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Mitochondrial dysfunction launches dexamethasoneinduced skeletal muscle atrophy via AMPK/FOXO3 signaling Jing Liu, Yunhua Peng, Xun Wang, Yingying Fan, Chuan Qin, Le Shi, Ying Tang, Hua Li, Jiangang Long, and Jiankang Liu Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.5b00516 • Publication Date (Web): 22 Nov 2015 Downloaded from http://pubs.acs.org on November 28, 2015

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Molecular Pharmaceutics

Mitochondrial dysfunction launches dexamethasone-induced skeletal muscle atrophy via AMPK/FOXO3 signaling Jing Liu1#, Yunhua Peng1#, Xun Wang1, Yingying Fan1, Chuan Qin 1, Le Shi1, Ying Tang1, Hua Li 1, Jiangang Long1*, Jiankang Liu1* 1 Center for Mitochondrial Biology and Medicine, The Key Laboratory of Biomedical Information Engineering of Ministry of Education, School of Life Science and Technology and Frontier Institute of Science and Technology, Xi’an Jiaotong University, Xi’an, 710049, China Running title: Mitochondrial dysfunction in dexamethasone-induced muscle atrophy # Contributed equally to this study *Correspondence should be addressed to: Jiangang Long, PhD and Jiankang Liu, PhD Center for Mitochondrial Biology and Medicine Xi’an Jiaotong University School of Life Science and Technology, Xi’an 710049, China Tel: +86-29-8266 5429 E-mail: [email protected] (J. Long), [email protected] (J. Liu)

ACS Paragon Plus Environment


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Abstract Graphic


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Abstract: Muscle atrophy occurs in several pathologic conditions such as diabetes and chronic obstructive pulmonary disease (COPD), as well as after long-term clinical administration of synthesized glucocorticoid, where increased circulating glucocorticoid accounts for the pathogenesis of muscle atrophy. Others and we previously reported mitochondrial dysfunction in muscle atrophy-related conditions, and that mitochondria-targeting nutrients efficiently prevent kinds of muscle

atrophy.

However,

whether

and

how

mitochondrial

dysfunction

involves

in

glucocorticoid-induced muscle atrophy remains unclear. Therefore, in the present study, we measured mitochondrial function in dexamethasone-induced muscle atrophy in vivo and in vitro, and found that mitochondrial respiration was compromised at 3 days after dexamethasone administration, earlier than the increases of MuRF1 and Fbx32, and dexamethasone-induced loss of mitochondrial components and key mitochondrial dynamics proteins. Furthermore, dexamethasone treatment caused intracellular ATP deprivation and robustly AMPK activation, which further activated FOXO3/Atrogenes pathway. By directly impairing mitochondrial respiration, FCCP leads to similar readouts in C2C12 myotube as dexamethasone does.

On the contrary, resveratrol, a mitochondrial nutrient, efficiently reversed

dexamethasone-induced mitochondrial dysfunction and muscle atrophy in both C2C12 myotube and mice, by improving mitochondrial function and blocking AMPK/FOXO3 signaling. These results indicate that mitochondrial dysfunction acts as a central role in dexamethasone-induced skeletal muscle atrophy, and nutrients or drugs targeting mitochondria might be beneficial in preventing or curing muscle atrophy. Keywords: Dexamethasone, Mitochondrial dysfunction, AMPK, Muscle atrophy, Resveratrol



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Abbreviations 4E-BP1: eukaryotic translation initiation factor 4E-binding protein 1 AMPK: AMP-activated protein kinase ATP: adenosine-triphosphate Atrogin-1: muscle atrophy F-box, MAFbx Drp1: dynamin-related protein 1 EGTA: ethylene glycol tetraacetic acid eIF-2B: eukaryotic initiation factor 2B FOXO1: forkhead box O 1 FOXO3a: forkhead box O 3a GSK3: glycogen synthase kinase 3 IFM: interfibrillar mitochondria IGF-1: insulin-like growth factor 1 JC-1: 5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethylbenzimidazolylcarbocyanine iodide KLF15: Kruppel-like factor 15 MAS: mitochondrial assay solution Mfn1: mitofusin 1 Mfn2: mitofusin 2 MMP: mitochondrial membrane potential MOPS: 3-[N-Morpholino]-propanesulfonic acid mTOR: mammalian target of rapamycin MuRF1: muscle RING finger 1 NMR: nuclear magnetic resonance OPA1: optic atrophy 1 PCr: phosphocreatine PGC-1: peroxisome proliferator-activated receptor-gamma coactivator 1 REDD1: regulated in development and DNA damage responses 1 ROS: reactive oxygen species S6K1: p70 ribosomal protein S6 kinase 1 SSM: subsarcolemmal mitochondria TA: tibia anterior TBST: Tris-buffered saline tween-20 TFAM: transcription factor A, mitochondria VDAC1: voltage-dependent anion channel 1


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Introduction In response to several stress circumstances, including fasting, sepsis, cachexia, and diabetes, the stress hormone glucocorticoid causes atrophy in skeletal muscle and respiratory muscle 1-5. Meanwhile, the synthesized glucocorticoid dexamethasone and its derivatives are widely applied to treat inflammation; however, the adverse effect of those glucocorticoids limits their clinical use. Last decades witnessed a large progress in understanding the cellular and molecular mechanisms of skeletal muscle atrophy caused by the catabolic actions of glucocorticoid, which helps to apply its anti-inflammatory effect while minimize its side effect. Same as in other muscle atrophy situations, both repressed protein synthesis and increased protein 6

proteolysis contribute to glucocorticoid-induced muscle atrophy

. The inhibitory effect of

glucocorticoid on protein synthesis results from 1) blocking the transport of amino acids into the muscle 7; 2) inhibiting the stimulatory action of insulin-like growth factor 1 (IGF-1) and amino acids on mammalian target of rapamycin (mTOR) by induction of regulated in development and DNA damage responses 1 (REDD1) and Kruppel-like factor 15 (KLF15) 8, and finally leading to weakened activity of eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) protein S6 kinase 1 (S6K1) decreasing β-catenin

11

10

9

and p70 ribosomal

; 3) stimulating glycogen synthase kinase 3 (GSK3), and subsequently

as well as eukaryotic initiation factor 2B (eIF-2B)

12

. The effect of

glucocorticoid on protein breakdown mainly bases on stimulating the transcription factors Forkhead box O 1 (FOXO1), Forkhead box O 3a (FOXO3a)

13, 14

, and GSK3β

15, 16

, which are associated with

increased muscle proteolysis through the ubiquitin proteasome system and autophagy

6, 17, 18

. The two

muscle atrophy related E3 ubiquitin ligases, muscle atrophy F-box (MAFbx/Atrogin-1) and muscle RING finger 1 (MuRF1), are also remarkably increased in the glucocorticoids-induced muscle atrophy 19, 20

. The pivotal role of mitochondrial dysfunction in the pathology of muscle atrophy is well

investigated in many muscle atrophy models, including disuse, diabetes, and aging

21-26

. Our previous

study showed that mitochondrial dysfunction, characterized by mitochondrial loss and sedentary dynamics, plays a key role in disuse-induced skeletal muscle atrophy nutrients

27, 28

24

. In addition, mitochondria

, which we defined improve mitochondria function by either eliminating reactive oxygen

species (ROS), or promoting mitochondrial biogenesis, or improving mitochondrial respiration function, efficiently protects rat from unloading-induced muscle atrophy 25. Given that glucocorticoid do not account for disuse or denervation induced atrophy

29, 30

, it is needed to identify whether

mitochondrial dysfunction also roots in glucocorticoids-induced muscle atrophy and whether ameliorating mitochondrial dysfunction or improving mitochondrial respiration helps to avoid glucocorticoid-induced muscle atrophy. Previous clinical and animal studies showed that chronic corticosteroid treatment disrupts mitochondrial morphology and oxidative capacity subsequently caused DNA oxidative damage

32

31-33

, which

. However, it is still controversial about the role of

mitochondrial dysfunction in glucocorticoid-induced muscle atrophy

34

. In the present study, we

determined whether dexamethasone induces mitochondrial dysfunction, and subsequently activates atrophy signaling. Furthermore, dexamethasone-treated mice were also administrated with resveratrol, a mitochondrial nutrient

35, 36

, to determine whether improving mitochondrial function could prevent

muscle atrophy. Methods Animal



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Experiment 1. Fourteen eight-week-old male C57 BL/J (21.4±0.19 g) mice were randomly divided into 2 groups (n=7 per group). Mice were subcutaneous injected with 5 mg/kg/day dexamethasone (Sigma Aldrich, St. Louis, MO, USA) or vehicle (0.9% saline) for 18 days to induce muscle atrophy. Experiment 2. Fifteen male mice were randomly divided into 5 groups (n=3 per group). Mice were subcutaneous injected with 5 mg/kg/day dexamethasone (Sigma Aldrich, St. Louis, MO, USA) for 0, 3, 6, 12, or 18 days. Experiment 3. Thirty male mice were randomly divided into 4 groups, namely control group (n=9, injected with vehicle), dexamethasone group (n=9, injected with 5 mg/kg/day dexamethasone), dexamethasone + 50 mg/kg/day resveratrol group (n=5, injected with 5 mg/kg/day dexamethasone, and gavage with 50 mg/kg/day of resveratrol), dexamethasone + 250 mg/kg/day resveratrol group (n=7, injected with 5 mg/kg/day dexamethasone, and gavage with 250mg/kg/day of resveratrol). The duration of dexamethasone treatment was 18 days and the gavage of resveratrol began 3 days before the injection of dexamethasone, and sustained for further 18 days when injecting dexamethasone. All the mice were housed at 22-28 °C and got access to food and water throughout the experiments. Body weight was recorded every day. All experimental procedures followed the principles of laboratory animal care, and the animal protocol was approved by the animal ethics committee of the School of Life Science, Xi’an Jiaotong University. Isolation of subsarcolemmal mitochondria (SSM) and interfibrillar mitochondria (IFM) After overnight fasting, mice were sacrificed and both lateral tibia anterior (TA) muscles were immediately dissected and weighed. Then, one lateral muscle was put in liquid nitrogen and then stored at -80 °C. The other lateral TA muscle was dissected, weighed, and immediately subjected to mitochondrial isolation as previously reported 37. Briefly, after removing connective tissue, TA muscle was directly mixed with 5 X volume of buffer A (100 mM KCl, 5 mM MgSO4, 1 mM adenosine-triphosphate (ATP), 1 mM ethylene glycol tetraacetic acid (EGTA), 50 mM 3-[N-Morpholino]-propanesulfonic acid (MOPS), pH 7.4), and homogenized with potter-elvehjem homogenizer for 5 passes. The homogenates were centrifuged for 10 min at 800 g. The pellet was washed with another 5 X volume of buffer A and centrifuged for 10 min at 800 g again. The two parts of supernatants were combined together and centrifuged for 10 min at 8,700 g, and the pellet of SSM was finally dissolved in 100 µl of buffer B (0.04% bovine serum albumin in buffer A). The pellet from the second centrifugation was resuspended with 8 X volume of buffer A supplemented with 1 mg/g nagarase (Sigma Aldrich) and incubated at 4 °C for 5 min, and then added with another 8 X volume of buffer A and immediately centrifuged for 10 min at 800 g. The supernatant was centrifuged for 10 min at 8,700 g, resulting in pellet of IFM that was further dissolved in 100 µl of buffer B. The concentration of mitochondrial protein was determined with a BCA Protein Assay kit (Pierce, Rockford, IL, USA) and the protein yields of SSM and IFM were calculated accordingly. Seahorse analysis for mitochondrial respiration The basal respiration of the two mitochondrial subpopulations was measured using the Seahorse XFe24 Extracellular Flux Analyzers (Seahorse Bioscience, Billerica, MA, USA), as described previously 38. Briefly, isolated SSM and IFM were concentrated by centrifuged at 10,000 g for 15 min, and the resuspended in a small amount of seahorse assay buffer. Then, 10 µg mitochondria (3 to 6 µl) were loaded at the center of the XF24 cell culture microplates (Seahorse Bioscience) on ice, and 50 µl of the substrates (5 mM pyruvate plus 5 mM malate) and 440 µl of mitochondrial assay solution (MAS, 70 mM sucrose, 220 mM mannitol, 5 mM KH2PO4, 5 mM MgCl2, 2 mM HEPES, 1 mM EGTA, and


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0.2% BSA, pH 7.4) were carefully added on the top. All the chemicals loaded in the Seahorse cartridge ports were diluted in MAS ( pH = 7.4)

39

. For C2C12 myotube, cells were seeded in XF 24-well

microplates (Seahorse Bioscience, Billerica, MA, USA), allowed for differentiation for 4 days, and then

the

oxygen

consumption

rate

(OCR)

was

measured

by

the

Seahorse

XFe24

Extracellular Flux Analyzers. The final concentrations of mitochondrial inhibitors were at 4 µM antimycin A, 4 µM FCCP, and 4 µM oligomycin. C2C12 culture and differentiation Mouse C2C12 myoblast was purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA) and maintained in growth media, namely Dulbecco's modified Eagle's medium with 10% fetal bovine serum (Invitrogen, Carlsbad, CA, USA). To initiate differentiation, the cells were grown to 100% confluence and replaced growth medium with differentiation media, namely Dulbecco's modified Eagle's medium containing 2% heat-inactivated horse serum (Invitrogen, Carlsbad, CA, USA). The differentiation lasted for 4 days and media was refreshed every day. The myotubes were incubated with 1 µM dexamethasone, 1 µM dexamethasone together with 10 µM compund C, 1 µM dexamethasone together with 10 µM of resveratrol, or 10 µM FCCP for indicated time before measurement. Cell experiments were all replicated 3 times. Intracellular ATP level After treatment with 1 µM of dexamethasone for indicated time, C2C12 myoblast was lysed using ATP lysis buffer (0.5% Triton X-100, 100 mmol/l glycine, pH 7.4) and then centrifuged for 10 min at 15,000 g. The supernatant was collected and subjected to intracellular ATP level measurement by using the ATP bioluminescent assay kit (Sigma, St. Louis, MO, USA) according to the manufacturer’s instruction. Western blotting Muscle samples and cells were lysed with Western and IP lysis buffer (Beyotime, Jiangsu, China). The lysates were homogenized and the homogenates were centrifuged at 13,000 g for 15 min at 4 °C. The supernatants were collected and the protein concentrations were determined with a Pierce BCA Protein Assay kit (Thermo Fisher Scientific, Rockford, IL, USA). Equal aliquots of samples were loaded onto 10% SDS-PAGE gels, transferred to pure nitrocellulose membranes (PerkinElmer Life Sciences, Boston, MA, USA), and blocked with 5% nonfat milk in tris-buffered saline tween-20 (TBST) buffer. The membranes were incubated with anti-mitofusin 1 (Mfn1, 1:1000; Santa Cruz Biotechnology), anti-mitofusin 2 (Mfn2, 1:1000; Santa Cruz Biotechnology), anti-optic atrophy 1 (OPA1, 1:1000; BD Biosciences, San Diego, CA, USA), anti-peroxisome proliferator-activated receptor-gamma coactivator 1 (PGC-1, 1:500; Santa Cruz Biotechnology), anti- transcription factor A, mitochondria (TFAM, 1:1000; Santa Cruz Biotechnology), anti-voltage-dependent anion channel 1 (VDAC1; 1:1000; Santa Cruz Biotechnology), Myosin heavy chain (MYH, 1:1000; Santa Cruz Biotechnology), anti-FOXO3 (1:1000; Cell Signaling Technology), anti-p-AMPK (1:1000; Cell Signaling Technology), anti-AMPK (1:1000; Cell Signaling Technology), anti-dynamin-related protein 1 (Drp1; 1:2000; BD Biosciences=) or anti-complex I, II, III, IV, or V (1:10,000; Invitrogen) at 4 °C overnight. Then, the membranes were incubated with anti-goat, anti-mouse, or anti-rabbit secondary antibodies conjugated with HRP at room temperature for 1 h. Chemiluminescence detection was performed using an ECL Western blotting detection kit (Pierce, Rockford, IL, USA). The density for Western blot band was analyzed using Quantity One software with Actin or GAPDH as internal control, and then the band density was normalized to vehicle control. Extraction of RNA and real-time PCR



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Page 8 of 25

Total RNA was extracted using TRIzol reagent according to the manufacturer's protocol. One microgram of RNA was reverse transcribed into cDNA. Quantitative PCR was performed using a real-time PCR system (Eppendorf, Hamburg, Germany). The reactions of quantitative PCR were performed with SYBR green master mix (TaKaRa, Dalian, China) using gene-speciﬁc primers. The primers were as follows: Atrogin-1, 5’-CTGGCAGCAGCAGCTGAATAG-3’ (forward) and 5’CACATGCAGGTCTGGGGCTGC-3’ (reverse); (forward)

and

MuRF1,

5’-TGGAAACGCTATGGAGAACC-3’

5’-ATTCGCAGCCTGGAAGATG-3’

5’-AGGCCCAGAGCAAGAGAGGTA-3’ (forward)

and

(reverse);

Actin,

5’-CCATGTCGTCCCAGTTGGTAA-3’

(reverse). The mRNA levels were normalized to the mRNA of Actin as a housekeeping gene and were expressed as relative values using the 2−∆∆CT method. Statistical analysis All data are reported as the means ± SEM. All statistical analysis was performed using Student t-test or One-way ANOVA. In all comparisons, significance was defined as P< 0.05. Results Dexamethasone induces muscle atrophy in vivo To evaluate the efficiency of dexamethasone administration (5 mg/kg/day) for inducing muscle atrophy, body weights of the mice were recorded every day for 19 days. The significant loss of body weight began at day 12 and continued declining afterwards (Fig. 1A). Consistent with the body weight loss, dexamethasone treatment significantly reduced the total tibia anterior (TA) muscle weight and the ratio of TA muscle weight to body weight (Fig. 1B and C). Furthermore, the two muscle atrophy biomarkers, Atrogin-1 and MuRF-1 were robustly induced in the TA muscle of dexamethasone-treated mice (Fig.1D). Dexamethasone induces mitochondrial dysfunction, characterized by mitochondrial loss, compromised respiration, and dynamics disorder To determine whether dexamethasone induces mitochondrial dysfunction, we firstly determined the expressions of key subunits of mitochondrial respiratory electron transport chain complexes and mitochondrial outer membrane anion channels in TA muscle, and found that mitochondrial components, including complex II, IV, and V, as well as voltage-dependent anion-selective channel proteins 1 (VDAC1) were significantly reduced after dexamethasone administration (Fig. 2A and B). Given that mitochondrial subpopulations IFM in skeletal muscle accounts for 80 % of total mitochondrial volume and are more sensitive to apoptosis than subsarcolemmal mitochondria (SSM), we further isolated IFM and SSM, measured the mitochondrial yield and mitochondrial respiration using the Seahorse XFe24 Extracellular Flux Analyzers. Results showed that mitochondrial yield of IFM, but not SSM, was significantly lower in dexamethasone-treated mice (Fig. 2C). Consistently, the basal mitochondrial respiration ability (oxygen consumption rate, OCR) of IFM but not SSM was impaired after dexamethasone administration (Fig. 2D). Mitochondrial homeostasis is maintained by several well-organized process, namely, mitochondrial biogenesis, mitochondrial fusion, fission, and mitophagy

40

. To determine whether

dexamethasone affects mitochondrial biogenesis and dynamics, the key molecules involved in these processes were examined. Mitochondrial fusion proteins, Mfn2 and OPA1 that are respectively responsible for fusion of outer and inner mitochondrial membranes, fission protein Drp1, and mitophagy protein parkin were all downregulated in dexamethasone treated mice (Fig. 2E and F). However, PGC-1α and Tfam, which are the key molecules related to mitochondrial biogenesis, were


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not affected (Fig.2E and F). Time-dependent effect of dexamethasone on mitochondrial function and muscle atrophy signaling To determine whether mitochondrial dysfunction is a causal factor or just the secondary effect of dexamethasone administration, we further treated mice with dexamethasone in a time-dependent manner, namely 0, 3, 6, 12 and 18 days. As shown in figure 3, mitochondrial respiration that measured by Seahorse XFe24 Extracellular Flux Analyzers, was compromised as early as 3 days after dexamethasone administration, and continued declining in 6, 12, and 18 days (Fig. 3A). Whereas, the TA muscle weight and the ratio of TA muscle weight to body weight declined at 6 and 18 days after dexamethasone administration, respectively (Fig. 3B and C). Also, the levels of biomarkers of muscle atrophy were also elevated 6 days after dexamethasone administration, while the key mitochondrial components levels were lowered 12 and 18 days following treatment, which was later than the change of atrophy biomarkers (Fig. 3D). These results indicated that mitochondrial dysfunction precedes muscle atrophy signaling, and in turn further leads to mitochondrial loss. Mitochondrial dysfunction activates AMPK/FOXO3 signaling in dexamethasone-induced muscle atrophy To further explore how mitochondrial dysfunction activates the atrophic signaling in muscle, we measured the intracellular ATP level in differentiated C2C12 myotubes, and found that dexamethasone caused robustly ATP deprivation (Fig. 4A). AMPK activity is regulated by the ratio of intracellular AMP to ATP, and ATP deprivation usually leads to robust AMPK activation. Therefore, the level of activated AMPK, p-AMPK, was markedly increased in an time-dependent manner and was higher after 18 days of dexamethasone treatment, resulting in induction of transcription factor FOXO3, which has been recognized as the master regulator of muscle atrophy for its role in regulating the two E3 ligases Atrogin-1 and MuRF1 14 (Fig. 4B-D). To determine whether AMPK activation contributes to protein degradation in dexamethasone treated muscle, the differentiated C2C12 myotubes were treated with AMPK specific inhibitor compound C in the presence of dexamethasone. We found that compound C blocked dexamethasone-induced AMPK activation, FOXO3 induction, and MYH loss (Fig. 4E and F). In addition, the mRNA levels of Atrogin-1 and MuRF1 were induced by dexamethasone and then restored by co-incubation with compound C (Fig. 4G). Dexamethasone-induced muscle atrophy depends on mitochondrial dysfunction and can be ameliorated by improving mitochondrial function To confirm the direct role of mitochondrial dysfunction in dexamethasone-induced muscle atrophy, we treated differentiated C2C12 myotubes with FCCP, a mitochondrial uncoupler that deprives intracellular ATP turnover, and found that FCCP mimicked the effects of dexamethasone, namely robustly ATP deprivation (Fig. 5A), AMPK activation, FOXO3 induction, MYH loss (Fig. 5B and C), and increased mRNA expressions of Atrogin-1 and MuRF1 (Fig. 5D). We then measured mitochondrial respiration by using the Seahorse XF Extracellular Flux Analyzer in differentiated C2C12 myotubes with or without dexamethasone treatment. We found that, similar with FCCP, dexamethasone treatment also led to declines in mitochondrial basal respiration, ATP-linked oxygen consumption, and maximal respiration (Fig. 5E and F). In contrast, resveratrol, an activator of mitochondrial function, significantly reversed the dexamethasone-induced oxygen consumption rate, as indicated by the increased basal respiration and ATP -linked oxygen consumption (Fig. 5E and F). By improving mitochondrial respiratory function, resveratrol ameliorates the



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dexamethasone induced atrophy in C2C12 myotubes by reducing the mRNA levels of Atrogin-1 and MuRF1 (Fig. 5G). We further treated mice with resveratrol together with dexamethasone to see whether resveratrol is useful in preventing muscle atrophy in vivo. The mitochondrial function studies using Seahorse XF24 analyzer showed that dexamethasone administration compromised mitochondrial respiration, resulting in about 30% of reduction in basal respiration, which was completely prevented by resveratrol treatment (Fig. 6A). As a result, the decreases of muscle weight and muscle weight/ body weight after dexamethasone administration were also reversed by resveratrol treatment (Fig. 6B and C). Consistent with that, the levels of p-AMPK, FOXO3, MuRF1, and Atrogin-1 were all induced after dexamethasone administration and reduced after resveratrol treatment. These results suggest that resveratrol is efficient in preventing dexamethasone-induced muscle atrophy, mainly by inhibiting mitochondrial dysfunction and further blocking the AMPK/FOXO3/Atrogene pathway. Discussion Mitochondrial dysfunction precedes muscle atrophy signaling induced by dexamethasone Muscle atrophy is a common clinical problem associated with several chronic diseases such as diabetes and chronic obstructive pulmonary disease (COPD), and it significantly affects the quality of life and accelerates the pathological progression of patients. Growing evidence has indicated the key role of mitochondrial dysfunction in the pathology of muscle atrophy induced by disuse and diseases 21, 22, 41, 42

. We and others have previously reported that hindlimb unloading induced dominant

mitochondrial dysfunction, characterized by mitochondrial loss, compromised mitochondrial respiration, and disrupted mitochondrial distribution and morphology, and that mitochondria-targeting therapy by promoting mitochondrial biogenesis and/or improving mitochondrial respiration is efficient in preventing or treating muscle atrophy 24, 25. Previous studies showed that patients receiving corticosteroids had significantly decreased mitochondrial complex I enzyme activity and oxidative damage of mtDNA32, 43. Short-term exposure to glucocorticoid (i.e. 6 mg/kg daily over a period of 3 days for rat) improves mitochondrial biogenesis and enzymatic activity of the respiratory chain complex IV

44, 45

, whereas prolonged exposure causes

disturbance in mitochondrial structure and respiratory function, resulting in ROS generation, apoptosis, and cell death, depending on the energy requirements of the target tissue and the developmental stage of the organism 31, 33, 45-48. Chronic exposure to glucocorticoid also increases lactate production after aerobic exercise, owing to mitochondrial dysfunction, oxidative damage to the mitochondrial and nuclear DNA in skeletal muscle 43, which could be prevented by antioxidant compounds 49. Moreover, a mitochondrial E3 ubiquitin protein ligase (Mul1) has been reported to be induced by dexamethasone and other muscle wasting stimuli, and Mul1 suppression partially rescued muscle wasting 41. In our study, we found that components loss and dysfunction of interfibrillar mitochondria (IFM) occurred in dexamethasone-induced muscle atrophy, and more importantly, mitochondrial dysfunction happened 3 days after dexamethasone administration, which is earlier than the induction of atrogin-1 and MuRF1, and loss of muscle weight. By using FCCP to directly induce mitochondrial dysfunction, it leads to robustly ATP deprivation and AMPK activation, which subsequently activates FOXO3/Atrogenes pathway. These evidences suggest that, similar as in other muscle atrophy situations, mitochondrial dysfunction plays a key role in the pathogenesis of dexamethasone-induced muscle atrophy. Dexamethasone affects IFM rather than SSM


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Muscle mitochondria can be defined as two distinct subpopulations according to the mitochondrial morphologies and functions, namely IFM and SSM. In the present study, we firstly determined that several important mitochondrial enzymes and anion channel were robustly reduced in muscle by dexamethasone. Therefore, we further separated the two mitochondrial subpopulations, IFM and SSM, calculated their protein yields, and measured their respiratory functions using the XF Extracellular Flux Analyzer. After dexamethasone administration, the mitochondrial protein yield and the respiration function of individual mitochondrion were reduced in IFM but not in SSM. SSM, accounting for 20% of mitochondria within skeletal muscle, is beneath the sarcolemma and mainly provides ATP for membrane events, such as signaling transduction and transportation of ions and other substrates, as well as participating in apoptosis signaling regulation. On the other hand, accounting for 80% of total mitochondria, IFM locates between filaments and mainly produces ATP for muscle contraction

50

.

Therefore, the state III respiration is 2.3 to 2.8 fold greater in IFM than that in SSM, and the ATP production rate is 3 fold greater in IFM than that in SSM 51. We found in the present study that rather than SSM, IFM is more susceptible to dexamethasone treatment, characterized by retarded respiratory function and ATP deprivation. Some previous reports showed that the mitochondrial function in liver but not in muscle was affected by dexamethasone administration at 5 mg/kg/day for 3 days in rat 52, 53, suggesting that skeletal mitochondria are of some resistance to glucocorticoid. These labs also demonstrated that none of the respiratory complex activities, oxygen consumption, degree of coupling of oxidative phosphorylation, and the proton leakage are affected in the SSM in muscle

52, 53

. However, when applying

31

P nuclear

magnetic resonance (NMR), they found that dexamethasone administration shifted the reduced phosphocreatine (PCr): ATP ratio and induced PCr to γ-ATP flux in rat TA muscle

54

. These

controversial readouts of mitochondrial activities might be due to functional diversities in mitochondrial subpopulations, glucocorticoid type, exposure duration, and strains of animals in glucocorticoid–induced muscle atrophy. Therefore, the underlying mechanism of diverse responses from each mitochondrial subpopulation needs to be further defined. Mitochondrial loss was further exacerbated due to degradation of key proteins in mitochondrial dynamics Mitochondrial homeostasis depends on several processes, e.g. biogenesis, fusion, fission, and mitophagy, synergistically responsible for mitochondrial quality control. In response to certain metabolic demands, mitochondria constantly fuse and divide

55

. Moreover, depolarized mitochondrial

fraction is eliminated from the fusion-fission cycle, and the unrepaired one is further degraded by mitophagy. Disturbance in any of these processes leads to mitochondrial dysfunction. We have previously reported that mitochondrial biogenesis is significantly repressed in disuse-induced atrophic muscle

24

, while enforced mitochondrial biogenesis ameliorates muscle atrophy by administration of

micro-nutrient formula

25

. Blocked mitochondrial fusion as well as enforced mitochondrial fission

cause muscle atrophy, via inducing mtDNA instability and AMPK activation, respectively Mitophagy also participates in muscle atrophy regulation

22, 41, 56

22, 42

.

. Previous reports showed that in L6

myotubes, dexamethasone stimulates autophagy depending on AMPK and Drp1 in an early stage as a quality control process, while blocking mitochondrial fission by mdivi-1, an inhibitor of Drp1, disrupted dexamethasone-induced autophagy/mitophagy, resulting in atrophy program 56. In the present study, we found that at a later stage of dexamethasone treatment, the levels of key proteins in mitochondrial dynamics were reduced, which should be a secondary effect and be attributed to overall reduction of protein synthesis and elevated protein degradation.



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Resveratrol can reverse dexamethasone-induced muscle atrophy by improving mitochondrial function Mitochondria are the main organelles for energy production in cells, and disturbance of mitochondrial function should firstly lead to insufficient energy supply, and activation of several intracellular signaling pathways, including AMPK activation, autophagy, and/or apoptosis. AMPK is a key energy sensor, and it is activated immediately during intracellular ATP deprivation, and subsequently leads to mitochondrial biogenesis by phosphorylating PGC-1α or autophagy by phosphorylating

57, 58

. Previous studies illustrated that AMPK can directly regulate FOXO3 via

phosphorylation on 6 regulatory sites of FOXO3

59

, and the AMPK/FOXO3 pathway participates in

muscle atrophy induced by enforced mitochondrial fission

22

. However, it is unclear whether and how

mitochondrial dysfunction involves in AMPK/FOXO3 signaling in dexamethasone-induced muscle atrophy. Here, with both in vivo and in vitro evidences, we demonstrated that dexamethasone robustly induced component loss and respiratory dysfunction of IFM, resulting in intracellular ATP deprivation, which in turn activates AMPK /FOXO3 signaling. Furthermore, activated protein degradation in turn reduces key proteins for mitochondrial dynamics and exacerbates mitochondrial dysfunction. On the other hand, resveratrol, which has been reported efficiently in preventing muscle atrophy in several models

60, 61

, can effectively reverse the mitochondrial dysfunction and subsequently muscle atrophy

induced by dexamethasone both in vitro and in vivo. Conclusion Taken together, we found that mitochondrial dysfunction and ATP deprivation precedes muscle atrophy after dexamethasone administration, and leads to atrophy by AMPK/FOXO3/agtrogenes signaling, which in turn exacerbate mitochondrial loss (Fig. 7). And, resveratrol is efficient in preventing dexamethasone-induced muscle atrophy by improving mitochondrial respiration. This finding provides new insight for mitochondria-centered action in dexamethasone-induced muscle atrophy, and suggests a promising strategy that targeting mitochondria to improve mitochondrial function might prevent and treat dexamethasone-induced muscle atrophy. Acknowledgement The study was supported by the Major State Basic Research Development Program (2015CB856302), Opening Foundation of the State Key Laboratory of Space Medicine Fundamentals and Application, the China Astronaut Research and Training Center (Grant SMFA15K01), the Merieux Research Starting Grant, the Fundamental Research Funds for the Central Universities(08143008, 08143101), Tianjin Applied Basic and Frontier Tech Major Project (12JCZDJC34400), Tianjin higher Education Sci-tech Development Project (20112D05). We thank Dr. Dan Gao for reading the manuscript and valuable advices.

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Figure legend Figure 1. Dexamethasone induces muscle atrophy in vivo. A. During the 19 days of dexamethasone treatment, body weight losing became significent from the day 12 and kept on losing afterwards in dexamethasone group compared to the control group. B. Tibia anterior muscle weight decreased significantly in dexamethasone group compared to the control group. C. The ratio of TA muscle weight to body weight was lower in dexamethasone group than those in the control group. D. mRNA levels of Atrogin-1 and MuRF1 were higher in dexamethasone-treated mice than those in control mice. The body weight results were statistical analyzed using one-way ANOVA, and other results were statistical analyzed using Student t-test. *p

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