Contractile activity-induced oxidative stress: cellular origin and adaptive responses

A. McArdle, D. Pattwell, A. Vasilaki, R. D. Griffiths, and M. J. Jackson

Department of Medicine, University of Liverpool, Liverpool L69 3GA, United Kingdom


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Previous studies have reported that oxidizing free radical species are generated during exercise, and there has been considerable interest in the potential effects of these on exercising tissues. We hypothesized that contracting skeletal muscle was a major source of oxidizing free radical species and that untrained skeletal muscle would adapt to the oxidative stress of a single short period of contractile activity by upregulation of the activity of cytoprotective proteins in the absence of overt cellular damage. Fifteen minutes of aerobic contractile activity was found to induce a rapid release of superoxide anions from mouse skeletal muscle in vivo, and studies with contracting cultured skeletal muscle myotubes confirmed that this was due to release from myocytes rather than other cell types present within muscle tissue in vivo. This increased oxidant production caused a rapid, transient reduction in muscle protein thiol content, followed by increases in the activities of superoxide dismutase and catalase and in content of heat shock proteins. These changes occurred in the absence of overt damage to the muscle cells.

superoxide; redox regulation; stress proteins


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

IT HAS BEEN SUGGESTED for some time that increased amounts of oxidizing free radical species are generated by exercising tissues (3, 9, 11, 21), and there has subsequently been considerable interest in the possibility that these species are involved in the pathogenesis of exercise-induced muscle damage or fatigue. This possibility remains unproven (see Ref. 20 for a review). The majority of evidence for an increased generation of free radicals during exercise is indirect, although some workers have attempted to identify the potential radical species involved. Reid and coworkers (41) and Kolbeck et al. (27) both reported that isolated strips of diaphragm released superoxide anions into the external medium during contraction. Initial evidence that contracting diaphragm may also produce hydroxyl radicals was presented by Diaz et al. (10), whereas O'Neill et al. (36) demonstrated that contracting cat skeletal muscle generated hydroxyl radicals that were detectable in the muscle microvasculature. The cellular origin of these radical species has not been defined because, in addition to the skeletal muscle cells, muscle tissue in vivo contains numerous other cell types, including endothelial cells, fibroblasts, and lymphocytes, that may potentially contribute to the generation of free radical species.

Skeletal muscle is known to be extremely responsive to changes in a variety of external stimuli, such as neural stimulation patterns, workload, or stretch. This well-recognized process of "training" of skeletal muscle is achieved by numerous structural and biochemical changes in the muscle cell and surrounding tissues (16). Several studies have examined the possibility that the ability of skeletal muscle to prevent damage by free radicals is influenced by training. Published data are conflicting, but Ohno et al. (35) concluded that levels and activities of superoxide dismutase (SOD) were generally elevated after acute and chronic exercise. Similar data have also been presented for changes in catalase activity (1, 4, 47), although such changes have not been universally observed (26, 39). Skeletal muscle also produces stress or heat shock proteins (HSPs) in response to some forms of contractile activity (43). These proteins act to prevent tissue damage induced by oxidative and other stresses.

The mechanisms underlying these adaptive changes in SOD and catalase activities and HSP content are unclear, but it was recently recognized that oxidizing free radical species can act as signals to modify gene expression. In particular, oxidizing free radicals appear to activate or modify the activity of a number of redox-sensitive transcription factors, such as nuclear factor-kappa B (23), metal response element-binding factor-1 (8), activator protein-1, and heat shock factor (44).

Many previous studies have described apparent adaptive responses in skeletal muscle antioxidant enzymes in response to substantial episodes of damaging exercise or serial training regimens; however, our previous data (21) and that of others (36) suggests that muscle cells generate free radical species rapidly during types of exercise associated with little or no cell damage. This current study has examined the time course of release of an oxidizing free radical species (superoxide anion) and of the adaptive responses of skeletal muscle after a short period of nondamaging aerobic activity. We hypothesized that the single short period of contractile activity by skeletal muscle would induce a rapid increase in the release of superoxide radicals, followed by an oxidation of muscle proteins and a subsequent adaptive response in the activities of antioxidant enzymes and HSPs. Furthermore, these changes would occur in the absence of overt damage to the cell.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Stimulation of skeletal muscle in vivo. Four- to six-month-old female BALB/c mice were anesthetized with pentobarbital sodium (65 mg/100 g ip). The muscles of one hindlimb were stimulated to contract by surface electrodes placed around the upper limb and ankle to induce isometric contractile activity in vivo. Stimulation was performed for 15 min with square wave pulses of 0.1 ms in duration at 100 Hz and 70 V for 0.5 s every 5 s. Control BALB/c animals were anesthetized without limb stimulation. Animals were allowed to recover and were killed at the times specified in RESULTS by overdose of pentobarbital sodium. Blood samples were removed from the aorta, and the gastrocnemius, anterior tibialis (AT), soleus, and extensor digitorum longus (EDL) muscles from the hindlimbs were rapidly dissected, frozen in liquid nitrogen, and stored at -70°C until analyzed. For histological analysis, portions of the EDL and soleus muscles were mounted on cork blocks, surrounded by optimum cutting temperature mounting medium, and frozen in isopentane precooled in liquid nitrogen.

Cultures of skeletal muscle myotubes. Primary mouse satellite cell cultures were prepared from 4- to 8-wk-old BALB/c mice as described by Veal and Jackson (46). Cells were grown in six-well plates in Dulbecco's modified Eagle's medium (DMEM) with 0.45% (wt/vol) glucose, 2 mM glutamine, 50 IU/ml penicillin, and 50 µg/ml streptomycin to which 20% fetal calf serum was added until the cells reached 80% confluence. Fusion was then induced by replacing the medium with DMEM containing 2% horse serum (GIBCO). At 5-7 days after the addition of horse serum, the cells were washed with Dulbecco's phosphate-buffered saline (D-PBS). Cells were then incubated in 2 ml of D-PBS containing 50 µM cytochrome c (Sigma, Poole, UK). Myotubes were stimulated for 15 min via platinum electrodes with the use of a modification of the method described by Thelen et al. (45). Stimulation was performed for 15 min with square wave pulses of 2 ms in duration at 1 Hz and 30 V/well. Nonstimulated cells acted as controls.

Superoxide release into cell culture supernatant and muscle interstitial fluid. The release of superoxide was measured by the reduction of cytochrome c as described by Green and Hill (17) and Reid et al. (41). To assess extracellular release of superoxide from cultures of skeletal muscle myotubes, we replaced the culture medium with D-PBS containing 50 µM cytochrome c. The medium was collected every 15 min and replaced with fresh D-PBS containing cytochrome c. Samples were analyzed with the use of scanning visible spectrometry. The superoxide content was calculated from the absorbance at 550 nm compared with that at the isobestic wavelengths of 542 and 560 nm and from a molar extinction coefficient of 21,000 as described by Green and Hill (17). To ensure the specificity of the assay, we stimulated selected cultures of myotubes in the presence of 1,000 U/ml SOD (Cu,Zn SOD; Sigma).

Superoxide anion in the interstitial fluid of the gastrocnemius muscles of both the stimulated and contralateral control legs was measured via a novel technique with the use of in vivo microdialysis. Microdialysis probes (MAB 3.8.10; Metalant) were placed in the gastrocnemius muscles of anesthetized mice and perfused with sterile isotonic saline containing 50 µM cytochrome c at a flow rate of 4 µl/min. Samples were collected for 15 min (60 µl) and diluted (1:4) with sterile isotonic saline before the reduction of cytochrome c was analyzed by spectrophotometry as described above.

Biochemical analyses. The total thiol content of EDL and soleus muscle proteins was analyzed by titrating a sulfosalicylic acid precipitate of muscle proteins with 5,5'-dithio-bis(2-nitrobenzoic acid) as described by Di Monte et al. (12). For analysis of stress proteins, muscle samples were homogenized in a range of protease inhibitors. Samples were centrifuged at 4°C, and the supernatant was analyzed for total protein content with the bicinchoninic acid method (Sigma). Fifty micrograms of total protein were separated on SDS-PAGE, followed by Western blotting. The content of heat shock protein 60 (HSP60), constitutively expressed heat shock protein 70 (HSC70), and inducible heat shock protein 70 (HSP70) was analyzed by using monoclonal antibodies obtained from Bioquote, Sigma, and Amersham Life Sciences (Amersham, UK) (32). Bands were visualized on X-ray film with the use of the ECL enhanced chemiluminescent detection system (Amersham Life Sciences). Membranes were exposed to film for three to four different exposure times to ensure that saturation of film had not occurred. Samples from each experiment were applied to the same gel, the intensity of staining for individual HSPs was quantified by densitometry, and the content of each HSP was expressed as a percentage of the preexercise content.

The AT muscle was homogenized in 100 mM phosphate buffer, pH 7.0, and the homogenate was analyzed for catalase activity in accordance with the kinetic decomposition of hydrogen peroxide followed spectrophotometrically at 240 nm by using a method derived from Claiborne (5). Total SOD activity was measured according to the method of Crapo et al. (6).

Plasma creatine kinase activities of control mice and animals subjected to the in vivo muscle contraction protocol were analyzed for up to 7 days after the protocol according to the spectrophotometric method of Jones et al. (25). Eight-micrometer sections of the blocked muscle tissue were stained with hematoxylin and eosin and then examined by light microscopy for evidence of overt muscle damage.

Statistical analysis. Data are presented as means ± SE. Comparisons between means was undertaken by repeated analysis of variance, followed by the Bonferroni post hoc test for multiple comparisons where appropriate.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Detection of superoxide in muscle extracellular space. The microdialysis probe placed in the resting gastrocnemius muscle detected ~0.5 nmol of superoxide anion in 15 min (Fig. 1). This did not change significantly in the control, nonstimulated gastrocnemius during the 135 min of the experiment. During the 15-min period of stimulation, a mean amount of 1.2 nmol of superoxide was recovered from the contracting gastrocnemius. After the stimulation was ended, the superoxide release declined rapidly (within 15 min) to control levels.


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Fig. 1.   In vivo release of superoxide anion measured by reduction of cytochrome c in a microdialysis probe placed in both gastrocnemius muscles of anesthetized mice. After placement of the probe, the muscle remained at rest for 60 min (4× 15-min collections of microdialysate); the test muscle (open bars) was then stimulated for 15 min and remained at rest for the remainder of the experiment. The contralateral control muscle (filled bars) remained at rest throughout the experiment. Values are means ± SE; n = 6 animals in each group. *P < 0.05 vs. control nonstimulated muscles at the same time point.

Skeletal muscle as the source of superoxide radicals. Primary myotubes were found to release very little superoxide at rest, with many cultures showing no detectable reduction of extracellular cytochrome c at rest (Fig. 2). When stimulated with low-frequency (1 Hz) pulses, the cells released superoxide at ~40 times basal levels over the 15-min stimulation period. After the stimulation was ended, the amount of reduction of cytochrome c declined but was still significantly higher than basal levels at 75 min after stimulation was completed.


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Fig. 2.   Superoxide release by primary skeletal muscle myotubes at rest (filled bars) and after repetitive electrically stimulated contractions during the 15-min period shown (open bars). The effect of addition of superoxide dismutase (1,000 U/ml) to the medium during the contraction period is also shown (). Data are means ± SE from 6 stimulated and 6 nonstimulated wells.*P < 0.05 vs. control nonstimulated cells at the same time point.

Addition of 1,000 U/ml Cu,Zn SOD to the medium prevented the stimulation-induced increase in cytochrome c reduction (Fig. 2), confirming that the reduction of cytochrome c was attributable to the production of superoxide. Control experiments in which stimulation of medium alone (i.e., with no myotubes present) was undertaken showed no significant reduction in cytochrome c, demonstrating that electrolysis did not contribute to the data obtained (results not shown in detail).

Oxidation of muscle thiol groups. A loss of protein thiol groups was apparent in both the soleus and EDL muscles after the contractile activity (Fig. 3). This change was seen immediately after the stimulation was ended in the soleus muscles (Fig. 3A) but not until 15 min later in the EDL muscles (Fig. 3B). In both cases, the reduction was transient, because total protein thiols were not significantly different from baseline values by 60 min after the contractile activity.


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Fig. 3.   Protein thiol content of soleus (A) and extensor digitorum longus (EDL) (B) muscles immediately (imm) and at specified times after the 15-min contractile activity. Values are means ± SE; n = 4-6 animals at each time point. *P < 0.05 vs. control nonstimulated muscles.

Time course of adaptive responses to oxidative stress. Muscle SOD activity was found to increase transiently after the contractile activity (Fig. 4). Activities increased postcontraction, peaking at ~12 h after the activity ended, when the mean activity was elevated ~3.5-fold compared with baseline. Activities then generally declined, although they were still significantly higher than baseline at 72 h postcontraction. Catalase activities also increased, but with a slower time course. These were significantly greater than control activities at 48 and 72 h postcontraction (Fig. 4).


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Fig. 4.   Superoxide dismutase (SOD) (A) and catalase (B) activities of anterior tibialis muscles at specified times after the 15-min contractile activity. Values are means ± SE; n = 4-6 animals at each time point. *P < 0.01 vs. control nonstimulated muscles.

Muscle HSP content also increased dramatically, but transiently, after the contractile activity (Fig. 5), with the pattern of changes dependent on the muscle examined. In the primarily oxidative soleus muscle (Fig. 5A), HSP70 content increased very rapidly, with significant changes by 2 h after the contractile activity. Mean values peaked at 18-24 h postcontraction before returning to control values. Comparable changes were seen in the primarily mitochondrially located HSP60, although with larger proportionate increases, with the content of this protein also peaking at 18-24 h postcontraction. HSC70 content did not vary significantly from control values in the exercised soleus muscles.


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Fig. 5.   Content of heat shock protein 60 (HSP60), constitutively expressed heat shock protein 70 (HSC70), and inducible heat shock protein 70 (HSP70) in soleus (A) and EDL (B) muscles at specified times after the 15-min contractile activity. Values are means ± SE; n = 4-6 animals at each time point. *P < 0.05 vs. control nonstimulated muscles.

In the fast-twitch EDL muscles (Fig. 5B), HSP70 content generally increased, reaching significantly different values at 18-48 h postcontraction. No significant changes in HSP60 or HSC70 content were seen, although both proteins showed a tendency to increase after contractile activity.

No evidence of damage to skeletal muscle was seen for up to 7 days after the period of contractile activity. Plasma creatine kinase activities were 158 ± 22 U/l in the control animals and showed no significant changes in the animals killed at each time point up to 7 days postcontraction, and no overt damage was seen on representative hematoxylin/eosin-stained histological sections (data not shown in detail).


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The short period of contractile activity studied here induced a significant rise in the superoxide level detected in muscle interstitial fluid by microdialysis techniques (Fig. 1) but did not induce any significant damage to skeletal muscle fibers. Previous data have shown that this pattern of stimulation induces a rise in the free radical signal seen on electron spin resonance examination of skeletal muscle (21). This has been claimed to primarily derive from a semiquinone radical within muscle mitochondria (22). Superoxide release from contracting skeletal muscle in vivo does not appear to have been described previously, although Reid et al. (41) originally demonstrated superoxide anion release from an isolated rat diaphragm preparation. Our data from contracting myotubes confirm that skeletal muscle cells per se, rather than other cells in the tissue, are likely to be the source of exercise-induced superoxide release. Skeletal muscle myotubes displayed a large and rapid increase in superoxide production during stimulation, which declined relatively slowly after stimulation was terminated.

The subcellular source(s) of the superoxide released cannot be defined from our current work. Various studies have suggested that the superoxide released is likely to be derived from muscle mitochondria (20, 40), although it also has been argued that mitochondria are unlikely to release superoxide to the cytosol in vivo (14). In the current studies, it seems unlikely that superoxide released within mitochondria would diffuse across both mitochondrial and plasma membranes to the extracellular space (18). The greater proportionate release of superoxide from stimulated skeletal muscle myotubes (Fig. 2) compared with that from muscle in vivo (Fig. 1) also argues against mitochondria as a potential source. Relatively immature myotubes contain few mitochondria (37) as potential sources of radical generation compared with mature muscle.

Alternative potential sources of superoxide production in skeletal muscle include membrane-bound oxidases or cytosolic xanthine oxidase (41). The primary potential source of xanthine oxidase in muscle tissue is within endothelial cells (19), and these cells would have been absent or only present in small numbers in the cultures. The data obtained therefore strongly suggest that this enzyme is not the source of the superoxide detected in these studies. However, the possible involvement of contraction-activated, membrane-bound oxidase(s) in superoxide release cannot be discounted.

The novel technique of microdialysis appears to hold considerable promise as a means of assessing the generation of free radical species in vivo (34). Attempts to measure reactive free radical species directly in vivo usually have been inhibited by the presence of large macromolecules that either inhibit the reaction of the molecules with the detection agent (in this case cytochrome c) or with which the free radicals react too rapidly to be detected. Microdialysis offers the advantage that any free radical species that can diffuse across the microdialysis membrane will be removed from such molecules and will be able to react with the detector molecule(s) in the dialysate.

Data from microdialysis experiments are only semiquantitative unless specific information relating to the recovery of the analyte across the microdialysis membrane is available (2, 31). In the case of superoxide, such data are not available because of the instability and reactivity of the molecule and a lack of applicable generation and detection systems. Both Forman and Azzi (14) and Reid and coworkers (41) comment that the apparent rates of production and diffusion of superoxide anion are influenced by the presence of trapping agents/scavengers, and preliminary experiments that we have undertaken to attempt to quantify recovery of superoxide across the microdialysis probe membrane have not been successful.

A comparison of the relative superoxide release by myotubes in culture (Fig. 2) and that detected by microdialysis measurements from muscle in vivo (Fig. 1) reveals that myotubes released very little superoxide at rest but had a much greater proportionate increase on stimulation than that observed from muscle tissue. One explanation for these data is that a significant proportion of the basal reduction of cytochrome c detected in the muscle microdialysate is not due to superoxide released from muscle and, hence, is unaffected by contractile activity. This might be due to superoxide released from other cells (e.g., white cells) or to alternative low-molecular-weight substances found in interstitial fluid that are capable of reducing cytochrome c. This latter possibility is supported by experiments in which increasing amounts of SOD (either Cu,Zn SOD or Mn SOD; Sigma) were added to the microdialysis perfusate to attempt to demonstrate the specificity of the assay; basal levels of cytochrome c reduction were reduced by a maximum of 50% on addition of the enzymes (results not shown in detail). Because of potential alternative reductants found in interstitial fluid, the absolute superoxide values calculated from the reduced cytochrome c measured in the microdialysis experiments may be subject to some error. Nevertheless, our experiments with SOD-treated stimulated myotubes (Fig. 2) clearly indicate that these cells are a major source of superoxide release during muscle contraction.

Previous data have indicated that contraction of skeletal muscle leads to oxidation of lipids, etc. (e.g., see Ref. 9). Muscle protein thiol content was measured here because oxidation of protein thiols has been shown to be part of a signaling mechanism leading to induction of HSP expression in other cell types (15). The contractile activity caused a transient oxidation of muscle proteins with loss of free thiol groups in both predominantly oxidative and glycolytic muscles (Fig. 3). Such changes have been reported previously after damaging contractile activity (33), although the transient nature of the changes reported here does not appear to have been observed previously. Despite this oxidation of muscle proteins, no evidence of overt cellular damage or loss of membrane viability leading to cytosolic enzyme efflux was seen. Reid (40) hypothesized that transient loss of reduced thiol groups due to contraction-induced oxidative stress might lead to a loss of intracellular calcium homeostasis, but we obtained no evidence of this. The period of contractile activity used here stimulated an adaptive response by the muscle with a consequent increase in the activity of SOD and catalase and an increase in the cellular content of two HSPs (Figs. 4 and 5). The increases in SOD and catalase activities occurred with differing time courses such that SOD activity was elevated within 12 h of completion of the contraction protocol, whereas catalase activities peaked 48-72 h after the end of the contraction protocol. Such changes would be expected to help protect the tissue against any subsequent exposure to oxidative stress and are fully compatible with the changes known to occur with longer term exercise-training regimens (24, 42, 47).

Previous data have also shown that chronic exercise leads to an increase in the HSP content of animal muscle (28, 43). These proteins are known to provide short-term cytoprotection to the cell and to facilitate adaptations involving de novo protein synthesis (29, 44). In tissues such as the heart, upregulation of HSP expression by exposure to a short period of nondamaging oxidative stress results in protection against a subsequent bout of (normally lethal) oxidative stress (induced by a prolonged period of ischemia and reperfusion) (7, 13, 29, 48), and preliminary data indicate that HSPs may have a similar cytoprotective role in skeletal muscle (32). The transient increase in HSPs observed after short-term contractile activity therefore also appears to be part of a coordinated response to protect the muscle against potential damage induced by further contractile activity-induced oxidative stress. Contractile activity induced similar rises in the HSP70 content of soleus and EDL muscles (Fig. 5), but differential effects on HSP60 content were seen. HSP60 is predominantly mitochondrial in origin, and its significant rise after contractile activity in oxidative soleus muscles is likely to be due to the greater mitochondrial numbers in this muscle compared with the EDL. The mechanisms by which HSPs provide cytoprotection has not been fully evaluated but may involve renaturation or refolding of denatured, misfolded, or aggregated proteins (38).

It is relevant to consider the possible stimuli for the adaptive changes seen after the contractile activity. Although it is known that exercise can lead to an increase in muscle temperature, no such rise was observed when a thermocouple probe was inserted into the contracting muscle (results not shown in detail), but, as previously indicated, oxidation of protein thiol groups has been shown to result in the induction of HSP expression in other cell types (15). We speculate that similar mechanisms are likely to be active in contracting skeletal muscle and that the oxidizing free radical species generated by contracting skeletal muscle may play a role in the signaling processes by which this tissue adapts to changes in the pattern or duration of contractile activity.


    ACKNOWLEDGEMENTS

We thank the Wellcome Trust (Grant 043364/Z) and the Mersey Kidney Research Fund for financial support and Drs. F. McArdle and J. Faulkner (University of Michigan) for helpful discussions. Dr. J. M. McCord (University of Colorado) provided helpful advice concerning the interpretation of microdialysis data.


    FOOTNOTES

Address for reprint requests and other correspondence: Prof. M. J. Jackson, Dept. of Medicine, Univ. of Liverpool, Liverpool L69 3GA, UK (E-mail: mjj{at}liv.ac.uk).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 8 May 2000; accepted in final form 10 October 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Cell Physiol 280(3):C621-C627
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