd et al. 1989; Yasuda et al. 1997). In this study, we hypothesized that eccentric exercise would result in persistent changes in SR Ca2+ uptake, Ca2+ release and Ca2+-ATPase activity that would occur immediately after exercise, with the most pronounced changes between 24 and 48 h postexercise and with a return to baseline levels some days later. Moreover, eccentric exercise would effect changes in SR membrane integrity, as indicated by the ratio of Ca2+-ATPase activity with ionophore to that without ionophore.
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Methods |
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Male Sprague–Dawley rats were used in this study. Upon arrival at the animal house, the rats were placed in standard rat cages (2–3 animals per cage) in a light- (12 h dark–12 h light; light–dark cycle was opposite to day–night) and temperature (21–22°C)-controlled environment. A 7 day acclimation period was allowed, during which ad libitum standard rat chow and water were offered. None of the rats had been on a treadmill before the acute experiment. At the time of killing, the animals weighed 395 ± 5.9 g (mean ± S.E.M.). Animal holding facilities and all experimental protocols and procedures were approved by the University of Sydney Animal Ethics Committee.
Exercise protocol
In all experiments, one to three rats were exercised simultaneously on a motor-driven treadmill at 15 m min–1 down a 16 deg decline continuously for 90 min. All experiments were performed at approximately the same time of day. It was usually necessary to use some mild electrical shocks to stimulate the animals to run during the initial part of the exercise, but every effort was made to keep this form of stimulation to a minimum. However, since the tail was usually the only part of the animal body to make contact with the electrical grid, we assumed that muscles chosen for study in this protocol (red vastus muscles) would not be affected by the infrequent electrical stimulation. Similar exercise protocols previously showed evidence of damage to the deep slow extensor muscles of rats (Armstrong et al. 1983; Duan et al. 1990).
All rats were randomly assigned to sedentary control (n = 7) and eccentric exercise groups. After the downhill exercise, the exercised rats were subdivided randomly into one of six subgroups (n = 7), which were killed immediately, or at 4, 24, 48, 72 or 144 h after the cessation of exercise. All exercised rats, except those assigned to the subgroup of immediate postexercise, were placed back in their cages with free access to food and water until they were killed for muscle tissue sampling.
Measurement of rat rectal temperature
Rat rectal temperature was measured after induction of general anaesthesia by insertion of a thermometer probe into the rectum to a depth of approximately 5 cm in sedentary control rats and those assigned to the subgroup of immediate postexercise. The probe consisted of thermocouple wire inserted into a 2.5 mm plastic tube. The thermocouple was connected to a dataTaker DT500 (Melbourne, Australia), which acquired data every 30 s. The thermometer remained in place for 
2 min and the highest temperature was recorded.
Sample preparation
The rats were anaesthetized by intraperitoneal injection of ketamine/xylazine at a dose of 104.3/15.6 mg (kg body weight)–1, and then humanely killed by excision of the heart. Muscles were immediately removed from both hindlimbs and immediately placed on a Petri dish with ice-cold physiological saline. The muscles removed were the predominantly red parts of m. vastus. After dissection, the muscle sample was freed from fat and connective tissue. Approximately 70–80 mg of muscle were weighed, diluted 1:10 (w/v) in cold homogenizing buffer (40 mM Tris, 0.3 M sucrose, pH 7.9) and then homogenized on ice at 18 000 r.p.m. three times for 15 s with 15 s rests (Omni 2000; Omni International, Warrington, Virginia, USA). This process resulted in a suspension of fragmented SR membranes which resealed into numerous small vesicles, allowing direct measurements of the rates of Ca2+ uptake, Ca2+ release and Ca2+-ATPase activity (Simonides & van Hardeveld, 1990; Ruell et al. 1995). The homogenizing procedure for each sample was completed within 5 min and the suspension kept cool by packing the sample in ice before, during and after homogenization. The homogenate was then rapidly frozen in liquid nitrogen for later analyses of SR Ca2+-ATPase activity, Ca2+ uptake and Ca2+ release. It has previously been shown that homogenates are stable to freezing and that SR function is only minimally altered (Ruell et al. 1995). Muscle homogenate protein content was determined in triplicate according to the methods of Markwell et al. (1978). On any given day, seven samples from each subgroup (control, 0, 4, 24, 48, 72 and 144 h postexercise) were analysed together. All measurements were completed within 50 min after thawing of the sample.
Ca2+-ATPase activity
The SR Ca2+-ATPase activity was measured in triplicate on mucle homogenate samples using an enzyme-linked reaction (Simonides & van Hardeveld, 1990). The assays were performed at 37°C using a spectrophotometer (UV-1601PC; Shimadzu, Tokyo, Japan) at 340 nm as previously described (Ruell et al. 1995). The assay buffer consisted of 18 mM Hepes buffer, pH 7.5, 180 mM KCl, 13 mM MgCl2, 1 mM EGTA, 9 mM NaN3, 0.3 mM NADH, 9 mM phosphoenolpyruvate, 22 U ml–1 lactate dehydrogenase, 16 U ml–1 pyruvate kinase and 4 mM ATP. When the intactness of SR vesicles was assessed, the assay mixture additionally contained 2.6 µM Ca2+ ionophore A23187 [GenBank] (Boehringer Mannheim GmbH, Mannheim, Germany). Ionophore stimulation was determined to evaluate vesicle integrity by calculating the ratio of Ca2+-ATPase activities in the presence and absence of A23187 [GenBank] .
Muscle homogenate (10 µl) was added to 1 ml assay buffer, and the reaction was initiated by the addition of 1.26 mM CaCl2. This concentration was determined from a pilot experiment, in which the calcium concentration producing the highest total activity was determined. The basal ATPase activity was then measured after the addition of 15 µl of 2 M CaCl2, giving a final concentration of 26.8 mM CaCl2, which completely inhibits the Ca2+-ATPase activity. The SR Ca2+-ATPase activity was determined from the total minus basal activities. The SR Ca2+-ATPase activity was corrected for protein content in the muscle homogenate and expressed as nmoles per minute per milligram of muscle protein.
Rates of Ca2+ uptake and Ca2+ release
The rates of oxalate-supported Ca2+ uptake and silver nitrate (AgNO3)-induced Ca2+ release were analysed at 37°C using the Ca2+-fluorescent dye indo 1 on a dual-emission luminescence spectrofluorometer (Series 2, Aminco Bowman, SLM Instruments, Urbana, IL, USA) in a reaction medium (2.2 ml) that consisted of 20 mM Hepes, 150 mM KCl, 10 mM NaN3, 6.8 mM oxalate, 4.5 mM MgATP, 5 µM N,N,N,'N'-tetrakis (2-pyridylmethyl)ethylenediamine (TPEN), and 1 µM indo-1, pH 7.0 (O'Brien, 1990; Ruell et al. 1995). Addition of extra CaCl2 was not necessary as the Ca2+ in the assay buffers gave a starting free [Ca2+] of approximately 1 µM. The excitation wavelength was 349 nm and the emission wavelength alternated between 410 and 485 nm (for Ca2+-bound and Ca2+-free indo-1, respectively). Excitation and emission bandpass widths were set at 1 and 8 nm, respectively. Once the homogenate was added, ratiometric data were collected every 1 s for the next 100 s. The decrease in [Ca2+] due to uptake by the SR was determined from the ratio of emission signals at 410 and 485 nm according to the equation of Grynkiewicz et al. (1985). A Ca2+–indo-1 dissociation constant of 170 nM was used (Ruell et al. 1995).
The maximal rate of Ca2+ release was determined by the addition of AgNO3 (141 µM) once free [Ca2+] had declined to a plateau, approximately 100 s after homogenate addition (Fig. 1), as previously described (Ruell et al. 1995). Minimum and maximum ratios were determined at the completion of the assay by the addition of EGTA (3.5 mM) and CaCl2 (3.8 mM), respectively. Maximal rates of Ca2+ uptake and release were calculated as previously described (Hill et al. 2001) and were corrected for protein content and expressed as nmoles per minute per milligram of muscle protein.
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Transmission electron microscopy was performed for qualitative ultrastructural analysis of red vastus muscle in rats following the eccentric exercise. Muscle tissues were immediately removed after dissection and fixed in Karnovski's fixative overnight, subsequently postfixed with 2% osmium tetroxide, dehydrated through an ascending series of ethanol, and embedded in Spurr's epoxy resin. Ultrathin sections (70 nm) of muscle tissue were double-contrasted with uranyl acetate and lead citrate, viewed and photographed with a Philips CM12 BioTwin transmission electron microscope.
Statistical analysis
All data are expressed as means ± S.E.M. unless otherwise stated. A one-way analysis of variance (ANOVA) was used in order to calculate whether there were significant differences between control and exercised conditions. If significance was reached using ANOVA, the difference between individual groups was determined using Duncan's post hoc analysis. Significance of the difference of rat rectal temperature between control and immediately postexercise groups was assessed through the Student's unpaired t test. All statistics were calculated using the Statistical Package for the Social Sciences (SPSS 10). For all comparisons, the level of significance was set at P < 0.05.
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Results |
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There was no difference observed in the muscle protein content of muscle samples of red vastus muscles before and following the eccentric exercise, as shown in Table 1.
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In rat red vastus muscle, the rate of SR Ca2+ uptake for whole muscle homogenate from control animals was 19.25 ± 1.38 nmol min–1 (mg protein)–1. The Ca2+ uptake rate was significantly (P < 0.05) depressed by 29% immediately following the eccentric exercise, further reduced (P < 0.05) by 36% at 4 h postexercise, and remained depressed (P < 0.05) by 20% at 24 h postexercise (Fig. 2).
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In the red vastus muscle, the rate of Ca2+ release from SR for whole muscle homogenate from control animals was 31.06 ± 2.36 nmol min–1 (mg protein)–1. The Ca2+ release rate followed a similar profile in exercise and recovery to Ca2+ uptake rate. Similar to Ca2+ uptake rate, Ca2+ release rate was decreased (P < 0.05) by 37% immediately following exercise, further reduced (P < 0.05) by 39% at 4 h postexercise, and remained depressed (P < 0.05) by 26% at 24 h postexercise (Fig. 3).
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Ca2+-ATPase activities, measured in homogenates with the ionophore A23187 [GenBank] , were found to be decreased by 31 and 24% at 4 and 24 h postexercise, respectively (P < 0.05; Table 2). When measured in the absence of the ionophore, Ca2+-ATPase activity in red vastus decreased at 4 h postexercise only. To determine whether SR membrane integrity is altered by eccentric exercise in this study, Ca2+-ATPase activity was assessed with and without the Ca2+ ionophore A23187. [GenBank] The ratio of Ca2+-ATPase activity with the ionophore to that without the ionophore (+/–) was calculated to provide an index of membrane alterations. A 2.2-fold increase in Ca2+-ATPase activity with ionophore was observed in homogenate from the control group (Table 2). From the results, there was no change in the ratio, hence membrane integrity was not altered by exercise for all experimental groups.
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Electron micrographs of the red vastus revealed a normal striated myofilament pattern in all sedentary control rats. No abnormalities were seen in any of the muscle samples taken from sedentary control rats (Fig. 4A). Qualitative morphological damage at the ultrastructural level was evident in red vastus muscle following an acute prolonged downhill exercise bout. Alterations of SR in the red vastus muscle samples taken immediately postexercise were indicated by dilated SR vesicles (Fig. 4B and D). Focal dilatations of the SR were also observed in the muscle samples taken at 4 h following downhill exercise (Fig. 4C). However, such SR distension at the ultrastructural level became less evident at 24, 48, 72 and 144 h following exercise. In some focal damaged areas from the 4 h group (Fig. 4C), there was occasionally complete disruption of the architecture, showing that the myofilaments were disorganized and out of register. The red vastus muscles in the 4 h group showed evidence of the early stages of muscle damage (Fig. 4C) compared with muscles from the control group (Fig. 4A). In the 24 and 48 h groups, the red vastus muscles showed substantial disruption of the myofilaments and loss of Z-bands occurring in more widespread areas, indicating progressive fibre degeneration (Fig. 5A and B).
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Discussion |
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Similar downhill exercise protocols were previously shown to result in marked injury to fibres in the deep slow extensor muscle of rats (Armstrong et al. 1983; Schwane & Armstrong, 1983; Duan et al. 1990; Lynn & Morgan, 1994). Armstrong et al. (1983) showed that a prolonged bout of decline running by untrained rats produced histological and biochemical changes in muscles, particularly the vastus intermedius, the deep red layer of the knee extensors, indicative of cell damage. They concluded that these postural muscles undergo eccentric contractions while lowering the animal down the decline, acting as ‘brakes’, but undergo concentric contractions during incline running. Comparisons between experiments with extremely different exercise protocols should be made with caution, since high-intensity exercise will change the recruitment order of motor units, when the fast-twitch glycolytic fibres are presumably recruited (Armstrong & Laughlin, 1985). This is evident with a voluntary high force eccentric exercise protocol and with evoking maximal eccentric contractions via electrical stimulation, where the damage was confined to the type II muscle fibres in general (Friden et al. 1983; Lieber & Friden, 2002).
In this study, immediately following downhill exercise, red vastus muscle fibres showed disruption of the normal banding pattern in localized areas. Similar morphological alterations were also seen 4 h after exercise, while in samples taken 24 and 48 h after exercise there were more extensive areas of damage where the degenerating fibres were undergoing necrosis. These initial and progressive changes of damaged muscles subjected to eccentric exercise are in agreement with most of the evidence from previous studies (Armstrong et al. 1983; Friden et al. 1983; Newham et al. 1983; Ogilvie et al. 1988). The fact that eccentric exercise causes progressive delayed damage indicates that there is continued active myofibrillar protein degradation (Armstrong et al. 1991). It has been suggested that the initial injury, which may take the form of mechanical disruption of the sarcolemma, myofilaments, T-tubules and SR, or chemical alteration of the cell membrane, could cause loss of intracellular Ca2+ homeostasis and then activate various proteolytic and degradative pathways intrinsic to the muscle cell (Armstrong et al. 1991; Morgan & Allen, 1999; Yeung et al. 2002).
In a recent study of rats subjected to downhill running, the triceps brachii muscles showed a number of ultrastructural abnormalities, including more longitudinal T-tubule segments, changes in the direction and disposition of triads, caveolar clusters and apposition of multiple T-tubule segments with terminal cisternae elements (Takekura et al. 2001). Lannergren et al. (2000) reported that vacuoles originating from the T-tubular system developed after fatigue produced by repeated, short tetanic contractions in Xenopus but not in mouse fibres. In their experiment, no clear correlation was found between the presence of vacuoles and force depression. However, development of T-tubular vacuoles in eccentrically damaged mouse muscle fibres has been observed in a recent investigation (Yeung et al. 2002). These investigators suggested that T-tubules are susceptible to rupture during eccentric contraction, probably as a result of the relative movement associated with the inhomogeneity of sarcomere lengths. Shearing damage to T-tubules by eccentric contraction was proposed to result in an increase in intracellular [Na+] and [Ca2+], which would cause localized swelling of the T-system and activation of proteases and phospholipases (Yeung et al. 2002). A similar explanation could also be applied to the dilatation of SR in the present study. It has been suggested that sarcomere length inhomogeneities during eccentric contractions could adversely affect adjacent SR segments (Armstrong et al. 1991; Morgan & Allen, 1999). Damage to SR could also produce an elevation in calcium concentration within the fibres, activating proteolytic enzymes (Armstrong et al. 1991; Morgan & Allen, 1999). This suggestion is supported by the findings of Friden & Lieber (1996), who showed that eccentric contractions resulted in formation of crystalline structures within the Z-disk and swelling of the SR. Similar dilatation of SR has also been observed with high-intensity exercise in an early study (McCutcheon et al. 1992). The swelling in the SR may be the result of damage to sarcolemmal membranes and imbalances in normally maintained ionic gradients, or may be the result of an uptake of water to balance the ionic shift (Byrd, 1992; McCutcheon et al. 1992).
The present study is the first to report the effects of downhill exercise on Ca2+ uptake, Ag+-induced Ca2+ release and Ca2+-ATPase activity in rat skeletal muscle, both in terms of magnitude and time course. Both silver nitrate (AgNO3) and 4-chloro-m-cresol (4-CMC) have been used to induce calcium release, and it has been shown that these agents exert their release effects differently (Tupling & Green, 2002). Nevertheless, the rate of release was reduced to a similar extent in fatigue studies comparing Ag+- and 4-CMC-induced Ca2+ release (Williams et al. 1998; Schertzer et al. 2004). An alternative method used in vivo nerve stimulation to induce eccentric muscle damage (Ingalls et al. 1998), resulting in depressions in SR Ca2+ uptake and release with AgNO3 used to stimulate Ca2+ release. With the more physiologically relevant animal model of voluntary exercise employed in the present study, which consisted of 90 min of downhill exercise, we have demonstrated that depressions in SR Ca2+ uptake and Ca2+ release in rat red vastus muscles as measured in vitro occur immediately following the exercise. In addition, interesting findings were the further reduction in both variables at 4 h postexercise, with both remaining depressed at 24 h of the recovery period. Ca2+-ATPase activity with ionophore in muscle homogenate showed significant depression during the recovery period (at 4 and 24 h postexercise).
These results support our main hypothesis advanced, namely that a disturbance in SR Ca2+ regulation would occur in rat red vastus muscles as a consequence of performing prolonged eccentric exercise. However, the ratio of Ca2+-ATPase activity in the presence and absence of ionophore A23187 [GenBank] in the exercised muscles was not significantly different from that of control animals (1.9 versus 2.2, immediately postexercise versus control), which indicated that membrane integrity was not changed by downhill exercise. It has been previously shown that SR membrane damage was induced in an animal model, in which maximal eccentric contractions were elicited via electrical stimulation (Yasuda et al. 1997). This disparity may result from several factors, for example different protocol and experimental duration. It appears that the present exercise intensity and/or volume were insufficient to change the SR membrane integrity.
It is well known that SR Ca2+ uptake is higher in muscles that are predominantly fast twitch, with a correlation between the percentage type II myosin heavy chain and SR Ca2+ uptake (Nielsen et al. 2005). There are no previous studies comparing SR function in slow and fast-twitch muscle following eccentric exercise; however, differences have been noted following concentric exercise. Sarcoplasmic reticulum Ca2+ uptake was depressed to a greater extent in soleus muscle compared with plantaris muscle following an acute bout of concentric exercise to exhaustion, and this was thought to result from the recruitment order of the muscles (Inashima et al. 2003).
In general, the major protein responsible for Ca2+ transport is the 105 kDa ATPase, which translocates 2 mol of Ca2+ across the SR bilayer membrane upon the hydrolysis of 1 mol of ATP (Luckin et al. 1991). A depression in Ca2+ uptake may not only affect relaxation but also reduce Ca2+ loading into the SR, resulting in less Ca2+ being available for release during subsequent action potentials (Zhu & Nosek, 1991). Moreover, inefficiency of Ca2+ transport or slowed Ca2+ uptake during repeated muscle contractions would expose the fibres to increased free [Ca2+] for prolonged periods of time, consequently activating Ca2+-sensitive proteolytic and phospholipolytic degradative pathways (e.g. calpain, phospholipase) involved in myofibrillar degradation (Armstrong et al. 1991; Belcastro et al. 1998).
Elevation in free [Ca2+] is one of the pivotal mechanisms underlying the muscle damage induced by eccentrically biased exercise. Investigators have hypothesized that when actin and myosin are pulled apart as a result of the tensions generated during eccentric exercise, the surface membrane is damaged, allowing entry of extracellular Ca2+ into the cytosol down its concentration gradient, resulting in an increased level of calcium within the muscle cell (Armstrong et al. 1991). Our observations showed that disruption of the normal banding pattern in the localized areas could presumably result in damaged surface membrane and trigger an increase of Ca2+ in the cytosol, since eccentric contractions produce higher forces per unit cross-sectional area. In addition, these increases of Ca2+ in the cytosol could also result from a defect in the ability of the SR to reuptake Ca2+ observed in our study. Belcastro et al. (1998) hypothesized that the influx of extracellular Ca2+ could be the primary factor underlying the activation of the non-lysosomal, calcium-activated neutral protease, calpain, which may be involved in the skeletal muscle protein breakdown response to eccentric exercise (calpain hypothesis). Also, it has been proposed that calpain could produce a characteristic partial fragmentation of the Ca2+ release channel and may modify its properties (Gilchrist et al. 1992). Therefore, elevation in free [Ca2+] is not only a consequence of SR dysfunction but may in turn also cause further damage to SR function that may be long lasting. In the present study, long-term decreases in Ca2+ uptake and Ca2+ release, which were extended up to 24 h postexercise, plus delayed depression in Ca2+-ATPase activity from 4 to 24 h after the exercise, could match this effect temporally and may be attributed, at least partly, to the activated protease process.
Although muscle temperature was not recorded in the present animal study owing to technical limitations, the rat rectal temperature increased from 37.62 to 39.39°C after the eccentric exercise. Thus a higher temperature in muscle compared with rectal temperature of the rat should occur because of higher heat production within the lengthening muscle. In addition, intramuscular temperatures of up to 42°C have been recorded previously in human skeletal muscles with eccentric exercise (Nadel et al. 1972). It has been suggested that the increased temperature could result in protein unfolding, exposing hydrophobic domains and leading to oligomerization, which was defined as aggregation from more active, low-molecular-weight aggregates to less active, high-molecular-weight aggregates (Senisterra et al. 1997). Schertzer et al. (2002) showed that exposure of homogenates to a temperature (41°C) typically experienced in exercise resulted in a reduction in the coupling ratio that was reflected in large reductions of SR Ca2+ uptake and only modest reductions in maximal Ca2+-ATPase activity, owing to an increase in membrane permeability to Ca2+. In the present study, Ca2+ uptake was depressed immediately after exercise with no reduction in Ca2+-ATPase activity; however, this cannot be attributed to alterations in membrane permeability resulting from elevated muscle temperature, since the effect of Ca2+ ionophore did not change after exercise.
In conclusion, this study has shown that a bout of low-intensity, prolonged downhill exercise results in long-lasting depression in SR function in rat red vastus muscles that is not fully restored after 2 days of recovery. However, changes in SR membrane integrity were not evident, which may be due to the relatively low intensity of the downhill exercise in this study. Focal dilatations of the SR were observed at the ultrastructural level in red vastus muscle immediately and 4 h following exercise. These changes could be the result of stress from sarcomere length inhomogeneities during eccentric contractions.
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