Proteomic analysis revealed different responses to hypergravity of soleus and extensor digitorum longus muscles in mice

Journal Pre-proof Proteomic analysis revealed different responses to hypergravity of soleus and extensor digitorum longus muscles in mice

Takashi Ohira, Yoko Ino, Yusuke Nakai, Hironobu Morita, Ayuko Kimura, Yoichi Kurata, Hiroyuki Kagawa, Mitsuo Kimura, Kenji Egashira, Shunsuke Moriya, Kyoko Hiramatsu, Masao Kawakita, Yayoi Kimura, Hisashi Hirano PII:

S1874-3919(20)30054-3

DOI:

https://doi.org/10.1016/j.jprot.2020.103686

Reference:

JPROT 103686

To appear in:

Journal of Proteomics

Received date:

22 November 2019

Revised date:

30 January 2020

Accepted date:

12 February 2020

Please cite this article as: T. Ohira, Y. Ino, Y. Nakai, et al., Proteomic analysis revealed different responses to hypergravity of soleus and extensor digitorum longus muscles in mice, Journal of Proteomics (2020), https://doi.org/10.1016/j.jprot.2020.103686

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© 2020 Published by Elsevier.

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Proteomic analysis revealed different responses to hypergravity of soleus and extensor digitorum longus muscles in mice

Takashi Ohira1* , Yoko Ino1* , Yusuke Nakai1 , Hironobu Morita2 , Ayuko Kimura1 , Yoichi

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Kurata1 , Hiroyuki Kagawa1 , Mitsuo Kimura1 , Kenji Egashira1 , Shunsuke Moriya3 ,

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Kyoko Hiramatsu3 , Masao Kawakita3 , Yayoi Kimura1 , Hisashi Hirano1

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*: Takashi Ohira and Yoko Ino have contributed equally to this work.

Advanced Medical Research Center, Yokohama City University, Kanagawa, Japan

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Department of Physiology, Gifu University Graduate School of Medicine, Gifu, Japan

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Department of Advanced Research for Biomolecules, Tokyo Metropolitan Institute of

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Medical Science, Tokyo, Japan

Corresponding authors: Hisashi Hirano, Ph.D. Advanced Medical Research Center, Yokohama City University Fukuura 3-9, Kanazawa-ku, Yokohama, 236-0004, Japan Phone: +81-45-787-2519, FAX: +81-45-787-2787

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E-mail: [email protected]

Yayoi Kimura, Ph.D. Advanced Medical Research Center, Yokohama City University

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Phone: +81-45-787-2519, FAX: +81-45-787-2787

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Fukuura 3-9, Kanazawa-ku, Yokohama, 236-0004, Japan

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E-mail: [email protected]

Abstract

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Investigating protein abundance profiles is important to understand the differences in

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the slow and fast skeletal muscle characteristics. The profiles in soleus (Sol) and extensor digitorum longus (EDL) muscles in mice exposed to 1 g or 3 g for 28 d were compared. The biological implications of the profiles revealed that hypergravity exposure activated a larger number of pathways involved in protein synthesis in Sol. In contrast, the inactivation of signalling pathways involved in oxidative phosphorylation were conspicuous in EDL. These results suggested that the reactivity of molecular pathways in Sol and EDL differed. Additionally, the levels of spermidine synthase and spermidine, an important polyamine for cell growth, increased in both muscles

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following hypergravity exposure, whereas the level of spermine oxidase (SMOX) increased in EDL alone. The SMOX level was negatively correlated with spermine content, which is involved in muscle atrophy, and was higher in EDL than Sol, even in the 1 g group. These results indicated that the contribution of SMOX to the regulation of

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spermidine and spermine contents in Sol and EDL differed. However, contrary to

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expectations, the difference in the SMOX level did not have a significant impact on the

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growth of these muscles following hypergravity exposure.

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proteomic analysis

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Keywords: soleus, extensor digitorum longus, hypergravity, polyamine, quantitative

Abbreviations: soleus (Sol), extensor digitorum longus (EDL), gastrocnemius (GA), tibialis anterior (TA), phosphatidylinositol 3-kinase (PI3K), peroxisome proliferator-activated receptor (PPAR), α/retinoid X receptor (RXR), spermine oxidase (SMOX), ornithine decarboxylase (ODC), spermidine synthase (SRM), spermine synthase (SMS), S-adenosylmethionine decarboxylase (AdoMetDC)

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1. Introduction Weight-bearing activity is important for maintaining and improving skeletal muscle mass and properties. However, various types of skeletal muscles have been reported to exhibit different responses to changes in gravitational load. Exposure to a hypergravity

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environment induces hypertrophy in slow-twitch soleus (Sol) muscles, but not

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fast-twitch gastrocnemius (Gast) and tibialis anterior (TA) muscles in mice [1,2]. In

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contrast, prolonged conditions of weightlessness, such as spaceflight and hindlimb

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unloading, induce marked atrophy in Sol muscles compared with that in fast-twitch extensor digitorum longus (EDL), TA, and Gast muscles in rodents [3−13]. It was

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reported that ankle plantar flexors were more susceptible to weightlessness than ankle

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dorsal flexors in humans and suggested that muscle atrophy was primarily caused by the decrease in gravitational loading and not muscle activity [14−16]. Although changes in skeletal muscle transcriptomics [7,11] and proteomics [10,13] in space-flown mice have been reported in previous studies, the specific molecular mechanisms related to the differing susceptibilities to gravitational changes in various types of skeletal muscles, to the best of our knowledge, have not been determined. Proteomic analysis is a powerful tool to comprehensively investigate intracellular and/or intra-organ changes, the results of which are useful here for

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elucidating the molecular mechanisms of muscle adaptations to changes in gravitational load. However, the wide dynamic range of protein abundance levels in skeletal muscles is a major issue for proteomic analysis using LC-MS/MS [17]. Generally, protein fractionation approaches using HPLC or gel electrophoresis are effective for reducing

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the complexity of the protein samples and increasing the number of proteins identified

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by LC-MS/MS analysis [18−20]. However, as the number of protein or peptide fractions

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and therefore sample size increases, the throughput of proteomic analysis decreases.

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In this study, we investigated changes in the protein abundance profiles of mouse Sol and EDL muscles in response to 1 g or 3 g environment exposure over 28 d.

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To increase the coverage of proteins identified by LC-MS/MS analysis, proteins in Sol

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and EDL muscles were extracted by a two-step solubilisation method using 50 mM Tris- hydrochloric acid (HCl) buffer (pH 7.5) containing protease inhibitor, and a lysis buffer containing 8 M urea, 50 mM NH4 HCO 3 , 4% sodium deoxycholate, and protease inhibitor. Then, the effects of hypergravity exposure on protein abundance profiles in each supernatant, i.e., SUP-1 and -2, were evaluated by LC-MS/MS analysis and label-free quantitation.

2. Materials and Methods 5

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2.1. Experimental animals All experimental procedures were conducted in accordance with the Guide for the Care and Use of Laboratory Animals of the Japanese Physiological Society and the National Institutes of Health guide for the care and use of Laboratory animals. This study was

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approved by the Committees on Animal Care and Use of the Japan Aerospace

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Exploration Agency (accreditation no.: 017-006A) and Gifu University (accreditation

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no.: 28-31). Male C57BL/6J mice (9–10 weeks old; Chubu Kagaku Shizai Co., Ltd.,

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Nagoya, Japan) were utilised in this study. Twelve mice were randomly divided into two groups: the 1 g group (N = 6) and 3 g group (N = 6). Three mice in each group were

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housed in plastic cages (18 cm width × 30 cm length × 14 cm height) over 28 d. A 3 g

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environment was created by centrifugation using a custom- made gondola-type rotating box with a 1.5- m arm length (Shimadzu, Kyoto, Japan), similar to that of previous studies [1,21]. The temperature and humidity in the animal room were controlled at approximately 24°C and 55%, respectively, with a 12:12-h light-dark cycle. All mice had free access to a solid diet (CE-2; Nihon CLEA, Tokyo, Japan) and water.

2.2. Muscle preparation After 28 d, the mice were euthanised by exsanguination under isoflurane anaesthesia

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and perfused transcardially with PBS (pH 7.4; Life Technologies Corp., Paisley, UK). Sol and EDL muscles were removed bilaterally, and wet weights of these muscles were measured. The muscles were frozen immediately in liquid nitrogen and stored at –80°C

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until subsequent analysis.

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2.3. Proteomic analysis

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As shown in Figure S1A, Sol and EDL muscles isolated from a left hindlimb, N = 6 in

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each group, were homogenised using Sample Grinding Kit (GE Healthcare UK Ltd., Buckinghamshire, UK) in 50 mM Tris-HCl (pH 7.5) containing protease inhibitor

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(Roche Diagnostics GmbH, Mannheim, Germany). Homogenised samples were

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centrifuged at 21,600 g for 10 min at 4°C, and the supernatants were used as SUP-1 (Fig. S1A). Precipitates were washed five times with 50 mM Tris-HCl (pH 7.5) containing protease inhibitor and rehomogenised using Sample Grinding Kit in a lysis buffer containing 8 M urea, 50 mM NH4 HCO 3 , 4% sodium deoxycholate, and protease inhibitor. Rehomogenised samples were centrifuged at 21,600 g for 10 min at 4°C, and the supernatants were used as SUP-2. Protein concentrations of each sample were determined using BCA Protein Assay kit (Thermo Fisher Scientific Inc., MA, USA). The extracted proteins in SUP-1 and -2 from Sol and EDL muscles, N = 5 in

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each group, were reduced individually with 10 mM DTT and subsequently alkylated with 25 mM 2- iodoacetamide. Each protein solution was diluted to 2 M urea in 50 mM NH4 HCO 3 (pH 8.0) and then incubated with trypsin (Promega, Madison, WI, USA) at 37°C for 16 h. The ratio protein:trypsin was 20:1 (w/w). The resulting peptides were

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desalted by purification using a reverse-phase C18 tip column (Stage Tip) [22]. Desalted

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peptides were resuspended in 0.3% formic acid. Then, 6 μl/vial of each sample was

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injected individually and analysed once using Orbitrap Elite, Hybrid Ion Trap-Orbitrap

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Mass Spectrometer (Thermo Fisher Scientific Inc.). The mass spectrometer was operated using LTQ Tune Plus (ver.2.7.0.1112 SP2; Thermo Fisher Scientific Inc.).

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The analytical conditions were as follows: Trap column (Acclaim PepMapT M

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100; 100 µm × 20 mm, C18, 5 μm, 100Å, No.164564), Nano HPLC capillary column

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(75 µm × 125 mm, C18, 3 μm, cat. no. NTCC-360/75-3-125; Nikkyo Technos Co., Ltd, Tokyo, Japan); temperature = Room temperature (22 ± 1.5ºC); mobile phase = (A) 0.1% formic acid and 2% ACN, (B) 0.1% formic acid and 95% ACN; gradient, A:B = 98:2 (0 min), 67:33 (120 min), 5:95 (120–130 min); flow rate = 350 nL/min; mass scan range = m/z 350–1,500; mass resolution = 120,000 at m/z 400; charge state screening = enable; unassigned charge states = 2+ and 3+ were not rejected; normalised collision energy = 35.0 %; AGC value = Ion Trap (Full MS = 3.0 × 10 4 , MS/MS = 1.0 × 104 ), Orbi FT

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(Full MS = 5.0 × 105 , MS/MS = 5.0 × 104 ); injection time = Auto Adjustment; lock mass = 391.284290, 445.120030; and dynamic exclusion = 60 sec. Quantitative data analysis was performed using Progenesis QI for proteomics software (version 2.0; Nonlinear Dynamics, Newcastle, UK) with the following parameters: alignment of

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two-dimensional feature maps = automatic alignment; exclusion of charge = 1 and more

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than 6.

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In each supernatant, i.e., SUP-1 and -2, the MS/MS data from Sol (N = 5) and

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EDL (N = 5) muscle proteins in 1 g and 3 g groups were integrated and analysed in two separate experiments by Progenesis QI for proteomics software. In other words, the

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normalised abundance of peptides in SUP-1 and -2 was quantified individually, and the

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same peptides in each supernatant were analysed in both Sol and EDL muscles. This protocol enabled us to evaluate peptides and proteins for which abundance levels were markedly different in Sol and EDL muscles. Database searches to identify proteins were performed using MASCOT (version 2.5.1; Matrix Science, London, UK) against Mus musculus protein sequences (17,034 sequences) in the UniProt Knowledgebase database released November, 2019 with the following parameters: enzyme, trypsin; peptide mass tolerance, ± 5 ppm (Orbitrap); fragment mass tolerance, ± 0.5 Da (Ion Trap); maximum missed cleavages, 2; variable modifications, acetyl (protein N-term), carbamidomethyl

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(C), oxidation (M), carbamyl (K), carbamyl (N-term); and ion score cut-off, 30. For the protein identification, peptides with a false discovery rate of less than 1% was used. The protein abundance levels were quantified using the abundance levels of unique peptides to each protein.

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From ANOVA, significantly altered SUP-1 and -2 protein abundances (p <

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0.05) in 3 g-exposed Sol and EDL muscles were interpreted individually using

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Ingenuity Pathway Analysis Version 2019 December (Qiagen, Hilden, Germany). P

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values in the Ingenuity Pathway Analysis indicated the coverages of components in each category. The activation or inactivation of each category was presumed based on

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the z score, which was calculated by comparing alteration pattern of the abundance

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levels of proteins engaging in each signalling pathway with the database. Furthermore, hypergravity responsive proteins were selected based on p values (< 0.01) and fold changes (> 2.0).

2.4. Immunoblot analysis The extracted proteins in SUP-1 and -2 from Sol and EDL muscles, N = 6 in each group, were mixed with 2× sample buffer with 100 mM DTT for SDS-PAGE and heated at 95ºC for 5 min. Equal amounts of protein (10 µg) were separated on SDS-PAGE gels

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(SuperSep Ace, 5−20%; FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) and transferred to PVDF membranes using a Trans-Blot Turbo Blotting System (Bio-Rad, CA, USA). Precision Plus Protein Dual Color Standards (Bio-Rad) were used to estimate protein molecular weight (10–250 kDa). Membranes were blocked with 5%

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skim milk diluted in TBS containing 0.05% Tween 20 (TBS-T) and then incubated with

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primary antibodies diluted at 1:1,000 using TBS-T overnight at room temperature. The

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following primary antibodies were used for immunoblot analysis: anti-spermidine

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synthase (cat. no. 19858-1-AP; Proteintech Group, IL, USA), anti-spermine synthase (cat. no. 15979-1-AP; Proteintech Group); and anti-spermine oxidase (cat. no.

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15052-1-AP; Proteintech Group). Membranes were then incubated with secondary

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antibody: goat anti-rabbit IgG-horseradish peroxidase (cat. no. sc-2004; Santa Cruz Biotechnology, CA, USA) diluted at 1:5,000 using Blocking One (Nacalai Tesque Inc., Kyoto, Japan) for 1 h at room temperature. Immunoblot images separated for Sol and EDL muscle proteins were obtained using a LAS-4000 UV mini system (FUJIFILM Corporation, Tokyo, Japan) and ECL Select Western Blotting Detection Reagent (GE Healthcare UK Ltd.) under the same exposure time. Then, using Multi Gauge software (ver.3.11; FUJIFILM Corporation), signal intensities of each target protein were calculated by the following formula: the signal intensity = (signal intensity of target

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protein [square pointed by arrowhead in Fig. S3]) – (signal intensity of background on the same membrane [square only in Fig. S3]). In this study, we did not normalise the signal intensities of each target protein because there is no consensus on suitable markers for normalisation of skeletal muscle proteins. Instead, after signal detection, we

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stained PVDF membranes with SimplyBlue SafeStain (Thermo Fisher Scientific Inc.)

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containing 25% methanol to confirm that equal amounts of proteins in each sample

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were separated by SDS-PAGE and transferred to the membrane (Fig. S3).

2.5. Measurement of polyamine content

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Polyamine contents in Sol and EDL muscles isolated from the right hindlimb, N = 6 in

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each group, were analysed as previously described [23]. Samples were prepared using the highest purity reagents as follows. Reagents and materials without annotations were purchased from FUJIFILM Wako Pure Chemical Corporation. As previously reported, muscles were homogenised in cold 10% TCA using BioMasher II (Nippi, Inc., Tokyo, Japan) with Power Homogeniser (AS ONE Corporation, Osaka, Japan) and centrifuged at 20,400 g for 15 min at 4°C [24,25]. Stable isotope- labelled polyamines, i.e., [1,2,4- 13 C3 ] putrescine (13 C3 -Put; 1 nmol), spermidine [1,1,4,4-d4 ] (Spd-d4 ; 5 nmol), and [1,4,8,12- 15N4 ] spermine [5,5,8,8-d4 ] 12

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(15 N4-Spm-d4 ; 5 nmol), were added to the supernatant as an internal standard. The labelled polyamines were produced at Tokyo Metropolitan Institute of Medical Science (Hiramatsu group). Samples were fractionated into a putrescine fraction, and a spermidine and spermine fraction using a 0.3- mL CM-cellulose column. Each sample

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was applied to the column, which was equilibrated with 3 mL of 0.01 M pyridine/acetic

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acid buffer. Next, after washing the column with 3 mL of 0.1 M pyridine/acetic acid

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buffer, putrescine, or spermidine and spermine were eluted from the column by adding 1

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mL of 0.33 M and 1 M pyridine/acetic acid buffer. After evaporating to dryness in vacuo, acetate in the residues was changed to hydrochloride by adding 0.1 mL of 0.1 M HCl in

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70% ethanol. This procedure was important to avoid a mixed anhydride formation,

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which could cause artifacts. Subsequently, each sample was evaporated to dryness in

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vacuo again and reacted with 20 μL heptafluorobutyric anhydride (HFB; Sigma-Aldrich, Tokyo, Japan) at 100°C for 30 min in 0.1 mL ACN. The reaction products were dried by spraying nitrogen gas. The residues were dissolved in 0.3 mL of an infusion solvent containing

50% ACN,

0.05%

formic acid,

and

0.1% ammonium acetate

(Sigma-Aldrich), and MS analysis was performed. For MS analysis, an Agilent 6530 Accurate-Mass quadrupole time-of-flight LC/MS system (Agilent Technologies, Inc., Tokyo, Japan) was used. A LC column was not used for injecting samples into the

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system; this method is commonly called flow injection analysis. A solution with the same composition as the infusion solvent was used as the mobile phase. MS of each ammonium ion adduct for HFB-derivatised polyamines was determined. The m/z of putrescine,

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C3-Put, spermidine, Spd-d4 , spermine, and

N4-Spm-d4 were 498, 501,

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751, 755, 1004, and 1012, respectively. The contents of polyamines, putrescine,

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spermidine, and spermine were determined using the relative ion intensity (peak area) of

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the endogenous polyamines to the labelled polyamines, which were added as an internal

2.6. Statistical analysis

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standard using MassHunter Software (Agilent Technologies, Inc.).

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The effects of hypergravity exposure on SUP-1 and -2 protein abundance levels in Sol and EDL muscles were evaluated individually by ANOVA using Progenesis QI for proteomics software. In this software, users can only select “ANOVA” for statistical analysis, even for comparisons of two groups. However, when there are only two groups, “ANOVA” in this software has the same meaning as “unpaired t-test”. The 438 proteins identified in both SUP-1 and -2 were analysed in each fraction individually. All data in Figures 1, 3, and 4 are presented as means ± SDs. For body weight data, two-way ANOVA was performed using GraphPad Prism (Version 7.02; GraphPad

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Software, CA, USA) to evaluate the significant main effects of hypergravity (1 g versus 3 g) and day (day 0 versus day 28). For other data, the effects of hypergravity exposure on Sol and EDL muscles were evaluated individually by unpaired t-tests using GraphPad Prism. Differences with p values of less than 0.05 were considered

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statistically significant.

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3. Results

hypergravity exposure

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3.1. Increase in muscle mass was higher in Sol muscles following

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The mean body weights of mice in the 1 g and 3 g groups increased from 23.0 ± 0.4 g to

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26.5 ± 0.8 g and from 24.6 ± 1.2 g to 26.0 ± 1.5 g, respectively, after 28 d (Fig. 1A). A two-way ANOVA showed significant differences in body weights between day 0 and 28 (p < 0.0001), but not between the 1 g and 3 g groups (p = 0.06). Results of unpaired t-tests showed that the absolute (Fig. 1B) and relative wet weights of Sol and EDL muscles to body weight (Figs. 1C) significantly increased following 3 g exposure for 28 d. The effects of hypergravity on muscle absolute wet weights was stronger in Sol (+29.9%) than in EDL (+8.5%) muscles (Fig. 1B). In addition, the amounts of proteins

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extracted in SUP-1 and -2 from Sol and EDL muscles were significantly higher in the 3 g group than in the 1 g group (Fig. 1D).

3.2.

Protein abundance profiles in Sol and EDL muscles were altered

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differently following 28-d hypergravity exposure

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The two-step solubilisation of skeletal muscle proteins using 50 mM Tris-HCl buffer

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(pH 7.5) containing protease inhibitor, and a lysis buffer containing 8 M urea, 50 mM

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NH4 HCO 3 , 4% sodium deoxycholate, and protease inhibitor (Fig. S1A) reduced the sample complexity (Fig. S1B) and increased the number of proteins identified by

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LC-MS/MS analysis (Table S1). In addition, proteomic analysis using this fractionation

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method showed low variation and high reproducibility in protein detection and peptide quantification (Fig. S1C and D). The numbers of proteins identified by LC-MS/MS analysis in SUP-1 and -2 were further increased by the integration of the MS/MS data from Sol and EDL muscle proteins in 1 g and 3 g groups, N = 5 each. As a result, 1,050 and 720 proteins were identified in SUP-1 and -2, respectively, and 438 proteins were identified in both supernatants (Fig. S2A). Raw MS data and analysis files were deposited in the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the jPOST partner

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repository (https://jpostdb.org) with the data set identifier PXD017240 (preview URL for reviewers: https://repository.jpostdb.org/preview/4853861005e27d4fb780b3, Access key: 6429). Quantified proteomics data are also shown in the supplementary datasets, S1−S4. Volcano plots for the identified proteins in SUP1 and -2 showed that Sol

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muscles were more responsive to hypergravity than EDL muscles (Fig. S2B). Results of

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the ANOVA using Progenesis QI for proteomics software showed that, in SUP-1, the

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abundance levels of 359 proteins in Sol muscles and 113 proteins in EDL muscles were

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significantly (p < 0.05) altered after 28-d of 3 g exposure. Moreover, significant alterations (p < 0.05) in the abundance levels of 251 and 105 proteins in Sol and EDL

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muscles, respectively, were observed in SUP-2. Among them, the altered abundances of

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proteins detected in both SUP-1 and -2 in Sol and EDL muscles are shown in Table S2 separately. In addition to the SUP-1 and -2 specific alterations, the alterations in common and opposite manners to SUP-1 and -2 were observed in each muscle. In this study, protein abundance levels in SUP-1 and -2 were probably altered by not only the regulation of transcription, translation, and protein degradation, but also shifts of proteins into different supernatants. Ingenuity Pathway Analysis for the significantly altered protein abundance profiles (p < 0.05) indicated that signalling pathways contributing to response to the

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increase in a mechanical stimulus resulting in muscle growth, i.e., actin cytoskeleton and integrin signalling, were activated, and those contributing to cell death were inactivated by 3 g exposure in both Sol and EDL muscles (Table 1A and B). The suppression of cell death in both muscles was evident by increases in heat shock

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proteins and anti-apoptotic proteins, such as Bax and Bag3 (Table S3), while this was

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more conspicuous in 3 g-exposed Sol than EDL muscles. In Sol muscles, the activation

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of signalling pathways that are known to enhance protein synthesis and skeletal muscle

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maturation, e.g., Cdc42, eukaryotic initiation factor 2 (EIF2), phosphatidylinositol 3-kinase (PI3K)/AKT, and RhoA signalling and signalling by Rho family GTPases, was

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also observed. Additionally, hypergravity exposure activated oxidative phosphorylation,

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and inactivated glycolysis in Sol muscles. In contrast, multiple pathways involved in

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oxidative phosphorylation, i.e., fatty acid -oxidation I, glutaryl-CoA degradation, oxidative phosphorylation, peroxisome proliferator-activated receptor (PPAR) α/retinoid X receptor (RXR) α activation, and TCA cycle II (eukaryotic), were remarkably inactivated in 3 g-exposed EDL muscles. Although, these results of Ingenuity Pathway Analysis were affected by the separate analysis of the significantly altered Sol and EDL muscle proteins in SUP-1 and -2, the biological implications for SUP-1 and -2 were consistent.

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As shown in supplementary dataset S3 and S4, eight myosin isoforms, i.e., myosin-1, -3, -4, -6, -7, -7B, -8, and -9, were identified in SUP-2 by LC-MS/MS analysis. The different protein abundance levels of myosin isoforms in 1-g and 3-g Sol muscles indicated that hypergravity exposure increased myosin-7, -8, and -9 and

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decreased myosin-4 (Table S4). On the other hand, the protein abundance level of

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myosin-4 in EDL muscles was significantly increased by 28-d of 3 g exposure.

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Hypergravity-responsive proteins in Sol and EDL muscles are shown in Table 2.

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These proteins were selected based on p values (< 0.01) and fold changes (> 2.0), which were obtained from the statistical comparison of the 1 g and 3 g groups using

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Progenesis QI for proteomics software. In SUP-1, the abundance levels of 14 proteins

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were increased, and those of four proteins were decreased in Sol muscles following exposure to 3 g over 28 d (Table 2A). Additionally, only spermine oxidase (SMOX) level was increased in 3 g-exposed EDL muscles. Our quantified proteomics data showed that the normalised abundances of SMOX protein in SUP-1 in Sol muscles in the 1 g and 3 g groups were 0 and 59.4 ± 132.9, respectively. These levels in EDL muscles in the 1 g and 3 g groups were 522.7 ± 181.6 and 2,302.0 ± 1,277.4, respectively, and were dramatically higher than those in Sol muscles. In contrast, in SUP-2, the abundance levels of 14 and four proteins were increased and decreased,

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respectively, in 3 g-exposed Sol muscles, and no responsive proteins were identified in 3 g-exposed EDL muscles (Table 2B).

3.3. The biosynthetic and interconversion pathway of polyamines was

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responsive to hypergravity exposure, and polyamine contents in

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Sol and EDL muscles were regulated via different mechanisms

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We performed additional analyses to elucidate the differences in original protein

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abundance of SMOX levels in Sol and EDL muscles and the responses of the biosynthetic and interconversion pathway of polyamines to hypergravity exposure.

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Spermine oxidase, spermidine synthase (SRM), and spermine synthase (SMS) are

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involved in regulating polyamine, i.e., putrescine, spermidine, and spermine, content in cells (Fig. 2). The protein abundance levels of SRM in Sol and EDL muscles were significantly increased to 158% and 166%, respectively, following 28-d exposure to 3 g (Fig. 3A and S3A). The levels of SMS in these muscles were not affected by hypergravity exposure (Fig. 3B and S3B). In the both 1 g and 3 g groups, the levels of SRM and SMS in Sol muscles were higher than those in EDL muscles (Fig. 3A and B). In contrast, the protein abundance levels of SMOX were significantly increased to 235% only in EDL muscles following hypergravity exposure (Fig. 3C and S3C).

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Spermine oxidase was highly expressed in EDL muscles whereas the protein abundance of SMOX in Sol muscles in the both 1 g and 3 g groups was undetectable by immunoblot analysis (Fig. 3C). Moreover, putrescine (Fig. 4A), spermidine (Fig. 4B), and spermine (Fig. 4C)

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contents in Sol muscles increased significantly to 142%, 186%, and 116%, respectively,

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following 3 g exposure over 28 d. Similarly, in EDL muscles, hypergravity exposure

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increased putrescine (Fig. 4A) and spermidine (Fig. 4B) contents to 141% and 164%,

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respectively, although no increase in spermine content (Fig. 4C) was observed. Spermine content in EDL muscles were 37% lower than that in Sol muscles, even in the

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1 g group (Fig. 4C). Additionally, the spermidine/spermine contents in Sol and EDL

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muscles in the 3 g group were 60.8% and 68.4% higher than those in the 1 g group, respectively (Fig. 4D).

4. Discussions We evaluated the effects of 1 g and 3 g exposure on protein abundance profiles in mouse Sol and EDL muscles. Food intake in rodents transiently decreases in response to hypergravity exposure, and their body weights decrease accordingly during the initial phase [26−28]. It is possible that the food intake of the mice in this study also

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transiently decreased at the beginning of hypergravity exposure. However, this did not significantly affect body weights of mice in the 3 g group after 28 d. Changes in wet weights and volcano plots of protein abundance profiles indicated that Sol muscles were more susceptible to hypergravity exposure than EDL muscles. Ingenuity Pathway

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Analysis for the altered protein abundance profiles indicated that a larger number of

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pathways involved in protein synthesis and skeletal muscle maturation were activated in

e-

Sol muscles following 3 g exposure over 28 d; this was further supported by the

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prominent increase in the wet weights of Sol muscles in the 3 g group. These results were also consistent with the results of previous studies, in which mice were exposed to

al

a 3 g environment for 28 d or a 2 g environment for 30 d [1,2]. Moreover, Kawao et al.

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[1] performed an experiment using the same procedure as in our study and reported that abundance levels of genes involved in protein degradation via the ubiquitin-proteasome system and autophagy were not changed in mouse Sol muscles following 3 g exposure over 28 d. In addition, they showed that changes in hindlimb muscle wet weights and fibre cross-sectional areas were positively correlated. Therefore, it is possible that the growth of muscle fibres in Sol and EDL muscles was also promoted by hypergravity exposure in our study. Higher amounts of proteins in SUP-1 and -2 in both muscles in the 3 g group than in the 1 g group supported this estimation.

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The number of hypergravity-responsive proteins in EDL muscles was lower than that in Sol muscles (Table 2). However, as with the Sol muscles, the biological implications of altered protein abundance profiles in EDL muscles indicated that hypergravity exposure activated actin cytoskeleton and integrin signalling in SUP-1

oo

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(Table 1A). These signalling pathways are involved in muscle adaptation to the increase

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in a mechanical stimulus and activated in response to exercise and chronic loading

e-

[29,30]. Integrin signalling contributes to skeletal muscle hypertrophy [29,31−33].

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Therefore, the activation of these signalling pathways would have contributed to the increase in wet weights of not only Sol but also EDL muscles following hypergravity

al

exposure. For SUP-2 of EDL muscle proteins, inactivation of the categories related to

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cell death, i.e., cell death of muscle cells and organismal death, was also indicated (Table 1B). Furthermore, in contrast to Sol muscles, the multiple categories related to oxidative phosphorylation in cells for energy production were significantly inactivated in 3 g-exposed EDL muscles (Table 1A and B). These results suggested that the reactivity of molecular pathways to hypergravity exposure differed between EDL and Sol muscles, not that EDL muscles were insensitive to hypergravity. Twenty-eight-day exposure to a 3 g environment would have suppressed oxidative phosphorylation for energy production in EDL muscles, which were

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predominantly composed of fast-twitch glycolytic muscle fibres [4,5,7,8,11,13]. This biological interpretation was supported by the significant increase in myosin-4 (type IIB) in EDL muscles following 3 g exposure (Table S4). This result suggested that the muscle fibres containing myosin-4 (type IIB), which were fast-twitch glycolytic fibres,

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would have probably increased in EDL muscles by 28-d of hypergravity exposure. In a

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previous study, 2 g exposure over 3 months resulted in the decrease in proteins involved

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in mitochondrial energy production in mouse neck muscles, which are predominantly

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composed of fast-twitch glycolytic muscle fibres, similar to EDL muscles [10]. Additionally, Martin and Romond [34] reported that fast glycolytic fibres increased,

al

whereas fast oxidative glycolytic fibres decreased in fast-twitch plantaris muscles in rats

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exposed to a 2 g environment from birth for 3 months. In contrast, increases in mean O 2 consumption, CO 2 production, and resting energy expenditure were observed in rats during exposure to 2.3 g or 4.1 g environments [35]. However, these results were obtained as comprehensive changes in the whole body; the changes in metabolic properties during hypergravity exposure may differ in each skeletal muscle according to alterations in muscle activity patterns. In fact, unlike in EDL muscles, oxidative phosphorylation was enhanced, and glycolysis was suppressed in Sol muscles by 3 g exposure (Table 1A). These biological interpretations were supported by the significant

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increase in myosin-7 (type I) and decreased in myosin-4 (type IIB) in 3 g-exposed Sol muscles (Table S4). The changes in myosin isoform abundances suggested that slow-twitch oxidative and fast-twitch glycolytic muscle fibres would have probably increased and decreased, respectively, in Sol muscles following by 28-d of hypergravity

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exposure. Similarly, Chi et al. [36] reported that enzymes characteristic of slow twitch

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muscles, i.e., hexokinase, mitochondrial thiolase, β-hydroxyacyl CoA dehydrogenase,

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and citrate synthase, decreased in TA muscles, but not in Sol, following 2 g exposure

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over 14 d.

Our results suggested that Sol and EDL muscles develop different systems to

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regulate intramuscular spermidine and spermine contents by SMOX. Polyamines,

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including putrescine, spermidine, and spermine, are essential factors for cell growth and survival in mammals [37−40]. The general biosynthetic and interconversion pathway of polyamines in mammalian cells has been extensively reported [37,39,40]. Putrescine is produced from ornithine by ornithine decarboxylase (ODC). The conversions of putrescine to spermidine and spermidine to spermine are facilitated by SRM and SMS, respectively.

Spermidine

synthase

and

SMS

also

convert

decarboxylated

S-adenosylmethionine, which is produced by S-adenosylmethionine decarboxylase (AdoMetDC), to spermidine and spermine, respectively; SMOX converts spermine to

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spermidine. The contents of putrescine, spermidine, and spermine in Sol muscles significantly increased following 3 g exposure; these hypergravity-induced changes in polyamine levels in Sol muscles may be due to the activation of components involved in the entire biosynthetic and interconversion pathway of polyamines, i.e., ODC,

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AdoMetDC, SRM, and SMS, but not SMOX. Turchanowa et al. [41] reported that a

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single bout of resistance or endurance exercise enhanced ODC activity and increased

e-

putrescine, spermidine, and spermine levels in Sol and EDL muscles in rats. However,

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in this study, spermine content was lower in EDL than Sol muscles, even in the 1 g group and did not increase in EDL muscles after 3 g exposure for 28 d. The low

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spermine content in EDL muscles could be attributed to high SMOX protein abundance.

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Further studies are needed to identify the mechanism resulting in low SMOX protein abundance through in Sol muscles and the meaning of muscle-specific mechanisms regulating polyamine contents. We consider that the differences in the composition of fibre types in Sol and EDL muscles might be related to their SMOX protein abundances. However, Sol muscles in C57BL6J mice contain more fast-twitch fibres than the muscles in rats; Sol muscles in 3- month-old male C57BL6J mice contain 37.42 ± 8.2% type I fibres [42]. Thus, other factors would also be involved in the control of SMOX protein abundance in these muscles.

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Recent studies have shown that SMOX protein abundance levels are positively correlated with skeletal muscle mass [43,44]. Bonger et al. [43] showed that limb immobilisation, fasting, and denervation decreased SMOX protein levels and caused atrophy in TA muscles in mice, which was ameliorated by the forced increase in SMOX

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protein. Lang et al. [19] also observed that SMOX protein abundance levels were

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decreased in Gast muscles during denervation- induced atrophy. Therefore, increased

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SMOX protein abundance may be involved in the increase in the wet weights of 3

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g-exposed mouse EDL muscles. However, our results showed that the protein abundance level of SMOX in Sol muscles was much lower than that in EDL muscles, in

al

the both 1 g and 3 g groups. The differences in the gene expression levels of SMOX in

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Sol and EDL muscles have also been observed by a microarray analysis [45]. Moreover, SMOX protein in Sol muscles did not increase following 3 g exposure for 28 d, despite the prominent increase in the wet weights of that was observed. Accordingly, the positive effects of SMOX protein on controlling muscle mass may not be universal in skeletal muscle tissues in the body. In contrast, increased spermidine levels are likely to universally contribute to skeletal muscle growth in response to hypergravity exposure and exercise because spermidine content was elevated in both hypertrophied Sol and EDL muscles in the 3 g

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group. Muscle mass is regulated by the net balance between protein synthesis and degradation [46]. Spermidine is essential for protein synthesis induced by eukaryotic translation initiation factor 5A [40,47−49]; elevated SMOX protein abundance increases spermidine content by activating the conversion from spermine to spermidine

oo

f

[37,39,40]. However, the increase in wet weights following hypergravity exposure was

pr

more prominent in Sol than EDL muscles, suggesting that differences in the

e-

mechanisms regulating intramuscular spermidine and spermine contents by SMOX

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were not likely to affect growth in Sol and EDL muscles in a 3 g environment. Hence, differences in Sol and EDL muscle growth in the hypergravity environment could be

al

attributed to other mechanisms, e.g., different reactivities of Cdc42, EIF2, PI3K/AKT,

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and RhoA signalling pathways and signalling by Rho family GTPases (Table 1), which are known to be involved in protein synthesis and skeletal muscle maturation [50−54]. We speculated that differences in the mechanisms controlling spermidine and spermine contents by SMOX in Sol and EDL muscles may be related to resistance to muscle atrophy under weightlessness conditions, such as spaceflight and hindlimb unloading. Prolonged conditions of weightlessness induce marked atrophy in Sol muscles compared with that in EDL, TA, and Gast muscles in rodents [4,5,8,11]. Spermidine content decreased in rat Sol muscles, but significantly increased in EDL

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muscles following 14-d hindlimb unloading [55,56]. In addition, increased spermine content and decreased SMOX protein abundance were observed in atrophied TA muscles by fasting, denervation, and aging in mice [43]. These data suggest that decreases in spermidine/spermine levels were likely to be related to muscle atrophy. It is

oo

f

possible that, in EDL muscles, high SMOX protein abundance might suppress the

pr

decrease in spermidine levels or the increase in spermine levels under weightlessness

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e-

condition, resulting in resistance to muscle atrophy.

5. Conclusions

al

Twenty-eight-day exposure to a 3 g environment increased wet weights of Sol

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and EDL muscles in mice, but this increase was more prominent in Sol muscles. Ingenuity Pathway Analysis for the protein abundance profiles in 1 g and 3 g groups indicated that actin cytoskeleton and integrin signalling in both Sol and EDL muscles was activated. However, the activation of pathways involved in protein synthesis and skeletal muscle maturation, and the inactivation of pathways involved in cell death were more conspicuous in 3 g-exposed Sol than EDL muscles. Additionally, hypergravity exposure significantly

inactivated

glycolysis

in Sol muscles and

oxidative

phosphorylation in EDL muscles. These results suggested that reactivity of molecular

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pathways in Sol and EDL muscles to hypergravity exposure differed. O ur results also demonstrated that the biosynthetic and interconversion pathway of polyamines in Sol and EDL muscles was responsive to hypergravity exposure, and spermidine and spermine contents in each muscle were regulated via different mechanisms by SMOX

oo

f

even in the 1 g group. Highly expressed SMOX protein in EDL muscles contributed to

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the low spermine contents. However, differences in SMOX abundance levels in Sol and

e-

EDL muscles did not affect the increase in spermidine contents following hypergravity

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exposure. Furthermore, our results indicated that the difference in the mechanism regulating polyamine contents is unlikely to have a significant effect on the difference

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in Sol and EDL muscle growth in 3-g exposed mice.

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precursor, Proc. Natl. Acad. Sci. U. S. A. 78 (1981) 2869−2873. https://doi.org/10.1073/pnas.78.5.2869. [50] S.C. Bodine, T.N. Stitt, M. Gonzalez, W.O. Kline, G.L. Stover, R. Bauerlein, E. Zlotchenko, A. Scrimgeour, J.C. Lawrence, D.J. Glass, G.D. Yancopoulos,

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Akt/mTOR pathway is a crucial regulator of skeletal muscle hypertrophy and can

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prevent muscle atrophy in vivo, Nat. Cell Biol. 3 (2001) 1014−1019.

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[51] B.A. Bryan, D. Li, X. Wu, M. Liu, The Rho family of small GTPases: crucial regulators of skeletal myogenesis, Cell. Mol. Life Sci. 62 (2005) 1547−1555.

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https://doi.org/10.1074/jbc.R110.200329. [53] M.H. Stipanuk, Leucine and protein synthesis: mTOR and be yond, Nutr. Rev. 65 (2007) 122−129. https://doi.org/10.1111/j.1753-4887.2007.tb00289.x. [54] L. Wei, W. Zhou, J.D. Croissant, F.E. Johansen, R. Prywes, A. Balasubramanyam, R.J. Schwartz, RhoA signaling via serum response factor plays an obligatory role in myogenic

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https://doi.org/10.1074/jbc.273.46.30287. [55] I.K. Abukhalaf, D.A. von Deutsch, L.E. Wineski, N.A. Silvestrov, S.A. Abera, S.W. Sahlu, D.E. Potter, Effect of hindlimb suspension and clenbuterol treatment on polyamine levels in skeletal muscle, Pharmacology. 65 (2002) 145−154.

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[56] D.A. von Deutsch, I.K. Abukhalaf, L.E. Wineski, N.A. Silvestrov, M.A. Bayorh,

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unloading and clenbuterol treatment, Can. J. Physiol. Pharmacol. 81 (2003) 28−39.

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https://doi.org/10.1139/y02-169.

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Figure Legends Figure 1: Changes in body weight and muscle wet weight in response to 1 g or 3 g exposure for 28 d. Data are presented as the mean ± SD (N = 6). Data of body weight at Day 0 and 28, absolute wet weights of soleus (Sol) and extensor digitorum longus

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(EDL) muscles at Day 28, and relative wet weights of Sol and EDL muscles to body

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weight at Day 28 are shown in A–C, respectively. The amounts of extracted protein in

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SUP-1 and -2 in Sol and EDL muscles are shown in D. *: p < 0.05 vs 1 g group.

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Figure 2: The biosynthetic and interconversion pathway of polyamines.

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Figure 3: Changes in abundance levels of proteins regulating polyamine content in skeletal muscles in response to 1 g or 3 g exposure for 28 d. Results of immunoblots and quantified data of protein abundance levels for spermidine synthase (SRM), spermine synthase (SMS), and spermine oxidase (SMOX) in Sol and EDL muscles at Day 28 are shown in A–C, respectively. Results of immunoblots were cropped and horizontally reversed using Adobe PhotoShop (CS5.1; Adobe Systems Inc, CA, USA) to align the display orders of data sets with the graphs. Raw data of full- length immunoblots are presented in Figure S3A–C. The images separated for Sol and EDL

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muscles were obtained and processed in parallel. Quantified data are presented as the mean ± SD (N = 6). *: p < 0.05 vs 1 g group. See Fig. 1 for other abbreviations.

Figure 4: Changes in polyamine content in skeletal muscles in response to 1 g or 3

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g exposure for 28 d. Data are presented as the mean ± SD (N = 6). Data of putrescine,

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spermidine, and spermine content, and the spermidine/spermine content in Sol and EDL

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muscles at Day 28 are shown in A–D, respectively. *: p < 0.05 vs 1 g group. See Fig. 1

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for other abbreviations.

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Table 1: Biological implications for hypergravity-induced changes in protein abundance profiles in SUP-1 (A) and SUP-2 (B). Effects of 3 g exposure on "canonical pathway" and "diseases and bio function" in Sol and EDL muscles were evaluated by the Ingenuity Pathway Analysis. P values in the Ingenuity Pathway

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Analysis indicated the coverages of components in each index. The activation or

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inactivation of each index was presumed based on z score. Z score was calculated by

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comparing alteration pattern of abundance levels of proteins engaging in each signalling

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pathway with the database. *: P values (< 0.05) and z score (> 2.0) were considered significant. Significantly activated and inactivated indices in red and blue, respectively.

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Sol

Canonical Pathway

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A

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N.D.: not determined.

14-3-3-mediated signalling Actin cytoskeleton signalling Apelin adipocyte signalling pathway BAG2 signalling pathway

P value

EDL Z score

−6

*4.57×10

−7

2.24

*1.02×10

2.18

*1.55×10−2

2.24

−3

*1.15×10

−2

2.24

Cdc42 signalling

*4.37×10

2.45

Fatty acid β-oxidation I

1.23×10−1

N.D.

−9

P value 1.73×10

Z score

−1

*8.51×10

−3

2.04×10

−3

*3.24×10−8 −4

−2.83

3.89×10

Glutaryl-CoA degradation

2.64×10−1

N.D.

*2.95×10−8

*5.89×10

−4

−2.83

8.32×10

−3 −3

Integrin signalling

*1.45×10

2.31

*7.76×10

Isoleucine degradation I

3.24×10−2

N.D.

*1.95×10−10

Oxidative phosphorylation PI3K/AKT signalling

−3

*2.95×10 *4.57×10−2 45

2.65 2.65

N.D.

N.D.

*8.71×10

Glycolysis I

2.24

N.D.

Gluconeogenesis I

−9

N.D.

2.24×10 3.80×10

−2 −3

−2.45 N.D. −2.24 N.D. 2.24 −2.45 N.D. 1.34

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2.24×10−2

PPARα/RXRα activation Regulation of actin-based motility by Rho RhoA signalling

−6

*5.25×10 *2.51×10−6 −5

*1.20×10

Signalling by Rho family GTPases

−2

TCA cycle II (Eukaryotic) tRNA charging

2.83 3.46 3.32

6.92×10

N.D.

*7.41×10−5

2.45

−1

Tryptophan degradation III (Eukaryotic) Unfolded protein response

0.38

3.56×10

N.D.

−8

*7.24×10

−4

−1.00

3.16×10

1.78×10 5.62×10 1.69×10

−3 −3 −1

*2.19×10

−7

−2.45 2.00 2.00 N.D. −2.24

N.D.

*2.19E-07 4.37×10

−3

*5.01×10

−14

−2.24 N.D. −2.83

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Valine degradation I

2.65

*7.76×10−4

Sol

Diseases and Bio Functions

Z score

P value

*4.71×10

−4

−2.11

N.D.

*4.72×10−4 *1.46×10−4

−3.46

N.D.

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P value

EDL

−3.46

N.D.

−4

−3.16

N.D.

*1.27×10−5

−2.89

N.D.

*2.68×10

−4

−2.89

N.D.

Cell movement of embryonic cells

*8.52×10−3

2.43

N.D.

Death of embryo

*5.84×10−3

−2.36

N.D.

*9.59×10

2.42

N.D.

*4.16×10−3

2.06

Cell death of cancer cells Cell death of malignant tumor Cell death of osteosarcoma cells Cell death of tumor

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Cell death of tumor cells

Endocytosis

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Endocytosis by eukaryotic cells Engulfment of cells

Genitourinary tumor

Immune response of cells Lung cancer

Morbidity or mortality Necrosis Necrosis of tumor Organismal death Phagocytosis of cells

*2.00×10

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Apoptosis

−4

−3

*3.14×10

4.79×10−2

2.59

N.D.

*1.08×10

2.41

N.D.

*9.75×10−3

2.01

N.D.

−6

−2

*1.43×10

2.18

3.72×10−2

*2.77×10

−3

−6.10

*1.11×10−6

−2.20

−5

−2.89

N.D.

−3

−6.09

N.D.

2.55

N.D.

*3.67×10 *2.60×10

−2

*1.88×10

B

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Z score

−0.28

1.97

N.D. 3.83×10

−5

0.65

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Sol

Canonical Pathway P value

Z score

−11

*3.98×10

Actin cytoskeleton signalling Apelin cardiomyocyte signalling pathway

−5

*4.27×10

−2

*1.17×10 *4.27×10−4

Cardiac hypertrophy signalling Cdc42 signalling

−2

*2.04×10

CXCR4 signalling

−12

*3.98×10 *3.24×10−6

Oxidative phosphorylation

3.55×10

−4

*1.45×10 *1.91×10−7

−10

RhoA signalling

Signalling by Rho family GTPases

1.71×10−1

2.24 2.45 2.11

2.25×10 2.69×10 2.63×10

−1 −2 −2

2.45 2.33

*1.95×10 4.03×10 8.13×10

2.67

1.38×10

*1.91×10−5 *1.00×10−7

−3.16

7.08×10

−2

*1.82×10

3.87 2.65

1.00×10 2.30×10

−10

−1 −2 −1 −2 −2 −1

Z score 2.00 N.D. N.D. N.D. N.D. N.D. 2.00 −3.16 N.D. N.D. N.D. N.D. N.D. N.D.

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Synaptogenesis signalling pathway

−1

2.12

*5.01×10

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RhoGDI signalling

1.00×10

−1

3.65×10

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Regulation of actin-based motility by Rho

2.88×10

−2

2.65

−0.38

−3

PAK Signalling

2.12

P value

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Integrin signalling

2.67

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EIF2 signalling

EDL

Apoptosis

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Diseases and Bio Functions

Cell death of cancer cells Cell death of muscle cells Cell death of osteosarcoma cells Cell death of tumor cells Contractility of cardiac muscle Contractility of ventricular myocardium

Sol P value

EDL Z score

−3

P value

*3.32×10

−2.99

N.D.

*6.02×10−12

−2.56

N.D.

−14

−4.69

N.D.

−5

−1.83

*2.62×10 1.47×10

*1.26×10−4

−17

−4.58

−12

−4.79

N.D.

2.54

N.D.

*3.98×10 *2.21×10

*3.34×10−9 −7

*7.64×10

6.01×10

−3

2.79

N.D.

Damage of heart

*1.33×10

−3

−2.19

N.D.

Death of embryo

*2.12×10−3

−2.83

N.D.

Function of muscle Motor dysfunction or movement disorder Movement disorders Necrosis Necrosis of muscle

−11

*4.47×10

2.42

4.92×10

−5

*1.14×10

−3

−2.33

N.D.

*3.61×10−3

−2.51

N.D.

−10

*2.12×10

−4.51

*5.80×10−6

−2.03

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1.15×10

−3

N.D.

Z score

−2.60 −1.00

N.D.

−1.69

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*8.50×10−14

Necrosis of tumor

−4

*1.03×10

−6.25

2.15×10−2

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−2

*2.93×10

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Organismal death

−4.79

−1.00 −2.27

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Table 2: List of hypergravity-responsive proteins identified in SUP-1 (A) and SUP-2 (B) by proteomic analysis. Proteins were selected based on p values (< 0.01) and fold changes (> 2.0). A Sol

Q8CI43

P value

Fold change

f

Increased P50462

Description (Gene name)

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Accession

Cysteine and glycine-rich protein 3 (Csrp3) Myosin light chain 6B (Myl6b)

4.41 × 10−8 6.35 × 10

−6 −6

2.91 2.89

Heat shock 70 kDa protein 1B (Hspa1b) BAG family molecular chaperone regulator 3 (Bag3)

9.77 × 10 2.51 × 10−5

2.26 2.05

Q9R0P9

Ubiquitin carboxyl-terminal hydrolase isozyme L1 (Uchl1)

3.58 × 10−5

2.63

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P17879 Q9JLV1

−5

Annexin A1 (Anxa1) Leukocyte elastase inhibitor A (Serpinb1a)

5.55 × 10 7.52 × 10−5

2.20 4.03

P30412 O70373

Peptidyl-prolyl cis-trans isomerase C (Ppic) Xin actin-binding repeat-containing protein 1 (Xirp1)

1.10 × 10−4 2.57 × 10−4

2.62 2.01

6.75 × 10−4

2.19

9.69 × 10−4

2.55

Q4VAA2 P19096 P49813 Decreased Q0P5V9

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(Tppp3) Elongation factor 1-alpha 1 (Eef1a1)

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P10126

Tubulin polymerization-promoting protein family member 3

Protein CDV3 (Cdv3)

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Q9CRB6

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P10107 Q9D154

3.12 × 10

−3 −3

2.03

Fatty acid synthase (Fasn) Tropomodulin-1 (Tmod1)

6.76 × 10 7.19 × 10−3

2.43 2.42

Solute carrier family 45 member 4 (Slc45a4)

1.64 × 10−4

2.01

−4

P70695 P24549

Fructose-1,6-bisphosphatase isozyme 2 (Fbp2) Retinal dehydrogenase 1 (Aldh1a1)

9.88 × 10 2.30 × 10−3

2.09 2.19

P32848

Parvalbumin alpha (Pvalb)

2.35 × 10−3

2.73

Description (Gene name)

P value

Fold change

Spermine oxidase (Smox)

2.01 × 10−3

4.40

EDL Accession Increased Q99K82

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B Sol Accession

Description (Gene name)

P value

Fold change

P50462 P09541

Cysteine and glycine-rich protein 3 (Csrp3) Myosin light chain 4 (Myl4)

3.11 × 10−7 3.42 × 10−6

2.30 3.35

Q9DCN2

NADH-cytochrome b5 reductase 3 (Cyb5r3)

1.68 × 10−5

2.32

Increased

−5

Heat shock 70 kDa protein 1B (Hspa1b) Microtubule-associated protein tau (Mapt)

2.39 × 10 5.23 × 10−5

2.71 2.81

Q7TMM9

Tubulin beta-2A chain (Tubb2a) Musculoskeletal embryonic nuclear protein 1

8.24 × 10−5

2.16

1.56 × 10−4

2.12

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Q99JI1

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P17879 P10637

(Mustn1)

Fibulin-5 (Fbln5) Myosin light chain 6B (Myl6b)

1.74 × 10−4 1.91 × 10−4

2.06 2.01

Q80XB4

Nebulin-related-anchoring protein (Nrap)

3.00 × 10−4

2.29

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Q9WVH9 Q8CI43

−3

Elongation factor 1-alpha 1 (Eef1a1) Actin-related protein 3 (Actr3)

1.18 × 10 1.37 × 10−3

2.11 2.24

Q9EQK5 P62270

Major vault protein (Mvp) 40S ribosomal protein S18 (Rps18)

1.37 × 10−3 5.62 × 10−3

2.50 2.07

2.84 × 10−6

2.44

O88990 Q9D9K3

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Q8C494

PDZ and LIM domain protein 7 (Pdlim7) Proline-rich protein 33 (Prr33)

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Decreased Q3TJD7

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P10126 Q99JY9

Alpha-actinin-3 (Actn3) Cell death regulator Aven (Aven)

50

2.96 × 10

−3 −3

3.03 × 10 6.81 × 10−3

2.80 2.10 8.59

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Fundings This study was supported by funding provided by the Japan Aerospace Exploration Agency (JAXA) for space biomedical research projects to H.M., Japan Society for the Promotion of Science KAKENHI Grant Numbers JP16K18994 to T.O., JP16K19210 to

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S.M., JP16K08957 to K.H., JP19H03774 to Y. Kimura, and the Special Coordination

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Fund for Promoting Science and Technology, “Creation and Innovation Centres for

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Advanced Interdisciplinary Research Areas” to H.H. from the Ministry of Education,

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Culture, Sports, Science and Technology/Japan Science and Technology Agency. The sources of funding had no role in the study design, collection of data, analyses, decision

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to publish, or preparation of the manuscript. The MS analyses were performed at

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Clinical Mass Spectrometer Platform (Yokohama City University) supported by the Ministry of Education, Culture, Sports, Science and Technology, Japan.

Acknowledgements The authors wish to thank Ms. Kayano Moriyama, and Dr. Shinsuke Kataoka (Yokohama City University), and Dr. Chie Matsuda, Dr. Satoshi Furukawa (JAXA) for their support of this work. We also want to thank Dr. Hideharu Taira, Dr. Keijiro Samejima (Tokyo Metropolitan Institute of Medical Science), and Dr. Chikara Abe, Dr.

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Koji Obata (Gifu University Graduate School of Medicine) for their technical assistance. Finally, we would like to thank Professor Clive S. Langham (Nihon University School of Dentistry) and Editage (www.editage.jp) for English language editing.

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Author contributions

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T.O., I.Y., Y. Kimura, and H.H. contributed to conception and design of the current

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study; H.M. reared mice; T.O., Y.N., M.K., and K.E. collected samples; Y.I, Y.N., A.K.,

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Y. Kurata, H.K., Y. Kimura, and H.H. performed proteomic and immunoblot analysis; Y.I. Y.N., and Y. Kimura deposited proteomics data; T.O., S.M., K.H., and M.K.

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measured polyamine contents in skeletal muscles; T.O. performed statistical analysis;

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T.O., Y.I., Y. Kimura, and H.H. interpreted results; T.O. and Y.I. prepared figures and tables; T.O., Y.I., Y. Kimura and H.H. drafted the manuscript; a ll authors revised and approved the final version of the manuscript.

Competing interests We declare that none of the authors have competing financial or non-financial interests.

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Significance The skeletal muscle-specific protein abundance profiles result in differences in the characteristics of slow and fast skeletal muscles. We investigated differences in the profiles in mouse slow-twitch Sol and fast-twitch EDL muscles following 28-d of 1 g

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and 3 g exposure by LC-MS/MS analysis and label-free quantitation. A two-step

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solubilisation of the skeletal muscle proteins increased the coverage of proteins

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identified by LC-MS/MS analysis. Additionally, this method reduced the complexity of

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samples more easily than protein or peptide fractionation by SDS-PAGE and offline HPLC while maintaining the high operability of samples and was reproducible. A larger

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number of hypergravity-responsive proteins as well as a prominent increase in the wet

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weights was observed in Sol than EDL muscles. The biological implications of the difference in the protein abundance profiles in 1 g and 3 g groups revealed that the reactivity of each molecular pathway in Sol and EDL muscles to hypergravity exposure differed significantly. In addition, we found that the biosynthetic and interconversion pathway of polyamines, essential factors for cell growth and survival in mammals, was responsive to hypergravity exposure; spermidine and spermine contents in Sol and EDL muscles were regulated by different mechanisms even in the 1 g group. However, our results indicated that the difference in the mechanism regulating polyamine contents is

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unlikely to have a significant effect on the differences in Sol and EDL muscle growth

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following hypergravity exposure.

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Highlights ·

Proteins in Sol and EDL muscles were extracted by a two-step solubilisation method. Protein abundance profiles in each muscle changed following 28-d exposure to 3 g.

·

Pathways involved in protein synthesis were activated prominently in Sol muscles.

·

Pathways involved in oxidative phosphorylation were inactivated in EDL muscles.

·

Polyamine contents in Sol and EDL muscles were regulated differently via SMOX.

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·

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Figure 1

Figure 2

Figure 3

Figure 4