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CELLULAR AND MOLECULAR

Greater Susceptibility of the Sarcoplasmic Reticulum to H₂O₂ Injuries in Diaphragm Muscle from mdx Mice

Université de Nantes, Centre National de la Recherche Scientifique, Unité Mixte Recherche 6204, Biotechnologie, Biocatalyse et Biorégulation, Faculté des Sciences et des Techniques, Nantes, Cedex, France

Received for publication February 21, 2006
Accepted June 23, 2006.

	Abstract

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The aim of the present study was to investigate the direct effects of a reactive oxygen species, H₂O₂, on the contractile function and sarcoplasmic reticulum properties of dystrophin-deficient diaphragm using chemically skinned fibers and sarcoplasmic reticulum vesicle preparations. The results obtained using Triton X-100-skinned fibers demonstrate that exposure to 1mMH₂O₂ had similar effects on the maximal Ca²⁺-activated tension and on the Ca²⁺ sensitivity of the contractile apparatus of diaphragm fibers in Bl10 and mdx mice. The effects of H₂O₂ were also assessed on sarcoplasmic reticulum function using saponin-skinned fibers and sarcoplasmic reticulum vesicle preparations. We found that H₂O₂ induced changes in sarcoplasmic reticulum properties, particularly in the Ca²⁺ pump function. The most important finding was that diaphragm muscle from mdx mice displayed increased sensitivity to the oxidant. Furthermore, in isolated superfused diaphragm muscle from mdx mice, the data demonstrate that the amount of superoxide anion produced under fatiguing conditions was increased. Our study shows that the sarcoplasmic reticulum, and the Ca²⁺ pump in particular, in dystrophin-deficient muscles display increased susceptibility to H₂O₂ injuries. This suggests that free radicals might, therefore, be involved in the pathophysiological pathway and dysregulation of Ca²⁺ homeostasis of muscular dystrophy.

In dystrophin-deficient skeletal muscles, nitric-oxide synthase is displaced from the plasma membrane (Brenman et al., 1995). In healthy muscles, this enzyme is associated with the dystrophin-glycoprotein complex and insures the synthesis of nitric oxide (NO), which is known to provide protection against the cell injuries and cytotoxicity induced by reactive oxygen species (ROS), by acting as an antioxidant (Wink et al., 1993). These properties involve the ability of NO to bind to intensely reactive species and thus to reduce their reactivity. The most important ROS include hydrogen peroxide (H₂O₂), the superoxide anion radical (), and the hydroxyl radical (^·OH). In skeletal muscle, the generation of oxygen-derived free radicals is a normal process that occurs both at rest and during contraction (Kolbeck et al., 1997; Smith and Reid, 2006). Under physiological conditions, low levels of these intermediates seem to be necessary for optimal skeletal muscle function. However, it has been postulated that during prolonged exercise or in certain disorders, ROS production may become excessive, leading to increased oxidative stress and tissue injury and, consequently, to muscle dysfunction (Kolbeck et al., 1997; Supinski, 1998). A number of studies have suggested that alterations in nitric-oxide synthase expression and localization may contribute to the pathogenesis of DMD as a result of the reduction in NO-mediated fiber protection combined with increased cellular susceptibility to oxidative stress (Rando, 2002). It has been shown that dystrophin-deficient mdx muscles display oxidative injury before the onset of frank muscle pathology and also display greater susceptibility to oxidative stress than normal muscles. Although it is thought that the combination of reduced NO-mediated cell protection and increased oxidative damage might contribute to the pathogenesis of muscular dystrophy, there has still been little investigation of the effects of free radicals on excitation-contraction coupling and Ca²⁺ homeostasis in dystrophin-deficient skeletal muscle.

Therefore, the purpose of the present study was to investigate the direct effects of H₂O₂ on contractile function and on the sarcoplasmic reticulum (SR) properties of dystrophin-deficient skeletal muscle using chemically skinned fibers and SR vesicle preparations. Experiments were performed on diaphragm muscle from mdx mice rather than using limb skeletal muscle because of the physiological importance of the diaphragm in sustaining life and the fact that it exhibits a high degree of necrosis without any regeneration process in this mouse model. Furthermore, numerous studies have identified the diaphragm as a potential target of free radical-mediated injury in several pathophysiological states. Using Triton X-100-skinned fibers, we specifically investigated the effects of H₂O₂ on the maximal Ca²⁺-activated force (T_max) and Ca²⁺ sensitivity of the contractile proteins. The effects of H₂O₂ were also assessed on SR function using saponin-skinned fibers and SR vesicle preparations.

	Materials and Methods

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All experiments were conducted in accordance with the guidelines of the French Council on Animal Care. Diaphragm muscles were isolated from male C57BL/10 wild-type mice (Bl10) and C57BL/10 mdx dystrophic mice (mdx), which had been killed by cervical dislocation at 16 weeks of age [31.4 ± 0.6 (n = 18) and 32.3 ± 0.5 g (n = 20), respectively].

Experimental Protocol for Skinned Fiber Experiments
Small bundles of two to five fibers were isolated manually from freshly isolated muscles, and with the aid of a microscope, short portions were excised (100-250 µm in diameter; 2-3 mm in length). For each preparation, the diameter was directly measured under a microscope and used for the determination of the cross-sectional area. Chemical skinning was carried out immediately after dissection using either Triton X-100 or saponin. The skinned fibers were transferred to a chamber and mounted between two stainless steel tubes that supported a metal target facing the sensor of a transducer (model 0.5 SU, Displacement Measuring System KD 2300; Kaman Instrumentation, Colorado Springs, CO) according to the protocol described previously by Huchet and Leoty (1993). At the beginning of each experiment, the fiber was adjusted to its slack length and then stretched progressively until the tension developed at pCa 4.5 became maximal, generally reaching at the resting length plus 20%. All experiments were performed at 22°C.

Tension/pCa Relationships in Triton X-100-Skinned Fibers
Preparations were incubated for 1 h in relaxing solution (pCa 9.0; see composition below) containing 1% Triton X-100 (v/v) to solubilize the sarcolemma and the SR membranes and subsequently washed several times in relaxing solution without detergent. After skinning, the fibers were stored at -20°C in relaxing solution containing 50% glycerol (v/v). Tension/pCa relationships (pCa =-log₁₀[Ca²⁺]) were obtained by exposing Triton X-100-skinned fibers sequentially to solutions with decreasing pCa values until the T_max was reached (at pCa 4.5); the fibers were then returned to a low [Ca²⁺] solution (pCa 9.0). A full set of solutions containing different [Ca²⁺] solutions was prepared, and each solution was then divided into the aliquots, one serving as the control and the other containing hydrogen peroxide (H₂O₂; 1 mM) or caffeine (2.5, 10 mM). The isometric tension was recorded continuously using a chart recorder (model 1200; Linear, Reno, NV), and the baseline tension was established at the steady-state measured in relaxing solution. To obtain the Ca²⁺ sensitivity curve, data for the relative tension (T/T_max) were fitted using a modified Hill equation (Huchet and Leoty, 1993): T/T_max = [Ca²⁺]^nH/(K^nH + [Ca²⁺]^nH), where K is the [Ca²⁺] at which activation is half-maximal. pCa₅₀, the negative decadian logarithm of K, is a measure of the apparent Ca²⁺ sensitivity of the contractile proteins, and the Hill coefficient (n_H) is an estimate of the degree of cooperativity. For each experiment, a curve fit was performed with Origin 5.0 software (MicroCal Software, Northampton, MA), and the pCa₅₀ and the n_H were determined. n_H was calculated as the slope of the fitted straight lines. The tension obtained at each [Ca²⁺] was normalized for the cross-sectional area of the fiber.

SR Ca²⁺ Uptake and Release in Saponin-Skinned Fibers
The saponin-skinned fiber technique, used to assess the SR properties, was performed by incubating the preparations for 20 min in relaxing solution at pCa 9.0 containing 50 µg · ml^-1 saponin. The skinned fibers were then washed several times in relaxing solution without detergent. At this low concentration, saponin permeabilizes the sarcolemmal and T-tubule membranes, without affecting the ability of the SR to accumulate and release Ca²⁺. Controls were carried out to confirm the integrity of the SR (see below). The preparation was immersed sequentially in three different solutions (Table 1), initially to load the SR with Ca²⁺ and then to release it using pharmacological tools, such as caffeine, an activator of ryanodine receptors (Herrmann-Frank et al., 1999). The application of 10 mM caffeine generates a transient contracture. The ionic composition of these solutions was the same as that of the relaxing and activating solutions (pCa 9.0 and 4.5, respectively). However, the concentrations of EGTA, Mg²⁺, and Ca²⁺ were varied (Table 1). For these experiments, SR Ca²⁺ uptake was performed in a solution containing 1mMMg²⁺, a physiological concentration. In contrast, Ca²⁺ was released in a solution containing caffeine at a low [Mg²⁺] (0.1 mM) that favors the release of Ca²⁺ by caffeine (Kabbara and Stephenson, 1994; Fryer and Stephenson, 1996). At the beginning of each experiment, two or three 10 mM caffeine contractures were generated to check the integrity of the SR after the saponin-skinning treatment, and control caffeine-induced contractures were recorded at regular intervals (4 min). The amplitude of the control caffeine contractures did not change significantly, suggesting that the SR remained in a functional state. After successive control caffeine applications, the fibers where the decrease in the contracture amplitude was more than 5% were discarded. The concentration/response relationship for caffeine-induced contractures was assessed using several concentrations of caffeine (0.5, 1.0, 1.5, 2.5, 5.0, and 10 mM). The data were fitted to a sigmoid equation, which made it possible to estimate the caffeine sensitivity on the basis of the concentration of caffeine that produced 50% of the maximal contracture (EC₅₀).

The effects of H₂O₂ were then investigated by adding this oxidant to all of the solutions used, and the experimental protocol consisted of performing a control cycle in the absence of H₂O₂ followed by a series of seven successive transient caffeine-induced contractures (2.5 or 10 mM caffeine) in the presence of 1 mM H₂O₂. The amplitude of the caffeine-induced force response was used for the analysis, and the data were expressed for each caffeine contracture as a percentage of the contraction amplitude versus the control contracture without the oxidant H₂O₂.

Solutions and Chemicals
The physiological solution contained 140 mM NaCl, 6 mM KCl, 3 mM CaCl₂, 2 mM MgCl₂, 5 mM glucose, and 5 mM HEPES, with pH 7.4 adjusted with Tris base. The composition of the skinned fiber solutions was calculated according to Godt and Nosek (1989). The relaxing (pCa 9.0) and activating (pCa 4.5) solutions consisted of 10 mM EGTA, 30 mM imidazole, 30.6 mM Na⁺, 1 mM Mg²⁺, 3.16 mM MgATP, and 12 mM phosphocreatine, with pH adjusted to 7.1 by adding HCl or NaOH. The ionic strength of these solutions was 160 mM. In saponin-skinned fiber experiments, the solutions also contained phosphocreatine kinase (17.5 IU · ml^-1) and sodium azide (1 mM). For both Triton X-100- and saponin-skinned fiber experiments, solutions with intermediate [Ca²⁺] were obtained by mixing the pCa 9.0 and 4.5 solutions in appropriate proportions. EGTA, phosphocreatine, and H₂O₂ were obtained from Sigma (St Louis, MO).

Experimental Protocol for SR Vesicle Experiments
Isolation of SR Membrane Vesicles. SR vesicles were prepared from pooled diaphragm muscles from several 16-week-old C57BL/10 and dystrophic animals, according to the method described by Saito et al. (1984). As described in our previous work (Divet et al., 2005), skeletal tissue was homogenized in a solution containing 400 mM sucrose, 5 mM HEPES, and 5 mM Tris-HCl, titrated to pH 7.2 (homogenization medium). The homogenate was centrifuged at 8400 rpm (7700g; Biofuge 28 RS; Heraeus, Osterode, Germany) for 20 min, and then the pellet was discarded. The supernatant was filtered through four layers of gauze and centrifuged at 35000 rpm (100,000g; Sorvall UltraPro 80; Sorvall, Asheville, NC) for 90 min. The final pellet, enriched in SR membranes was resuspended in homogenization medium, frozen rapidly in liquid nitrogen, and stored at -80°C until use. The protein concentration was determined according to the method of Bradford (1976) using bovine serum albumin as standard. All isolation procedures were performed at 4°C.

Measurement of SR Ca²⁺-Dependent ATPase Activity. Ca²⁺-ATPase activity was measured by following the rate of decrease in NADH absorbance at 340 nm (UVIKON XS; Bio-Tek Instruments, Winooski, VT) according to the protocol described by Schwinger et al. (1995). The reaction was carried out as described previously (Divet et al., 2005) in a volume of 1 ml at 22°C, and the SR vesicles (final concentration, 50 µg · ml^-1) were suspended in the reaction mixture containing 0.003 mM A23187 [GenBank] , 0.035 mM CaCl₂, 0.06 mM EGTA, 21 mM 3-morpholinopropanesulfonic acid, 4.9 mM NaN₃, 100 mM KCl, 3 mM MgCl₂, 1 mM ATP, 1 mM phosphoenolpyruvate, 0.2 mM NADH, and 8.4/12 IU of coupled-enzyme assay pyruvate kinase/lactate dehydrogenase. The pH of the solution was adjusted to 7.2 by adding Tris base. The Ca²⁺-dependent ATPase activity corresponded to the difference measured between the linear rates in the presence and absence of Ca²⁺ (with 4 mM EGTA). The activity of the SR Ca²⁺-ATPase was expressed in micromoles · minute^-1 of ATP per milligram of SR protein. The effects of 1 mM H₂O₂ on the SR Ca²⁺-ATPase activity were tested by adding the oxidant to the reaction medium containing the SR preparations, with or without an incubation period lasting 10 min before the start of the reaction by adding ATP.

Measurements of Ca²⁺ Uptake in SR Vesicle Preparations. Ca²⁺ uptake was assessed at room temperature in a medium containing 100 mM KCl, 0.1 µM CaCl₂ (pCa 7.0), 0.06 mM EGTA, 30 mM imidazole, 30.6 mM Na⁺, 1 mM Mg²⁺, 3.16 mM MgATP, 12 mM phosphocreatine, 1 mM NaN₃, and 17.5 IU · ml^-1 phosphocreatine kinase, with pH adjusted to 7.2 with Tris base. Experiments were conducted in the absence or presence of 3 mM potassium oxalate. Accumulation of Ca²⁺ by the SR vesicles (1 mg · ml^-1) was measured using the Ca²⁺-sensitive indicator Indo-1 (10 µM) that can be used to monitor the extravesicular [Ca²⁺]. Excitation wavelength (365 nm) was provided via a 75-watt Xenon arc lamp with a monochromator, and both resulting emissions (405 and 480 nm) were selected by appropriate filters and transmitted to photomultiplier tubes. Variations in the levels of free Ca²⁺ were estimated from the ratio of 405/480-nm emissions that corresponded to the Ca²⁺-bound and the Ca²⁺-free forms of the probe, respectively. The fluorescence signal was calibrated with solutions containing various concentrations of free Ca²⁺. For each experiment, the extravesicular [Ca²⁺] remaining at the end of the Ca²⁺ uptake process was determined and described as the Ca²⁺ min, which was used to determine the amount of Ca²⁺ loaded into the SR lumen. The SR Ca²⁺ uptake rate, which corresponds to the amount of Ca²⁺ loaded in SR vesicles (nanomoles of Ca²⁺ · second^-1 · mg^-1 SR proteins), are calculated using the points of the linear part of the curves before the steady state was achieved. The data concerning the effects of oxidant on the SR Ca²⁺ uptake rate were normalized to those obtained for Bl10 diaphragm SR vesicle preparations under control conditions without oxidant. In addition, the time constant (T) was calculated using experimental data fitted with a monoexponential equation. The effects of H₂O₂ (1 mM) on the SR Ca²⁺ uptake were tested with or without an incubation period lasting 10 min before starting the accumulation of Ca²⁺.

Superoxide Anion Production and Stimulation Procedures
The cytochrome c assay was used to measure the extracellular release of the superoxide anion () by the diaphragm muscle, employing the combination of methods described previously (Kolbeck et al., 1997; Zuo et al., 2000). The detection of is based on its demonstrated ability to reduce cytochrome c through a one-electron transfer reaction resulting in an increase in the absorbance peak at 550 nm. Intact diaphragm muscles were isolated from Bl10 and mdx mice and washed several times in physiological solution to eliminate blood and serum contamination. The diaphragm muscle was then placed in the physiological solution containing 10 µM cytochrome c (Sigma). The experiments were performed at room temperature and in a darkened room to prevent the photobleaching of cytochrome c. Cytochrome c was added to the solution 15 min before to begin the fatiguing stimulation in order to obtain equilibration. The diaphragms were then subjected to a 1-min fatiguing protocol consisting of successive contractile responses obtained by electrical stimulation (stimulator Isostim A320) through two platinum electrode rings, with a constant voltage of 50 V and 1-ms 80-Hz trains delivered at a periodicity of 1/s. The experiment was concluded by a recovery period of 14 min without any stimulation. In the course of this protocol, aliquots of the solution were collected at 2-min intervals, and the spectrum of the cytochrome c was measured using a spectrophotometer (UVIKON XS). The reduction of cytochrome c was determined as the absolute magnitude of the 550 peak (P_{OD 550}), which was calculated as the difference between the absorbance measured at 550 nm and the average of the absorbance found at 540- and 560-nm wavelengths, as described by Kolbeck et al. (1997), to exclude measurement errors due to variations in absorbance not specific to cytochrome c. To study the time-dependent production of the peroxide anion, the data were expressed as the variation of the optical density at 550 nm between two successive solution aliquots collected at 2-min intervals (P_{OD 550}).

View larger version (14K): [in this window] [in a new window]   Fig. 1. Caffeine dose-response curves on saponin-skinned fibers from C57BL/10 (n = 7) and mdx (n = 9) mice. Values are mean ± S.E.M. The solid lines show the fits of the data using a sigmoidal equation. n represents the number of fibers. *, p < 0.05 Bl10 versus mdx.

View larger version (11K): [in this window] [in a new window]   Fig. 2. Force-pCa relationships in Triton X-100-skinned diaphragm fibers from C57BL/10 (n = 18) and mdx (n = 18) mice. Mean values are fitted to sigmoidal (Hill) curves by nonlinear regression (see Materials and Methods), and Hill fit parameters are given in Table 2. n represents the number of fibers. *, p < 0.05 Bl10 versus mdx.

	Results

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Effects of Caffeine on Saponin- and Triton-X-100-Skinned Fibers from mdx Diaphragm. In saponin-skinned fibers, in which the SR was functional, caffeine was used to release the Ca²⁺ from the SR and induced a transient contracture reflecting the release of a specific amount of Ca²⁺ from the SR. In our experiments, six concentrations of caffeine (0.5-10 mM) were used to compare the caffeine sensitivity of the SR Ca²⁺-release mechanism in healthy C57BL/10 and mdx mice. Caffeine induced a concentration-dependent transient contracture and, for the concentration of 1.5 mM caffeine and greater, the amplitude of the contractile response was lower in mdx fibers than in C57BL/10 fibers (Fig. 1). However, the concentration of caffeine that produced a contracture equal to 50% of the maximal caffeine response (EC₅₀) did not differ significantly between Bl10 (EC₅₀ = 2.59 ± 0.15 mM, n = 7) and mdx (EC₅₀ = 2.87 ± 0.19 mM, n = 9) diaphragm fibers.

Previous reports have shown that caffeine has various side effects, including an increase in the Ca²⁺ sensitivity of the contractile proteins (Wendt and Stephenson, 1983). Accordingly, we tested the effects of 2.5 and 10 mM caffeine on the properties of the contractile apparatus in Triton X-100-skinned fibers from Bl10 and dystrophic diaphragm muscles. The results show that, under control conditions (without caffeine), from 1 µMCa²⁺ (pCa 6.0), the isometric tensions developed by mdx fibers were lower than those developed by Bl10 muscle (Fig. 2). For example, the tension generated by the contractile proteins when maximally activated by Ca²⁺ (T_max) was lower in mdx fibers than in Bl10 fibers, on average by 40% (Table 2). Moreover, in both Bl10 and mdx fibers, the presence of 2.5 mM caffeine induced a small but significant decrease in the maximal Ca²⁺-activated tension and Hill coefficient (n_H), whereas no change was observed in the apparent Ca²⁺ sensitivity (pCa₅₀) (Table 2). These effects on the T_max and n_H were intensified when 10 mM caffeine was added, and they were associated with an increase in the pCa₅₀ value, indicating an increase in the Ca²⁺ sensitivity. As illustrated in Table 2, these effects of 2.5 and 10 mM caffeine on the contractile protein properties were similar in diaphragm fibers from dystrophic and Bl10 mice.

Taken together, these results show that even though Ca²⁺-activated force was lower in mdx muscle, the dystrophic process did not modify the caffeine sensitivity of the SR or the side effects of this ryanodine receptor activator on contractile proteins. Therefore, we used caffeine to investigate the effects of H₂O₂ on the SR properties in diaphragm from mdx mice.

Effects of H₂O₂ on Contractile Proteins in Triton X-100-Skinned Fibers from mdx Diaphragm. The effects of H₂O₂ on the T_max and pCa₅₀ of the contractile proteins were tested in Triton X-100-skinned fibers from Bl10 and mdx diaphragm muscles. As shown in Table 2, exposure to 1 mM H₂O₂ caused a slight decrease in the T_max exhibited by both Bl10 and dystrophic muscles compared with the values obtained under control conditions in the absence of H₂O₂. Specifically, with 1 mM H₂O₂, the T_max corresponded to 94 and 94.5% of the initial force in Bl10 and dystrophic fibers, respectively. When we examined the effects of exposure to H₂O₂ on the Ca²⁺ sensitivity of the contractile apparatus, no significant changes in the pCa₅₀ were observed in either Bl10 or dystrophic fibers. Furthermore, the analysis of the Hill coefficient also showed that H₂O₂ treatment induced no significant differences among the various groups. These findings demonstrate that exposure to 1 mM H₂O₂ had similar effects on the T_max and the Ca²⁺ sensitivity of the contractile apparatus of diaphragm fibers in Bl10 and mdx mice.

View larger version (16K): [in this window] [in a new window]   Fig. 3. Representative caffeine-induced contractures (2.5 mM) under control conditions and when preparations were exposed to 1 mM H2O2 in all solutions (contractile responses 1 and 7 correspond to the number of contractures in the presence of the oxidant) in saponin-skinned diaphragm fibers from C57BL/10 and mdx mice.

The presence of H₂O₂ first induced an increase in the amplitude of the 10 mM caffeine contracture, which was followed by a marked decrease in the response to caffeine in both Bl10 and dystrophic diaphragm fibers. However, when the data were expressed as a percentage of the amplitude of the control caffeine contracture obtained without oxidant, the results showed that the presence of 1 mM H₂O₂ during successive caffeine contractures produced similar effects in both Bl10 and mdx diaphragm. For example, the increases in tension in the presence of H₂O₂ of 12.0 ± 4.1% (n = 14) in Bl10 and of 16.4 ± 5.7% (n = 14) in mdx were not significantly different.

Because 10 mM caffeine is a sufficient concentration to release all of the Ca²⁺ from the SR, thus leading to the maximal amplitude of the contracture, the increase in the amplitude of the first caffeine contracture observed in the presence of 1 mM H₂O₂ could be then underestimated. As illustrated in Fig. 3, we therefore carried out similar experiments to test the effect of 1 mM H₂O₂ on contractures elicited by the application of 2.5 mM caffeine, a concentration that produced a response with an amplitude around 50% of the maximal 10 mM caffeine contracture (Fig. 1). The result shows that the tension developed during each successive caffeine contracture was significantly lower in mdx than in Bl10 diaphragm muscles (Fig. 4A). In addition, 1 mM H₂O₂ induced a similar pattern to that observed with 10 mM caffeine; i.e., there was a rise in the amplitude of the caffeine contracture followed by a progressive decrease for both Bl10 and dystrophic muscles (Fig. 4A). Nevertheless, as illustrated in Fig. 4B, the effects of 1 mM H₂O₂ on contracture induced by 2.5 mM caffeine were significantly greater for diaphragm muscle from dystrophic mice than that from their Bl10 counterparts. Indeed, the increase in the amplitude of the first caffeine contracture in the presence of H₂O₂ compared with the control contracture without oxidant was 52% for mdx versus only 17% for Bl10 diaphragm muscles. In the same way, the decrease observed for the seventh contracture was 65 and 45% for mdx and Bl10 muscles, respectively. Taken together, these findings indicate that the effects of H₂O₂ on diaphragm muscle from both Bl10 and mdx mice were qualitatively similar, but that mdx fibers seemed to be more sensitive to H₂O₂ than wild-type fibers.

View larger version (24K): [in this window] [in a new window]   Fig. 4. Effects of 1 mM H2O2 on 2.5 mM caffeine-induced contractures. Amplitude of the contractile responses induced by the application of 2.5 mM caffeine (A) and relative amplitude (B) expressed as percentage of control contracture amplitude without the oxidant in saponin-skinned diaphragm fibers from C57BL/10 (n = 12) and mdx (n = 16) mice. Caffeine contractures were obtained under control conditions in the absence and in the presence of the oxidant in all solutions. The horizontal bar represents the number of successive contractures in the presence of H2O2. n represents the number of fibers. *, p < 0.05 Bl10 versus mdx;, p < 0.05 control conditions versus conditions with H2O2.

SR Ca²⁺ Uptake in the Presence of H₂O₂. To investigate the effects of H₂O₂ on SR Ca²⁺ uptake, experiments were performed on SR vesicle preparations from B10 and mdx diaphragm muscles. The time course of Ca²⁺ uptake was measured at pCa 7.0 in the absence and in the presence of oxalate, a Ca²⁺-precipitating anion used to prolong and maximize Ca²⁺ pumping by the SR. The results indicated that, in both conditions (with and without oxalate), SR Ca²⁺ uptake was slower in mdx than in Bl10 muscles (Table 4; Fig. 5). For example, in the presence of oxalate, the time constant was 48% greater in mdx diaphragm than in Bl10 muscles, without any change in the values of Ca²⁺ min (Table 4; Fig. 6A). This finding is consistent with previous data showing that the ATP-dependent activity of the SR Ca²⁺ pump is lower in dystrophic diaphragm muscle.

View larger version (24K): [in this window] [in a new window]   Fig. 5. Representative time course of Ca2+ uptake in isolated SR vesicles obtained from C57BL/10 and mdx diaphragm under control conditions without oxidant and after 1 mM H2O2 had been present for 10 min. SR vesicles were suspended in a cuvette that contains 3 mM oxalate, and extravesicular Indo-1 fluorescence ratio 405/480 nm was monitored over time.

View larger version (17K): [in this window] [in a new window]   Fig. 6. Effects of 1 mM H2O2 on Ca2+ uptake by isolated SR vesicles obtained from C57BL/10 (n = 9) and mdx (n = 10) diaphragm in the presence of oxalate. Values (mean ± S.E.M.) of the time constant (A) and Ca2+ uptake rate (B) were obtained under control conditions in the absence or in the presence of the oxidant, 0 and 10 min corresponding to the incubation time with H2O2. Values of the SR Ca2+ uptake rate in percentage are normalized to the data obtained for Bl10 diaphragm SR vesicle preparations under control conditions without oxidant. n is the number of experiments. For each experiment, three and four diaphragms were pooled for the C57BL/10 and mdx mice, respectively. *, p < 0.05 Bl10 versus mdx;, p < 0.05 control conditions versus conditions with H2O2.

The effects of 1 mM H₂O₂ on the SR Ca²⁺ accumulation were tested in solutions containing 3 mM oxalate. When H₂O₂ was added to the reaction medium at the beginning of the experiment, without a preliminary incubation period, the Ca²⁺ uptake rate was significantly increased in both Bl10 and mdx diaphragm SR vesicles. The data clearly showed that H₂O₂ had a more pronounced potentiating effect on mdx than on Bl10 muscles. Indeed, SR Ca²⁺ uptake time constants were decreased by 38 and 24% in dystrophic and Bl10 vesicle preparations, respectively (Fig. 6A). We then investigated the effect of 1 mM H₂O₂ over a more prolonged period by introducing a 10-min incubation period before to activate the Ca²⁺ uptake mechanism. Under these conditions, the presence of H₂O₂ induced a slowing down of the SR Ca²⁺ uptake rate in mdx muscles (Fig. 5). As illustrated in Fig. 6A, the SR Ca²⁺ uptake time constant was significantly increased by 52% in dystrophic diaphragm SR vesicles but showed no significant modification in Bl10 muscles. In the presence of 1 mM H₂O₂, the SR Ca²⁺ uptake rate was significantly increased in the SR vesicles of both Bl10 and mdx diaphragm (Fig. 6B). When the oxidant had been present for a longer period, the SR Ca²⁺ uptake rate decreased in both types of muscle, but this effect was more pronounced in dystrophic muscle. In addition, exposure of the SR vesicles to H₂O₂, with or without an incubation period, did not induce any significant change in Ca²⁺ min, which ruled out the possibility that this decrease was attributable to a change in the amount of Ca²⁺ loaded into either Bl10 and mdx muscles. These findings suggest that the dual time-dependent effect of H₂O₂ observed in saponin-skinned fibers, i.e., the increase and the decrease in the amplitude of caffeine-induced contractures, might be mediated by a stimulation or inhibition of the SR Ca²⁺ uptake.

Superoxide Anion Production during Fatiguing Stimulation. Extracellular superoxide anion () production was measured on the basis of its ability to reduce cytochrome c. As described previously under Materials and Methods, the P_{OD 550} of cytochrome c was determined in samples collected during the equilibration, fatiguing, and recovery periods. A plot of P_{OD 550} of the solution bathing the muscle (expressed as a percentage of the mean value for each muscle after equilibration) is shown in Fig. 7. The results show that, for both Bl10 and mdx diaphragm muscles, the fatiguing stimulation induced a significant increase in the P_{OD 550}, followed by a progressive decrease in P_{OD 550} during the recovery period. However, the P_{OD 550} values measured during the fatiguing stimulation and during the recovery period were significantly higher for dystrophic diaphragm than for Bl10 muscles. After the fatiguing protocol, P_{OD 550} values had returned to the values at equilibrium after 2 min of recovery for Bl10 muscles, whereas in mdx muscles, the values remained elevated throughout the recovery period.

View larger version (19K): [in this window] [in a new window]   Fig. 7. Superoxide anion produced during fatiguing stimulation. A plot of POD 550 of the muscle incubating solution expressed as a percentage of the mean equilibration value for each muscle, during the equilibration, fatiguing (—), and recovery periods were obtained for C57BL/10-(n = 5) and mdx-living (n = 3) diaphragm muscles. Values (mean ± S.E.M.) reflect the oxidation state of exogenous cytochrome c added to the muscle bathing solution. n represents the number of muscles. *, p < 0.05 Bl10 versus mdx;, p < 0.05 before versus after stimulation.

	Discussion

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This study compared the effects of the exogenous application of H₂O₂ on the contractile properties of skeletal muscle, more specifically, on the SR function in Bl10 and dystrophic diaphragm muscles. Our results show that dystrophin-deficient muscles displayed greater susceptibility to H₂O₂ injuries, which involved the SR compartment. This suggests that free radicals might be involved in the pathophysiological pathway and dysregulation of Ca²⁺ homeostasis that occurs in muscular dystrophy.

We have chosen to perform the experiments on the diaphragm muscle of mdx mice that are characterized by a progressive decline in muscle function, which closely resembles the human DMD phenotype, linked to the fact that the regeneration process no longer compensates for the degeneration (Petrof et al., 1993; Lynch et al., 1997). The mdx mouse model is characterized by a mild phenotype, in which an equilibrium is maintained between degeneration and regeneration, as the result of a high regenerative capacity. However, diaphragm muscle in the mdx mouse is severely affected and displays a early-onset progressive pathological process that contrasts with the late-onset progressive process observed in limb muscles (Stedman et al., 1991).

Our results show that the mechanisms involved in SR Ca²⁺ uptake were strikingly altered by the dystrophic process. Indeed, the Ca²⁺ uptake properties investigated in SR vesicles and saponin-skinned fibers have shown that the amplitude of the caffeine contracture was reduced, that SR Ca²⁺-ATPase activity was halved, and that the rate of Ca²⁺ uptake was lower in dystrophic than in Bl10 diaphragm muscle. Moreover, our results, obtained with saponin- and Triton X-100-skinned fibers, demonstrate that the differences between Bl10 and mdx fibers for the amplitudes of caffeine-induced contractions were not attributable to a change in the sensitivity to caffeine or to the side effects of caffeine on contractile proteins. The possibility that mdx fibers suffer from impaired myoplasmic Ca²⁺-removal mechanisms has previously been raised; for instance, Kargacin and Kargacin (1996) reported that the maximal velocity of SR Ca²⁺ uptake is lower in mdx than in normal fibers. Furthermore in a previous work using saponin-skinned fibers, we have shown that the SR was involved in the alteration of Ca²⁺ homeostasis in fast- and slow-twitch muscles from mdx mice during the period of necrosis (Divet and Huchet-Cadiou, 2002; Divet et al., 2005). The reduced ability of the SR Ca²⁺-ATPase to load Ca²⁺ would clearly disturb intracellular Ca²⁺ homeostasis, probably prolonging the presence of this cation, and this could activate some pathological processes. There is overwhelming evidence that, in their final stages, dystrophin-deficient muscle fibers are overloaded with Ca²⁺ (Ruegg et al., 2002) and that secondary Ca²⁺-mediated damage is responsible for their degeneration. Probably because of different technical procedures and types of cells used in the experiments, the values of intracellular Ca²⁺ concentration ([Ca²⁺]_i) reported have sometimes been inconsistent. However, as some studies have demonstrated, the [Ca²⁺]_i decay rate presented a significantly higher time constant in mdx fibers than in Bl10 values (Collet et al., 1999). Thus, our results show in diaphragm muscle from mdx mice an important dysfunction in the Ca²⁺ homeostasis, and this mdx muscle provides a useful model for the study of the pathophysiological process of human DMD.

The degenerative process, which involves cycles of necrosis and regeneration in muscle from DMD patients and mdx mice, is usually caused by alterations in Ca²⁺ homeostasis, as previously exposed, but also by shifts in the redox balance. It has also been found that dystrophic muscle cells seem to be inherently more susceptible to oxidative stress than normal muscle cells and that this susceptibility correlates inversely with the amount of residual dystrophin expression (Disatnik et al., 2000). Evidence for the involvement of ROS has been provided by observations that biological byproducts of oxidative stress were higher than normal (lipid peroxidation and protein carbonyls), that cellular antioxidants were lower than normal (glutathione and vitamin E), and that concentrations of antioxidant enzymes were altered. In this way, our study shows that, in isolated diaphragm muscles from both Bl10 and mdx mice, the amount of superoxide anion produced under fatiguing conditions was increased. However, during the fatigue stimulation, this increase in superoxide production was greater in muscles from dystrophic mice and was maintained longer during the recovery period. Thus, it is possible that, during the dystrophic process, the increased production of free radicals may have injured subcellular membranes and organelles when muscles were repetitively stimulated.

Free radicals are known to produce chemical and molecular damage of DNA, proteins, lipids, and cell membrane structure. Moreover, studies performed on isolated muscle fibers indicate that myofilaments are sensitive to direct redox modification and that their function is impaired by exposure to either ROS or NO (Andrade et al., 1998; Posterino et al., 2003). Under our experimental conditions, the results obtained in Triton X-100-skinned fibers show that 1 mM H₂O₂ slightly decreased the maximal Ca²⁺-activated tension without affecting the Ca²⁺ sensitivity of the contractile proteins; these effects were similar in Bl10 and dystrophic diaphragm.

The contractile changes mediated by ROS are likely to involve more than one molecular target. The diversity of redox-sensitive proteins that are involved in contractile regulation argues against a single site of action. It has been also recognized that several proteins located in the SR, including the ryanodine-sensitive Ca²⁺ release channel and the Ca²⁺ pump, are sensitive to redox modulation (Favero, 1999; Hamilton and Reid, 2000). Our results demonstrate that, in saponin-skinned fibers and in SR vesicles, the presence of H₂O₂ modified SR Ca²⁺ uptake mechanisms. In Bl10 and dystrophic diaphragm muscles, the presence of this oxidant over a prolonged period clearly decreased the amplitude of the caffeine contracture and reduced the SR Ca²⁺ uptake rate. However, our findings show that SR properties and SR Ca²⁺ uptake mechanisms, in particular, were more sensitive to the effects of H₂O₂ in preparations from dystrophic diaphragm than in those from normal muscles. Previous studies have shown that a potential target of ROS action is the SR Ca²⁺-ATPase, whose activity was altered in extreme redox conditions. Oxidative stress inhibits SR Ca²⁺ pump function, leading to a slowing down of the reuptake of Ca²⁺ into the SR, via effects on regulatory sulfhydryls located near the Ca²⁺-ATPase active site (Scherer and Deamer, 1986).

Moreover, our data suggest that the effects of H₂O₂ were use- and time-dependent. In the short-term, H₂O₂ led to an increase in the amplitude of the caffeine contracture developed by saponin-skinned fibers, but in the long term, the effect was reversed with a decrease in the amplitude. These results demonstrate that H₂O₂ affected SR properties, and it can be proposed that these effects could result from a direct action on the Ca²⁺ pump. This dual action in saponin-skinned fibers, as well as in SR vesicle preparations, could be explained if it can act as an activator or as an inhibitor of the activity of Ca²⁺-ATPase. Although this work was focused on the SR function in the presence of H₂O₂, our results were in agreement with the finding obtained in intact skeletal muscle (Oba et al., 1996; Andrade et al., 1998). Indeed, in mammalian and batrachian, skeletal muscles, an exposure to H₂O₂ altered the [Ca₂ ⁺]_i, tetani, and twitch forces, and then these effects support a fundamental role for an alteration of the SR function by the oxidant.

This study demonstrates the predominant role of Ca²⁺ in the progression of muscular dystrophy in diaphragm muscles from mdx mice and shows that it implicates the SR, the main system that regulates Ca²⁺ in skeletal muscle. Our findings support the hypothesis that intracellular Ca²⁺ homeostasis is disrupted in dystrophic muscle and demonstrate a major dysfunction of the SR Ca²⁺ pump. These results also high-light the potential role of ROS in the dystrophic process and show that these molecules, largely produced during fatigue, could amplify the disruption of Ca²⁺ intracellular homeostasis by maintaining an elevated [Ca²⁺]_i in dystrophic muscular cells over a long period of time. It could be then suggested that the prevention of oxidative stress in mdx mice may provide protection against disease progression.

	Footnotes

 
doi:10.1124/jpet.106.103291.

ABBREVIATIONS: DMD, Duchenne muscular dystrophy; SR, sarcoplasmic reticulum; NO, nitric oxide; ROS, reactive oxygen species.

¹ This work was performed as part of the Ph.D. requirement.

Address correspondence to: Dr. Corinne Huchet-Cadiou, Université de Nantes, Centre National de la Recherche Scientifique, UMR 6204, Biotechnologie, Biocatalyse et Biorégulation, Faculté des Sciences et des Techniques, 2 rue de la Houssinière, BP 92208, F-44322 Nantes, Cedex 03, France. E-mail: corinne.cadiou{at}univ-nantes.fr

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