The precise mechanisms underlying skeletal muscle damage in Duchenne muscular dystrophy (DMD) remain ill-defined. Functional ischemia during muscle activation, with subsequent reperfusion during rest, has been documented. Therefore, one possibility is the presence of increased oxidative stress. We applied a model of acute hindlimb ischemia/reperfusion (I/R) in mdx mice (genetic homolog of DMD) to evaluate dynamic in vivo responses of dystrophic muscles to this form of oxidative stress. Before the application of I/R, mdx muscles showed: 1) decreased levels of total glutathione (GSH) with an increased oxidized (GSSG)-to-reduced (GSH) glutathione ratio; 2) greater activity of the GSH-metabolizing enzymes glutathione peroxidase (GPx) and glutathione reductase; and 3) lower activity levels of NADP-linked isocitrate dehydrogenase (ICDH) and aconitase, two metabolic enzymes that are sensitive to inactivation by oxidative stress and also implicated in GSH regeneration. Interestingly, nondystrophic muscles subjected to I/R exhibited similar changes in total glutathione, GSSG/GSH, GPx, ICDH, and aconitase. In contrast, all of the above remained stable in mdx muscles subjected to I/R. Taken together, these results suggest that mdx muscles are chronically subjected to increased oxidative stress, leading to adaptive changes that attempt to protect (although only in part) the dystrophic muscles from acute I/R-induced oxidative stress. In addition, mdx muscles show significant impairment of the redox-sensitive metabolic enzymes ICDH and aconitase, which may further contribute to contractile dysfunction in dystrophic muscles.
- reduced glutathione
- glutathione reductase
- Duchenne muscular dystrophy
duchenne muscular dystrophy (DMD) is caused by mutations in the dystrophin gene, which normally encodes a large (427 kDa) subsarcolemmal cytoskeletal protein. Several mechanisms have been implicated in the pathogenesis of skeletal muscle fiber necrosis triggered by the loss of dystrophin in DMD (30), including the presence of increased oxidative stress. Skeletal muscles of DMD patients or dystrophin-deficient mice (mdx) show increases in various markers of oxidative stress such as protein carbonyls (18) and lipid peroxidation by-products (34). Moreover, dystrophin-deficient myotubes appear to be abnormally susceptible to cellular injury when exposed to reactive oxygen species in vitro (12, 36). Some investigators also reported an upregulation of antioxidant systems within dystrophic muscles in vivo (12, 34), which was presumed to represent physiological compensation for increased oxidative stress. More recently, it has been shown that skeletal muscles of DMD patients and mdx mice experience recurrent bouts of functional ischemia during muscle contraction because of a loss of neuronal nitric oxide synthase (nNOS) from its normal subsarcolemmal location (38, 41). To the extent that reperfusion after ischemia is a well-established cause of oxidative stress, these findings further reinforce the idea that increases in the level of exposure to oxidative stress, as well as a potentially greater intrinsic vulnerability to its effects, could be major contributors to the pathophysiology of dystrophin deficiency.
The glutathione system is one of the most important antioxidant systems for most tissue types in vivo (17). In skeletal muscle, deficiencies of either glutathione or selenium (the latter is required for the activity of glutathione peroxidase, GPx) lead to the development of severe muscle fiber degeneration (24, 39). Glutathione is required for the antioxidant function of GPx and is also able to scavenge various free radical species directly (17, 46). The ratio of oxidized to reduced glutathione (GSSG/GSH) is a widely used indicator of oxidative stress within different cell types (17). Under normal circumstances, GSH levels within the cell are maintained via recycling of GSSG by the NADPH-dependent enzyme glutathione reductase (GR). In muscle cells, the main source of NADPH for GR is NADP+-specific isocitrate dehydrogenase (ICDH; see Ref. 3, 22, and 26). Another metabolic enzyme, aconitase, supplies isocitrate for the ICDH reaction (10). Interestingly, both metabolic enzymes are also known to be highly susceptible to inactivation by free radical species (2, 9, 32, 43, 44). Therefore, the metabolic pathways that supply the glutathione antioxidant system in skeletal muscle are themselves potential targets for adverse modifications by oxidative stress. Conceivably, this could initiate a vicious cycle, because studies have shown that cells or organelles depleted of NADP-ICDH are abnormally susceptible to oxidative stress (20, 23).
In the present study, our main objective was to examine the status of different components of the glutathione system, including critical metabolic pathways that are required for it to function efficiently, in the skeletal muscles of dystrophin-deficient mice. Because ischemia-reperfusion (I/R) is a form of oxidative stress that is particularly relevant to the pathogenesis of dystrophin deficiency (27, 38, 41), we also sought to characterize the dynamic in vivo responses of the skeletal muscle glutathione system to an acute challenge by I/R. Overall, our data suggest that dystrophin-deficient muscles adapt to chronic ongoing oxidative stress by upregulating glutathione-associated antioxidant enzymes and that this affords a degree of protection against acute bouts of oxidative stress induced by I/R. However, despite these adaptations, we also find evidence for persistent ongoing oxidative stress in dystrophic muscles. Furthermore, accessory metabolic pathways that feed the glutathione system (i.e., NADP-ICDH and aconitase) are compromised in dystrophin-deficient muscles, suggesting another possible factor underlying the inability of dystrophic muscle fibers to restore a normal redox status in the face of chronic oxidative stress.
MATERIALS AND METHODS
I/R is a well-established model for inducing oxidative stress (4, 17, 47). To induce ischemia in the tibialis anterior muscle, a locking plastic pull-tie tourniquet was applied just above the knee of the right hindlimb in deeply anesthetized (130 mg/kg ketamine, 20 mg/kg xylazine by im injection) mice. Sponge padding (3 mm thickness) was applied between the skin and the tourniquet to prevent skin damage. Cessation of blood flow was confirmed by a notable decrease in temperature and visible pallor of the lower limb. After 90 min of ischemia, the tourniquet was removed, and the appearance of the lower limb quickly returned to normal. Animals were killed 1 h after the removal of the tourniquet. The periods of ischemia and reperfusion used here are similar to those employed by other investigators studying short-term I/R injury in mouse hindlimb skeletal muscles (8, 42). Sham-treated (control) mice were subjected to the same procedures as the I/R mice, except that no tourniquet was applied to the hindlimb. Dystrophin-deficient (mdx) and normal dystrophin-expressing (C57BL6/10) mice were purchased from The Jackson Laboratory (Bar Harbor, ME). Sedentary animals were studied at 6–8 wk of age and were exclusively males. The mice were provided with food and water ad libitum, and all experimental protocols were approved by the McGill University Animal Care and Use Committee.
Frozen muscles were homogenized in 20 μl of 5% (wt/vol) 5-sulfosalicylic acid (SSA)/mg tissue and centrifuged at 11,500 rpm for 5 min at 4°C. The supernatant was diluted 1:5.5 in ddH2O. Separate aliquots of 400 μl were taken for total glutathione and GSSG determination. Total glutathione was determined by Tietze's GR recycling method adapted for the Cobas Mira spectrophotometer (Roche Diagnostics). The Cobas Mira was programmed to pipette 250 μl of 0.3 mM NADPH, 30 μl of 6.0 mM 5,5′-dithiobis(2-nitrobenzoic acid), and 95 μl of either sample, standard, or 5% SSA in cuvettes. The reaction mixture was allowed to incubate at 37°C for 4 min. Next, 15 μl of GR enzyme (1.0 U/100 μl) was added, and the reaction was monitored at 405 nm every 24 s for a period of 12 min. Using these conditions, the GR method is linear for glutathione concentrations ranging from 0 to 6 μM. Finally, the concentration of total glutathione in each sample was determined from a calibration curve produced using known glutathione standards. Reproducibility of total glutathione determination using this method is very high, with an intra-assay coefficient of variation of <2%. The supernatant sample for the GSSG assay was derivatized in 50 μl of the following solution: 45.5% triethanolamine, 0.4% SSA, and 9% 2-vinylpyridine in ddH2O. The mixture was vortexed vigorously for 10 min at room temperature. To quantify the amount of GSSG, known standards of 2.5 μM GSH, 2.5 μM GSH + 0.1 μM GSSG, and 2.5 μM GSH + 0.5 GSSG were derivatized and assayed. The samples were analyzed as for total glutathione above. A regression curve was used to obtain the value of the GSSG recovered in each assay, and from this, a correction factor was obtained to calculate the GSSG for each unknown.
GPx and GR activity assays.
GPx and GR activities were measured using commercial kits (RS505 and GR2368, respectively; Randox Laboratories, San Francisco, CA). For the GR activity assay, muscles were homogenized in PBS (pH 7.4) containing 0.1 mM EDTA. For the GPx activity assay, muscles were homogenized in the same PBS buffer except that 1 mM dithiothreitol was added. In both cases, ∼200 mg of muscle were homogenized per milliliter of buffer. Homogenates were spun at 8,500 g for 10 min at 4°C, and the supernatant was collected. For the GPx activity assay, 5 μl of sample were added to 220 μl reagent buffer (4 mM GSH, 0.5 U/l GR, 0.28 mM NADPH in phosphate/EDTA buffer) and mixed at 37°C. Next, 10 μl cumene hydroperoxide (0.18 mM in ddH2O) were added, and the change in absorbance at 340 nm was followed for 2 min at 37°C. Background interference was assessed by assaying each sample using ddH2O instead of cumene hydroperoxide. For the GR activity assay, 10 μl of sample were added to 250 μl GSSG substrate (2.2 mM in potassium phosphate/EDTA buffer) and incubated for 5 min at 37°C. Next, 30 μl NADPH (0.17 mM in ddH2O) were added, and the change in absorbance at 340 nm was followed for 5 min at 37°C. Background interference is assessed by assaying each sample using buffer instead of substrate.
ICDH and aconitase activity assays.
Skeletal muscle tissue (50 mg) was homogenized on ice in 1 ml of buffer containing 0.1 mM Tris·HCl and 15 mM tricarboxylic acid (pH 7.8) and then centrifuged at 6,000 g for 10 min at 4°C. Maximal enzymatic activity was assayed in supernatants by monitoring the kinetics of optical density of NADPH (340 nm) measured with a spectrophotometer (Cobas Fara; Roche). NADP+-ICDH activity was measured using a commercial kit (Sigma Diagnostics, St. Louis, MO), and the procedure of Nulton-Persson and Szweda (29) was slightly modified for measurements of aconitase activity. Supernatants were added to an incubation mixture containing 5 mM citrate, 0.5 mM MgCl2, 1 mM NADP+, and 1 U/ml ICDH (pH 7.4). The protein content of the samples was determined using the Bradford method (Bio-Rad), with BSA serving as a standard. Enzymatic activities are expressed in units per milligram of protein, where 1 unit is defined as the amount of enzyme necessary to catalyze the conversion of 1 mmol substrate/min at 37°C.
All data are reported as means ± SE and were analyzed with the statistical analysis program SigmaStat (SPSS, Chicago, IL). Differences between groups were initially tested by ANOVA, with post hoc application of the Tukey test where appropriate. Statistical significance was defined as P < 0.05.
Glutathione status in prenecrotic dystrophic muscles.
Because an abnormal glutathione status could reflect a primary change in dystrophic muscles or the secondary consequence of necrotic cell death, we first performed glutathione analysis on 14-day-old mice. Because the onset of muscle fiber necrosis in the mdx mouse occurs at ∼4 wk of age (1, 40), the use of this younger age group allows one to eliminate the potential confounding effects of necrosis. As shown in Fig. 1A, there were no significant differences in total glutathione content between 14-day-old dystrophic (mdx) and normal (C57) muscles. However, a greatly increased GSSG/GSH ratio was observed in the mdx group even at this early age (Fig. 1B). Therefore, these results indicate that an abnormal glutathione status is a precocious feature of dystrophy in the mdx mouse that cannot be solely attributed to muscle fiber necrosis or associated inflammation.
Glutathione status in adult dystrophic muscles.
In adult mice, glutathione analysis revealed that dystrophic muscles had significantly less total glutathione than the normal C57 muscles (Fig. 2A). In addition, there was a significantly higher GSSG/GSH within mdx muscles (Fig. 2B) that was similar to that observed in the younger mdx animals. Accordingly, these observations in adult mice again point to the existence of an abnormal redox status within dystrophic muscles under basal conditions.
To determine the in vivo response to an acute oxidative stress challenge, the adult mice were subjected to I/R. In normal muscles, there was a significant reduction in total glutathione levels after the imposition of I/R. In marked contrast, no significant change in total glutathione occurred within mdx muscles under the same conditions (Fig. 2A). No significant changes in the GSSG/GSH ratio were observed in either mouse strain after being subjected to I/R, although there was a trend toward greater GSSG/GSH in the C57 group that was not seen in the mdx muscles (Fig. 2B).
GPx and GR activities.
We next examined whether modifications in the activities of GPx or GR could play a role in producing the abnormal glutathione status of adult mdx mouse muscle noted above. Figure 3 shows that, under baseline conditions, the mdx muscles exhibited significantly greater GPx activity than the C57 group. The imposition of I/R caused a small but significant increase in GPx activity in C57 muscles, whereas no significant change in GPx activity was observed in mdx muscles under the same conditions.
Figure 4 shows that, under basal conditions, the mdx muscles also demonstrated significantly greater GR activity than the C57 group. After the imposition of I/R, there was no change in GR activity in the C57 group. However, in contrast to the C57 group, the mdx muscles showed a significant reduction in GR activity after I/R.
ICDH and aconitase activities.
To evaluate the potential involvement of metabolic enzymes supplying the glutathione system, NADP-ICDH and aconitase activities were measured. These enzymes can also serve as markers of oxidative stress because of their known susceptibility to inactivation by free radical species. Figure 5 shows that, under basal conditions, ICDH activity was decreased significantly in the adult mdx muscles relative to the C57 group. However, I/R suppressed ICDH activity to a substantial degree in the C57 group, whereas the ICDH activity did not change from its baseline value in the mdx group after I/R.
A similar picture was observed for aconitase activity, as shown in Fig. 6. Once again, the basal level of aconitase activity was significantly lower in the adult mdx muscles than in the C57 group. However, the mdx group demonstrated a better ability to maintain its basal level of metabolic enzyme activity when challenged with I/R. Hence, aconitase activity was decreased after I/R in the C57 group only, whereas it was not significantly altered from its baseline value in the mdx group.
Glutathione is one of the most abundant and physiologically important antioxidants in skeletal muscle (17, 37). Despite this prominent role, a comprehensive examination of the key components of the glutathione system in dystrophin-deficient muscles and the dynamic responses of these different components to an acute increase in oxidative stress have not been reported. In this study, we have performed a detailed analysis of GSH, GSSG, and their associated regulatory enzymes (GPx and GR), as well as metabolic pathways that are implicated in the regeneration of GSH. Overall, our findings indicate that, compared with normal muscles, the following changes are present within dystrophic muscles: 1) an increase in oxidative stress levels under basal conditions that precedes the onset of skeletal muscle necrosis; 2) significant disturbances of metabolic pathways that supply the reactions necessary for efficient GSH regeneration; and 3) despite the above, a paradoxically enhanced resistance of the glutathione system to the effects of oxidative stress induced by I/R. Therefore, our results suggest that, although there appears to be a chronically altered redox status within mdx muscles under baseline conditions, dystrophic muscles have developed compensatory mechanisms that confer a degree of protection against at least one form of acute oxidant challenge, i.e., the oxidative stress induced by I/R. This is particularly interesting since the latter has been specifically implicated in the pathogenesis of dystrophin-deficient muscle disease (27, 38, 41).
Baseline abnormalities in dystrophin-deficient muscle.
To our knowledge, this is the first study to directly measure glutathione levels and the GSSG/GSH status of mdx muscles. Our finding of an abnormally elevated GSSG/GSH ratio in prenecrotic mdx muscles strongly supports increased oxidative stress as an early phenomenon that is not dependent on the presence of necrosis or inflammation. Interestingly, in adult mdx muscles, the increased GSSG/GSH ratio was also accompanied by a relative decrease in total glutathione compared with normal muscles. Because the major rate-limiting enzyme for de novo synthesis of GSH is γ-glutamylcysteine synthetase (GCS), the decrease in total glutathione found in adult mdx muscles could have been due to a downregulation of this enzyme. However, in the setting of increased oxidative stress, GCS activity/expression is generally increased (15). GSH can also react with either nitric oxide (NO) or ONOO− to form S-nitrosoglutathione (GSNO; see Ref. 19), which is not taken into account by the method of glutathione quantification employed in our study. Therefore, another potential mechanism to explain the apparent loss of total glutathione in adult mdx muscles would be through the increased formation of GSNO. It is also conceivable that pathological muscle fiber membrane leakiness to large macromolecules, which is a hallmark feature of mdx muscles beyond the age of 3 wk (25), allows for abnormal glutathione efflux from the cell along the strong concentration gradient that exists between the intracellular (mM) and extracellular (μM) compartments (15, 17).
To further ascertain the effects of dystrophin deficiency on glutathione metabolism, we measured the activity levels of GPx and GR, two of the major enzymes that regulate glutathione status. GPx plays an antioxidant role by using GSH as a cofactor for the removal of peroxides, whereas GR recycles GSSG back to GSH. We found that the baseline activity levels of both GPx and GR in adult mdx mice were greater than normal, suggesting an attempt to compensate for increased oxidative stress through upregulation of these antioxidant enzymes. The increased GPx activity found here is in general agreement with other reports (13, 14), whereas conflicting data have been published regarding the status of GR in mdx muscles (34, 35). It should be noted that the method for determining GR activity used in our study involves adding NADPH to the reaction buffer, such that the measured activity level reflects the inherent properties of the enzyme but not the availability of cofactors such as NADPH, which are needed for its optimal function in vivo. In vivo, this NADPH is provided mainly through the oxidative pentose phosphate pathway in many cell types (17), but in skeletal and cardiac muscle, this is accomplished primarily by NADP-ICDH (3, 22, 26), which catalyzes the oxidative decarboxylation of isocitrate to α-ketoglutarate.
In the present study, we observed that the activity level of NADP-ICDH was decreased in adult mdx muscles under basal conditions. Aconitase, which supplies isocitrate for the ICDH reaction (10), also exhibited a significant decrease in activity within mdx muscles. Because skeletal muscle ICDH provides the reducing equivalent (NADPH) required for the regeneration of GSH from GSSG by GR in vivo, these findings collectively suggest that GSH regeneration by GR could be impaired within mdx skeletal muscles in vivo because of a deficiency of ICDH-mediated NADPH production. This could help account for the elevated GSSG/GSH ratio observed in dystrophic muscles. Because both enzymes are known to be highly susceptible to inactivation by free radical species and have even been used as markers of oxidative stress in previous studies (5), these findings provide additional evidence to support chronically increased oxidative stress levels within dystrophic muscles under basal conditions. Finally, in addition to these proposed effects on the glutathione system, the decreased activity of these metabolic enzymes could contribute to dystrophic pathophysiology in other ways. For example, aconitase, being an integral enzyme in the tricarboxylic acid cycle, plays an important role in ATP production via the oxidation of acetyl-CoA. To the extent that decreased ATP production can make cells more susceptible to membrane injury (21), this could serve to exacerbate the muscle membrane damage associated with dystrophin deficiency. The adverse effects on muscle energy production of these metabolic enzyme disturbances would be expected to be most prominent during exercise as a result of the associated increase in energy demands and could play a role in the increased susceptibility of dystrophic muscles to exercise-induced damage (31).
Differential responses to an oxidative stress challenge induced by I/R.
In normal skeletal muscle, dystrophin exists as part of a multimolecular complex. This complex is destabilized when dystrophin is absent, and most of its members are consequently lost from the sarcolemma. One such example is nNOS, which is displaced away from the sarcolemma to an abnormal location within the cytosol of dystrophin-deficient muscles, where its enzymatic activity is reduced significantly (6, 7). This has been shown to result in a failure of normal NO-mediated vasodilation during muscle contraction, which leads to episodes of functional ischemia during muscle activity, followed by reperfusion during rest (38, 41). Accordingly, recurrent episodes of I/R (albeit of less severity than in our experimental model) are likely to occur in dystrophin-deficient muscles during the initial phases of the disease when exercise is still possible and could thus be an important contributor to early disease progression.
In this study, we show that dystrophin-deficient muscles of the mdx mouse do not respond in the same manner as normal muscles to I/R-induced oxidative stress. Interestingly, the imposition of I/R in normal skeletal muscles led to an altered redox state that closely resembled the mdx muscles at baseline (i.e., mdx not subjected to I/R). Hence, in normal muscles, I/R triggered decreases in total glutathione, as well as ICDH and aconitase activities. On the other hand, mdx muscles demonstrated a large degree of resistance to these acute changes, suggesting an enhanced ability of glutathione system components to cope with I/R. In this regard, it is well known that brief periods of ischemic stress in either cardiac (11) or skeletal (16, 33) muscle are capable of inducing cellular adaptations that are protective against subsequent I/R insults. This phenomenon of ischemic “preconditioning” appears to be mediated via several different signaling pathways (28, 45), with the downstream effectors believed to include antioxidant enzymes such as superoxide dismutase, catalase, and GPx (11). Therefore, our findings of increased GPx and GR activities within mdx muscles under basal conditions are consistent with the notion that previous bouts of I/R in dystrophic muscles (related to nNOS deficiency and functional ischemia, as noted above) may induce changes consistent with ischemic preconditioning. This would serve to limit the amount of oxidative damage triggered by such episodes and could be an important endogenous mechanism for slowing disease progression. In conclusion, similarities in the glutathione system between normal muscles subjected to acute I/R on the one hand and the basal status of dystrophic muscles on the other support functional I/R as a potential source of increased oxidative stress in dystrophin-deficient muscles. Furthermore, the decreased activity of the oxidative stress-sensitive enzymes, ICDH and aconitase, suggests that oxidative stress-induced metabolic disturbances may also contribute to the dystrophic process. Compensatory mechanisms in mdx muscle appear to provide a degree of protection against acute changes in redox status and further inhibition of metabolic enzyme activity after acute exposure to I/R. However, these compensatory mechanisms are not able to completely restore a normal redox status to dystrophic muscles, suggesting that antioxidant protective mechanisms are ultimately overwhelmed by the presence of chronically increased oxidative stress.
This investigation was supported by grants from the Canadian Institutes of Health Research, the Canadian Lung Association, and the Muscular Dystrophy Association (USA).
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