Neutralizing mitochondrial ROS does not rescue muscle atrophy induced by hindlimb unloading in female mice

Abstract

Excess reactive oxygen species (ROS) induced by physical inactivity is associated with muscle atrophy and muscle weakness. However, the role of mitochondrial ROS on disuse-induced muscle atrophy is not fully understood. The purpose of this study was to utilize a genetic strategy to examine the effect of neutralizing mitochondrial ROS on disuse-induced skeletal muscle atrophy. This was accomplished by placing wild-type (WT) and mitochondrial-targeted catalase-expressing (MCAT) littermate mice on 7 days of hindlimb unloading. After assessment of body weight and composition, muscles were analyzed for individual muscle mass, force-generating capacity, fiber type, cross-sectional area, and mitochondrial function, including H₂O₂ production. Despite a successful attenuation of mitochondrial ROS, MCAT mice were not protected from muscle atrophy. No differences were observed in body composition, lean mass, individual muscle masses, force-generating capacity, or muscle fiber cross-sectional area. These data suggest that neutralizing mitochondrial ROS is insufficient to suppress disuse-induced loss of skeletal muscle mass and contractile function.

NEW & NOTEWORTHY The premise of this study was to examine the efficacy of genetic suppression of mitochondrial reactive oxygen species (ROS) to attenuate disuse-induced muscle atrophy and muscle weakness. Neutralization of mitochondrial ROS by MCAT expression was insufficient to rescue muscle atrophy and muscle weakness.

INTRODUCTION

Skeletal muscle inactivity or mechanical unloading are known to cause muscle atrophy and impair contractile function (10, 33). Hindlimb unloading in mice represents an experimental animal model that is known to reproducibly induce robust muscle atrophy (4). Muscle atrophy following hindlimb unloading leads to lower protein synthesis rates and increased protein breakdown (5, 24, 46). However, the upstream mechanism by which these changes occur is not well understood.

Oxidative stress accumulates in prolonged skeletal muscle inactivity (20, 34, 37). A primary source of oxidative stress is the generation of superoxide anions by the mitochondrial electron transport chain (ETC), xanthine oxidase, and nicotinamide adenine dinucleotide phosphate oxidase. Hindlimb unloading is known to increase oxidative stress and reduce antioxidant capacity (24). In particular, the rise in mitochondrial reactive oxygen species (ROS) production has been speculated to trigger proteolysis and reduce protein synthesis with disuse (38, 54). Min et al (31) and Talbert et al. (45) demonstrated that treatment of rats with a mitochondrial-targeted antioxidant reduced mitochondrial H₂O₂ production and prevented casting-induced muscle atrophy. Similar findings were seen in muscle atrophy in aging (17, 49). These findings suggest that oxidative stress arising from elevated mitochondrial ROS production and impaired antioxidant defense may be involved in muscle atrophy. However, these compounds may exhibit biological activities other than rescuing mitochondrial ROS to prevent atrophy (3, 14).

Expression of catalase targeted to the mitochondria (MCAT) promotes attenuation of mitochondrial-induced oxidative stress in conditions such as insulin resistance and muscle weakness (12). In rats, MCAT expression via in vivo electroporation increased catalase activity and attenuated cast immobilization-induced muscle atrophy compared with control limbs (8). In contrast, some data suggest that mitochondrial ROS may not be important for loss of muscle mass or function associated with aging, dystrophy, or congenital mitochondrial disorders (27, 43). Electroporation of skeletal muscle is known to promote highly unpredictable and heterogenous expression in skeletal muscle tissues. To test a model system in which MCAT is expressed in all myofibers across the animal, we placed MCAT mice (18, 42) on 7-day hindlimb unloading. We hypothesized that this genetic neutralization of mitochondrial ROS would protect mice from disuse-induced skeletal muscle atrophy.

METHODS

Animals and experimental design.

Mitochondrial-targeted catalase (MCAT) transgenic mice were purchased from The Jackson Laboratory [stock no. 016197, B6.Cg-Tg(CAG-OTC/CAT) Prab/J]. The transgenic lines used in this study were maintained as hemizygous and bred onto noncarrier (C57BL/6J background). For genotyping, DNA was extracted with NaOH from the ear and purified by 40 mM Tris·HCl. Genotype was determined using the PCR as recommended by The Jackson Laboratory. Eight-month-old female MCAT and wild-type (WT) littermates were used for all experiments. Animals were assigned to one of four experimental groups (n = 5–11/group): 1) WT mice without unloading (WT non-HU; n = 5), 2) MCAT mice without unloading (MCAT non-HU; n = 5), 3) WT mice with hindlimb unloading (WT HU; n = 10), and 4) MCAT with hindlimb unloading (MCAT HU; n = 11). Non-HU animals were able to freely ambulate in their cage (3 or 4 animals/cage) and had ad libitum access to food and water. Mice were maintained in a temperature-controlled room on a 12-h light/dark cycle. All animal procedures used in this study were approved by the Institutional Animal Care and Use Committee at the University of Utah. For an unclear reason, despite MCAT expression being driven by a β-actin promoter, these mice are known to exhibit increased MCAT protein in heart and skeletal muscles but not in other tissues (12, 42). Body mass were measured every day during HU. Body composition measurements were taken immediately before terminal experiments with a Bruker Minispec MQ20 nuclear magnetic resonance (NMR) analyzer (Bruker, Rheinstetten, Germany).

Hindlimb unloading.

WT and MCAT mice underwent 7 days of HU (2 mice/cage) using a previously described protocol (21, 30, 40) based on the traditional Morey-Holton design to study disuse atrophy in rodents. Along with daily monitoring of body mass, food intake was monitored every other day to ensure that the mice did not experience excessive weight loss due to malnutrition or dehydration. Following 7 days of HU, mice were fasted for 4 h and given an intraperitoneal injection of 80 mg/kg ketamine and 10 mg/kg xylazine, after which tissues were harvested. Tibialis anterior (TA), extensor digitorum longus (EDL), soleus (SOL), plantaris (PLA), and gastrocnemius (GAS) were carefully dissected for weight measurements.

Assessments of ex vivo skeletal muscle contractility.

Maximal force production in EDL and SOL muscles were measured using an ex vivo small animal muscle contraction apparatus (Aurora Scientific), as previously described (11, 50). Briefly, the EDL and SOL muscles were carefully excised from anesthetized mice. Muscles were then attached to the anchor and force transducer of the apparatus and submerged in oxygenated Krebs-Henseleit buffer (118 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO₄, 1.25 mM CaCl₂, 1.2 mM KH₂PO₄, 25 mM NaHCO₃, and 11 mM glucose) at 37°C. The optimal length was determined by maximum twitch force (20 V, 0.1-ms single pulse). After the muscle was set to an optimal length for maximal contraction, the buffer solution was replaced with freshly oxygenated Krebs-Henseleit Buffer (KHB). At the end of the 5-min equilibration, muscles were stimulated with pulses at 10, 20, 30, 40, 60, 80, 100, 125, 150, and 200 Hz (20 V, 0.2-ms pulse, 350-ms train duration) at 2-min intervals.

Muscle fiber-type and cross-sectional area.

The TA and SOL muscle tissues were embedded in optimal cutting temperature (OCT) compound and sectioned (10 μm) with a cryostat (Microtome Plus). Muscle sections were used for myosin heavy chain (MHC) isoform immunofluorescence (IF). The sections were incubated with MHC I (BA-D5), MHC IIa (SC-71), and MHC IIb (BF-F3) (all 3 from Developmental Studies Hybridoma Bank, University of Iowa). Negative-stained fibers were considered to be MHC IIx. Myofiber cross-sectional area was quantified using semiautomatic muscle analysis using segmentation of histology, a MATLAB application (SMASH) alongside ImageJ software.

Mitochondrial H₂O₂ measurements.

Mitochondrial H₂O₂ production was measured using the Horiba Fluoromax-4, as previously described (15, 18). Briefly, skeletal muscle was minced in mitochondria isolation medium (300 mM sucrose, 10 mM HEPES, 1 mM EGTA) and subsequently homogenized using a Teflon glass system. Homogenates were then centrifuged at 800 g for 10 min, after which the supernatant was taken and centrifuged at 12,000 g for 10 min. The resulting pellet was carefully resuspended in mitochondria isolation medium. JH₂O₂ was measured in buffer Z (MES potassium salt; 105 mM, KCl 30 mM, KH₂PO₄ 10 mM, MgCl₂ 5 mM, and BSA 0.5 mg/ml) supplemented with 10 mM Amplex UltraRed (Invitrogen) and 20 U/mL CuZnSOD in the presence of the following substrates: 10 mM succinate, 100 µM 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), and 1 µM auranofin. The appearance of the fluorescent product was measured with excitation/emission at 565/600 nm.

Mitochondrial respiration measurements.

Mitochondrial O₂ utilization was measured using the Oroboros O2K Oxygraphs, as previously described (18, 19). Isolated mitochondria were added to the oxygraph chambers containing buffer Z. Respiration was measured in response to the following substrate concentrations: 0.5 mM malate, 5 mM pyruvate, 2 mM ADP, 10 mM succinate, and 1.5 μM FCCP.

Western blotting.

Muscle lysates or isolated mitochondria (described above) were subjected to Western blotting analyses. For muscle lysates, frozen GAS muscles were homogenized using a glass tube and mechanically driven pestle grinder in an ice-cold buffer containing 50 mM Tris (pH 7.6), 5 mM EDTA, 150 mM NaCl, 0.1% SDS, 0.1% sodium deoxycholate, and 1% Triton X-100 with a protease inhibitor cocktail. Samples were centrifuged at 4°C for 10 min at 12,000 g. Supernatant protein concentration was determined using the BCA Protein Assay Kit (Thermo Scientific). Equal amounts of protein were then mixed with Laemmeli sample buffer and loaded onto 4–15% gradient gels (Bio-Rad). Proteins were then transferred onto nitrocellulose membranes and Ponceau S stained and imaged to ensure equal protein loading between lanes. Thereafter, membranes were blocked for 1 h at room temperature with 5% bovine serum albumin in Tris-buffered saline with 0.1% Tween 20 (TBST) and subsequently treated with primary antibodies for 4-hydroxynonenal (4-HNE; ab48506; Abcam), catalase (C0979; Sigma), citrate synthase (ab96600; Abcam), SOD2 (sc-137254; Santa Cruz Biotechnology), GPx4 (ab125066; Abcam) or total OXPHOS Rodent Cocktail (ab110413; Abcam). Following incubation, blots were washed in TBST, incubated in appropriate secondary antibodies (Anti-rabbit IgG, 7074, Cell Signaling Technology; or anti-mouse IgG, 31450, Thermo Scientific), and washed in TBST. Western Lightning Plus-ECL (PerkinElmer) were used to facilitate blot detection, and blots were scanned and quantified using FluorChem E imager (ProteinSimple).

Statistical analysis.

Values are expressed as means ± SE. Statistical comparisons were performed using an unpaired two-tailed Student’s t test for two-group analyses and two-way ANOVA with Tukey’s post hoc test for multiple comparisons (GraphPad Prism 8.1.0).

RESULTS

Body mass, food consumption, and body composition.

Without HU, body mass and body compositions of WT and MCAT mice were not different (Fig. 1, A–D), consistent with our previous work (18) and other published studies (12, 25). These studies also showed no differences in muscle weights and force-generating capacity between the groups. WT and MCAT mice lost weight similarly during the 7-day HU period (Fig. 1B). The reduction in body mass was largely accounted for by reduced lean mass (Fig. 1C), with no effect on fat mass (Fig. 1D). No difference was observed in food consumption during HU (Fig. 1E).

Fig. 1.

Mitochondrial-targeted catalase (MCAT) does not rescue the loss of lean mass induced by hindlimb unloading (HU). A: body mass from wild-type (WT; open bars) and MCAT (black bars) mice (WT non-HU, n = 5; MCAT non-HU, n = 5; WT HU, n = 10; MCAT HU, n = 11). B: time course changes in body mass during HU (WT HU, n = 10; MCAT HU, n = 11). C: lean mass. D: fat mass (WT non-HU, n = 5; MCAT non-HU, n = 5; WT HU, n = 10; MCAT HU, n = 11). E: daily food consumption during HU (WT HU, n = 10; MCAT HU, n = 11). *Main effect of HU (P < 0.05). Values are means ± SE.

Muscle mass.

Without HU, muscle weights between WT and MCAT were not different for any of the five muscles that were studied. HU significantly reduced muscle mass in TA, SOL, and GAS muscles, but not in EDL or PLA muscles (Fig. 2, A–E). After 7 days of HU, muscle weights between WT and MCAT mice were not different for any of the five muscles.

Fig. 2.

Mitochondrial-targeted catalase (MCAT) does not rescue the loss of muscle mass induced by hindlimb unloading (HU). A–E: absolute mass of several muscle tissue from wild-type (WT; open bars) and MCAT (black bars) mice with or without HU (WT non-HU, n = 5; MCAT non-HU, n = 5; WT HU, n = 10; MCAT HU, n = 11). EDL, extensor digitorum longus; GAS, gastrocnemius; PLA, plantaris; SOL, soleus; TA, tibialis anterior. *Main effect of HU (P < 0.05). Values are means ± SE.

Contractile function.

Force-generating capacity was analyzed for SOL (Fig. 3, A–D) and EDL (Fig. 3, E–H). Without HU, twitch and tetanic muscle force production were not different between WT and MCAT mice. As expected, HU significantly reduced twitch and tetanic force production in both WT and MCAT mice. After 7 days of HU, twitch and tetanic force production between WT and MCAT mice were not different. In the force-frequency curves, reduction in maximal tetanic force production induced by HU also coincided with increases in the midpoint of this relationship both in soleus (from 20 to 25 Hz to 30–35 Hz; Fig. 3C) and in EDL (from ∼60 Hz to 65–70 Hz; Fig. 3G).

Fig. 3.

Mitochondrial-targeted catalase (MCAT) does not rescue contractile dysfunction induced by hindlimb unloading (HU). A: peak twitch force for soleus (SOL) muscle in wild-type (WT; open bars) and MCAT (filled bars) mice with or without HU. B: representative twitch force tracings in SOL muscles with or without HU. C: force frequency curves for SOL muscles with or without HU. D: representative tetanic force tracings (200 Hz) for SOL muscles with or without HU (WT non-HU, n = 4; MCAT non-HU, n = 5; WT HU, n = 5; MCAT HU, n = 8). E: peak twitch force for extensor digitorum longus (EDL) muscles with or without HU. F: representative twitch force tracings in EDL muscles with or without HU. G: force-frequency curves for EDL muscles with or without HU. H: representative tetanic force tracings (200 Hz) for EDL muscles with or without HU (WT non-HU, n = 5; MCAT non-HU, n = 4; WT HU, n = 6; MCAT HU, n = 8). *Main effect of HU (P < 0.05). Values are means ± SE.

Muscle fiber-type and cross-sectional area.

Consistent with previous studies (48), fiber cross-sectional area (CSA) was not different between WT and MCAT mice in TA or SOL muscles (Fig. 4, B and E). HU reduced fiber CSA in both TA and SOL (except for MHC IIa in TA) in both WT and MCAT mice. After 7 days of HU, fiber CSA was not different between WT and MCAT mice in TA or SOL muscles. HU did not significantly alter fiber type composition in TA or SOL muscles (Fig. 5, C and F). Independent of HU, MCAT overexpression appeared to lower the proportion of type I fibers in SOL muscle (main effect of genotype). No such difference was observed in TA muscles, where type I fibers were virtually absent.

Fig. 4.

Mitochondrial-targeted catalase (MCAT) has no effect on muscle fiber cross-sectional area. A: representative images of myosin heavy chain (MHC) immunofluorescence for tibialis anterior (TA) muscles in wild-type (WT) and MCAT mice with or without HU. B: muscle fiber cross-sectional area by fiber type for TA muscles. C: fiber-type composition for TA muscles (WT non-HU, n = 4; MCAT non-HU, n = 4; WT HU, n = 6; MCAT HU, n = 6). D: representative images of MHC immunofluorescence for soleus (SOL) muscles. E: muscle fiber cross-sectional area by fiber type for SOL muscles. F: fiber type composition for SOL muscles (WT non-HU, n = 3; MCAT non-HU, n = 3; WT HU, n = 7; MCAT HU, n = 8). *Main effect of HU (P < 0.05); #main effect of genotype (P < 0.05). Values are means ± SE.

Fig. 5.

Mitochondrial-targeted catalase (MCAT) suppresses mitochondrial oxidative stress. A: representative Western blots of catalase and citrate synthase (CS; used as a loading control) in isolated skeletal muscle mitochondria from wild-type (WT) and MCAT mice with or without hindlimb unloading (HU). B: superoxide dismutase 2 (SOD2) protein abundance in skeletal muscle isolated mitochondria from WT (open bars) and MCAT (black bars) mice with or without HU (WT non-HU, n = 3; MCAT non-HU, n = 3; WT HU, n = 3; MCAT HU, n = 3). C: glutathione peroxidase 4 (GPx4) protein abundance in isolated skeletal muscle mitochondria (WT non-HU, n = 3; MCAT non-HU, n = 3; WT HU, n = 3; MCAT HU, n = 3). D: rate of mitochondrial H₂O₂ production in response to 10 mM succinate (WT non-HU, n = 5; MCAT non-HU, n = 5; WT HU, n = 7; MCAT HU, n = 7). E and F: representative images and quantification of muscle 4-hydroxynonenal (4-HNE) at ∼50 and ∼20 kDa in whole skeletal muscle tissues (WT non-HU, n = 3; MCAT non-HU, n = 3; WT HU, n = 3; MCAT HU, n = 3). *Significantly different from WT (P < 0.05); #significantly different from non-HU (P < 0.05). Values are means ± SE.

Mitochondrial H₂O₂ production and O₂ consumption.

Western blotting was used to confirm MCAT expression (Fig. 5A). WT and MCAT did not differ in the amount of antioxidant enzymes SOD2 and GPx4 with or without HU (Fig. 5, B and C). As expected, H₂O₂ production was successfully attenuated in muscles with mitochondrial-enriched catalase compared with WT muscles (Fig. 5D). Surprisingly, rates for H₂O₂ production (succinate driven) were not different between non-HU and HU groups. In contrast, oxidative stress measured by 4-HNE antibody showed marked increase with HU in WT mice (Fig. 5, E and F) that was completely removed with MCAT overexpression (18). No significant differences were observed in skeletal muscle mitochondrial respiration stimulated under a variety of substrates (Fig. 6A), nor were they observed in abundance of respiratory enzymes (Fig. 6, B and C) by genotype or with/without HU.

Fig. 6.

Mitochondrial-targeted catalase (MCAT) has no effect on oxygen consumption in the mitochondria. A: rates of oxygen consumption measured in isolated skeletal muscle mitochondria with Krebs cycle substrates from wild-type (WT; open and dark gray bars) and MCAT (black and light gray bars) mice without (non-HU) or with hindlimb unloading (HU). ADP, adenosine diphosphate; FCCP, carbonyl cyanide-p-trifluoromethoxyphenylhydrazone; Mal, malate; Pyr, pyruvate (WT non-HU, n = 3; MCAT non-HU, n = 3; WT HU, n = 3; MCAT HU, n = 3). B: representative Western blot of the Electron Transport System (ETS) protein complexes in isolated skeletal muscle mitochondria. C: quantification of ETS protein complexes (WT non-HU, n = 3; MCAT non-HU, n = 3; WT HU, n = 3; MCAT HU, n = 3). Values are means ± SE.

DISCUSSION

Oxidative stress has been implicated in disuse-induced muscle atrophy (31, 37, 45). Mitochondria in particular are responsible for generating a significant amount of ROS in the form of superoxide anions derived from electron leakage from the ETC (47). Previous studies show that antioxidants targeted to mitochondria can attenuate muscle atrophy and weakness, suggesting that mitochondrial ROS may directly induce muscle atrophy (31, 45). However, these compounds may exhibit alternative biological activity to other cellular systems to attenuate atrophy. Thus, the premise of this study was to use a genetic model to suppress mitochondrial ROS to more definitively conclude its role in mediating disuse-induced muscle atrophy. Surprisingly, neutralization of mitochondrial ROS (specifically H₂O₂) by MCAT expression did not alter the propensity for HU-induced loss of skeletal muscle mass or contractile function. These findings suggest that skeletal muscle mitochondrial ROS is not the mechanism by which disuse promotes skeletal muscle atrophy and weakness.

Mitochondrial ROS is thought to induce signaling events to promote muscle atrophy in various models of disuse (39). In response to denervation, whole body CuZn-superoxide dismutase (SOD1)-knockout mice show higher levels of oxidative damage and accelerated muscle atrophy and weakness (22, 32). However, a more recent study shows that muscle-specific knockout of SOD1 mice are not more prone to denervation-induced muscle atrophy (1). In another study, a mimetic of superoxide dismutase and catalase (EUK-134) attenuated HU-induced muscle atrophy (23), although this could be mediated by the action of these compounds in nonmuscle tissues. Directly relevant to the current study, in vivo electroporation of MCAT attenuated casting-induced muscle atrophy in rats (8). The differences are likely contributed by differences in the animal model, mode of disuse, and mode of overexpression. In corroboration with our previous work (18), MCAT expression in mice was sufficient to suppress H₂O₂ emission without affecting the capacity for mitochondrial oxidative phosphorylation (Fig. 6). In aging, some studies have also shown that mitochondrial oxidative stress may not be causal for age-related muscle atrophy (26, 28).

MCAT overexpression likely only neutralizes mitochondrial ROS (specifically H₂O₂), not those produced by cytosolic proteins. In this study, rates for H₂O₂ production were measured in isolated mitochondria. This was done so to validate that MCAT successfully attenuates H₂O₂ production that occurs specifically in mitochondria. Meanwhile, HU did not increase mitochondrial JH₂O₂, potentially suggesting that disuse may promote ROS production largely outside of mitochondria. We do not believe this is the case. HU robustly promoted an increase in whole muscle 4-HNE confirming that whole cellular ROS is increased with HU. Furthermore, MCAT overexpression completely neutralized the HU-induced increase in 4-HNE, indicating that ROS induced by HU occurs in mitochondria. Whole muscle 4-HNE likely better reflects ROS observed in vivo, whereas JH₂O₂ measurements were taken in isolated mitochondria that lost some of the three-dimensional structure present in vivo, studied only with succinate as the substrate.

SOD catalyzes the reaction by which superoxide anions are converted into hydrogen peroxides. Consistent with previous report (20), muscle SOD levels were not altered with MCAT overexpression or with HU. Previous studies suggest that skeletal muscle contains excess mitochondrial SOD that effectively neutralizes superoxides into hydrogen peroxide (16, 35), but we cannot completely rule out a possibility that superoxide anions, which are not scavenged by MCAT, are involved in signaling events that promote muscle atrophy and weakness.

Prolonged periods of mechanical unloading can result in muscle weakness and atrophy (2, 10, 13). Loss of contractile function in skeletal muscle has been linked to increases in oxidative stress and mitochondrial ROS production (1, 36, 44). A study using SS-31, a mitochondria-targeted antioxidant, showed no effect on fatigue-induced reduction in contractile force (7). SS-31 mostly scavenges the superoxide anion (41), suggesting that signaling from superoxide anions is likely not essential for this effect. Similar findings were observed with other antioxidants (6, 52), but it is important to recognize that each of these antioxidants may act primarily on different components of cellular ROS. Nevertheless, examination of force production in mice selectively overexpressing catalase, CuZnSOD, and MnSOD in the diaphragm also revealed no differences (29). In this study, no significant differences were observed in force-generating capacity between MCAT and WT mice following 7 days of HU. Taken together, these studies demonstrate that the involvement of oxidative stress to loss of muscle contractile function is likely intervention dependent.

After HU, muscles from WT and MCAT mice did not differ in fiber CSA or mass of individual muscles. Consistent with these findings, a previous study showed that mitochondrial antioxidant has no effect on muscle CSA (48). There was no difference in fiber type composition in fast/slow-mixed TA muscles. In contrast, the proportion of MHC I fibers from SOL muscles was slightly lower in MCAT mice compared with WT mice as a result of HU. Hindlimb unloading can induce a slow-to-fast transition of MHC isoforms in skeletal muscle, but this is largely a shift within MHC II subisoforms from IIa to IIx and IIb (9, 51, 53). Thus, the difference in fiber type composition is likely influenced by genetics during development, perhaps by the presence of MCAT in the myofibers or α-motor neurons. Regardless, these observations show that neutralization of mitochondrial ROS does not protect loss of muscle mass, even at the microscopic level.

Conclusions.

In conclusion, neutralization of mitochondrial ROS by expression of mitochondrial-targeted catalase was insufficient to rescue disuse-induced muscle atrophy and muscle weakness induced by hindlimb suspension. Consistent with our previous reports (18), the present study questions the notion that suppression of mitochondrial ROS protects muscles from loss in muscle mass or contractile function. Thus, increases in skeletal muscle oxidative stress may be coincidental, and not causal, to atrophy. It is noteworthy that MCAT only neutralizes oxidative stress derived in the mitochondria and that ROS production in other regions of cell, or ROS in other tissues for that matter, may contribute to disuse-induced muscle atrophy. Nevertheless, caution would need to be employed when utilizing mitochondrial-targeted antioxidant to treat muscle atrophy.

GRANTS

This work was supported by National Institutes of Health Grants DK-107397, DK-109888, and AG-063077 to K.F., AG-050781 to M.J.D., and HL-139451 to Z.S.M., a Larry H. & Gail Miller Foundation Grant to P.J.F., American Heart Association Grants 18PRE33960491 to A.R.P.V. and 19PRE34380991 to J.M.J, and the Uehara Memorial Foundation to H.E.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

H.E., P.S., M.J.D., and K.F. conceived and designed research; H.E., P.S., Z.S.M., J.M.J., P.J.F., A.R.V., and A.S. performed experiments; H.E., P.S., Z.S.M., J.M.J., P.J.F., A.R.V., A.S., M.J.D., and K.F. analyzed data; H.E., P.S., Z.S.M., J.M.J., A.R.V., A.S., M.J.D., and K.F. interpreted results of experiments; H.E., P.S., A.S., and K.F. prepared figures; H.E., P.S., and K.F. drafted manuscript; H.E., P.S., J.M.J., P.J.F., A.R.V., M.J.D., and K.F. edited and revised manuscript; K.F. approved final version of manuscript.