Beta-lapachone attenuates immobilization-induced skeletal muscle atrophy in mice

Experimental Gerontology 126 (2019) 110711

Contents lists available at ScienceDirect

Experimental Gerontology journal homepage: www.elsevier.com/locate/expgero

Beta-lapachone attenuates immobilization-induced skeletal muscle atrophy in mice Soyoung Parka,b,1, Min-Gyeong Shina,b, Jae-Ryong Kimb,c, So-Young Parka,b,

T

⁎,1

a

Department of Physiology, College of Medicine, Yeungnam University, Daegu, Republic of Korea Smart-aging Convergence Research Center, College of Medicine, Yeungnam University, Daegu, Republic of Korea c Department of Biochemistry and Molecular Biology, College of Medicine, Yeungnam University, Daegu, Republic of Korea b

A R T I C LE I N FO

A B S T R A C T

Section Editor: Christiaan Leeuwenburgh

Skeletal muscle atrophy reduces quality of life and increases morbidity and mortality in patients with chronic conditions. Oxidative stress is a key factor contributing to skeletal muscle atrophy by altering both protein synthesis and protein degradation pathways. Beta-lapachone (Beta-L) is known to act as a pro-oxidant in cancer cells but suppresses oxidative stress in normal cells and tissues. In the present study, we examined whether BetaL (100 mg/kg body weight) prevents immobilization-induced skeletal muscle atrophy in male C57BL/6N mice. Skeletal muscle atrophy was induced by immobilization of left hindlimbs for two weeks, and right hindlimbs were used as controls. The muscle weights of gastrocnemius (0.132 ± 0.003 g vs. 0.115 ± 0.003 g in Beta-L and SLS, respectively, p < 0.01) and tibialis anterior (0.043 ± 0.001 vs. 0.027 ± 0.002 in Beta-L and SLS, respectively, p < 0.001) were significantly heavier in Beta-L-treated mice than that in SLS-treated mice in immobilization group, which was accompanied by improved skeletal muscle function as tested by treadmill exhaustion and grip strength test. Immobilization increased H2O2 levels, while Beta-L treatment normalized such levels (1.6 ± 0.16 μM vs. 2.7 ± 0.44 μM in Beta-L and vehicle, respectively, p < 0.05). Oxidative stress makers were also normalized by Beta-L treatment. Protein synthesis signaling pathways were unaltered in the case of both immobilization and Beta-L treatment. However, protein catabolic, ubiquitin-proteasomal, and autophagy-lysosomal pathways were stimulated by immobilization and were normalized by Beta-L treatment. Upregulation of transforming growth factor β and Smad 2/3 after immobilization was significantly diminished by Beta-L treatment. These results suggest that Beta-L attenuates the loss of muscle weight and function induced by immobilization through suppression of oxidative stress.

Keywords: Beta-lapachone Immobilization Skeletal muscle atrophy Oxidative stress

1. Introduction Skeletal muscle comprises approximately 40% of total body weight and plays crucial roles in locomotion and metabolism (Reid and Fielding, 2012; Zurlo et al., 1990). Chronic conditions such as aging, diabetes, cancer, prolonged bed rest, nerve injury and reduced weightbearing can induce muscle atrophy, leading to reduction in quality of life, and increases in morbidity and mortality (Cohen et al., 2015). Skeletal muscle atrophy occurs as a result of an imbalance between protein synthesis and degradation; decreased protein synthesis, increased protein degradation or both induces skeletal muscle atrophy (Goldspink et al., 1983). A large body of evidence supports the important role of the insulin like growth factor 1 (IGF-1) and downstream phosphatidylinositol 3 kinase (PI3K) pathways in the protein synthetic pathway. Activation of PI3K by IGF-1 results in cascade stimulation of

the AKT/mTOR/p70S6K/4EBP pathway, leading to increased protein translation in cells and experimental animals (Egerman and Glass, 2014). A reduction in the protein synthesis pathway produces skeletal muscle atrophy in mice (Goncalves et al., 2010; Risson et al., 2009). Protein degradation is mediated by two main proteolytic signaling pathways: the ubiquitin-proteasome system and the autophagy-lysosome pathways (Mammucari et al., 2007). Forkhead box O1 (FOXO1) and Forkhead box O3 (FOXO3) are transcriptional factors that regulate the expression of atrophy-related genes, including muscle specific-RING finger protein-1 (MuRF1) and muscle atrophy F-box (atrogin1) in C2C12 cells and skeletal muscle of mice (Sandri et al., 2004; Waddell et al., 2008; Senf et al., 2010). MuRF1 and atrogin1, two muscle-specific E3 ubiquitin ligases, have been considered to be crucial regulators of skeletal muscle atrophy and are upregulated in various animal models of muscle atrophy (Gomes et al., 2001; Dehoux et al., 2003).

⁎

Corresponding author at: Hyunchoongro 170, Namgu, Daegu 42415, Republic of Korea. E-mail address: [email protected] (S.-Y. Park). 1 So-Young Park and Soyoung Park are different people with the same name. https://doi.org/10.1016/j.exger.2019.110711 Received 7 March 2019; Received in revised form 25 July 2019; Accepted 23 August 2019 Available online 24 August 2019 0531-5565/ © 2019 Elsevier Inc. All rights reserved.

Experimental Gerontology 126 (2019) 110711

S. Park, et al.

a 12:12 h light/dark cycle, and fed a standard chow diet with free access to water. The mice were anesthetized by intraperitoneal injection of avertin (> 1.0 g/kg) at the end of experiments. Blood was collected from the retro-orbital plexus using capillary tubes coated with heparin. Skeletal muscles were excised, weighed, and stored at −80 °C. The study was conducted in strict accordance with the guidelines and protocols for laboratory animals approved by the Institutional Animal Care and Use Committee of Yeungnam University College of Medicine (YUMC-AEC2016-014).

FOXOs also regulate the autophagy-lysosomal pathway in human cell lines and skeletal muscle of mice (Webb and Brunet, 2014), and this pathway is primarily responsible for the degradation of long-lasting proteins, aggregated proteins, and cellular organelles (Lilienbaum, 2013). This degradation pathway involves the production of an autolysosome, which is the result of the fusion of a lysosome with an autophagosome (Eskelinen and Saftig, 2009). This fusion is dependent on lysosomal-associated membrane protein 1 (LAMP1) and light chain 3 (LC3) which is localized in the autophagosomal membrane. Both of these molecules are markers of the autophagy-lysosomal pathway (Eskelinen and Saftig, 2009). Tissue transforming growth factor-β (TGFβ) is also known to cause skeletal muscle atrophy by inducing MuRF1 expression and oxidative stress enhances TGF-β activity and the expression of its downstream signaling molecules Smad2/3 in C2C12 cells and skeletal muscle of mice (Abrigo et al., 2016). Reactive oxygen species (ROS) are naturally occurring molecules that play important roles in physiological signaling and are particularly critical in skeletal muscle adaptation to exercise (Reid, 2001). However, ROS overproduction is known to be closely associated with skeletal muscle atrophy in mice (Qiu et al., 2018). Oxidative stress induced by excessive ROS triggers non-specific, large-scale oxidative damage to proteins, lipids, and DNA (Costa et al., 2007) and is a key factor that promotes proteolytic signaling and reduces protein synthesis (Pomies et al., 2016; Bae et al., 2012). Recent evidence suggests that mitochondrial dysfunction induced by prolonged skeletal muscle inactivity increases ROS production, leading to skeletal muscle atrophy (Hyatt et al., 2019; Powers et al., 2012). Moreover, reduced oxidative stress prevents immobilization-induced skeletal muscle atrophy in mice (Talbert et al., 2013). Beta-lapachone (Beta-L) is a natural ortho-naphthoquinone compound found in the bark of the lapacho tree. Beta-L is reduced to highly unstable hydroquinone by NAD(P)H:quinone oxidoreductase 1 (NQO1), and hydroquinone is oxidized back to semiquinone or quinone (Pink et al., 2000). This redox cycle produces substantial amounts of ROS, leading to DNA damage and cell death, especially in NQO1-expressing cancer cells (Pardee et al., 2002). In contrast to these pro-oxidant effects of Beta-L in cancer, Beta-L treatment reduces oxidative stress in non-cancer cells and tissues by activating sirtuin 1 (SIRT1) and AMPactivated protein kinase (AMPK) (Park et al., 2016; Lu, 2014). An increase in the NAD+/NADH ratio by Beta-L activates sirtuin 1 (SIRT1), leading to AMPK activation and nuclear factor erythroid-derived 2-related factor 2 (Nrf2) expression in rat primary astrocytes and hypertensive rats (Park et al., 2016; Kim et al., 2014). Nrf2 increases the expression of antioxidant enzymes, including heme oxygenase 1 (HO1), NQO1, and glutathione peroxidase 1 (GPX1), by binding to antioxidant response elements in target gene promoter regions in mice (Miller et al., 2012; Lee et al., 2005; Dong et al., 2008). SIRT1 also induces several antioxidant enzymes through FOXOs (Olmos et al., 2013; Salminen et al., 2013). Beta-L treatment significantly improves oxidative stress, renal dysfunction and tubular damage and apoptosis caused by ischemia/reperfusion injury in the kidney of mice (Gang et al., 2014). Cisplatininduced renal damage and NADPH oxidase expression are also abrogated by Beta-L treatment in mice (Oh et al., 2014). Therefore, based on these previous results regarding Beta-L in normal tissues, we hypothesized that Beta-L may prevent skeletal muscle atrophy by suppressing oxidative stress. To address this hypothesis, we examined whether Beta-L administration prevents skeletal muscle atrophy and oxidative stress induced by immobilization in mice.

2.2. Beta-L treatment To measure the effect of Beta-L on immobilization, Beta-L was administered by oral gavage at a concentration of 100 mg/kg body weight. The chemical was dissolved in 0.1% sodium lauryl sulfate (SLS) solution on the day of the experiment. The same amount of pure 0.1% SLS solution was administered to control mice. Beta-L administration was conducted at the same time (11 am) every day during the immobilization period. The first gavage was commenced one day before the immobilization procedure. After oral gavage, mice were observed to monitor if there was any vomiting of the solution. Body weight and food intake were measured each day, just before oral gavage. The mice were euthanized 1 h after the final Beta-L treatment. To explore the effect of Beta-L on the expression of antioxidant molecules, mice were injected with Beta-L (100 mg/kg body weight) intraperitoneally and euthanized 7 h after injection. To measure the effect of Beta-L on the protein synthesis signaling pathways in a timedependent manner, mice immobilized for two weeks were euthanized at 1, 3, and 7 h after the last Beta-L administration. 2.3. Immobilization The left hindlimbs of the mice were immobilized using a Manipler AZ (MANI. Inc.; Utsunomiya, Tochigi, Japan). The foot of each mouse was folded until the foot dorsum reached the shin, and then the foot was fixed with a staple. The right hindlimbs were used as controls. The immobilization status of the mice was confirmed daily. If the staple was removed from the mouse ankle, immobilization was reapplied immediately. Immobilization was maintained for two weeks. 2.4. Muscle function test 2.4.1. Treadmill exhaustion test Protocols for acclimation and the exhaustion test referred to a previous study (Gan et al., 2011). Mice were acclimated to a motorized treadmill (Ugo Basile; Gemonio, VA, Italy) for 2 consecutive days (10 m/min for 9 min and 20 m/min for 1 min at 0 degrees) before the treadmill test. On the day of the experiment, mice were unstapled 1 h prior to the treadmill test and allowed to move freely in cages. Mice were then run for 1 h at a speed of 10 m/min at 0 degrees. The incline was increased by 5 degrees every 15 min until it reached 15 degrees and the speed was increased by 3 m per minute until the mice were exhausted. Exhaustion was defined as mice staying on the electric shock grids for 5 consecutive seconds. 2.4.2. Grip strength test The inverted-cling grip test was performed as previously described (Cha et al., 2019). Left legs of mice were unstapled 1 h prior to the test. Three trials were performed with a 1-hour inter-trial interval, and the average score was used as the result.

2. Materials and methods 2.5. Hydrogen peroxide measurement 2.1. Animals Hydrogen peroxide levels in the gastrocnemius muscle were determined using the ferric-sensitive dye, xylenol orange (Sigma–Aldrich; St. Louis, MO, USA) (Gay et al., 1999). Briefly, muscle samples were

Nine-week old C57BL/6N male mice were purchased from KOATECH (Seoul, South Korea). The mice were housed in a room with 2

Experimental Gerontology 126 (2019) 110711

S. Park, et al.

2.9. Cell culture

mixed with 25 mM H2SO4, 25 mM ferrous ammonium sulfate and 100 μM xylenol orange in a volume of 1 ml and incubated for 30 min in the dark at room temperature. The absorbance was measured at 550 nm.

2.9.1. Cell culture C2C12 mouse muscle cells (ATCC, Manassas, VA, USA) were cultured in DMEM containing 10% fetal bovine serum, 100 U/ml penicillin and 100 μg/ml streptomycin. The cells were grown in an atmosphere with 5% CO2 at 37 °C.

2.6. Electron microscopy Skeletal muscle samples for electron microscopy were prepared as previously described (Heo et al., 2018). The muscle tissues were observed under an H-7000 transmission electron microscope (Hitachi, Tokyo, Japan), and cross-sectional area was measured using Olympus Soft Imaging Solution.

2.9.2. Cell viability and ROS levels The MTT assay using thiazolyl blue tetrazolium (Sigma-Aldrich) was carried out to measure cell viability as described elsewhere (Kim et al., 2010). C2C12 cells were plated in 96-well plates at a seeding density of 1 × 104. H2O2 or Beta-L was added into each well, and the MTT assay was then carried out. To measure ROS levels, flow cytometry was used as described previously (Kim et al., 2010). Cells were plated in 12-well plates at a seeding density of 1.6 × 105, then 30 μM carboxy-H2DCFDA (Invitrogen; Carlsbad, CA, USA) was added, and the plates were incubated for 30 min at 37 °C. After harvesting the cells, flow cytometry was performed on a BD Accuri C6 Plus (BD Biosciences; Franklin Lakes, NJ, USA).

2.7. Western blotting Antibodies against AKT, phosphorylated AKT (pAKT), mTOR, pmTOR, p70S6K, pp70S6K, 4EBP1, p4EBP1, FOXO1, pFOXO1, FOXO3, pFOXO3, LC3, AMPK, and pAMPK were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies targeting atrogin1, ubiquitin, NQO1, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), Smad2/3, and SIRT1 were purchased from Santa Cruz Biotechnology (Dallas, Texas, USA). Antibodies against malondialdehyde (MDA), MuRF1, TGF-β, and LAMP1 were obtained from Abcam (Cambridge, UK). Antibodies against GPX1 and nitrotyrosine were obtained from Young-In Frontier (Geumcheon-gu, Seoul, Korea), and an antibody directed against HO-1 was purchased from ENZO Life Sciences (Farmingdale, NY, USA). Muscle samples (25 mg) were homogenized in lysis buffer containing 150 mM NaCl, 50 mM HEPES, 50 mM NaF, 1 mM benzamide, 1 mM EGTA, 1 mM EDTA, 1 mM dithiothreitol (DTT), 1 mM sodium ortho-vanadate (Na3VO4), 1 mM phenylmethylsulfonyl fluoride (PMSF), 1% NP40, 10% glycerol, 0.22% β-glycerophosphate, and a protease inhibitor cocktail (Santa Cruz Biotechnology). Protein samples were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). The resolved proteins were then transferred to 0.45 μm polyvinylidene fluoride (PVDF; Millipore) membranes. The membranes were blocked with 5% skim milk in Tris buffered saline with tween 20 (TBST) (150 mM NaCl, 10 mM Tris-HCl (pH 7.4) and 0.1% Tween-20) and were subsequently incubated overnight with primary antibodies at 4 °C. However, this was not the case for the GAPDH antibody, where membranes were incubated for 1 h at room temperature. Specific primary antibody binding was detected using sheep antirabbit IgG secondary antibody conjugated to horseradish peroxidase or goat anti-mouse IgG secondary antibody conjugated to horseradish peroxidase (Bio-Rad, Hercules, California, USA) for 1 h at room temperature. After the addition of a chemiluminescence detection reagent (Millipore, Burlington, Massachusetts, USA), the signals were recorded using an LAS-4000 image analyzer and quantified using Multi Gauge 3.0 software (Fujifilm, Minato-ku, Tokyo, Japan). GAPDH was used as the loading control since levels of GAPDH were not significantly different among the experimental groups.

2.10. Statistical analyses Prism version 7 (Graph Pad Software; La Jolla, CA, USA) was utilized for all statistical analyses. The results are expressed as the means ± standard error (SE). Differences between two groups were assessed via Student's t-test. Statistical analyses of three or more groups were assessed via one-way or two-way analysis of variance (ANOVA), followed by Tukey's HSD test. P values below 0.05 were deemed statistically significant. 3. Results 3.1. One-leg immobilization induces atrophy without hypertrophy of the contralateral leg Immobilization of the left hindlimb for two weeks reduced the gastrocnemius muscle weight of the left leg compared with that of the contralateral right leg (0.142 ± 0.003 g vs. 0.123 ± 0.003 g in the right and left legs, respectively; p < 0.05). No significant difference was observed in the gastrocnemius muscle weight between the right legs of immobilized mice and non-immobilized mice (Fig. 1). These results suggest that one-leg immobilization induces muscle atrophy without significant hypertrophy of the contralateral leg and is a suitable animal model for skeletal muscle atrophy.

*

**

2.8. Quantitative real-time PCR (qRT-PCR) Total RNA was extracted from 25-mg tissue samples using TRIzol (Invitrogen, Carlsbad, California, USA). qRT-PCR was conducted using the Real-time PCR 7500 System and Power SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA) as previously described (Cha et al., 2019). The expression levels of ribosomal protein lateral stalk subunit P0 (36B4) were used for sample normalization. Each reaction mixture was incubated at 95 °C for 10 min followed by 45 cycles of 95 °C for 15 s, 55 °C for 20 s, and 72 °C for 35 s. The primer sequences were as follows: 36B4 (72-bp PCR product: forward, 5′-CACTGGTCTA GGACCCGAGAA-3′; reverse, 5′-GGTGCCTCTGGAGATTTTCG-3′), Nrf2 (199-bp product: forward, 5′-TTCTTTCAGCAGCATCCTCTCCAC-3′; reverse, 5′-ACAGCCTTCAATAGTCCCGTCCAG-3′).

Fig. 1. One-leg immobilization induces muscle atrophy without contralateral hypertrophy. Data are expressed as the means ± SE (n = 5 per group). Statistical analyses were performed using two-way ANOVA, followed by Tukey's HSD test. *p < 0.05 and **p < 0.01. 3

Experimental Gerontology 126 (2019) 110711

S. Park, et al.

A

B

C ***

0.1% SLS Beta-L

** *** **** ***

D

** **** p=0.08

Control Immob

Control Immob

Control Immob

Control Immob

Gastrocnemius

Tibialis Anterior

EDL

Soleus

Beta-L

0.1% SLS

**

Control Immobilization

* ****

* 2 m

2 m

2 m

2 m

Fig. 2. Beta-L attenuates the reduction in muscle mass induced by immobilization in mice. (A) Body weight. (B) Food intake. (C) Muscle weight. (D) Electron microscope images of myofibrils in red gastrocnemius muscle (×10,000) and cross-sectional area of myofibrils. Data are expressed as the means ± SE for 5–13 experimental cases. Statistical analyses were performed using Student's t-test in A and B and two-way ANOVA, followed by Tukey's HSD test in C and D. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. Beta-L, beta-lapachone; EDL, extensor digitorum longus; Immob, immobilization; SLS, sodium lauryl sulfate.

significantly attenuated the reduction in muscle weight of gastrocnemius (0.132 ± 0.003 g vs. 0.115 ± 0.003 g in Beta-L and SLS, respectively, p < 0.01) and tibialis anterior (0.043 ± 0.001 vs. 0.027 ± 0.002 in Beta-L and SLS, respectively, p < 0.001). Muscle weights of the extensor digitorum longus (p = 0.18) and soleus (p = 0.08) were also increased in Beta-L treatment without statistical significance (Fig. 2C). The ultracellular structure of skeletal muscle

3.2. Beta-L attenuates the immobilization-induced reduction in muscle mass Body weight and food intake were similar after immobilization in 0.1% SLS- and Beta-L-treated mice (Fig. 2A and B). Immobilization reduced the muscle weight of lower hindlimb muscles; gastrocnemius (p < 0.001), tibialis anterior (p < 0.0001), extensor digitorum longus (P < 0.0001), and soleus (p < 0.0001). Treatment with Beta-L 4

Experimental Gerontology 126 (2019) 110711

S. Park, et al.

B

A

** *

*

****

*

Immobilization

Fig. 3. Beta-L improves skeletal muscle function. (A) Treadmill exhaustion test. (B) Grip strength test. Data are expressed as the means ± SE for 4–9 experimental animals per group. Statistical analyses were performed using one-way ANOVA, followed by Tukey's HSD test, *p < 0.05, **p < 0.01, and ****p < 0.0001.Beta-L, beta-lapachone; SLS, sodium lauryl sulfate.

Immobilization

fibers and myofibrils was examined under an electron microscope and Beta-L did not cause any significant changes in cellular structure (data not shown). Cress-sectional area of myofibrils is smaller in atrophic skeletal muscle in a previous study (Priit Kaasik et al., 2012). In this study, electron microscopic analysis of muscle fibers also revealed that the cross-sectional area of myofibrils in red gastrocnemius muscle was significantly larger in Beta-L-treated mice than that in SLS-treated mice (1.3 ± 0.05 μm2 vs.1.0 ± 0.07 μm2 in Beta-L and SLS, respectively, p < 0.05) in the immobilization group (Fig. 2D).

(p < 0.05), respectively in SLS-treated mice following immobilization (Fig. 5B and C). As observed for oxidative stress markers, the protein levels of antioxidants, HO-1 and GPX1 were also increased by 2.2-fold (p < 0.05) and 1.4-fold (p < 0.05), respectively in immobilized mice, and beta-L treatment normalized these levels (Fig. 5D and E). The interaction between drug administration and immobilization was statistically significant in MDA and GPX1 (p = 0.0054 for MDA and p = 0.0359 for GPX1, two-way ANOVA). These results suggest that Beta-L suppresses oxidative stress induced by immobilization.

3.3. Beta-L improves skeletal muscle function reduced by immobilization

3.6. Beta-L does not alter protein synthesis pathway

Since Beta-L treatment attenuated the reduction of muscle mass by immobilization, we tested whether it also improved the loss of skeletal muscle function induced by immobilization. Treadmill exhaustion test showed that SLS-treated immobilized mice ran a significantly less distance than non-immobilized mice, but Beta-L treatment normalized the running distance (1871 ± 39 m vs. 1580 ± 106 in Beta-L and SLS, respectively; p < 0.05) (Fig. 3A). In the grip strength test, the length of time suspended on a grid wire was significantly lower in SLS-treated immobilized mice than non-immobilized mice, which was partially restored by Beta-L treatment (119 ± 21.4 s vs. 66 ± 8.1 s in Beta-L and SLS, respectively; p < 0.05). These results suggest that Beta-L treatment improves the loss of skeletal muscle function induced by immobilization in mice.

To examine whether Beta-L affects protein synthesis pathway, we assessed activities of AKT/mTOR/p70S6K/4EBP by measuring the levels of their phosphorylated forms in the gastrocnemius muscle. After two weeks of immobilization, the protein levels of pAKT, pmTOR, pp70S6K, and p4EBP did not significantly differ between SLS- and BetaL-treated mice (Fig. 6A–D). In addition, the levels of pAKT, pmTOR, pp70S6K, and p4EBP at 1, 3, and 7 h after Beta-L administration were not significantly different between SLS- and Beta-L-treated mice in the immobilization group (Fig. 6E). These data suggest that the signaling pathway for protein synthesis might not be altered by Beta-L treatment. 3.7. Beta-L suppresses protein degradation pathway Since skeletal muscle mass is determined by a balance between protein synthesis and protein degradation, we next measured the signaling pathway involved in protein degradation. The levels of pFOXO1 and pFOXO3 proteins were not significantly affected by immobilization. Moreover, they did not show a significant difference between the SLS- and Beta-L-treated mice, in either the control or immobilization groups (Fig. 7A and B). FOXO1 protein levels significantly increased following immobilization in SLS-treated mice but Beta-L treatment normalized FOXO1 protein levels (Fig. 7C). FOXO1 protein levels in Beta-L-treated mice were significantly lower than those in SLS-treated mice in the immobilization group (1.1-fold vs. 1.8-fold in Beta-L and SLS, respectively; p < 0.01). The interaction between drug administration and immobilization was statistically significant (p = 0.0139, two-way ANOVA). On the other hand, FOXO3 protein levels significantly increased by 1.7-fold following immobilization both in SLS(p < 0.05) and Beta-L-treated (p < 0.05) mice and were similar in SLS- and Beta-L-treated mice in the control and immobilization groups (Fig. 7D). TGF-β protein levels were significantly increased by immobilization in SLS-treated mice (2.1-fold; p < 0.01) but were normalized by Beta-L treatment. The protein levels of TGF-β (p < 0.001) and Smad2/3 (p < 0.05) were significantly lower in Beta-L treated mice than in SLS-treated mice in the immobilization group (Fig. 7E and F). The interaction between drug administration and immobilization for TGF-β was statistically significant (p = 0.0158, two-way ANOVA). The levels of MuRF1 and atrogin1 proteins significantly increased

3.4. Beta-L up-regulates the pAMPK and Nrf2 signaling pathways Since Beta-L improved the loss of muscle mass and function induced by immobilization, we tried to investigate its molecular mechanisms. We measured mRNA and protein levels of SIRT1 and AMPK, and antioxidant-related genes such as Nrf2, NQO1, GPX1 and HO-1 after a single injection of Beta-L in non-immobilized control mice. The levels of SIRT1 and phosphorylated AMPK proteins in gastrocnemius muscle were 1.4- and 1.7-fold higher (p < 0.05), respectively in Beta-L-treated mice than in SLS-treated mice (Fig. 4A and B). Beta-L treatment also increased Nrf2 mRNA levels (p < 0.05) and the protein levels of NQO1 (p < 0.01), GPX1 (p < 0.05) and HO-1 (p < 0.05) (Fig. 4C–F). These data suggest that Beta-L induces upregulation of antioxidant molecules. 3.5. Beta-L suppresses immobilization-induced oxidative stress To investigate the effects of Beta-L on ROS accumulation during immobilization, we measured the levels of H2O2, oxidative stress markers and antioxidant expression in SLS- and Beta-L-treated mice. Beta-L treatment significantly reduced H2O2 levels in the gastrocnemius muscle that were increased by immobilization (1.6 ± 0.16 μM vs 2.7 ± 0.44 μM in Beta-L and SLS, respectively; p < 0.05) (Fig. 5A). Beta-L treatment normalized the protein levels of nitrotyrosine and MDA which significantly increased by 2.5-fold (p < 0.01) and 1.9-fold 5

Experimental Gerontology 126 (2019) 110711

S. Park, et al.

A

B 0.1% SLS

Beta-L

SIRT1 GAPDH

C 0.1% SLS

Beta-L

*

pAMPK AMPK

*

D

*

E 0.1% SLS

Beta-L

NQO1 GAPDH

F 0.1% SLS

Beta-L

HO-1 GAPDH

0.1% SLS

Beta-L

GPX1 GAPDH

*

**

*

Fig. 4. Single administration of Beta-L intraperitoneally increases antioxidant and related molecules in the gastrocnemius muscles of mice. (A) NAD-dependent deacetylase sirtuin 1 (SIRT1) protein levels. (B) Phosphorylated AMP-activated protein kinase (AMPK) protein levels. (C) Nuclear factor erythroid-derived 2-related factor 2 (Nrf2) mRNA levels. (D) NAD(P)H:quinone acceptor oxidoreductase 1 (NQO1) protein levels. (E) Heme-oxygenase 1 (HO-1) protein levels. (F) Glutathione peroxidase 1 (GPX1) protein levels. Data are expressed as the means ± SE for 6 experimental animals per group. Statistical analyses were performed using Student's ttest. *p < 0.05 and **p < 0.01. Beta-L, beta-lapachone; SLS, sodium lauryl sulfate.

2, 2.5, 5 and 10 μM (p < 0.0001) after incubation for 4 h (Fig. 9A). Cell viability was not significantly affected by 1 μM Beta-L after incubation for 1, 4 and 6 h but was significantly reduced at 24 h (p < 0.05) (Fig. 9B). To identify the maximum tolerated dosage and time for H2O2 treatment, cells were treated with varying doses of H2O2 for the indicated times. H2O2 did not significantly affect cell viability at all doses after incubation for 1 h (Fig. 9C). While H2O2 at 100 μM and 150 μM did not significantly reduce cell viability, H2O2 at 300 μM (p < 0.01) and 500 μM (p < 0.0001) reduced cell viability after incubation for 4 h. H2O2 at 100 μM reduced cell viability at 6 h (p < 0.05) and 24 h (p < 0.0001). Therefore, cells were treated with 150 μM H2O2 and 1 μM Beta-L for 4 h to assess ROS levels in this experiment. Beta-L at 1 μM did not increase ROS levels. H2O2 at 150 μM increased ROS levels by 2-fold (p < 0.0001) but 1 μM Beta-L suppressed ROS accumulation (p < 0.0001) (Fig. 9D). The interaction between drug administration and H2O2 for ROS levels was statistically significant (p < 0.0001, twoway ANOVA). An AMPK inhibitor, Compound C at 2.5 μM (p < 0.001) or an Nrf2 inhibitor, ML385 at 5 μM (p < 0.001) inhibited the suppression of ROS accumulation by Beta-L. These results suggest that AMPK and Nrf2 are involved in the suppression of oxidative stress by Beta-L.

by 1.6-fold (p < 0.01) and 1.3-fold (p < 0.05), respectively following immobilization, but Beta-L treatment normalized their expression (Fig. 8A and B). The levels of MuRF1 (p < 0.05) and atrogin1 (p < 0.01) proteins were significantly lower in Beta-L-treated mice than in SLS-treated mice in the immobilization group. The interactions between drug administration and immobilization were statistically significant for MuRF1 and atrogin1 (p = 0.0028 for MuRF1 and p = 0.0102 for atrogin1, two-way ANOVA). The levels of ubiquitinated proteins were increased by immobilization in SLS-treated mice (1.8fold; p < 0.0001), whereas they were not significantly affected by Beta-L treatment (Fig. 8C). The levels of ubiquitinated proteins were significantly lower in Beta-L-treated mice (p < 0.001) than those in SLS-treated mice in the immobilization group (p = 0.0074 for the interaction between drug administration and immobilization, two-way ANOVA). The levels of LAMP1 and LC3 II proteins significantly increased 4-fold (p < 0.001) and 2.6-fold (p < 0.0001), respectively following immobilization, but Beta-L treatment normalized their levels (Fig. 8D and E). The levels of LAMP1 (p < 0.05) and LC3 II (p < 0.001) proteins were significantly lower in Beta-L-treated mice than in SLS-treated mice. The interaction effect between drug administration and immobilization was statistically significant for LAMP1 and LC3 II (p = 0.0168 and p = 0.0010, respectively, two-way ANOVA). These data suggest that Beta-L suppressed protein degradation induced by immobilization.

4. Discussion In the present study, we demonstrated that immobilization of hindlimb for two weeks increases ROS accumulation and oxidative stress, which results in loss of muscle weight and function in mice. Beta-L treatment normalizes ROS levels, leading to suppression of oxidative stress, and thus attenuates skeletal muscle atrophy and improves muscle function. The findings of this study suggest that Beta-L could be a potential therapeutic agent against skeletal muscle atrophy. Increased ROS production and/or reduced anti-oxidant capacity

3.8. Beta-L suppresses ROS accumulation via pAMPK and Nrf2 in C2C12 cells In addition to animal models, we tested the effects of Beta-L in the mouse myoblast C2C12 cell line. The effect of Beta-L on C2C12 cell viability was measured as a function of dosage and time. Beta-L did not affect cell viability at 0.2, 0.5 and 1 μM but reduced cell viability at 1.5, 6

Experimental Gerontology 126 (2019) 110711

S. Park, et al.

A

B

Control Beta-L

0.1% SLS

**

Immobilization 0.1% SLS

Beta-L

Nitrotyrosine GAPDH

*

**

**

C ***

*

Control

Immobilization

Beta-L

0.1% SLS

0.1% SLS

Beta-L

MDA

GAPDH

D

Control 0.1% SLS

Beta-L

Immobilization 0.1% SLS

E

Control

Beta-L

0.1% SLS

Beta-L

Immobilization 0.1% SLS

Beta-L

GPX1 GAPDH

HO-1 GAPDH

*

*

*

***

Fig. 5. Beta-L reduces the levels of H2O2, oxidative stress markers and antioxidant expression in the gastrocnemius muscles of immobilized mice. (A) H2O2. (B) Nitrotyrosine levels. (C) Malondialdehyde (MDA) levels. (D) Heme-oxygenase 1 (HO-1) levels. (E) Glutathione peroxidase 1 (GPX1) levels. Data are expressed as the means ± SE for 9–13 experimental animals per group. Statistical analyses were performed using two-way ANOVA, followed by Tukey's HSD test. *p < 0.05, **p < 0.01, and ***p < 0.001. Beta-L, beta-lapachone; SLS, sodium lauryl sulfate.

immobilization for two weeks in mice increased H2O2 accumulation and the levels of nitrotyrosine and MDA, suggesting that immobilization increases oxidative stress. It appears that antioxidant enzyme levels increase to suppress oxidative stress in immobilized legs. However, Beta-L treatment normalized the levels of H2O2 and oxidative stress markers. Therefore, it is reasonable to believe that Beta-L attenuates skeletal muscle atrophy by suppressing oxidative stress. Previously, one-leg immobilization for two weeks in healthy young men was shown to increases H2O2 production by 1.7-fold in the mitochondria (Gram

result in oxidative stress which leads to skeletal muscle atrophy. Sources of ROS production in skeletal muscle undergoing atrophy include mitochondria, NADPH oxidase, and xanthine oxidase. Among these, mitochondria are a major contributor (Powers et al., 2016). Mitochondrial dysfunction induced by skeletal muscle inactivity is closely associated with increased ROS production (Diaz et al., 2012; Gram et al., 2015). Reduction of ROS by antioxidant treatment attenuates immobilization-induced skeletal muscle atrophy (Talbert et al., 2013). In this study, we observed that reduced weight loading by 7

Experimental Gerontology 126 (2019) 110711

S. Park, et al.

A

Control 0.1% SLS

Beta-L

B

Immobilization 0.1% SLS

pAKT AKT

C

Control 0.1% SLS

Beta-L

Beta-L

Immobilization 0.1% SLS

Beta-L

pmTOR mTOR

Control 0.1% SLS

Beta-L

D

Immobilization 0.1% SLS

Control

Beta-L

0.1% SLS

pp70S6K p70S6K

Beta-L

Immobilization 0.1% SLS

Beta-L

p4EBP 4EBP

E

1h

3h

7h

Fig. 6. Beta-L does not alter protein synthesis signaling pathways in the gastrocnemius muscles of immobilized mice. Phosphorylated levels of (A) AKT, (B) mTOR, (C) p70S6K, and (D) 4EBP. (E) Phosphorylated levels of AKT, mTOR, p70S6K and 4EBP at 1, 3, and 7 h after the last Beta-L administration. Statistical analysis was performed using two-way ANOVA in A-D, followed by Tukey's HSD test and Student's t-test in E. Data are expressed as the means ± SE for 4–9 experimental animals per group. Beta-L, beta-lapachone; SLS, sodium lauryl sulfate.

beneficial effect of Beta-L on muscle atrophy. Although we did not measure the source of H2O2 in this study, mitochondria could be a major source of ROS production (Powers et al., 2016). Previously, it was recognized that Beta-L has dual functions in redox biology; a pro-oxidant function involving the futile hydroquinone-quinone cycle and an inhibitory effect on oxidative stress by activating

et al., 2015), which is similar to our observation that H2O2 levels in immobilized muscle were 1.9-fold higher than levels in control muscle. Exercise for 6 weeks normalized H2O2 levels following immobilization in humans (Gram et al., 2015), indicating that Beta-L could be an effective therapeutic agent for skeletal muscle atrophy. Improved muscle functions by Beta-L treatment in the immobilized mice also support this 8

Experimental Gerontology 126 (2019) 110711

S. Park, et al.

A

Control 0.1% SLS

Beta-L

B

Immobilization 0.1% SLS

Control 0.1% SLS

D

Immobilization

Beta-L

0.1% SLS

Beta-L

Control 0.1% SLS

FOXO1 GAPDH

0.1% SLS

Beta-L

Immobilization

Beta-L

0.1% SLS

Beta-L

FOXO3 GAPDH

**

E

Immobilization

Beta-L

pFOXO3 FOXO3

pFOXO1 FOXO1

C

Control 0.1% SLS

Beta-L

Control 0.1% SLS

Beta-L

**

*

F

Immobilization 0.1% SLS

Beta-L

Control 0.1% SLS

Beta-L

*

Immobilization 0.1% SLS

Beta-L

Smad2/3 GAPDH

TGFβ GAPDH

**

***

**

*

Fig. 7. Beta-L suppresses the protein levels of FOXO1 and TGF-β in the gastrocnemius muscles of immobilized mice. Phosphorylated levels of (A) forkhead box O1(FOXO1) and (B) FOXO3. Protein levels of (C) FOXO1 and (D) FOXO3. (E) Tissue transforming growth factor-β (TGF-β). (F) Smad2/3. Data are expressed as the means ± SE. Experimental cases are 11–13 per group from A to D and 6–7 in E and F. Statistical analyses were performed using two-way ANOVA, followed by Tukey's HSD test. *p < 0.05, **p < 0.01, and ***p < 0.001. . Beta-L, beta-lapachone; SLS, sodium lauryl sulfate.

SIRT1/AMPK (Park et al., 2016; Lu, 2014). Increased NAD+/NADH ratios induced by Beta-L activate AMPK phosphorylation, and activated AMPK increases antioxidant abundance via Nrf2 in rat astrocytes (Park et al., 2016). Beta-L treatment suppresses oxidative stress in an experimental animal model of cisplatin-induced renal damage by activating SIRT1 (Oh et al., 2014). Similar to these previous studies, we

also observed that a single administration of Beta-L increased antioxidant levels and related proteins, including SIRT1, pAMPK, NQO-1, HO-1 and GPX1. Therefore, we assume that the increases in SIRT1/ AMPK/Nrf2 may mediate the suppressive effect of Beta-L on oxidative stress. This notion is supported by our data indicating that the inhibitory effect of Beta-L on ROS accumulation was suppressed by

9

Experimental Gerontology 126 (2019) 110711

S. Park, et al.

A

Control 0.1% SLS

Beta-L

B

Immobilization 0.1% SLS

Control

Beta-L

0.1% SLS

MuRF1 GAPDH

Beta-L

Immobilization 0.1% SLS

Beta-L

Atrogin1 GAPDH

*

**

*

****

***

**

C Control 0.1% SLS

Beta-L

Immobilization 0.1% SLS

Beta-L

Ubiquitin

GAPDH

D

Control 0.1% SLS

Beta-L

E

Immobilization 0.1% SLS

Beta-L

Beta-L

Immobilization 0.1% SLS

Beta-L

LC 3 I LC 3 II GAPDH

LAMP1 GAPDH

***

Control 0.1% SLS

****

*

***

Fig. 8. Beta-L suppresses the levels of E3 ligase, ubiquitinated protein, LAMP1 and LC3II in the gastrocnemius muscles of immobilized mice. (A) Muscle RING-finger protein-1 (MuRF1) protein levels. (B) Atrogin1 protein levels. (C) Ubiquitinated protein levels. (D) Lysosomal-associated membrane protein 1 (LAMP1) protein levels. (D) LC3II protein levels. Data are expressed as the means ± SE for 11–13 experimental animals per group. Statistical analyses were performed using two-way ANOVA, followed by Tukey's HSD test. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. Beta-L, beta-lapachone; SLS, sodium lauryl sulfate.

oxidative stress are increased. Consistent with this study, while mRNA levels of GPX1 and HO-1 are increased by short-term administration of resveratrol, they are no longer significantly enhanced by long-term administration in the liver of mice, and oxidative stress is closely correlated with this differential response (Fusser et al., 2011). FOXOs are transcription factors that play critical roles in oxidative stress and protein degradation (Webb and Brunet, 2014). Oxidative stress increases FOXOs activity and thus enhances the ubiquitin-proteasomal pathway by inducing E3 ligase expression (Zhao et al., 2011).

inhibition of the AMPK and Nrf2 in C2C12 cells. This hypothesis can be confirmed in vivo by measuring time-dependent changes in the expression of these molecules or using genetically modified knockout mice in further study. The discrepancy in the expression of antioxidant molecules between a single injection and repeated administration of Beta-L is unclear in this study. It is possible that although Beta-L can induce the expression of antioxidant molecules, repeated administration of Beta-L does not continuously induce antioxidant molecules unless ROS levels and 10

Experimental Gerontology 126 (2019) 110711

S. Park, et al.

B

A ****

*

C **** *

**** **

****

( M)

D

E ****

**** ****

***

Fig. 9. Inhibition of 5′ AMP-activated protein kinase (AMPK) and nuclear factor erythroid 2-related factor 2(Nrf2) abrogates the suppressive effect of Beta-L on reactive oxygen species (ROS) accumulation in C2C12 cells. (A) Cells were treated with increasing concentrations of Beta-L for 4 h. (B) Cells were treated with 1 μM Beta-L for the indicated times. (C) Cells were treated with increasing concentrations of H2O2 for the indicate times. Cell viability was measured with the MTT assay from A to C. (D) Cells were treated with 1 μM Beta-L and 150 μM H2O2 for 4 h. (E) Cells were pretreated with 2.5 μM Compound C (AMPK inhibitor) or 5 μM ML385 (Nrf2 inhibitor) and then treated with 1 μM Beta-L and 150 μM H2O2 for 4 h. ROS levels were measured with flow cytometry in D and E. Data are expressed as the means ± SE for 5 separate experiments. Statistical analyses were performed using one-way ANOVA in A, B, C, and E and two-way ANOVA in D, followed by Tukey's HSD test. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. Beta-L, beta-lapachone.

(Urbanek and Klotz, 2017), further studies are needed to clarify the regulation of FOXOs protein levels in immobilized skeletal muscle. Unlike FOXO1, FOXO3 protein levels were not reduced by Beta-L treatment in this study. Consistent with our study, a previous report indicated that 8 weeks of training involving resistance exercise reduced FOXO1 expression, but not FOXO3 expression, in human skeletal muscle (Leger et al., 2006). In accordance with increased FOXO1 protein levels, the levels of MuRF1, atrogin1 and ubiquitinated protein were increased in the skeletal muscles of immobilized limbs. These results suggest that increased protein degradation by the ubiquitinproteasome pathway induced skeletal muscle atrophy in this study. However, Beta-L treatment normalized E3 ligase and ubiquitinated

Since shuttling of FOXOs between the cytoplasm and nucleus is regulated by phosphorylation-dephosphorylation (Sandri et al., 2004; Waddell et al., 2008; Zhao et al., 2011), we expected to observe alterations in pFOXO1/3 levels in this study. However, pFOXO1/3 levels were not altered, either by immobilization or by Beta-L administration. Instead, FOXO1 protein levels were increased by immobilization but were normalized by Beta-L treatment. Although the mechanisms by which immobilization increases FOXO1 expression are unclear in this study, it is possible that oxidative stress enhances the expression of E2F1 and p53 transcription factors, and that these transcription factors increase the expression of FOXOs (Klotz et al., 2015). Since post-transcriptional regulation is also reported in FOXOs protein synthesis

11

Experimental Gerontology 126 (2019) 110711

S. Park, et al.

protein levels, implicating a suppressive role of Beta-L in ubiquitinproteasomal pathways, with consequent skeletal muscle atrophy. TGF-β could be an additional regulator enhancing the E3 ligase expression in this study. TGF-β is a secretary protein that mediates a variety of physiological and pathological processes, including skeletal muscle atrophy via the intracellular Smad2/3 signaling pathway (Gumucio and Mendias, 2013). We also observed that the protein levels of TGF-β and Smad2/3 were increased by immobilization and that BetaL treatment normalized these expression levels. The increased abundance of FOXO1 and ROS possibly increased expression levels of TGF-β (Ponugoti et al., 2013). FOXO1 also regulates autophagy-lysosomal pathways (Webb and Brunet, 2014). In this study, we also observed that the lysosomal protein marker LAMP1, and the autophagy marker LC3II, were higher in immobilized legs. However, Beta-L treatment suppressed such protein level elevation. These results suggest that autophagy-lysosomal pathways are also involved in the preventive effect of Beta-L in skeletal muscle atrophy. Although protein degradation is mainly regulated by oxidative stress, protein synthesis is also regulated by ROS levels (Powers et al., 2016). AKT/mTOR/p70S6K/4EBP proteins are well known for their involvement in protein synthesis signaling pathways, and oxidative stress suppresses these pathways, leading to skeletal muscle atrophy (Powers et al., 2016). Interestingly, however, many previous studies reported no changes or even upregulation of these pathways (Kim et al., 2015; MacDonald et al., 2014). We also observed that the activities of these proteins were unaltered by immobilization. These inconsistent results may be caused by time-dependent changes in the activities of these proteins. A previous study reported that AKT activity is reduced at 6 h, returned to basal levels at 1 day, and then elevated at 5 days following denervation of skeletal muscle (Argadine et al., 2011). Therefore, it is possible that the protein synthesis pathways might be reduced during the early phase of immobilization and then returned to basal levels in this study. Beta-L treatment might not cause any significant effect on the protein synthesis pathways of immobilized mice since the activity of AKT/mTOR/p70S6K/4EBP proteins was not significantly altered by Beta-L at 1, 3, and 7 h after administration. In summary, oxidative stress plays a critical role in skeletal muscle atrophy, and suppression of oxidative stress attenuates such atrophy. However, no effective therapeutic agents for skeletal muscle atrophy are currently available. Beta-L treatment suppresses ROS accumulation and oxidative stress, which results in attenuation of skeletal muscle atrophy and improvement of muscle function in this study. Drug development based on Beta-L could lead to a new effective treatment for skeletal muscle atrophy.

aggravates aging-induced insulin resistance and declines muscle strength. J. Gerontol. A Biol. Sci. Med. Sci. 74, 147–154. Cohen, S., Nathan, J.A., Goldberg, A.L., 2015. Muscle wasting in disease: molecular mechanisms and promising therapies. Nat. Rev. Drug Discov. 14, 58–74. Costa, V., Quintanilha, A., Moradas-Ferreira, P., 2007. Protein oxidation, repair mechanisms and proteolysis in Saccharomyces cerevisiae. IUBMB Life 59, 293–298. Dehoux, M.J., van Beneden, R.P., Fernandez-Celemin, L., Lause, P.L., Thissen, J.P., 2003. Induction of MafBx and Murf ubiquitin ligase mRNAs in rat skeletal muscle after LPS injection. FEBS Lett. 544, 214–217. Diaz, F., Garcia, S., Padgett, K.R., Moraes, C.T., 2012. A defect in the mitochondrial complex III, but not complex IV, triggers early ROS-dependent damage in defined brain regions. Hum. Mol. Genet. 21, 5066–5077. Dong, J., Sulik, K.K., Chen, S.Y., 2008. Nrf2-mediated transcriptional induction of antioxidant response in mouse embryos exposed to ethanol in vivo: implications for the prevention of fetal alcohol spectrum disorders. Antioxid. Redox Signal. 10, 2023–2033. Egerman, M.A., Glass, D.J., 2014. Signaling pathways controlling skeletal muscle mass. Crit. Rev. Biochem. Mol. Biol. 49, 59–68. Eskelinen, E.L., Saftig, P., 2009. Autophagy: a lysosomal degradation pathway with a central role in health and disease. Biochim. Biophys. Acta 1793, 664–673. Fusser, M., Nesse, G.J., Khobta, A., Xia, N., Li, H., Klungland, A., et al., 2011. Spontaneous mutagenesis in Csb(m/m)Ogg1(−)(/)(−) mice is attenuated by dietary resveratrol. Carcinogenesis 32, 80–85. Gan, Z., Burkart-Hartman, E.M., Han, D.H., Finck, B., Leone, T.C., Smith, E.Y., et al., 2011. The nuclear receptor PPARbeta/delta programs muscle glucose metabolism in cooperation with AMPK and MEF2. Genes Dev. 25, 2619–2630. Gang, G.T., Hwang, J.H., Kim, Y.H., Noh, J.R., Kim, K.S., Jeong, J.Y., et al., 2014. Protection of NAD(P)H:quinone oxidoreductase 1 against renal ischemia/reperfusion injury in mice. Free Radic. Biol. Med. 67, 139–149. Gay, C., Collins, J., Gebicki, J.M., 1999. Hydroperoxide assay with the ferric-xylenol orange complex. Anal. Biochem. 273, 149–155. Goldspink, D.F., Garlick, P.J., McNurlan, M.A., 1983. Protein turnover measured in vivo and in vitro in muscles undergoing compensatory growth and subsequent denervation atrophy. Biochem. J. 210, 89–98. Gomes, M.D., Lecker, S.H., Jagoe, R.T., Navon, A., Goldberg, A.L., 2001. Atrogin-1, a muscle-specific F-box protein highly expressed during muscle atrophy. Proc. Natl. Acad. Sci. U. S. A. 98, 14440–14445. Goncalves, M.D., Pistilli, E.E., Balduzzi, A., Birnbaum, M.J., Lachey, J., Khurana, T.S., et al., 2010. Akt deficiency attenuates muscle size and function but not the response to ActRIIB inhibition. PLoS One 5, e12707. Gram, M., Vigelso, A., Yokota, T., Helge, J.W., Dela, F., Hey-Mogensen, M., 2015. Skeletal muscle mitochondrial H2 O2 emission increases with immobilization and decreases after aerobic training in young and older men. J. Physiol. 593, 4011–4027. Gumucio, J.P., Mendias, C.L., 2013. Atrogin-1, MuRF-1, and sarcopenia. Endocrine 43, 12–21. Heo, J.Y., Kim, J.E., Dan, Y., Kim, Y.W., Kim, J.Y., Cho, K.H., et al., 2018. Clusterin deficiency induces lipid accumulation and tissue damage in kidney. J. Endocrinol. 237, 175–191. Hyatt, H., Deminice, R., Yoshihara, T., Powers, S.K., 2019. Mitochondrial dysfunction induces muscle atrophy during prolonged inactivity: a review of the causes and effects. Arch. Biochem. Biophys. 662, 49–60. Lilienbaum, A., 2013. Relationship between the proteasomal system and autophagy. Int J Biochem Mol Biol 4, 1–26. Lu, C.Y., 2014. beta-Lapachone ameliorates murine cisplatin nephrotoxicity: NAD(+), NQO1, and SIRT1 at the crossroads of metabolism, injury, and inflammation. Kidney Int. 85, 496–498. Kim, J.E., Song, S.E., Kim, Y.W., Kim, J.Y., Park, S.C., Park, Y.K., et al., 2010. Adiponectin inhibits palmitate-induced apoptosis through suppression of reactive oxygen species in endothelial cells: involvement of cAMP/protein kinase A and AMP-activated protein kinase. J. Endocrinol. 207, 35–44. Kim, Y.H., Hwang, J.H., Kim, K.S., Noh, J.R., Gang, G.T., Oh, W.K., et al., 2014. Enhanced activation of NAD(P)H: quinone oxidoreductase 1 attenuates spontaneous hypertension by improvement of endothelial nitric oxide synthase coupling via tumor suppressor kinase liver kinase B1/adenosine 5′-monophosphate-activated protein kinase-mediated guanosine 5′-triphosphate cyclohydrolase 1 preservation. J. Hypertens. 32, 306–317. Kim, D.S., Cha, H.N., Jo, H.J., Song, I.H., Baek, S.H., Dan, J.M., et al., 2015. TLR2 deficiency attenuates skeletal muscle atrophy in mice. Biochem. Biophys. Res. Commun. 459, 534–540. Klotz, L.O., Sanchez-Ramos, C., Prieto-Arroyo, I., Urbanek, P., Steinbrenner, H., Monsalve, M., 2015. Redox regulation of FoxO transcription factors. Redox Biol. 6, 51–72. Lee, J.M., Li, J., Johnson, D.A., Stein, T.D., Kraft, A.D., Calkins, M.J., et al., 2005. Nrf2, a multi-organ protector? FASEB J. 19, 1061–1066. Leger, B., Cartoni, R., Praz, M., Lamon, S., Deriaz, O., Crettenand, A., et al., 2006. Akt signalling through GSK-3beta, mTOR and Foxo1 is involved in human skeletal muscle hypertrophy and atrophy. J. Physiol. 576, 923–933. MacDonald, E.M., Andres-Mateos, E., Mejias, R., Simmers, J.L., Mi, R., Park, J.S., et al., 2014. Denervation atrophy is independent from Akt and mTOR activation and is not rescued by myostatin inhibition. Dis. Model. Mech. 7, 471–481. Mammucari, C., Milan, G., Romanello, V., Masiero, E., Rudolf, R., Del Piccolo, P., et al., 2007. FoxO3 controls autophagy in skeletal muscle in vivo. Cell Metab. 6, 458–471. Miller, C.J., Gounder, S.S., Kannan, S., Goutam, K., Muthusamy, V.R., Firpo, M.A., et al., 2012. Disruption of Nrf2/ARE signaling impairs antioxidant mechanisms and promotes cell degradation pathways in aged skeletal muscle. Biochim. Biophys. Acta 1822, 1038–1050.

Acknowledgements This work was supported by the Medical Research Center Program (2015R1A5A2009124) of the National Research Foundation of Korea (NRF), funded by the Ministry of Science and ICT. Declaration of competing interest The authors report no conflict of interest. References Abrigo, J., Rivera, J.C., Simon, F., Cabrera, D., Cabello-Verrugio, C., 2016. Transforming growth factor type beta (TGF-beta) requires reactive oxygen species to induce skeletal muscle atrophy. Cell. Signal. 28, 366–376. Argadine, H.M., Mantilla, C.B., Zhan, W.Z., Sieck, G.C., 2011. Intracellular signaling pathways regulating net protein balance following diaphragm muscle denervation. Am. J. Physiol. Cell Physiol. 300, C318–C327. Bae, S.K., Cha, H.N., Ju, T.J., Kim, Y.W., Kim, H.S., Kim, Y.D., et al., 2012. Deficiency of inducible nitric oxide synthase attenuates immobilization-induced skeletal muscle atrophy in mice. J. Appl. Physiol. (1985) 113, 114–123. Cha, H.N., Park, S., Dan, Y., Kim, J.R., Park, S.Y., 2019. Peroxiredoxin2 deficiency

12

Experimental Gerontology 126 (2019) 110711

S. Park, et al.

Reid, M.B., 2001. Nitric oxide, reactive oxygen species, and skeletal muscle contraction. Med. Sci. Sports Exerc. 33, 371–376. Reid, K.F., Fielding, R.A., 2012. Skeletal muscle power: a critical determinant of physical functioning in older adults. Exerc. Sport Sci. Rev. 40, 4–12. Risson, V., Mazelin, L., Roceri, M., Sanchez, H., Moncollin, V., Corneloup, C., et al., 2009. Muscle inactivation of mTOR causes metabolic and dystrophin defects leading to severe myopathy. J. Cell Biol. 187, 859–874. Salminen, A., Kaarniranta, K., Kauppinen, A., 2013. Crosstalk between oxidative stress and SIRT1: impact on the aging process. Int. J. Mol. Sci. 14, 3834–3859. Sandri, M., Sandri, C., Gilbert, A., Skurk, C., Calabria, E., Picard, A., et al., 2004. Foxo transcription factors induce the atrophy-related ubiquitin ligase atrogin-1 and cause skeletal muscle atrophy. Cell 117, 399–412. Senf, S.M., Dodd, S.L., Judge, A.R., 2010. FOXO signaling is required for disuse muscle atrophy and is directly regulated by Hsp70. Am. J. Physiol. Cell Physiol. 298, C38–C45. Talbert, E.E., Smuder, A.J., Min, K., Kwon, O.S., Szeto, H.H., Powers, S.K., 2013. Immobilization-induced activation of key proteolytic systems in skeletal muscles is prevented by a mitochondria-targeted antioxidant. J. Appl. Physiol. (1985) 115, 529–538. Urbanek, P., Klotz, L.O., 2017. Posttranscriptional regulation of FOXO expression: microRNAs and beyond. Br. J. Pharmacol. 174, 1514–1532. Waddell, D.S., Baehr, L.M., van den Brandt, J., Johnsen, S.A., Reichardt, H.M., Furlow, J.D., et al., 2008. The glucocorticoid receptor and FOXO1 synergistically activate the skeletal muscle atrophy-associated MuRF1 gene. Am. J. Physiol. Endocrinol. Metab. 295, E785–E797. Webb, A.E., Brunet, A., 2014. FOXO transcription factors: key regulators of cellular quality control. Trends Biochem. Sci. 39, 159–169. Zhao, Y., Wang, Y., Zhu, W.G., 2011. Applications of post-translational modifications of FoxO family proteins in biological functions. J. Mol. Cell Biol. 3, 276–282. Zurlo, F., Larson, K., Bogardus, C., Ravussin, E., 1990. Skeletal muscle metabolism is a major determinant of resting energy expenditure. J. Clin. Invest. 86, 1423–1427.

Oh, G.S., Kim, H.J., Choi, J.H., Shen, A., Choe, S.K., Karna, A., et al., 2014. Pharmacological activation of NQO1 increases NAD(+) levels and attenuates cisplatin-mediated acute kidney injury in mice. Kidney Int. 85, 547–560. Olmos, Y., Sanchez-Gomez, F.J., Wild, B., Garcia-Quintans, N., Cabezudo, S., Lamas, S., et al., 2013. SirT1 regulation of antioxidant genes is dependent on the formation of a FoxO3a/PGC-1alpha complex. Antioxid. Redox Signal. 19, 1507–1521. Pardee, A.B., Li, Y.Z., Li, C.J., 2002. Cancer therapy with beta-lapachone. Curr. Cancer Drug Targets 2, 227–242. Park, J.S., Lee, Y.Y., Kim, J., Seo, H., 2016. Kim HS beta-Lapachone increases phase II antioxidant enzyme expression via NQO1-AMPK/PI3K-Nrf2/ARE signaling in rat primary astrocytes. Free Radic. Biol. Med. 97, 168–178. Pink, J.J., Planchon, S.M., Tagliarino, C., Varnes, M.E., Siegel, D., Boothman, D.A., 2000. NAD(P)H:quinone oxidoreductase activity is the principal determinant of beta-lapachone cytotoxicity. J. Biol. Chem. 275, 5416–5424. Pomies, P., Blaquiere, M., Maury, J., Mercier, J., Gouzi, F., Hayot, M., 2016. Involvement of the FoxO1/MuRF1/Atrogin-1 signaling pathway in the oxidative stress-induced atrophy of cultured chronic obstructive pulmonary disease myotubes. PLoS One 11, e0160092. Ponugoti, B., Xu, F., Zhang, C., Tian, C., Pacios, S., Graves, D.T., 2013. FOXO1 promotes wound healing through the up-regulation of TGF-beta1 and prevention of oxidative stress. J. Cell Biol. 203, 327–343. Powers, S.K., Wiggs, M.P., Duarte, J.A., Zergeroglu, A.M., Demirel, H.A., 2012. Mitochondrial signaling contributes to disuse muscle atrophy. Am. J. Physiol. Endocrinol. Metab. 303, E31–E39. Powers, S.K., Morton, A.B., Ahn, B., Smuder, A.J., 2016. Redox control of skeletal muscle atrophy. Free Radic. Biol. Med. 98, 208–217. Priit Kaasik, M.U., Alev, K., Selart, A., Seene, T., 2012. Fine architectonics and protein turnover rate in myofibrils of glucocorticoid-caused myopathic rats. J. Interdiscip. Hist. 1, 6. Qiu, J., Fang, Q., Xu, T., Wu, C., Xu, L., Wang, L., et al., 2018. Mechanistic role of reactive oxygen species and therapeutic potential of antioxidants in denervation- or fastinginduced skeletal muscle atrophy. Front. Physiol. 9, 215.

13