Loss of myostatin (mstn) function leads to a decrease in mitochondrial content, a reduced expression of cytochrome c oxidase, and a lower citrate synthase activity in skeletal muscle. These data suggest functional or ultrastructural mitochondrial abnormalities that can impact on muscle endurance characteristics in such phenotype. To address this issue, we investigated subsarcolemmal and intermyofibrillar (IMF) mitochondrial activities, skeletal muscle redox homeostasis, and muscle fiber endurance quality in mstn-deficient mice [mstn knockout (KO)]. We report that lack of mstn induced a decrease in the coupling of IMF mitochondria respiration, with significantly higher basal oxygen consumption. No lysis of mitochondrial cristae or excessive swelling were observed in mstn KO mice compared with wild-type (WT) mice. Concerning redox status, mstn KO gastrocnemius exhibited a significant decrease in lipid peroxidation levels (−56%; P < 0.01 vs. WT) together with a significant upregulation of the antioxidant glutathione system. In contrast, superoxide dismutase and catalase activities were altered in mstn KO, gastrocnemius and soleus with a reduction of up to 80% compared with WT animals. The force production observed after contractile endurance test was significantly lower in extensor digitorum longus and soleus muscles of mstn KO mice compared with the controls (17 ± 3 and 36 ± 5% vs. 28 ± 4 and 56 ± 5%, respectively, P < 0.05). Together, these findings indicate that, besides an increased skeletal muscle mass, genetic mstn inhibition has differential effects on redox homeostasis and mitochondrial function that would have functional consequences on muscle response to endurance exercise.
- skeletal muscle
- GDF-8 inactivation
- respiratory chain
- contractile properties
myostatin (mstn) is a member of the transforming growth factor-β superfamily of growth and differentiation factors that is expressed predominantly in skeletal muscle and acts to negatively regulate growth of this tissue (28, 35). Its targeted inhibition or deletion results in an up to twofold increase in skeletal muscle mass (9, 35). The identification of a hypermuscular child with a loss-of-function mutation in the mstn gene (52) has brought much interest on mstn inhibition for the treatment of muscle injuries and muscle wasting diseases (7, 11, 39, 42).
Despite muscle mass hypertrophy, mstn inhibition has physiological effects that impact on both skeletal muscle metabolism and function (2, 13). Several studies have reported that loss of mstn expression is associated with a decrease in mitochondrial content that reduces muscle oxidative capacity (3, 29, 51). This can be partly explained by the lower relative proportion of oxidative fibers in mstn-deficient muscle (20, 55). Besides mitochondrial content, capacity for oxidative metabolism also relies on mitochondrial activity. Using NRM imaging, Baligand et al. (5) reported that, following several bouts of electrostimulation, capacity for mitochondrial ATP synthesis was altered in mstn-deficient muscles. Moreover, within skeletal muscle, mitochondria are divided into subsarcolemmal (SS) and intermyofibrillar (IMF) populations located at different cellular compartments, with SS mitochondria generating ATP for cellular exchanges while IMF mitochondria are responsible for generating ATP for muscular contraction. Previous data have shown that each subpopulation exhibits specific morphological and biochemical properties and responds differentially to physiological stimuli (1, 12, 14, 17). It should thus be of interest to assess activity of both mitochondrial subfractions to have a better understanding of oxidative capacity of mstn-deficient muscle.
Besides ATP production, mitochondria are also a major source of superoxide anion that causes formation of reactive oxygen species (ROS) but possess an important stock of antioxidant enzymes, such as manganese superoxide dismutase (MnSOD), catalase, and glutathione peroxidase (GPx) (45). In type IIb muscle fibers compared with type I, these antioxidant enzyme activities are low (46), whereas mitochondrial ROS production is elevated (4). The increase in type IIb muscle fibers as well as the reduced content of mitochondria observed in mstn-deficient muscles could alter redox status homeostasis.
Impairment in mitochondria metabolism and/or redox status impacts skeletal muscle contractile properties, in particular muscle endurance. Indeed, mitochondria are considered as the main source of ATP production in skeletal muscle during aerobic exercise (30), and any imbalance in redox status, leading to increased oxidative stress, impairs muscle contractile properties during chronic repetitive contractions (49). Previous studies have shown that specific muscle force (ratio of force to muscle mass), a marker of muscle contractile efficiency, is reduced in mstn knockout (mstn KO) mice compared with wild-type (WT) mice (3, 37, 44). Strikingly, muscle endurance qualities have been poorly investigated in mstn-deficient muscles, especially the fatigue susceptibility to chronic repetitive contractions. Global aerobic exercise capacity was evaluated in mstn KO mice with maximal running incremental tests and showed a significantly reduced maximal running speed in mstn KO mice (40, 51). However, such exercise tests involved whole body activity and did not examine skeletal muscle endurance capacity. In this context, it appears relevant to evaluate the individual and intrinsic fatigue susceptibility to chronic repetitive contractions in mstn-deficient fibers to exclude nonmuscle determinants.
In this study, we investigated the impact of mstn deficiency on mitochondrial function and redox status regulation in mstn KO mice compared with WT. We asked if mstn deficiency differentially altered SS and IMF mitochondrial respiration and antioxidant enzyme activities. Additionally, to evaluate the physiological consequences of these modifications on muscle function, tolerance to chronic repetitive contractions was assessed using ex vivo contractile tests.
MATERIALS AND METHODS
Male mstn KO mice used in this study have been described previously and were generously provided by L. Grobet (Faculty of Veterinary Medicine, University of Liège, Belgium). These mice harbor a constitutive deletion of the third mstn exon leading to the deletion of the entire mature COOH-terminal region of mstn and were therefore null for mstn function (24). The mice were generated on a FVB/N-C57BL genetic background. WT and mstn KO mice (9 wk old) were produced from homozygous matings. Parental genotypage were determined by polymerase chain reaction analysis of tail DNA. Mice were fed ad libitum and kept under a 12:12-h light-dark cycle. The experimental protocols of this study were handled in strict accordance with European directives (86/609/CEE) and approved by the Ethical Committee of Region Languedoc Roussillon.
Mice were weighed and killed by cervical elongation. Gastrocnemius and soleus muscles were removed, frozen in liquid nitrogen, and then stored at −80°C for enzymatic analysis. Tibialis anterior, extensor digitorum longus (EDL), gastrocnemius, and quadriceps muscles were quickly excised and immediately placed in ice-cold buffer (100 mM KCl, 5 mM MgSO4, 5 mM EDTA, and 50 mM Tris·HCl, pH = 7.4). SS and IMF mitochondria were fractionated by differential centrifugation as described previously (14, 58). Briefly, muscles were freed of connective tissues, minced, and homogenized with a Potter-Elvehjem homogenizer. SS mitochondria were separated from IMF mitochondria by centrifugation at 800 g and pelleted from the supernatant at 9,000 g. IMF mitochondria were obtained from the initial pellet following a treatment with Subtilisin A (0.25 mg/g wet muscle) and subsequent centrifugations at 800 and 9,000 g. Mitochondria were resuspended in 100 mM KCl and 10 mM MOPS, pH 7.4. Mitochondrial protein content was determined using the Bradford (8) assay, and the yield was expressed as milligram of mitochondrial proteins per gram of muscle wet weight.
SS and IMF mitochondria oxygen consumption was measured using the high-resolution Oxygraph-2k (OROBOROS Instruments, Innsbruck, Austria). Mitochondrial subfractions were incubated in two sealed thermostated chambers (37°C) containing 2 ml of MIRO5 respiration medium [0.5 mM EGTA, 3 mM MgCl2·6H2O, 65 mM KCl, 20 mM taurine, 10 mM KH2PO4, 20 mM HEPES, 110 mM sucrose, and 1 g/l BSA, pH 7.1 (21)]. Resting rate (state 4) was evaluated in the presence of 2.5 mM malate, 5 mM glutamate, and 5 mM succinate; ADP-stimulated rate (state 3) was determined after addition of 0.5 mM ADP. Mitochondria integrity was checked using NADH addition during state 3 measurement. The increase in respiration was <10% and not significantly different between WT and KO mice, showing that mitochondria were fully functional (data not shown). Data acquisition and analysis was performed using Oxygraph-2k-DatLab software version 4.3 (OROBOROS Instruments). The respiratory control ratio (RCR) was set as the ratio of oxygen consumption at state 3 over oxygen consumption at state 4.
Mitochondrial ROS production.
ROS were measured as described previously (12). Briefly, mitochondrial subfractions (50 μg) were incubated in V̇o2 buffer (250 mM sucrose, 50 mM KCl, 25 mM Tris·HCl, and 10 mM K2HPO4, pH 7.4) in a 96-well black plate. ROS production was assessed at 37°C for 60 min during states 3 and 4 respiration, by adding respiration substrates before the addition of 50 μM dichlorodihydrofluorescein diacetate. ROS production is directly proportional to fluorescence emission monitored at λex 485 nm/λem 530 nm with a microplate fluorimeter (Synergy 2; Biotek Instruments). Microplate data were compiled and analyzed using Gen5 software (version 1.04), and results were expressed as arbitrary fluorescent units.
Mitochondrial respiratory complexes and citrate synthase activities in tissues.
Mitochondrial activities were measured in gastrocnemius and soleus muscles. Complex I activity was measured according to Ref. 25: the method is based on measuring spectrophotometric 2,6-dichloroindophenol reduction by electrons accepted from decylubiquinol, reduced after oxidation of NADH by complex I. Complex II and complex II + III activities were measured according to Ref. 50: succinate-ubiquinone reductase and succinate-cytochrome c reductase activity were, respectively, determined spectrophotometrically. Cytochrome c oxidase (COX) activity was measured according to Ref. 60: oxidation of reduced cytochrome c is followed spectrophotometrically. Citrate synthase (CS) activity was measured according to Ref. 53: the activity of the enzyme is measured by following the color of 5-thio-2-nitrobenzoic acid, which is generated from 5,5′-dithiobis-2-nitrobenzoic acid present in the reaction of citrate synthesis, and caused by the deacetylation of acetyl-CoA.
Ultrastructural evaluation of mitochondria.
Samples from gastrocnemius and soleus muscles were immersed in a solution of 2.5% glutaraldehyde in Sorensen's buffer (0.1 M, pH 7.4) overnight at 4°C. They were then rinsed in Sorensen's buffer and postfixed in 0.5% osmic acid for 2 h in the dark at room temperature. After two rinses in Sorensen's buffer, tissues were dehydrated in a graded series of ethanol solutions (30–100%). Tissues were embedded in EmBed 812 using an Automated Microwave Tissue Processor for Electronic Microscopy, Leica EM AMW. Thin sections (80 nm; Leica-Reichert Ultracut E) were collected at different levels of each block. These sections were counterstained with uranyl acetate and observed using a Hitachi 7100 transmission electron microscope in the Centre de Ressources en Imagerie Cellulaire de Montpellier (France). Micrographs were taken at ×10,000 magnification, and SS and IMF mitochondria ultrastructure were determined at ×50,000 magnification. Presence of mitochondrial cristae lysis and/or swelling was explored as previously described (56) on three different animals of each group with more 600 IMF and SS mitochondria observed by muscle.
Lipid peroxidation levels or thiobarbituric acid-reactive substances (TBARS) were measured in tissue homogenates (gastrocnemius and soleus) according to the method of Sunderman et al. (57). Protein oxidation was assessed by measurement of sulfhydryl groups according to Faure and Lafond (16). Concerning antioxidant activities, total superoxide dismutase (SOD) and MnSOD were measured in tissues according to the method of Marklund (33). Catalase activity was measured in tissues according to the method of Beers and Sizer (6). Total glutathione (GSH) was measured in tissues according to the method of Griffith (23). GPx was measured according to the method of Flohe and Gunzler (19). Glutathione reductase (GRx) was measured according to the method of Carlberg and Mannervik (10). Measurements are associated with a coefficient of variation <15%.
Muscle contractile studies.
The EDL and soleus muscles were carefully removed from another group of mice that was deeply anesthetized with a xylazine-ketamine mix [0.4% Rompun (xylazine)-5% Imalgene 500 (ketamine) vol/vol, 0.01 ml/g body mass]. Muscles were tied at the proximal and distal tendons with braided surgical silk and transferred to a custom-built Plexiglas bath filled with a standard solution containing (in mM): 121 NaCl, 5 KCl, 1.8 CaCl2, 0.5 MgCl2, 0.4 NaH2PO4, 24 NaHCO3, and 5.5 glucose. The solution was continually bubbled with 95% O2-5% CO2 (pH 7.4) and thermostatically maintained at 37°C. The distal tendon of the muscle was tied to an immovable pin, and the proximal tendon was attached to the lever arm of a dual-mode force transducer (model 305B; Cambridge Instruments, Aurora Scientific, Ontario, Canada). The EDL and soleus muscles were stimulated by supramaximal square-wave pulses and delivered via two platinum electrodes that flanked the length of the muscle to produce a maximum isometric contraction. All stimulation parameters and contractile responses were controlled and measured using custom-built applications of LabView software (National Instruments, Austin, TX). Optimum muscle length was determined from micromanipulations of muscle length during a series of isometric twitch contractions. Maximum isometric force was determined from the plateau of the frequency-force relationship (1 Hz up to 120 Hz) before and 3 min after a 5-min high-frequency repetitive contraction stimulation according to Ref. 31 (50-Hz trains of 700 ms delivered every 2 s). The deficit in force-producing capacity after the endurance test was calculated compared with initial force production, expressed in percent. The effect of N-acetylcysteine (NAC), a pharmacological antioxidant known to protect against muscle fatigue, was also measured. The stimulation protocol was also carried out with a 20 mM NAC-enriched perfusion solution. After being tested, the muscles were removed from the bath, trimmed of their tendons and nonmuscle tissue, and weighed on an analytical balance (22, 32).
All data are presented as means ± SE. A two-way ANOVA followed by Tukey's pairwise multiple-comparison procedure was used to determine the effects of muscle type, animal groups, and NAC-enriched perfusion on contractile properties and antioxidant activities. Other biological data were compared between the two groups of mice using an unpaired t-test or a Mann-Whitney rank sum test when normality was not obtained. The significance level was set at 0.05. The data were analyzed using the statistical package Statistica.
Animals and muscle characteristics.
As expected, mstn KO mice had a greater body and muscle weight than their WT littermates (Table 1). Compared with WT, muscle hypertrophy reached 221, 187 and 132% for EDL, gastrocnemius, and soleus, respectively.
Lack of mstn-induced mitochondrial depletion.
Significant mitochondrial depletion (−40%, P < 0.05) was found in mstn KO mice for both SS and IMF subfractions compared with WT (Table 2). In addition, microscopic qualitative analysis showed a reduction in IMF mitochondrial density (Fig. 1). Total CS activity in gastrocnemius muscle showed a 27% reduction in mstn KO mice compared with their WT littermates (P < 0.001; Fig. 2), while no alteration was detected in mitochondrial CS activity between animal groups (Table 2). These results confirmed a mitochondrial depletion in skeletal muscle of mstn KO mice.
Lack of mstn altered the function of IMF mitochondria without evidence of ultrastructural damage.
As previously described (1, 14), we found higher state 3 respiration rates for IMF mitochondria compared with SS in both animal groups (P < 0.05; Table 2). However, IMF subfraction respiration was altered in mstn KO mice. Indeed, all KO mice exhibited a significantly higher basal oxygen consumption measured at state 4 (P < 0.05; Table 2) compared with WT, whereas no change was observed at state 3. Consequently, a significant 17% decrease of the RCR was found in the IMF subfraction of mstn KO mice compared with WT (P < 0.05; Table 2). No differences were observed between mstn KO and WT mice in the SS subfraction for oxygen consumption and RCR. Because mitochondrial function is related to mitochondrial ultrastructure, we performed qualitative analysis by transmission electron microscope analysis of gastrocnemius muscles from mstn KO and WT mice (Fig. 1). We reported an absence of lysis of mitochondrial cristae, and no excessive swelling in SS or IMF mitochondria was observed.
Lack of mstn increased COX activity in skeletal muscle.
To explain the observed increase in mitochondrial respiration, we measured the activities of complexes of the mitochondrial respiratory chain in gastrocnemius muscle homogenates and normalized for mitochondrial content with total CS activity. A significant increase was observed for the COX activity in gastrocnemius muscle of mstn KO mice compared with WT (P < 0.05). No alteration was found for the other complexes (Fig. 2).
Lack of mstn induced a reducing redox basal state with differential effects on antioxidant activities.
ROS production was measured in both subfractions, and as previously described (1, 12) it was higher in SS compared with IMF mitochondria (P < 0.05; Table 2). In contrast, no alteration was detected between mstn KO and WT mice (Table 2).
Concerning skeletal muscle oxidation products, lipid oxidation assessed by the TBARS assay was significantly decreased (−56%, P < 0.01) in mstn KO gastrocnemius relative to WT (Table 3). No difference was found in soleus muscle. Protein oxidation assessed by the evaluation of thiol levels was not altered in mstn KO mice compared with WT in both gastrocnemius and soleus muscles (Table 3).
When muscles were compared, primary antioxidant activities of SOD, catalase, and GPx were significantly reduced in gastrocnemius compared with soleus (2-way ANOVA, P < 0.05; Table 3). In gastrocnemius muscle, the antioxidant GSH system content increased in mstn KO mice compared with WT (Table 3). The amounts of total GSH and the GPx activity were 35 and 22% higher, respectively, in mstn KO mice relative to WT, whereas the GRx activity showed a 56% decrease. No difference was observed for the GSH system in soleus muscle between mstn KO mice and WT (Table 3).
The antioxidant SOD and catalase enzyme activities assayed in gastrocnemius and soleus muscles from mstn KO mice were significantly altered. Notably, MnSOD activity was decreased by 35% and up to 88% in mstn KO soleus and gastrocnemius muscles, respectively (Table 3). Depletion in MnSOD activity persisted in mstn KO mice compared with WT in both muscles when normalized by the mitochondrial content (data not shown). Overall, a greater reduction in antioxidant SOD and catalase activities was observed in gastrocnemius relative to soleus muscles in mstn KO mice (Table 3).
Lack of mstn impaired not only specific force but also muscle fiber tolerance to chronic repetitive contractions.
As expected, in EDL and soleus muscles, absolute tetanic force was not different in mstn KO muscle, and mean specific force (per milligram of muscle) was significantly reduced compared with WT (Table 4). To evaluate the effect of mstn-induced hypertrophy on muscle response to endurance exercise, we compared maximum isometric force before and after a high-frequency stimulation protocol. The effect of NAC, a pharmacological antioxidant known to protect against muscle fatigue, was also measured. Illustrative layouts of the muscle contractile endurance test are presented in Fig. 3A. The force reduction caused by repetitive tetanic stimulations was significantly greater in mstn KO muscles than in WT (Fig. 3B). Three minutes after the endurance test, the mean EDL and soleus forces were significantly lower in mstn KO muscles compared with WT (17 ± 3 and 36 ± 5% vs. 28 ± 4 and 56 ± 5%, respectively, P < 0.05). No significant effect of NAC-enriched perfusion solution was observed on force reduction in both groups.
Morphologic analysis confirmed the hypermuscularity of our mstn KO mice model, in accordance with previous reports (3, 35, 37). Moreover, this increase of muscle mass was especially important in mixed or fast muscles like gastrocnemius and EDL muscles, which is in agreement with previous findings (37). Besides this increase in muscle mass, we provide evidence that mstn deficiency induces metabolic perturbations in the respiration of IMF mitochondria and redox homeostasis that would have functional consequences on skeletal muscle response to endurance exercise.
For the first time, mitochondrial content and function were studied according to their subcellular location in mstn KO skeletal muscle. Mitochondrial yield and electron microscope scans indicated a reduction in mitochondrial content in mstn-deficient skeletal muscle that affects both SS and IMF subfractions to the same extent. We confirmed this result with a reduction in CS activity in muscle homogenates, which was previously observed by others (3, 20, 51).
Concerning mitochondrial function, while SS mitochondria respiration rates did not differ between the two genotypes, IMF mitochondria basal respiration (state 4) was significantly increased in the absence of mstn. State 4, defined as oxygen consumption by mitochondria in the absence of ADP, is commonly associated with proton leak across the mitochondrial inner membrane. We hypothesized that this increase in state 4 oxygen consumption would lead to a decrease in ROS production, an adaptative mechanism considered as a first line of defense toward ROS-induced damages (27). In line with this assumption, no increase in IMF mitochondrial ROS production was noticed at state 4 in mstn KO mice despite the depletion in mitochondrial antioxidant activity (i.e., MnSOD). Furthermore, transmission electronic microscopy analysis on mstn-deficient gastrocnemius excluded mitochondrial ultrastructural damages. Despite this protective effect against ROS, the increase in state 4 oxygen consumption, which was not significant during state 3 respiration, could also lead to a decay in RCR in IMF mitochondria of mstn-deficient muscles. Thus, a reduced mitochondrial ATP production cannot be excluded. Moreover, alteration in ATP production in mstn KO muscles was previously reported using nuclear magnetic resonance imaging (5), and linked to the switch in muscle fiber type induced by mstn deletion. We therefore believed that both mitochondrial content and function may be involved in the decrease of ATP production in mstn KO muscles.
To gain insight on any alteration in mitochondrial respiratory chain function due to this increase in oxygen consumption, we explored the activities of the enzymatic complexes in a mixed muscle, i.e., gastrocnemius muscle. At the tissue level, our results showed an increase in gastrocnemius muscle COX activity normalized for mitochondrial content. We hypothesize that this moderate increase may reflect a compensatory event to overcome a potential decrease in mitochondrial ATP synthesis, underlined by both the lower IMF mitochondria RCR and decline in mitochondrial content.
Given the switch in muscle fiber types observed in mstn KO mice from oxidative to glycolytic types, a decrease in overall antioxidant activities could have been expected, especially in gastrocnemius muscle. Indeed, it is well described that SOD, catalase, and the GSH system are highly expressed in muscle fibers with high oxidative capacities (for review, see Ref. 45). In our study, when muscles are compared, primary antioxidant enzymes activities, i.e., SOD, catalase, and GPx, are significantly reduced in gastrocnemius compared with soleus. However, when the genotype is considered, we showed a differential alteration in regulation of antioxidant defenses in mstn KO mice. Indeed, mitochondrial SOD activity and catalase activity, to a lesser extent, are decreased in gastrocnemius muscle from mstn KO mice compared with WT. Interestingly, this result could not be explained by a decrease of mitochondrial content since mitochondrial MnSOD activity is even lower after normalization by citrate synthase. Recently, Sriram et al. (54) reported an increase in mRNA of SOD1 and catalase in young mstn KO gastrocnemius compared with WT, whereas we found a reduction of MnSOD (SOD2) and catalase activities. Such discrepancy may reflect different methods used in these studies (mRNA expression vs. activities) and that different SOD isoforms were studied [copper-zinc SOD (CuZnSOD) for mRNA expression and MnSOD for activity]. It is interesting to note the absence of increase in IMF mitochondrial ROS production in mstn KO mice despite the depletion in mitochondrial antioxidant activity (i.e., MnSOD). Therefore, we cannot exclude that CuZnSOD, whose fraction is localized in the mitochondrial intermembrane space (43), may contribute in maintaining a normal mitochondrial ROS level.
We noticed a reduction in lipid peroxidation in mstn KO muscles with no changes in oxidized protein content. This result, indicative of protection toward oxidative stress in KO mstn animals, could be related to the subsequent upregulation of GPx activity and GSH content. In this context, a high level of GRx is not required in mstn-deficient muscle. This increase in GSH involved a fine regulation in its biosynthesis that requires a thorough evaluation in mstn KO mice. Altogether, our results suggest that lack of mstn plays a critical role in integrating redox signaling and induces a reducing basal state in skeletal muscle. This may provide protection from the lack of MnSOD, which represents one of the main antioxidant mitochondrial systems (47).
Our work showed that tolerance to endurance exercise was reduced in EDL and soleus muscles of mstn KO mice (∼50% compared with WT) associated with an increased fatigability of EDL muscle compared with soleus. The ex vivo model used in the current work limited nonmuscle component interference, like oxygen delivery and cardiac output, in the evaluation of muscle aerobic capacity and demonstrated that endurance muscular work is clearly altered in the mstn KO model. This higher fatigability can be related with the switch in fiber type that accompanied the deletion of mstn gene (20, 55). However, muscle fiber contractile properties, and especially the response to chronic exercise, are notably regulated by mitochondrial content and function (30) and muscle redox homeostasis (48). Our data underlined mitochondrial dysfunction, rather than redox status, in the higher fatigability observed in KO mstn mice. Indeed, the reduced availability of ATP, as a result of IMF mitochondrial dysfunction observed in mstn KO mice, could contribute to the alteration in skeletal muscle resistance to fatigue, since high chronic levels of ATP are required for muscle repetitive contractions. Concerning redox homeostasis, several data suggest that mitochondria are not the primary source of ROS during chronic contractile activity (1, 15). Indeed, during state 3 respiration, which is considered as aerobic contractile activity respiration, mitochondria produced less ROS. Our data are consistent with this, since mitochondrial ROS production remained unchanged between mstn KO and WT animals despite the lack of MnSOD. In our study, the use of NAC, a GSH precursor (62) known to alleviate fatigue in isolated muscles in animals as well as humans (26, 36, 41, 61), did not reduce the force deficit observed during the endurance test in mstn KO muscles. This suggested that the elevated GSH content and GPx activity measured in mstn KO animals may provide a protective environment that partly preserves muscle against exercise-induced oxidative stress in mstn KO mice. Nevertheless, to exclude the lack of MnSOD as a component of reduced muscle tolerance to chronic repetitive contractions in mstn KO muscles, the effect of SOD-enriched extract should be investigated during endurance tests (38).
Altogether, our results demonstrated that, besides an increased skeletal muscle mass, constitutive inhibition of mstn has signaling effects on mitochondrial function and redox homeostasis with consequences on muscle function. The present study showed that Mstn deficiency had beneficial effects on muscle redox status, providing a protective environment toward ROS. In contrast, mitochondrial metabolism is altered, with an uncoupling in the respiration of IMF mitochondria in glycolytic muscles, and is associated with reduced muscle response to endurance exercise. In regard to our results, it would be relevant to test mitochondrial biogenesis inducers, such as AMP-activated protein kinase (18), together with exercise training (34, 51) to improve skeletal muscle endurance quality in mstn KO mice.
Investigations performed on animals with constitutive deletion of mstn gene have brought much interest on mstn inhibition for the treatment of muscle degenerative disorders. However, in humans, such treatments have to take place in mature skeletal muscle. Studies using anti-mstn antibodies in adult mice or postdevelopmental mstn KO did not reveal any alteration in mitochondrial content (20) or expression of mitochondrial proteins using transcriptomic analysis (59). This did not preclude any alteration in mitochondrial functioning or muscle redox status as explored in the present study. Further investigations with postdevelopmental inhibition of mstn, including such mitochondrial metabolism and redox status analyses, will be required to conclude on the beneficial effects of such a therapy on skeletal muscle metabolism.
This study was supported by funds from the Institut National de la Recherche Agronomique, the University of Montpellier 1, and the Association Française contre les Myopathies.
No conflicts of interest, financial or otherwise, are declared by the authors.
Author contributions: C.P., B.C., G.F., B.V., and C.R. performed experiments; C.P., B.C., G.F., C.F.-C., C.C., A.B., and C.R. analyzed data; C.P., B.C., C.F.-C., C.C., A.B., and C.R. interpreted results of experiments; C.P., B.C., and C.R. prepared figures; C.P., B.C., and C.R. drafted manuscript; C.P., B.C., C.F.-C., C.C., A.B., and C.R. edited and revised manuscript; C.P., B.C., G.F., B.V., C.F.-C., C.C., A.B., and C.R. approved final version of manuscript; B.C., A.B., and C.R. conception and design of research.
We thank the Centre de Ressources en Imagerie Cellulaire facility and are particularly grateful to Chantal Cazevieille, Cécile Sanchez, and Elodie Jublanc for technical assistance and interpreting data concerning mitochondria ultrastructural evaluation. We are grateful to Beatrice Bonafos and the animal care facility for animal handling and breeding. Special thanks go to Vincent Ollendorff for helpful discussions and Lisa Leung for critical reading of the manuscript.
- Copyright © 2012 the American Physiological Society