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Article
Mitochondrial dysfunction launches dexamethasone-
induced 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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Page 1 of 25 Molecular Pharmaceutics

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3 Mitochondrial dysfunction launches dexamethasone-induced skeletal muscle atrophy via
4 AMPK/FOXO3 signaling
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Jing Liu1#, Yunhua Peng1#, Xun Wang1, Yingying Fan1, Chuan Qin 1, Le Shi1, Ying Tang1, Hua Li 1,
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7 Jiangang Long1*, Jiankang Liu1*
8 1 Center for Mitochondrial Biology and Medicine, The Key Laboratory of Biomedical Information
9 Engineering of Ministry of Education, School of Life Science and Technology and Frontier Institute of
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11 Science and Technology, Xi’an Jiaotong University, Xi’an, 710049, China
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13 Running title: Mitochondrial dysfunction in dexamethasone-induced muscle atrophy
14 # Contributed equally to this study
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16 *Correspondence should be addressed to:
17 Jiangang Long, PhD and Jiankang Liu, PhD
18 Center for Mitochondrial Biology and Medicine
19 Xi’an Jiaotong University School of Life Science and Technology,
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21 Xi’an 710049, China
22 Tel: +86-29-8266 5429
23 E-mail: jglong@mail.xjtu.edu.cn (J. Long), j.liu@mail.xjtu.edu.cn (J. Liu)
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3 Abstract: Muscle atrophy occurs in several pathologic conditions such as diabetes and chronic
4 obstructive pulmonary disease (COPD), as well as after long-term clinical administration of
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synthesized glucocorticoid, where increased circulating glucocorticoid accounts for the pathogenesis of
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7 muscle atrophy. Others and we previously reported mitochondrial dysfunction in muscle
8 atrophy-related conditions, and that mitochondria-targeting nutrients efficiently prevent kinds of
9 muscle atrophy. However, whether and how mitochondrial dysfunction involves in
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11 glucocorticoid-induced muscle atrophy remains unclear. Therefore, in the present study, we measured
12 mitochondrial function in dexamethasone-induced muscle atrophy in vivo and in vitro, and found that
13 mitochondrial respiration was compromised at 3 days after dexamethasone administration, earlier than
14 the increases of MuRF1 and Fbx32, and dexamethasone-induced loss of mitochondrial components and
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16 key mitochondrial dynamics proteins. Furthermore, dexamethasone treatment caused intracellular ATP
17 deprivation and robustly AMPK activation, which further activated FOXO3/Atrogenes pathway. By
18 directly impairing mitochondrial respiration, FCCP leads to similar readouts in C2C12 myotube as
19 dexamethasone does. On the contrary, resveratrol, a mitochondrial nutrient, efficiently reversed
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21 dexamethasone-induced mitochondrial dysfunction and muscle atrophy in both C2C12 myotube and
22 mice, by improving mitochondrial function and blocking AMPK/FOXO3 signaling. These results
23 indicate that mitochondrial dysfunction acts as a central role in dexamethasone-induced skeletal muscle
24 atrophy, and nutrients or drugs targeting mitochondria might be beneficial in preventing or curing
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26 muscle atrophy.
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28 Keywords: Dexamethasone, Mitochondrial dysfunction, AMPK, Muscle atrophy, Resveratrol
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4 Abbreviations
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4E-BP1: eukaryotic translation initiation factor 4E-binding protein 1
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7 AMPK: AMP-activated protein kinase
8 ATP: adenosine-triphosphate
9 Atrogin-1: muscle atrophy F-box, MAFbx
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11 Drp1: dynamin-related protein 1
12 EGTA: ethylene glycol tetraacetic acid
13 eIF-2B: eukaryotic initiation factor 2B
14 FOXO1: forkhead box O 1
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16 FOXO3a: forkhead box O 3a
17 GSK3: glycogen synthase kinase 3
18 IFM: interfibrillar mitochondria
19 IGF-1: insulin-like growth factor 1
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21 JC-1: 5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethylbenzimidazolylcarbocyanine iodide
22 KLF15: Kruppel-like factor 15
23 MAS: mitochondrial assay solution
24 Mfn1: mitofusin 1
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26 Mfn2: mitofusin 2
27 MMP: mitochondrial membrane potential
28 MOPS: 3-[N-Morpholino]-propanesulfonic acid
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mTOR: mammalian target of rapamycin
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31 MuRF1: muscle RING finger 1
32 NMR: nuclear magnetic resonance
33 OPA1: optic atrophy 1
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PCr: phosphocreatine
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36 PGC-1: peroxisome proliferator-activated receptor-gamma coactivator 1
37 REDD1: regulated in development and DNA damage responses 1
38 ROS: reactive oxygen species
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S6K1: p70 ribosomal protein S6 kinase 1
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41 SSM: subsarcolemmal mitochondria
42 TA: tibia anterior
43 TBST: Tris-buffered saline tween-20
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TFAM: transcription factor A, mitochondria
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46 VDAC1: voltage-dependent anion channel 1
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3 Introduction
4 In response to several stress circumstances, including fasting, sepsis, cachexia, and diabetes, the
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stress hormone glucocorticoid causes atrophy in skeletal muscle and respiratory muscle 1-5. Meanwhile,
6
7 the synthesized glucocorticoid dexamethasone and its derivatives are widely applied to treat
8 inflammation; however, the adverse effect of those glucocorticoids limits their clinical use. Last
9 decades witnessed a large progress in understanding the cellular and molecular mechanisms of skeletal
10
11 muscle atrophy caused by the catabolic actions of glucocorticoid, which helps to apply its
12 anti-inflammatory effect while minimize its side effect.
13 Same as in other muscle atrophy situations, both repressed protein synthesis and increased protein
14 proteolysis contribute to glucocorticoid-induced muscle atrophy 6
. The inhibitory effect of
15
16 glucocorticoid on protein synthesis results from 1) blocking the transport of amino acids into the
17 muscle 7; 2) inhibiting the stimulatory action of insulin-like growth factor 1 (IGF-1) and amino acids
18 on mammalian target of rapamycin (mTOR) by induction of regulated in development and DNA
19 damage responses 1 (REDD1) and Kruppel-like factor 15 (KLF15) 8, and finally leading to weakened
20 9
21 activity of eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) and p70 ribosomal
10
22 protein S6 kinase 1 (S6K1) ; 3) stimulating glycogen synthase kinase 3 (GSK3), and subsequently
23 decreasing β-catenin 11
as well as eukaryotic initiation factor 2B (eIF-2B) 12
. The effect of
24 glucocorticoid on protein breakdown mainly bases on stimulating the transcription factors Forkhead
25 13, 14 15, 16
26 box O 1 (FOXO1), Forkhead box O 3a (FOXO3a) , and GSK3β , which are associated with
6, 17, 18
27 increased muscle proteolysis through the ubiquitin proteasome system and autophagy . The two
28 muscle atrophy related E3 ubiquitin ligases, muscle atrophy F-box (MAFbx/Atrogin-1) and muscle
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RING finger 1 (MuRF1), are also remarkably increased in the glucocorticoids-induced muscle atrophy
30 19, 20
31 .
32 The pivotal role of mitochondrial dysfunction in the pathology of muscle atrophy is well
33 investigated in many muscle atrophy models, including disuse, diabetes, and aging 21-26
. Our previous
34
study showed that mitochondrial dysfunction, characterized by mitochondrial loss and sedentary
35 24
36 dynamics, plays a key role in disuse-induced skeletal muscle atrophy . In addition, mitochondria
27, 28
37 nutrients , which we defined improve mitochondria function by either eliminating reactive oxygen
38 species (ROS), or promoting mitochondrial biogenesis, or improving mitochondrial respiration
39
function, efficiently protects rat from unloading-induced muscle atrophy 25. Given that glucocorticoid
40 29, 30
41 do not account for disuse or denervation induced atrophy , it is needed to identify whether
42 mitochondrial dysfunction also roots in glucocorticoids-induced muscle atrophy and whether
43 ameliorating mitochondrial dysfunction or improving mitochondrial respiration helps to avoid
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glucocorticoid-induced muscle atrophy. Previous clinical and animal studies showed that chronic
45 31-33
46 corticosteroid treatment disrupts mitochondrial morphology and oxidative capacity , which
32
47 subsequently caused DNA oxidative damage . However, it is still controversial about the role of
48 mitochondrial dysfunction in glucocorticoid-induced muscle atrophy 34
. In the present study, we
49
determined whether dexamethasone induces mitochondrial dysfunction, and subsequently activates
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51 atrophy signaling. Furthermore, dexamethasone-treated mice were also administrated with resveratrol,
35, 36
52 a mitochondrial nutrient , to determine whether improving mitochondrial function could prevent
53 muscle atrophy.
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56 Methods
57 Animal
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3 Experiment 1. Fourteen eight-week-old male C57 BL/J (21.4±0.19 g) mice were randomly
4 divided into 2 groups (n=7 per group). Mice were subcutaneous injected with 5 mg/kg/day
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dexamethasone (Sigma Aldrich, St. Louis, MO, USA) or vehicle (0.9% saline) for 18 days to induce
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7 muscle atrophy.
8 Experiment 2. Fifteen male mice were randomly divided into 5 groups (n=3 per group). Mice
9 were subcutaneous injected with 5 mg/kg/day dexamethasone (Sigma Aldrich, St. Louis, MO, USA)
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11 for 0, 3, 6, 12, or 18 days.
12 Experiment 3. Thirty male mice were randomly divided into 4 groups, namely control group (n=9,
13 injected with vehicle), dexamethasone group (n=9, injected with 5 mg/kg/day dexamethasone),
14 dexamethasone + 50 mg/kg/day resveratrol group (n=5, injected with 5 mg/kg/day dexamethasone, and
15
16 gavage with 50 mg/kg/day of resveratrol), dexamethasone + 250 mg/kg/day resveratrol group (n=7,
17 injected with 5 mg/kg/day dexamethasone, and gavage with 250mg/kg/day of resveratrol). The duration
18 of dexamethasone treatment was 18 days and the gavage of resveratrol began 3 days before the
19 injection of dexamethasone, and sustained for further 18 days when injecting dexamethasone.
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21 All the mice were housed at 22-28 °C and got access to food and water throughout the
22 experiments. Body weight was recorded every day. All experimental procedures followed the principles
23 of laboratory animal care, and the animal protocol was approved by the animal ethics committee of the
24 School of Life Science, Xi’an Jiaotong University.
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26 Isolation of subsarcolemmal mitochondria (SSM) and interfibrillar mitochondria (IFM)
27 After overnight fasting, mice were sacrificed and both lateral tibia anterior (TA) muscles were
28 immediately dissected and weighed. Then, one lateral muscle was put in liquid nitrogen and then stored
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at -80 °C. The other lateral TA muscle was dissected, weighed, and immediately subjected to
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31 mitochondrial isolation as previously reported 37. Briefly, after removing connective tissue, TA muscle
32 was directly mixed with 5 X volume of buffer A (100 mM KCl, 5 mM MgSO4, 1 mM
33 adenosine-triphosphate (ATP), 1 mM ethylene glycol tetraacetic acid (EGTA), 50 mM
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3-[N-Morpholino]-propanesulfonic acid (MOPS), pH 7.4), and homogenized with potter-elvehjem
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36 homogenizer for 5 passes. The homogenates were centrifuged for 10 min at 800 g. The pellet was
37 washed with another 5 X volume of buffer A and centrifuged for 10 min at 800 g again. The two parts
38 of supernatants were combined together and centrifuged for 10 min at 8,700 g, and the pellet of SSM
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was finally dissolved in 100 µl of buffer B (0.04% bovine serum albumin in buffer A). The pellet from
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41 the second centrifugation was resuspended with 8 X volume of buffer A supplemented with 1 mg/g
42 nagarase (Sigma Aldrich) and incubated at 4 °C for 5 min, and then added with another 8 X volume of
43 buffer A and immediately centrifuged for 10 min at 800 g. The supernatant was centrifuged for 10 min
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at 8,700 g, resulting in pellet of IFM that was further dissolved in 100 µl of buffer B. The concentration
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46 of mitochondrial protein was determined with a BCA Protein Assay kit (Pierce, Rockford, IL, USA)
47 and the protein yields of SSM and IFM were calculated accordingly.
48 Seahorse analysis for mitochondrial respiration
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The basal respiration of the two mitochondrial subpopulations was measured using the Seahorse
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51 XFe24 Extracellular Flux Analyzers (Seahorse Bioscience, Billerica, MA, USA), as described
52 previously 38. Briefly, isolated SSM and IFM were concentrated by centrifuged at 10,000 g for 15 min,
53 and the resuspended in a small amount of seahorse assay buffer. Then, 10 µg mitochondria (3 to 6 µl)
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were loaded at the center of the XF24 cell culture microplates (Seahorse Bioscience) on ice, and 50 µl
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56 of the substrates (5 mM pyruvate plus 5 mM malate) and 440 µl of mitochondrial assay solution (MAS,
57 70 mM sucrose, 220 mM mannitol, 5 mM KH2PO4, 5 mM MgCl2, 2 mM HEPES, 1 mM EGTA, and
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3 0.2% BSA, pH 7.4) were carefully added on the top. All the chemicals loaded in the Seahorse cartridge
4 ports were diluted in MAS ( pH = 7.4) 39
. For C2C12 myotube, cells were seeded in XF 24-well
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microplates (Seahorse Bioscience, Billerica, MA, USA), allowed for differentiation for 4 days, and
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7 then the oxygen consumption rate (OCR) was measured by the Seahorse XFe24
8 Extracellular Flux Analyzers. The final concentrations of mitochondrial inhibitors were at 4 µM
9 antimycin A, 4 µM FCCP, and 4 µM oligomycin.
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11 C2C12 culture and differentiation
12 Mouse C2C12 myoblast was purchased from the American Type Culture Collection (ATCC;
13 Manassas, VA, USA) and maintained in growth media, namely Dulbecco's modified Eagle's medium
14 with 10% fetal bovine serum (Invitrogen, Carlsbad, CA, USA). To initiate differentiation, the cells
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16 were grown to 100% confluence and replaced growth medium with differentiation media, namely
17 Dulbecco's modified Eagle's medium containing 2% heat-inactivated horse serum (Invitrogen, Carlsbad,
18 CA, USA). The differentiation lasted for 4 days and media was refreshed every day. The myotubes
19 were incubated with 1 µM dexamethasone, 1 µM dexamethasone together with 10 µM compund C, 1
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21 µM dexamethasone together with 10 µM of resveratrol, or 10 µM FCCP for indicated time before
22 measurement. Cell experiments were all replicated 3 times.
23 Intracellular ATP level
24 After treatment with 1 µM of dexamethasone for indicated time, C2C12 myoblast was lysed using
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26 ATP lysis buffer (0.5% Triton X-100, 100 mmol/l glycine, pH 7.4) and then centrifuged for 10 min at
27 15,000 g. The supernatant was collected and subjected to intracellular ATP level measurement by using
28 the ATP bioluminescent assay kit (Sigma, St. Louis, MO, USA) according to the manufacturer’s
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instruction.
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31 Western blotting
32 Muscle samples and cells were lysed with Western and IP lysis buffer (Beyotime, Jiangsu, China).
33 The lysates were homogenized and the homogenates were centrifuged at 13,000 g for 15 min at 4 °C.
34
The supernatants were collected and the protein concentrations were determined with a Pierce BCA
35
36 Protein Assay kit (Thermo Fisher Scientific, Rockford, IL, USA). Equal aliquots of samples were
37 loaded onto 10% SDS-PAGE gels, transferred to pure nitrocellulose membranes (PerkinElmer Life
38 Sciences, Boston, MA, USA), and blocked with 5% nonfat milk in tris-buffered saline tween-20 (TBST)
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buffer. The membranes were incubated with anti-mitofusin 1 (Mfn1, 1:1000; Santa Cruz
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41 Biotechnology), anti-mitofusin 2 (Mfn2, 1:1000; Santa Cruz Biotechnology), anti-optic atrophy 1
42 (OPA1, 1:1000; BD Biosciences, San Diego, CA, USA), anti-peroxisome proliferator-activated
43 receptor-gamma coactivator 1 (PGC-1, 1:500; Santa Cruz Biotechnology), anti- transcription factor A,
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mitochondria (TFAM, 1:1000; Santa Cruz Biotechnology), anti-voltage-dependent anion channel 1
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46 (VDAC1; 1:1000; Santa Cruz Biotechnology), Myosin heavy chain (MYH, 1:1000; Santa Cruz
47 Biotechnology), anti-FOXO3 (1:1000; Cell Signaling Technology), anti-p-AMPK (1:1000; Cell
48 Signaling Technology), anti-AMPK (1:1000; Cell Signaling Technology), anti-dynamin-related
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protein 1 (Drp1; 1:2000; BD Biosciences=) or anti-complex I, II, III, IV, or V (1:10,000; Invitrogen) at
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51 4 °C overnight. Then, the membranes were incubated with anti-goat, anti-mouse, or anti-rabbit
52 secondary antibodies conjugated with HRP at room temperature for 1 h. Chemiluminescence detection
53 was performed using an ECL Western blotting detection kit (Pierce, Rockford, IL, USA). The density
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for Western blot band was analyzed using Quantity One software with Actin or GAPDH as internal
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56 control, and then the band density was normalized to vehicle control.
57 Extraction of RNA and real-time PCR
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3 Total RNA was extracted using TRIzol reagent according to the manufacturer's protocol. One
4 microgram of RNA was reverse transcribed into cDNA. Quantitative PCR was performed using a
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real-time PCR system (Eppendorf, Hamburg, Germany). The reactions of quantitative PCR were
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7 performed with SYBR green master mix (TaKaRa, Dalian, China) using gene-specific primers. The
8 primers were as follows: Atrogin-1, 5’-CTGGCAGCAGCAGCTGAATAG-3’ (forward) and 5’-
9 CACATGCAGGTCTGGGGCTGC-3’ (reverse); MuRF1, 5’-TGGAAACGCTATGGAGAACC-3’
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11 (forward) and 5’-ATTCGCAGCCTGGAAGATG-3’ (reverse); Actin,
12 5’-AGGCCCAGAGCAAGAGAGGTA-3’ (forward) and 5’-CCATGTCGTCCCAGTTGGTAA-3’
13 (reverse). The mRNA levels were normalized to the mRNA of Actin as a housekeeping gene and were
14 expressed as relative values using the 2−∆∆CT method.
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16 Statistical analysis
17 All data are reported as the means ± SEM. All statistical analysis was performed using Student
18 t-test or One-way ANOVA. In all comparisons, significance was defined as P< 0.05.
19
20
21 Results
22 Dexamethasone induces muscle atrophy in vivo
23 To evaluate the efficiency of dexamethasone administration (5 mg/kg/day) for inducing muscle
24 atrophy, body weights of the mice were recorded every day for 19 days. The significant loss of body
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26 weight began at day 12 and continued declining afterwards (Fig. 1A). Consistent with the body weight
27 loss, dexamethasone treatment significantly reduced the total tibia anterior (TA) muscle weight and the
28 ratio of TA muscle weight to body weight (Fig. 1B and C). Furthermore, the two muscle atrophy
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biomarkers, Atrogin-1 and MuRF-1 were robustly induced in the TA muscle of dexamethasone-treated
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31 mice (Fig.1D).
32 Dexamethasone induces mitochondrial dysfunction, characterized by mitochondrial loss,
33 compromised respiration, and dynamics disorder
34
To determine whether dexamethasone induces mitochondrial dysfunction, we firstly determined
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36 the expressions of key subunits of mitochondrial respiratory electron transport chain complexes and
37 mitochondrial outer membrane anion channels in TA muscle, and found that mitochondrial components,
38 including complex II, IV, and V, as well as voltage-dependent anion-selective channel proteins 1
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(VDAC1) were significantly reduced after dexamethasone administration (Fig. 2A and B). Given that
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41 mitochondrial subpopulations IFM in skeletal muscle accounts for 80 % of total mitochondrial volume
42 and are more sensitive to apoptosis than subsarcolemmal mitochondria (SSM), we further isolated IFM
43 and SSM, measured the mitochondrial yield and mitochondrial respiration using the Seahorse XFe24
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Extracellular Flux Analyzers. Results showed that mitochondrial yield of IFM, but not SSM, was
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46 significantly lower in dexamethasone-treated mice (Fig. 2C). Consistently, the basal mitochondrial
47 respiration ability (oxygen consumption rate, OCR) of IFM but not SSM was impaired after
48 dexamethasone administration (Fig. 2D).
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Mitochondrial homeostasis is maintained by several well-organized process, namely,
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51 mitochondrial biogenesis, mitochondrial fusion, fission, and mitophagy . To determine whether
52 dexamethasone affects mitochondrial biogenesis and dynamics, the key molecules involved in these
53 processes were examined. Mitochondrial fusion proteins, Mfn2 and OPA1 that are respectively
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responsible for fusion of outer and inner mitochondrial membranes, fission protein Drp1, and
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56 mitophagy protein parkin were all downregulated in dexamethasone treated mice (Fig. 2E and F).
57 However, PGC-1α and Tfam, which are the key molecules related to mitochondrial biogenesis, were
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3 not affected (Fig.2E and F).
4 Time-dependent effect of dexamethasone on mitochondrial function and muscle atrophy
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6 signaling
7 To determine whether mitochondrial dysfunction is a causal factor or just the secondary effect of
8 dexamethasone administration, we further treated mice with dexamethasone in a time-dependent
9 manner, namely 0, 3, 6, 12 and 18 days. As shown in figure 3, mitochondrial respiration that measured
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11 by Seahorse XFe24 Extracellular Flux Analyzers, was compromised as early as 3 days after
12 dexamethasone administration, and continued declining in 6, 12, and 18 days (Fig. 3A). Whereas, the
13 TA muscle weight and the ratio of TA muscle weight to body weight declined at 6 and 18 days after
14 dexamethasone administration, respectively (Fig. 3B and C). Also, the levels of biomarkers of muscle
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16 atrophy were also elevated 6 days after dexamethasone administration, while the key mitochondrial
17 components levels were lowered 12 and 18 days following treatment, which was later than the change
18 of atrophy biomarkers (Fig. 3D). These results indicated that mitochondrial dysfunction precedes
19 muscle atrophy signaling, and in turn further leads to mitochondrial loss.
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21 Mitochondrial dysfunction activates AMPK/FOXO3 signaling in dexamethasone-induced muscle
22 atrophy
23 To further explore how mitochondrial dysfunction activates the atrophic signaling in muscle, we
24 measured the intracellular ATP level in differentiated C2C12 myotubes, and found that dexamethasone
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26 caused robustly ATP deprivation (Fig. 4A). AMPK activity is regulated by the ratio of intracellular
27 AMP to ATP, and ATP deprivation usually leads to robust AMPK activation. Therefore, the level of
28 activated AMPK, p-AMPK, was markedly increased in an time-dependent manner and was higher after
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18 days of dexamethasone treatment, resulting in induction of transcription factor FOXO3, which has
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31 been recognized as the master regulator of muscle atrophy for its role in regulating the two E3 ligases
32 Atrogin-1 and MuRF1 14 (Fig. 4B-D).
33 To determine whether AMPK activation contributes to protein degradation in dexamethasone
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treated muscle, the differentiated C2C12 myotubes were treated with AMPK specific inhibitor
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36 compound C in the presence of dexamethasone. We found that compound C blocked
37 dexamethasone-induced AMPK activation, FOXO3 induction, and MYH loss (Fig. 4E and F). In
38 addition, the mRNA levels of Atrogin-1 and MuRF1 were induced by dexamethasone and then restored
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by co-incubation with compound C (Fig. 4G).
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41 Dexamethasone-induced muscle atrophy depends on mitochondrial dysfunction and can be
42 ameliorated by improving mitochondrial function
43 To confirm the direct role of mitochondrial dysfunction in dexamethasone-induced muscle
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atrophy, we treated differentiated C2C12 myotubes with FCCP, a mitochondrial uncoupler that deprives
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46 intracellular ATP turnover, and found that FCCP mimicked the effects of dexamethasone, namely
47 robustly ATP deprivation (Fig. 5A), AMPK activation, FOXO3 induction, MYH loss (Fig. 5B and C),
48 and increased mRNA expressions of Atrogin-1 and MuRF1 (Fig. 5D).
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We then measured mitochondrial respiration by using the Seahorse XF Extracellular Flux
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51 Analyzer in differentiated C2C12 myotubes with or without dexamethasone treatment. We found that,
52 similar with FCCP, dexamethasone treatment also led to declines in mitochondrial basal respiration,
53 ATP-linked oxygen consumption, and maximal respiration (Fig. 5E and F). In contrast, resveratrol, an
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activator of mitochondrial function, significantly reversed the dexamethasone-induced oxygen
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56 consumption rate, as indicated by the increased basal respiration and ATP -linked oxygen consumption
57 (Fig. 5E and F). By improving mitochondrial respiratory function, resveratrol ameliorates the
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3 dexamethasone induced atrophy in C2C12 myotubes by reducing the mRNA levels of Atrogin-1 and
4 MuRF1 (Fig. 5G).
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We further treated mice with resveratrol together with dexamethasone to see whether resveratrol
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7 is useful in preventing muscle atrophy in vivo. The mitochondrial function studies using Seahorse
8 XF24 analyzer showed that dexamethasone administration compromised mitochondrial respiration,
9 resulting in about 30% of reduction in basal respiration, which was completely prevented by resveratrol
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11 treatment (Fig. 6A). As a result, the decreases of muscle weight and muscle weight/ body weight after
12 dexamethasone administration were also reversed by resveratrol treatment (Fig. 6B and C). Consistent
13 with that, the levels of p-AMPK, FOXO3, MuRF1, and Atrogin-1 were all induced after
14 dexamethasone administration and reduced after resveratrol treatment. These results suggest that
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16 resveratrol is efficient in preventing dexamethasone-induced muscle atrophy, mainly by inhibiting
17 mitochondrial dysfunction and further blocking the AMPK/FOXO3/Atrogene pathway.
18
19 Discussion
20
21 Mitochondrial dysfunction precedes muscle atrophy signaling induced by dexamethasone
22 Muscle atrophy is a common clinical problem associated with several chronic diseases such as
23 diabetes and chronic obstructive pulmonary disease (COPD), and it significantly affects the quality of
24 life and accelerates the pathological progression of patients. Growing evidence has indicated the key
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26 role of mitochondrial dysfunction in the pathology of muscle atrophy induced by disuse and diseases 21,
22, 41, 42
27 . We and others have previously reported that hindlimb unloading induced dominant
28 mitochondrial dysfunction, characterized by mitochondrial loss, compromised mitochondrial
29
respiration, and disrupted mitochondrial distribution and morphology, and that mitochondria-targeting
30
31 therapy by promoting mitochondrial biogenesis and/or improving mitochondrial respiration is efficient
32 in preventing or treating muscle atrophy 24, 25.
33 Previous studies showed that patients receiving corticosteroids had significantly decreased
34
mitochondrial complex I enzyme activity and oxidative damage of mtDNA32, 43. Short-term exposure to
35
36 glucocorticoid (i.e. 6 mg/kg daily over a period of 3 days for rat) improves mitochondrial biogenesis
44, 45
37 and enzymatic activity of the respiratory chain complex IV , whereas prolonged exposure causes
38 disturbance in mitochondrial structure and respiratory function, resulting in ROS generation, apoptosis,
39
and cell death, depending on the energy requirements of the target tissue and the developmental stage
40
41 of the organism 31, 33, 45-48. Chronic exposure to glucocorticoid also increases lactate production after
42 aerobic exercise, owing to mitochondrial dysfunction, oxidative damage to the mitochondrial and
43 nuclear DNA in skeletal muscle 43, which could be prevented by antioxidant compounds 49. Moreover, a
44
mitochondrial E3 ubiquitin protein ligase (Mul1) has been reported to be induced by dexamethasone
45
46 and other muscle wasting stimuli, and Mul1 suppression partially rescued muscle wasting 41.
47 In our study, we found that components loss and dysfunction of interfibrillar mitochondria (IFM)
48 occurred in dexamethasone-induced muscle atrophy, and more importantly, mitochondrial dysfunction
49
happened 3 days after dexamethasone administration, which is earlier than the induction of atrogin-1
50
51 and MuRF1, and loss of muscle weight. By using FCCP to directly induce mitochondrial dysfunction,
52 it leads to robustly ATP deprivation and AMPK activation, which subsequently activates
53 FOXO3/Atrogenes pathway. These evidences suggest that, similar as in other muscle atrophy situations,
54
mitochondrial dysfunction plays a key role in the pathogenesis of dexamethasone-induced muscle
55
56 atrophy.
57 Dexamethasone affects IFM rather than SSM
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3 Muscle mitochondria can be defined as two distinct subpopulations according to the mitochondrial
4 morphologies and functions, namely IFM and SSM. In the present study, we firstly determined that
5
several important mitochondrial enzymes and anion channel were robustly reduced in muscle by
6
7 dexamethasone. Therefore, we further separated the two mitochondrial subpopulations, IFM and SSM,
8 calculated their protein yields, and measured their respiratory functions using the XF Extracellular Flux
9 Analyzer. After dexamethasone administration, the mitochondrial protein yield and the respiration
10
11 function of individual mitochondrion were reduced in IFM but not in SSM. SSM, accounting for 20%
12 of mitochondria within skeletal muscle, is beneath the sarcolemma and mainly provides ATP for
13 membrane events, such as signaling transduction and transportation of ions and other substrates, as
14 well as participating in apoptosis signaling regulation. On the other hand, accounting for 80% of total
15 50
16 mitochondria, IFM locates between filaments and mainly produces ATP for muscle contraction .
17 Therefore, the state III respiration is 2.3 to 2.8 fold greater in IFM than that in SSM, and the ATP
18 production rate is 3 fold greater in IFM than that in SSM 51. We found in the present study that rather
19 than SSM, IFM is more susceptible to dexamethasone treatment, characterized by retarded respiratory
20
21 function and ATP deprivation.
22 Some previous reports showed that the mitochondrial function in liver but not in muscle was
23 affected by dexamethasone administration at 5 mg/kg/day for 3 days in rat 52, 53, suggesting that skeletal
24 mitochondria are of some resistance to glucocorticoid. These labs also demonstrated that none of the
25
26 respiratory complex activities, oxygen consumption, degree of coupling of oxidative phosphorylation,
52, 53 31
27 and the proton leakage are affected in the SSM in muscle . However, when applying P nuclear
28 magnetic resonance (NMR), they found that dexamethasone administration shifted the reduced
29 54
phosphocreatine (PCr): ATP ratio and induced PCr to γ-ATP flux in rat TA muscle . These
30
31 controversial readouts of mitochondrial activities might be due to functional diversities in
32 mitochondrial subpopulations, glucocorticoid type, exposure duration, and strains of animals in
33 glucocorticoid–induced muscle atrophy. Therefore, the underlying mechanism of diverse responses
34
from each mitochondrial subpopulation needs to be further defined.
35
36 Mitochondrial loss was further exacerbated due to degradation of key proteins in mitochondrial
37 dynamics
38 Mitochondrial homeostasis depends on several processes, e.g. biogenesis, fusion, fission, and
39
mitophagy, synergistically responsible for mitochondrial quality control. In response to certain
40 55
41 metabolic demands, mitochondria constantly fuse and divide . Moreover, depolarized mitochondrial
42 fraction is eliminated from the fusion-fission cycle, and the unrepaired one is further degraded by
43 mitophagy. Disturbance in any of these processes leads to mitochondrial dysfunction. We have
44
previously reported that mitochondrial biogenesis is significantly repressed in disuse-induced atrophic
45 24
46 muscle , while enforced mitochondrial biogenesis ameliorates muscle atrophy by administration of
25
47 micro-nutrient formula . Blocked mitochondrial fusion as well as enforced mitochondrial fission
48 cause muscle atrophy, via inducing mtDNA instability and AMPK activation, respectively 22, 42
.
49 22, 41, 56
Mitophagy also participates in muscle atrophy regulation . Previous reports showed that in L6
50
51 myotubes, dexamethasone stimulates autophagy depending on AMPK and Drp1 in an early stage as a
52 quality control process, while blocking mitochondrial fission by mdivi-1, an inhibitor of Drp1,
53 disrupted dexamethasone-induced autophagy/mitophagy, resulting in atrophy program 56. In the present
54
study, we found that at a later stage of dexamethasone treatment, the levels of key proteins in
55
56 mitochondrial dynamics were reduced, which should be a secondary effect and be attributed to overall
57 reduction of protein synthesis and elevated protein degradation.
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3 Resveratrol can reverse dexamethasone-induced muscle atrophy by improving mitochondrial
4 function
5
Mitochondria are the main organelles for energy production in cells, and disturbance of
6
7 mitochondrial function should firstly lead to insufficient energy supply, and activation of several
8 intracellular signaling pathways, including AMPK activation, autophagy, and/or apoptosis. AMPK is a
9 key energy sensor, and it is activated immediately during intracellular ATP deprivation, and
10
11 subsequently leads to mitochondrial biogenesis by phosphorylating PGC-1α or autophagy by
57, 58
12 phosphorylating . Previous studies illustrated that AMPK can directly regulate FOXO3 via
13 59
phosphorylation on 6 regulatory sites of FOXO3 , and the AMPK/FOXO3 pathway participates in
14 muscle atrophy induced by enforced mitochondrial fission 22
. However, it is unclear whether and how
15
16 mitochondrial dysfunction involves in AMPK/FOXO3 signaling in dexamethasone-induced muscle
17 atrophy. Here, with both in vivo and in vitro evidences, we demonstrated that dexamethasone robustly
18 induced component loss and respiratory dysfunction of IFM, resulting in intracellular ATP deprivation,
19 which in turn activates AMPK /FOXO3 signaling. Furthermore, activated protein degradation in turn
20
21 reduces key proteins for mitochondrial dynamics and exacerbates mitochondrial dysfunction. On the
22 other hand, resveratrol, which has been reported efficiently in preventing muscle atrophy in several
23 models 60, 61
, can effectively reverse the mitochondrial dysfunction and subsequently muscle atrophy
24 induced by dexamethasone both in vitro and in vivo.
25
26
27 Conclusion
28 Taken together, we found that mitochondrial dysfunction and ATP deprivation precedes muscle
29
atrophy after dexamethasone administration, and leads to atrophy by AMPK/FOXO3/agtrogenes
30
31 signaling, which in turn exacerbate mitochondrial loss (Fig. 7). And, resveratrol is efficient in
32 preventing dexamethasone-induced muscle atrophy by improving mitochondrial respiration. This
33 finding provides new insight for mitochondria-centered action in dexamethasone-induced muscle
34
atrophy, and suggests a promising strategy that targeting mitochondria to improve mitochondrial
35
36 function might prevent and treat dexamethasone-induced muscle atrophy.
37
38 Acknowledgement
39
The study was supported by the Major State Basic Research Development Program
40
41 (2015CB856302), Opening Foundation of the State Key Laboratory of Space Medicine Fundamentals
42 and Application, the China Astronaut Research and Training Center (Grant SMFA15K01), the Merieux
43 Research Starting Grant, the Fundamental Research Funds for the Central Universities(08143008,
44
08143101), Tianjin Applied Basic and Frontier Tech Major Project (12JCZDJC34400), Tianjin higher
45
46 Education Sci-tech Development Project (20112D05). We thank Dr. Dan Gao for reading the
47 manuscript and valuable advices.
48
49
50
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4 Figure legend
5
Figure 1. Dexamethasone induces muscle atrophy in vivo. A. During the 19 days of dexamethasone
6
7 treatment, body weight losing became significent from the day 12 and kept on losing afterwards in
8 dexamethasone group compared to the control group. B. Tibia anterior muscle weight decreased
9 significantly in dexamethasone group compared to the control group. C. The ratio of TA muscle weight
10
11 to body weight was lower in dexamethasone group than those in the control group. D. mRNA levels of
12 Atrogin-1 and MuRF1 were higher in dexamethasone-treated mice than those in control mice. The
13 body weight results were statistical analyzed using one-way ANOVA, and other results were statistical
14 analyzed using Student t-test. *p<0.05, **p<0.01,***p<0.001, vs control.
15
16
17 Figure 2. Dexamethasone induces mitochondrial loss and compromised mitochondrial
18 respiration. A. Representative western blots of complex I, II, III, IV, and V, VDAC1 and 2 in TA
19 muscle in dexamethasone treated mice and control mice. B. Quantifications of A, with Actin as loading
20
21 control. Protein expressions of complex II, IV, and V, and VDAC1 were significantly declined in
22 dexamethasone group compared to the control group. C. Mitochondrial yield in isolated IFM was lower
23 in dexamethasone-treated mice than in the control mice. Mitochondrial yield in SSM in each mice
24 group was comparable. D. The mitochondrial oxygen comsuption rate (OCR) was weakened in isolated
25
26 IFM but no in SSM from the muscle of dexamethasone-treated mice. E. Western blots of Mfn1 and 2,
27 OPA1, Drp1, Fis1, parkin, PGC-1α, and Tfam in TA muscle in dexamethasone treated mice and control
28 mice. F: Quantifications of E, with actin as loading control. Protein expressions of Mfn2, OPA1, Drp1,
29
and parkin were significantly declined in dexamethasone group compared to the control group. *p<0.05,
30
31 **p<0.01 ***p<0.001, vs control.
32
33 Figure 3. Mitochondrial dysfunction was induced by dexamethsone at an early stage. A. The basal
34
respiration measured by Seahorse XF24 analyzer was declined at 3 days after dexamethasone
35
36 administration, and further decreased along the duration of dexamethasone administration. B. TA
37 muscle weight started to decrease at 6 days after dexamethasone administration. C. The ratio of TA
38 muscle weight to body weight was lower at 18 days after dexamethasone administration. D. Induction
39
of MuRF1 and Atrogin-1 were at 6 days, while the decrease of II, IV, and V were at 12 and 18 days.
40
41 *p<0.05, **p<0.01 vs. control.
42
43 Figure 4. Dexamethasone causes ATP deprivation, and activates AMPK/FOXO3 signaling in
44
atrophic muscle. A: The intracellular ATP level was robustly reduced after dexamethasone incubation.
45
46 n=3. B. AMPK was activated at 3 days after dexamethasone administration, and further induced
47 FOXO3. C. Western blots of p-AMPK, AMPK, and FOXO3 in TA mucle. AMPK/FOXO3 signaling
48 was activated in TA muscle after 18 days of dexamethasone administration. D. Quantifications of C.
49
E. Representative western blots of p-AMPK, AMPK, FOXO3, and MYH in C2C12 myotubes treated
50
51 with dexamethasone alone or dexamethasone plus compound C. F. Quantifications of E, n=4.. G.
52 mRNA levels of Atrogin-1 and MuRF1 were induced in dexmethasone treated C2C12 myotubes and
53 blocked by AMPK inhibitor, compound C. A-D: * p<0.05, ** p<0.01, vs control. F-G: **p<0.01,
54
***p<0.001, vs.dexamethasone.
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57 Figure 5 Mitochondrial dysfunction induced by dexomethasone triggers
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3 AMPK/FOXO3/Atrogenes expression. A. FCCP induced intracellular ATP deprivation. B.
4 Representative western blots of p-AMPK, AMPK, FOXO3, and MYH in C2C12 myotubes treated with
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FCCP. C. Quantifications of B. n=3. D: mRNA levels of Atrogin-1 and MuRF1 in C2C12 myotubes
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7 treated with FCCP or vehicle. E: OCR in C2C12 myotubes treated with dexamethasone,
8 dexamethasone + resveratrol, FCCP, or vehicle, n=5. F: Basal respiration, ATP-linked oxygen
9 consumption, and maximal respiration were decreased by dexamethazone incubation. Resveratrol
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11 reversed the reduces in basal respiration and ATP-linked oxygen consumption caused by
12 dexamethasone. n=5. G: mRNA levels of Atrogin-1 and MuRF1 were induced in dexmethasone treated
13 C2C12 myotubes and reversed by reveratrol. The results in C and D were statistical analyzed using
14 Student t-test, and other results were statistical analyzed using one-way ANOVA. *p<0.05, **p<0.01,
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16 ***p<0.001 vs control.
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18 Figure 6. Resveratrol protected from dexamethasone-induced muscle atrophy in vivo. A: Basal
19 respiration of isolated mitochondria was compromised in dexamethasone-treated mice, and reversed by
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21 250 mg/kg/day of resveratrol treatment. B: The loss of TA muscle weight of dexamethasone-treated
22 mice was reversed by by 250 mg/kg/day of resveratrol. C: The decrease in ratio of muscle weight to
23 body weight in dexamethasone-treated mice was reversed by by 250 mg/kg/day of resveratrol. D:
24 Western blots of p-AMPK, AMPK, FOXO3, MuRF1, and Atrogin-1 in TA muscle from mice treated
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26 with dexamethasone or dexamethasone + resveratrol.
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28 Figure 7. Diagram of mitochondrial dysfunction in mediating dexamethasone-induced muscle
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atrophy. Dexamethasone induces mitochondrial dysfunction, resulting in ATP deprivation and
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31 subsequently AMPK activation, which further activates the FOXO3/atrogenes and eventually causes
32 protein degradation and muscle atrophy. By improving mitochondrial respiration, resveratrol efficiently
33 prevented muscle atrophy caused by dexamethasone.
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Figure 1. Dexamethasone induces muscle atrophy in vivo. A: During the 19 days of dexamethasone
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treatment, body weight losing became significent from the day 12 and kept on losing afterwards in
48 dexamethasone group compared to the control group. B: Tibia anterior (TA) muscle weight decreased
49 significantly in dexamethasone group compared to the control group. C: The ratio of TA muscle weight to
50 body weight was lower in dexamethasone group than that in the control group. D: mRNA levels of Atrogin-1
51 and MuRF1 were higher in dexamethasone-treated mice than those in control mice. The body weight results
52 were statistical analyzed using one-way ANOVA, and other results were statistical analyzed using Student t-
53 test. *p<0.05, **p<0.01,***p<0.001, vs. control.
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Figure 2. Dexamethasone induces mitochondrial loss and compromised mitochondrial respiration. A:
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Representative western blots of complex I, II, III, IV, and V, VDAC1 and 2 in TA muscle in dexamethasone
48 treated mice and control mice. B: Quantifications of A, with Actin as loading control. Protein expressions of
49 complex II, IV, V, and VDAC1 were significantly declined in dexamethasone group compared to the control
50 group. C: Mitochondrial yield in isolated IFM was lower in dexamethasone-treated mice than in the control
51 mice. Mitochondrial yield in SSM in each mice group was comparable. D: The mitochondrial oxygen
52 comsuption rate (OCR) was weakened in isolated IFM but not in SSM from the muscle of dexamethasone-
53 treated mice. E: Western blots of Mfn1 and 2, OPA1, Drp1, Fis1, parkin, PGC-1α, and Tfam in TA muscle in
54 dexamethasone treated mice and control mice. F: Quantifications of E, with actin as loading control. Protein
expressions of Mfn2, OPA1, Drp1, and parkin were significantly declined in dexamethasone group compared
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to the control group. *p<0.05, **p<0.01, ***p<0.001, vs. control.
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Figure 3. Mitochondrial dysfunction was induced by dexamethsone at an early stage. A. The basal respiration
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measured by Seahorse XF24 analyzer was declined at 3 days after dexamethasone administration, and
48 further decreased along the duration of dexamethasone administration. B. TA muscle weight started to
49 decrease at 6 days after dexamethasone administration. C: The ratio of TA muscle weight to body weight
50 was lower at 18 days after dexamethasone administration. D. Induction of MuRF1 and Atrogin-1 were at 6
51 days, while the decrease of II, IV, and V were at 12 and 18 days. *p<0.05, **p<0.01 vs. control.
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Figure 4. Dexamethasone causes ATP deprivation, and activates AMPK/FOXO3 signaling in atrophic muscle.
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A: The intracellular ATP level was robustly reduced after dexamethasone incubation. n=3. B. AMPK was
48 activated at 3 days after dexamethasone administration, and further induced FOXO3. C. Western blots of p-
49 AMPK, AMPK, and FOXO3 in TA mucle. AMPK/FOXO3 signaling was activated in TA muscle after 18 days of
50 dexamethasone administration. D. Quantifications of C. E. Representative western blots of p-AMPK, AMPK,
51 FOXO3, and MYH in C2C12 myotubes treated with dexamethasone alone or dexamethasone plus compound
52 C. F: Quantifications of E, n=4.. G. mRNA levels of Atrogin-1 and MuRF1 were induced in dexmethasone
53 treated C2C12 myotubes and blocked by AMPK inhibitor, compound C. A-D. *p<0.05, **p<0.01, vs. control.
54 F-G: **p<0.01, ***p<0.001, vs.dexamethasone.
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Figure 5. Mitochondrial dysfunction induced by dexomethasone triggers AMPK/FOXO3/Atrogenes expression.
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A: FCCP induced intracellular ATP deprivation. B: Representative western blots of p-AMPK, AMPK, FOXO3,
48 and MYH in C2C12 myotubes treated with FCCP. C: Quantifications of B. n=3. D: mRNA levels of Atrogin-1
49 and MuRF1 in C2C12 myotubes treated with FCCP or vehicle. n=3. E: OCR in C2C12 myotubes treated with
50 dexamethasone, dexamethasone + resveratrol, FCCP, or vehicle. n=5. F: Basal respiration, ATP-linked
51 oxygen consumption, and maximal respiration were decreased by dexamethazone incubation. Resveratrol
52 reversed the reduces in basal respiration and ATP-linked oxygen consumption caused by dexamethasone.
53 n=5. G: mRNA levels of Atrogin-1 and MuRF1 were induced in dexmethasone treated C2C12 myotubes and
54 reversed by reveratrol. The results in C and D were statistical analyzed using Student t-test, and other
results were statistical analyzed using one-way ANOVA. *p<0.05, **p<0.01, ***p<0.001 vs control.
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Figure 6. Resveratrol protected from dexamethasone-induced muscle atrophy in vivo. A: Basal respiration of
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isolated mitochondria was compromised in dexamethasone-treated mice, and reversed by 250 mg/kg/day of
48 resveratrol treatment. B: The loss of TA muscle weight of dexamethasone-treated mice was reversed by by
49 250 mg/kg/day of resveratrol. C: The decrease in ratio of muscle weight to body weight in dexamethasone-
50 treated mice was reversed by by 250 mg/kg/day of resveratrol. D: Western blots of p-AMPK, AMPK, FOXO3,
51 MuRF1, and Atrogin-1 in TA muscle from mice treated with dexamethasone or dexamethasone + resveratrol.
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Figure 7. Diagram of mitochondrial dysfunction in mediating dexamethasone-induced muscle atrophy.
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Dexamethasone induces mitochondrial dysfunction, resulting in ATP deprivation and subsequently AMPK
48 activation, which further activates the FOXO3/atrogenes and eventually causes protein degradation and
49 muscle atrophy. By improving mitochondrial respiration, resveratrol efficiently prevented muscle atrophy
50 caused by dexamethasone.
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