Ausrine Bartasyte
email: ausrine.bartasyte@gmail.com

El R Bouregba

Hadj Dogheche

Michel Boudard

G Poullain

Carmen Jiménez

V Plausinaitiene

Denis Remiens

Adulfas Abrutis

François Weiss

E Dogheche

C Jimenez

O Chaix-Pluchery

Z Saltyte

Ferroelectric PbTiO3 films grown by pulsed liquid injection MOCVD

Keywords: MOCVD, PbTiO 3 films, ferroelectric and piezoelectric properties

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Introduction

Ferroelectric PbTiO 3 (PTO) and PbTi 1-x Zr x O 3 (PZT) thin films are extensively studied since their properties offer a great promise for various applications [1]. High-quality epitaxial ferroelectric films are needed in prospect of new devices; moreover, ferroelectrics can be combined with other functional oxides, such as high temperature superconductors and magnetic oxides. Heterostructures of ferroelectrics with manganites (La 1-x Sr(Ca) x MnO 3 ) exhibiting colossal magnetoresistance are of interest due to the possibility of achieving electric-field-tuned metal-insulator phase transitions, an idea that is being explored for applications based on oxide channel field-effect transistors operating at room temperature [2]. For some applications, epitaxial ferroelectric films must be grown on electrode/substrate. Stresses in such lattice mismatched heterostructures significantly influence both the structural and electrical properties of the films [3]; therefore residual stresses should be evaluated in addition to electrical measurements.

Epitaxial PTO films have been grown by metalorganic chemical vapour deposition (MOCVD) [4,5], pulsed laser deposition [6] and rf-magnetron sputtering [7]. Among various deposition methods, MOCVD has been recognized as the most promising technique due to simple apparatus, excellent film uniformity, good control of composition and possibility to grow films on large area with high growth rate and good conformal step coverage. Most of PTO film studies are related to structural properties; some papers deal with ferroelectric and dielectric properties [8][9][10][11][12] and a few ones about piezoelectric properties are available [11,12].

In the present paper, we report on the role of system pressure, solution concentration and injection frequency on the growth rate and composition of PbTiO 3 films grown by pulsed liquid injection MOCVD. Further, the Pt/PbTiO 3 /La 1-x Sr x MnO 3 /LaAlO 3 (Pt/PTO/LSMO/LAO) heterostructures were grown and their microstructure, morphology and electrical properties were studied and discussed.

Experimental details

Film depositions were carried out in a vertical hot wall pulsed injection MOCVD reactor. This technique is largely described elsewhere [START_REF] Sénateur | Innovative processing of films and nanocrystalline powders[END_REF]. A computer controlled injector repeatedly injects microdroplets of organic solution containing a mixture of metalorganic precursors into a hot evaporation zone. After flash evaporation of microdroplet, resulting vapour is carried out to deposition zone by an Ar + O 2 gas flow. Deposition conditions of PTO and LSMO films are summarized in Table 1. More details and discussion on the influence of solution composition, deposition temperature, oxygen partial pressure and film annealing in different atmospheres on PTO film properties can be found in previous paper [START_REF] Bartasyte | [END_REF]. Details about deposition, structural and physical properties of LSMO films are given in Ref. 15.

Film composition was determined by wavelength dispersion spectroscopy (WDS) using Cameca Sx50. The film texture and microstructure were studied by XRD using a SIEMENS D5000 4-circle diffractometer with monochromatic CuK a radiation (l=0.15418 nm). An analysis of the microstructure was carried out through standard w-, c-, j-scans. More details concerning this analysis can be found in Ref. [START_REF] Bartasyte | [END_REF]. Percentage of a-axis oriented domains in the films was calculated by peak integration of (102) reflection in j-scans measured at different angles, corresponding to a-and c-axis oriented crystallites (i.e. a-and c-domains, respectively) [START_REF] Bartasyte | [END_REF]. The c-parameter of PTO c-domains was calculated from the position of three (00l) peaks in q/2q scans. 3D mapping of the reciprocal space was performed with an automatic Nonius Kappa CCD diffractometer (Mo K a radiation, l= 0.7071 Å). Film morphology was examined by atomic force microscopy (AFM) using a Digital Instruments Multimode Scanning Probe Microscope, in the taping mode. Thickness of films was determined from cross-section images obtained by SEM Philips XL30.

Raman spectra were collected using Jobin Yvon/Horiba Labram spectrometer equipped with liquid nitrogen cooled CCD detector. Experiments were conducted in the micro-Raman mode at room temperature, in the backscattering geometry. The 514.5 nm line of an Ar + laser was focused to a spot size smaller than 1 µm. Spectra from different experiments were calibrated using Si spectra at ambient temperature.

To characterise the ferroelectric properties of PTO films from an electrical point of view, 235 x235 mm 2 square capacitors were designed by UV photolithography using a metallic shadow, and then top contacts were patterned by lift off. Ferroelectric hysteresis loops were observed using a Sawyer-Tower circuit driven at 50 kHz by a sine wave. Capacitance-voltage (C-V) curves and dielectric constants e r were determined from small signal capacitance measurements performed at 50 kHz with a HP Agilent LCZ meter. Piezoelectric coefficients d 33 were measured by laser doppler vibrometry using a Polytec OFV512 as described in Ref. 16.

Results

Effects of film growth rate

As shown in our previous paper [START_REF] Bartasyte | [END_REF], Pb fraction in the film grown at higher temperatures (≥600°C) increases until near stoichiometric value and after remains almost stable with further increase of the Pb content in the solution; thus, Pb excess cannot be reached in the film. In order to observe the maximum Pb/Ti ratio in the film which can be reached by varying the growth rate, lead deficient films were studied. Series of PTO films were deposited at 600 ˚C by varying the total solution concentration (0.02 M, 0.04 M or 0.08 M) and injection frequency ( 1Hz or 2 Hz) while the precursors'ratio in solution (Pb/(Pb+Ti)=28.1%) and other deposition conditions were kept constant as given in Table1. The variation of film composition with growth rate is shown in Fig. 1. Pb content in the film increases with increasing growth rate from 4.9 to 10.9 nm/min and saturates when sufficiently high growth rates (>10.9 nm/min) are reached.

Deposition with low growth rate results in high Pb desorption from the film. For high growth rates, Pb loss highly decreases and film composition becomes defined mainly by Pb/Ti ratio in solution and precursor's decomposition rates at deposition temperature. In order to obtain Pb/Ti=1 in the films grown with rates >10.9 nm/min, Pb/(Pb+Ti) ratio in the precursor solution should be increased from 28.1% to 31.3% or more. The 0.04 M total concentration and 2 Hz injection frequency were chosen as optimal conditions for further deposition investigation.

Effects of deposition pressure

In order to test the influence of deposition pressure on PTO film growth, films were grown at 650 ˚C at three different pressures (2, 5 and 10 Torr). The surface of layers deposited at 2 Torr of pressure was completely covered by spherical particles, probably due to precursor decomposition in the reactor volume, thus this pressure was discarded from further investigation.

Stoichiometric film was reached using 0.020 M and 0.023 M Pb(thd) 2 concentrations (at 0.04 M total concentration of solution) for depositions carried at 5 torr and 10 Torr pressure, respectively.

The growth rates of stoichiometric PTO films at 5 Torr and 10 Torr were 20.6 nm/min and 18.8 nm/min, respectively. P. Lu et al. also reported a growth rate decrease of PZT films with increasing pressure [17]. As lower growth rate enhances Pb desorption, higher concentration of lead precursor was needed to obtain stoichiometric PTO at higher pressures.

The results of our present and previous studies on PTO deposition suggest the possibility to control accurately the film growth rate and film composition is important for reproducing stoichiometric PTO films. Therefore, PI MOCVD is a promising method as it offers an easy control of precursor feeding rate, vapour and film composition. Film surface roughness was evaluated by AFM. Higher roughness of PTO films in heterostructure (Ra = 4.3 nm), in comparison with PTO films on LAO (Ra = 0.96 nm), may result from LSMO film roughness (Ra = 2.6 nm). Roughness values are comparable to reported ones for similar heterostructures grown by pulsed laser deposition [19].

PbTiO 3 /La

Residual stresses

Polarized Raman spectra of PTO/LSMO/LAO heterostructure measured in crossed polarization configuration (VH) confirmed the presence of pure tetragonal PTO phase (Fig. 3), as only E(TO) modes are observed [20]. PTO E(1TO) soft mode is very pressure sensitive and, as a consequence, its wavenumber shifts as a function of increasing pressure according to the following relation:

w[E(1TO)] = w 0 + (∂w /∂P)P (1)
where w 0 = 89cm -1 is the soft mode wavenumber at zero pressure and room temperature, and ∂w /∂P = -5.8cm -1 GPa -1 is the pressure coefficient [21,22]. The shift value is used to evaluate the residual stress s in PTO films. PTO E (1TO) mode was observed at 81 cm -1 for PTO/LSMO/LAO and at 86.6 cm -1 for PTO/LAO. These values, as well as all other E(TO) mode wavenumbers are quite below those observed in single crystal (89 cm -1 for E(1TO)). This gives evidence that PTO films on LSMO/LAO and on LAO are under tensile stress in-plane in agreement with XRD results. Corresponding stress values calculated from Eq. 1 are closed to 1.38 and 0.41 GPa in PTO/LSMO/LAO and PTO/LAO, respectively. Despite bigger misfit strain in PTO/LAO system, PTO films on LSMO/LAO were more stressed than PTO/LAO films probably due to distortion induced by rhombohedral structure of LSMO.

Electrical properties

Ferroelectric properties of PTO films on LSMO/LAO were characterized by measuring polarization hysteresis (Fig. 4a). The films displayed hysteresis loops typical of ferroelectric materials for an applied voltage of 6 V. Remanent polarization and average coercive field values were 2.4 mC/cm 2 and 65 kV/cm, respectively. Quite low remanent polarization values comparing to reported ones [8,9,11] can be due to incomplete reversal of the switching domains [11].

However, increasing the applied voltage up to 10 V led to inflating loops typical of non linear lossy dielectrics [START_REF] Lines | Principles and Applications of Ferroelectrics and Related Materials[END_REF]. The saturation of polarization was not achievable and it was indeed difficult to reach the coercive field due to the leakage currents. The coercive fields are expected to be rather high (>100 kV/cm) due to the strain in the film resulting from epitaxial growth. This fact combined with the leakage current made difficult to observe large polarization in the range of applied voltage. Deposition of stoichiometric PTO films on LSMO/LAO at 10 Torr resulted in films characterized by elliptical hysteresis loops, which is characteristic of excessive leakage currents in the film [START_REF] Bouregba | [END_REF]. Hence, leakage current can be reduced by decreasing lead desorption and then probably the resulting defects.

PTO film deposited at lower pressure displayed capacitance-electric-field curve (Fig. 4b) typical of ferroelectric materials. The dielectric constant of this film reached 325, which is higher than reported values for bulk materials (210 perpendicular to and 120 along polarization direction [25,26]) and than values published by many authors for PTO films [8][9][10]. However, Kighelman et al. measured dielectric constant higher than 300 [11]. Higher dielectric constant in our films in comparison with bulk value may be consistent with an increased number of domain walls and with film stresses. Raman spectroscopy results confirmed that PTO films on LSMO/LAO are under tensile stress in the plane parallel to the substrate, despite negative misfit stress. Unlike our results, Haeni et al. reported enhancement of dielectrical permittivity of SrTiO 3 films in case of positive misfit strain but information about stresses was not specified [27]. Dielectric losses (tga = 0.02) obtained in our films were similar to the reported values.

Using the procedure described in Ref. 16 for an accurate measurement, a small top electrode and a clamped sample have been used to reduce the substrate bending and eliminate the resonant effects. We can illustrate the evolution of the piezoelectric properties of PTO thin films in Fig. 4c. The d 33 versus E dc loop, measured by laser Doppler vibrometry, was rectangular, with some asymmetry. The piezoelectric coefficient d 33 measured in PTO films was around 50 pm/V (V ac = 1 V, 48.5 kHz), which is comparable to values reported for PTO films [11] and even for Pb 1-x Ca x TiO 3 or PZT films [28,29]. The asymmetry of piezoelectric and ferroelectric hysteresis and C-V loop resulted from the different nature of bottom (LSMO) and top (Pt) electrodes. In fact, the film properties are correlated to the film orientation and control of the film growth results in optimum piezoelectric activity.

Conlusions

The growth rate and composition of PTO films were studied as a function of deposition pressure, solution concentration and injection frequency. It was shown, that Pb desorption can be governed by changing film growth rate. Epitaxial PTO/LSMO/LAO films were elaborated at optimized deposition conditions. PTO films in such heterostructure were under tensile stress inplane; they showed piezoelectric activity and very good dielectric properties, but remanent polarization values were quite low.

Figure captions

Fig. 1: PTO/LAO film composition evolution as a function of growth rate.

3. 3 . 1 .

 31 Microstructure and surface roughness XRD 2D reciprocal space mapping of this heterostructure (Fig.2a) shows successive epitaxial growth of probably rhombohedral LSMO and tetragonal PTO on LAO substrate along the [00l] direction (referred to pseudo-cubic settings for LSMO). More precise investigations are needed to confirm rhombohedral LSMO structure. Weak reflections in Fig.2bindicate the presence of PTO crystallites with a-axis oriented almost perpendicular to the substrate plane (adomains). The c-parameter (4.111±0.005 Å) of c-domains was reduced compared to the bulk value (4.1532 Å), indicating that PTO film is under tensile stress in-plane. As it is known, the aand c-domains are bounded by the (101) twin plane and this results in a tilt of domains by angle from the substrate plane normal depending on the cell tetragonality and domains fraction[18].The split into four peaks (Fig.2b) confirms the existence of twinning resulting in tilt of adomains by ~ 2 ˚ angle with respect to the substrate plane. No split was observed for the central reflection in Fig.2ccorresponding to c-domains of PTO. The volume fraction of a-domains in PTO film was 13.7 %. FWHM of PTO and LSMO rocking curves were 0.64 ˚ and 0.22 respectively, indicating a good texture quality of the heterostructure. Heterostructure has good inplane orientation, as FWHM of PTO and LSMO j-scans were 1.7° and 1° respectively.

Fig. 2 :

 2 Fig. 2: 2D sections of the reciprocal space of a PTO/LSMO/LAO structure: (a) k = 0 plane; (b) l = -3.9 plane; (c) l = -3.7 plane.

Fig. 3 :

 3 Fig. 3: Polarized Raman spectra of PTO/LAO, PTO/LSMO/LAO and LSMO/LAO films measured in VH polarization configuration.

Fig. 4 :

 4 Fig. 4: Electrical characterizations of PTO/LSMO/LAO films: (a) polarization hysteresis loop ; (b) capacitance-electric-field curve ; (c) piezoelectric displacement versus E dc loop.

Fig. 1

 1 Fig. 1

Fig. 2

 2 Fig. 2

Fig. 3

 3 Fig.3

Fig. 4

 4 Fig. 4

  1-x Sr x MnO 3 /LaAlO 3 heterostructures PTO/LSMO heterostructures consisting of stoichiometric 300 nm thick PTO film and 200 nm LSMO film were grown on LAO at 650 ˚C and 5 Torr pressure using optimal conditions described above and given in Table1. The microstructure, surface roughness, residual stresses and electrical properties of the PTO/LSMO/LAO heterostructure are presented and discussed below.

Table 1 :

 1 Deposition conditions for PbTiO 3 and La 1-x Sr x MnO 3 layers by PI MOCVD PbTiO 3 La 1-x Sr x MnO 3

	Substrates	LaAlO 3 (001) in a pseudo-cubic setting	LaAlO 3 (001) in a pseudo-cubic setting
	Substrate temperature (°C)	600 or 650	800
	Evaporation temperature (°C)	280	310
	Transport gas	Ar +O 2	Ar + O 2
	Total gas flow rate (l/h)	60	95
	Oxygen fraction (%)	37.7	35
	Total pressure (Torr)	2-10	5
	Precursors*	Pb(thd) 2 ,Ti(thd) 2 (O i Pr) 2	La(thd) 3 , Mn(thd) 3 , Sr(thd) 2
	Solvent	toluene	monoglyme
	Solution concentration (total)	0.02-0.08	0.049; with proportion of the
	(mol/l)		precursors: Sr(thd) 2 :La(thd) 3 :Mn(thd) 3
			= 1:1.5:1.58
	Injection frequency (Hz)	1or 2	2
	Thickness (nm)	200-340	200

* All precursors were synthesized in Vilnius University.


Kevin Nay

David J R Martin

Luz Orfila

Dany Saligaut

Brice Martin

Mathieu Horeau

Thibaut Cavey

Moussa Kenawi

Marie-Laure Island

Martine Ropert

Kévin Nay

Olivier Loréal

Christelle Koechlin-Ramonatxo

Frédéric Derbré

PhD Frederic Derbré
email: frederic.derbre@univ-rennes2.fr

Intermittent reloading does not prevent iron availability decrease and hepcidin upregulation caused by hindlimb unloading

Keywords: disuse, physical inactivity, crosstalk, myosin heavy chain, iron overload

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INTRODUCTION

During spaceflights, the astronauts' physical function is decreased, leading to reduced capacity to work, ability to carry out missions, and also complications during recovery. Lack of muscle activity due to microgravity considerably contributes to this functional decline through reduction of muscle mass and strength, impairment of cardiovascular performance, loss of bone mineral density, and also anemia [START_REF] Santo | Anemia and erythropoietin in space flights[END_REF][START_REF] Pavy-Le Traon | From space to Earth: advances in human physiology from 20 years of bed rest studies (1986-2006)[END_REF][START_REF] Pierre | From physical inactivity to immobilization: Dissecting the role of oxidative stress in skeletal muscle insulin resistance and atrophy[END_REF]. Astronauts and bedridden subjects also exhibit higher serum ferritin levels and lower serum iron and transferrin levels suggesting that microgravity increases iron store and reduces iron availability [START_REF] Smith | The nutritional status of astronauts is altered after long-term space flight aboard the International Space Station[END_REF][START_REF] Morgan | Bone metabolism and nutritional status during 30-day head-down-tilt bed rest[END_REF]Zwart et al., 2013). These iron metabolism alterations could play a key role in the appearance of anemia due to lower iron availability for erythropoiesis, but also in muscle atrophy and osteoporosis, because iron overload promotes oxidative stress in these organs through the Fenton reaction [START_REF] Reardon | Iron injections in mice increase skeletal muscle iron content, induce oxidative stress and reduce exercise performance[END_REF][START_REF] Tsay | Bone loss caused by iron overload in a murine model: importance of oxidative stress[END_REF].

Iron metabolism is mainly controlled by hepcidin, a 25 amino-acid peptide hormone that is secreted by liver and inhibits iron export through the degradation of the iron exporter ferroportin [START_REF] Zhang | Control of Systemic Iron Homeostasis by the Hemojuvelin-Hepcidin Axis12[END_REF]. In addition, hepcidin reduces iron absorption by duodenal enterocytes. Consequently, hepcidin induces a decrease of blood iron availability and an increase of iron storage in cells that express ferroportin (i.e., macrophages and enterocytes).

Although previous studies reported that serum ferritin levels are higher in astronauts and bedridden patients, indicative of increased iron storage [START_REF] Smith | The nutritional status of astronauts is altered after long-term space flight aboard the International Space Station[END_REF][START_REF] Morgan | Bone metabolism and nutritional status during 30-day head-down-tilt bed rest[END_REF]Zwart et al., 2013), the organs where iron accumulates remain unclear. Using the hindlimb unloading (HU) model to simulate microgravity in rats, we recently reported that . short-term exposure to microgravity (i.e., 7 days) decreases transferrin saturation and concomitantly increases iron concentration in spleen, but not in liver [START_REF] Cavey | Simulated microgravity decreases circulating iron in rats: role of inflammation-induced hepcidin upregulation[END_REF].

These changes were associated with increased hepcidin synthesis in liver [START_REF] Cavey | Simulated microgravity decreases circulating iron in rats: role of inflammation-induced hepcidin upregulation[END_REF], suggesting that this hormone could play a key role in iron distribution alterations in conditions of microgravity.

Hepcidin is mainly upregulated in response to: 1) iron accumulation in the liver that activates the bone morphogenetic protein/SMAD (BMP/SMAD) signaling pathway; 2) inflammation that activates the interleukin-6/Janus kinase 2/signal transducer and activator of transcription 3 (IL-6/JAK2/STAT3) signaling pathway; 3) increase of transferrin-bound iron that activates ERK1/2 [START_REF] Ramey | Cross-talk between the mitogen activated protein kinase and bone morphogenetic protein/hemojuvelin pathways is required for the induction of hepcidin by holotransferrin in primary mouse hepatocytes[END_REF][START_REF] Zhao | Iron regulation by hepcidin[END_REF]. In agreement, after 7 days of HU, we observed an increase of STAT3 activation in liver combined with Il6 mRNA upregulation in atrophied rat skeletal muscle [START_REF] Cavey | Simulated microgravity decreases circulating iron in rats: role of inflammation-induced hepcidin upregulation[END_REF]. Similarly to physical exercise known to upregulate hepcidin synthesis through an IL-6-dependent mechanism [START_REF] Banzet | Interleukin-6 contributes to hepcidin mRNA increase in response to exercise[END_REF], these data suggest that microgravity early induces systemic pro-inflammatory state promoting hepcidin synthesis. They also raise questions about the transient or permanent nature of this physiological response to microgravity. As simulated microgravity increases IL-6 levels in atrophied muscle both in humans and rodents [START_REF] Kang | Muscle immobilization and remobilization downregulates PGC-1α signaling and the mitochondrial biogenesis pathway[END_REF][START_REF] Kwon | MyD88 regulates physical inactivityinduced skeletal muscle inflammation, ceramide biosynthesis signaling, and glucose intolerance[END_REF][START_REF] Cavey | Simulated microgravity decreases circulating iron in rats: role of inflammation-induced hepcidin upregulation[END_REF], these findings finally suggest that IL-6 could be involved in a muscle-to-liver crosstalk to regulate iron metabolism.

Intermittent muscle reloading (IR) during HU is considered an efficient strategy to limit muscle mass loss as well as reduction in fiber cross-sectional area (CSA) and muscle strength in rodents [START_REF] Thomason | Activity influences on soleus muscle myosin during rodent hindlimb suspension[END_REF][START_REF] Hauschka | Periodic weight support effects on rat soleus fibers after hindlimb suspension[END_REF][START_REF] D'aunno | Centrifugal intensity and duration as countermeasures to soleus muscle atrophy[END_REF][START_REF] D'aunno | Intermittent acceleration as a countermeasure to soleus muscle atrophy[END_REF][START_REF] Bangart | Effect of intermittent weight bearing on soleus fiber force-velocity-power and force-pCa relationships[END_REF]. By modulating muscle plasticity, this experimental model constitutes an interesting tool to explore the role of skeletal muscle on iron metabolism regulation during simulated microgravity. Therefore, the aim of this study was to determine 1) whether medium-term HU affects iron distribution and hepcidin synthesis, and 2) whether IR (1h/day) could limit iron distribution alterations during HU by targeting skeletal muscle atrophy.

.

MATERIAL AND METHODS

Ethical approval

The experimental protocol and procedures used in this study were approved by the Rennes committee on Ethics in Research (authorization 8585-2017011709503125) and by the French Ministry of Higher Education.

Animals and experimental procedures

Male Wistar rats (7-week-old; Janvier Labs, Le Genest St Isle, France) were housed in a temperature-controlled room (21 ± 2°C) with a 12h:12h light/dark cycle and received standard rodent chow and water ad libitum. After 1 week of acclimatization, animals were randomly assigned to three experimental groups: non-suspended (control, n = 12), hindlimb unloading (HU, n = 12), and hind limb unloading with 1h reloading per day (HU-IR, n=12).

HU was performed according to the Morey's tail suspension protocol [START_REF] Morey-Holton | Hindlimb unloading rodent model: technical aspects[END_REF]. One 1h per day, HU-IR animals were removed from hindlimb suspension and free to perform ambulatory activity on their cage (i.e. 30cm x 30cm area). After 14 days, rats were deeply anaesthetized with a ketamine-xylazine-butorphanol cocktail. Liver, spleen, soleus and gastrocnemius were harvested, weighed, frozen in liquid nitrogen, or fixed in 4% paraformaldehyde (PFA). Intracardiac blood was collected in dry tubes. Hemoglobin concentration was determined using the HemoCue® 201+ system (HemoCue, AB, Meaux, France). Blood was then centrifuged (1 500 g, 10 min) for serum isolation. Rats were euthanized by exsanguination.

Biochemical methods

Serum iron and unsaturated iron binding capacity (UIBC) were measured using a Cobas 8000 analyzer (Roche®) with the Cobas® reagents 03183696 122 and 04536355 190, respectively (Roche Diagnostics, Meylan, France) at the biochemistry laboratory of Rennes Pontchaillou Hospital. Serum transferrin saturation was calculated as [serum iron/(serum iron + UIBC)] × 100 [START_REF] Cavey | Mouse genetic background impacts both on iron and non-iron metals parameters and on their relationships[END_REF]. Liver, spleen and gastrocnemius iron concentrations were quantified by inductively coupled plasma mass spectrometry, as previously described [START_REF] Cavey | Mouse genetic background impacts both on iron and non-iron metals parameters and on their relationships[END_REF]. Serum erythropoietin (EPO) and IL-6 concentrations were measured by ELISA, according to the manufacturer's instructions (BioLegend, London, UK).

.

Histological analyses

Soleus muscles were fixed in PFA and embedded in paraffin for 24 hours. Serial transverse sections of 4µm were cut from the wider part of each sample using a LEICA microtome, and were mounted on glass slides (3 sections/slide). Muscle sections were then stained using the Gomori's method to detect reticulin fibers. All stained sections were scanned (x20 objective) to measure the fiber CSA from at least 1000 fibers per muscle using the Image J software (NIH, Bethesda, MD).

Protein extraction

Protein extraction was performed from soleus muscle and liver in cold lysis buffer that contained 10.0mM Tris•HCl, pH 7.4, 0.5M sucrose, 50.0mM NaCl, 5.0mM EDTA, 30.0mM Na 4 P 2 O 7 , 1% NP-40, 0.25% sodium deoxycholate, 50.0mM NaF, 100.0µM sodium orthovanadate, and protease inhibitor cocktail (5µl/ml, P8340; Sigma, St. Louis, MO).

Samples were homogenized using a Polytron homogenizer at 4°C. Each sample was then incubated on ice for 30 min followed by sonication (3x10s). Homogenates were transferred to microcentrifuge tubes and centrifuged at 12,000g at 4°C for 12 min. Protein concentration in supernatants was determined with the Lowry assay using BSA as standard. Samples were then diluted in SDS-PAGE sample buffer (50mM Tris•HCl, pH 6.8, 2% SDS, 10% glycerol, 5% β-mercaptoethanol, and 0.1% bromophenol blue) and heated at 95°C for 5 min.

Western blotting

Samples containing 50-100µg of proteins were resolved on 12.5% SDS-PAGE. Proteins were transferred onto a 0.2µm nitrocellulose membrane at 240 mA for 90 min. Membranes were blocked with 5% BSA or nonfat dry milk in Tris-buffered saline/0.05% Tween-20 (TBST) at room temperature for 1 hour. Membranes were incubated at 4°C with the appropriate primary antibodies (Table 1) overnight. Then, membranes were washed with TBST and incubated at room temperature with infrared dye-conjugated secondary antibodies (LI-COR, Lincoln, NE) for 1 hour. After washing, blots were imaged with the Odyssey Imaging System (LI-COR).

All blots were scanned, and densitometric analysis was performed using a GS-800 Calibrated Imaging Densitometer and the Quantity One software. The phosphorylation-specific signal was normalized to the total signal to estimate the ratio of activated marker.

Myosin heavy chain isoform distribution

The muscle fiber phenotype was determined by assessing the presence of the different myosin heavy chain (MHC) isoforms (slow MHC I and fast MHC IIa, IId/x, IIb isoforms), as . previously described [START_REF] Talmadge | Electrophoretic separation of rat skeletal muscle myosin heavy-chain isoforms[END_REF][START_REF] Nay | Gut bacteria are critical for optimal muscle function: a potential link with glucose homeostasis[END_REF]. Protein samples (6 µg) were separated on high-glycerol-containing (30%) gels with an acrylamide-to-bis-acrylamide ratio of 50:1 for the separating gel (8% total acrylamide, pH 8.8) and for the stacking gel (4% total acrylamide, pH 6.8) using a Mini-Protean II dual slab cell apparatus (Bio-Rad, Hercules, CA). After electrophoresis (at 140V in a refrigerated room at 4°C for 22h), gels were stained with Coomassie blue (IRDye® Blue Protein Stain, LI-COR Biosciences, Lincoln, NE, USA) and the different MHC isoforms were identified according to their electrophoretic mobility pattern [START_REF] Derbré | Single Muscle Immobilization Decreases Single-Fibre Myosin Heavy Chain Polymorphism: Possible Involvement of p38 and JNK MAP Kinases[END_REF][START_REF] Nay | Gut bacteria are critical for optimal muscle function: a potential link with glucose homeostasis[END_REF].

RNA extraction and quantitative real-time PCR

Total RNA was extracted from liver (∼30 mg) and soleus (∼15 mg) with TRIzol® (Invitrogen, Vilvoorde, Belgium), according to the manufacturer's instructions. RNA quality and quantity were assessed by 1.5% agarose gel electrophoresis and Nanodrop® spectrophotometry. Reverse transcription was performed with the iScript cDNA Synthesis Kit (Bio-Rad) using 1 µg total RNA. Real-time PCR experiments were done on a MyIQ2 thermocycler (Bio-Rad) using the following conditions: 3 min at 95°C, followed by 35 cycles of 30 s at 95°C, 30 s at 60°C and 30 s at 72°C. Samples were analyzed in duplicate in 10 µl reaction volume containing 4.8 µl IQSybrGreen SuperMix (Bio-Rad), 0.1 µl of each primer (100nM final), and 5 µl cDNA. Primer sequences are in Table 2. Melting curves were systematically analyzed to ensure the amplification specificity. The expression level of the target genes was normalized using three reference genes according to geNorm [START_REF] Vandesompele | Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes[END_REF], and expressed relative to the control group. The references genes were ribosomal protein L4 (Rpl4), glyceraldehyde-3-phosphate dehydrogenase (Gapdh), and hypoxanthine phosphoribosyltransferase 1 (Hprt1).

Statistical analysis

All data are presented as the mean ± standard error of the mean (SEM). The normality of each distribution and homogeneity of variance were assessed with the Kolmogorov-Smirnov and Bartlett test, respectively. A one-way analysis of variance (ANOVA) was then performed. Significant interaction effects were analyzed with the Fisher's Least Significant Difference (LSD) post-hoc test. The Kruskal Wallis test was chosen when the normality and/or equal variance test failed. For all statistical analyses, the significance level was set at 0.05. Data were analyzed using the statistical package GraphPad Prism version 6.02 for Windows (GraphPad Software, La Jolla, California).

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RESULTS

Intermittent reloading prevents HU-induced soleus atrophy. Body weight and the muscle weight-to-body weight ratio are shown in Table 3. After 14 days of HU, soleus and gastrocnemius weight-to body weight ratio were reduced by 46% and 19%, respectively in the HU group compared with control rats (p<0.001, Table 3). The soleus muscle mass reduction was associated with a lower mean fiber CSA in the HU than control group (-62%, p<0.001, Fig. 1A andB). IR (HU-IR group) attenuates the reduction of soleus muscle mass and fiber CSA compared with HU alone (HU group) (+36%, p<0.001 and +58%, p<0.001 respectively) (Table 3 and Fig. 1A andB). Conversely, gastrocnemius weight-to body weight ratio was comparable in HU and HU-IR rats (Table 3).

Microgravity-induced muscle atrophy is associated with imbalance between muscle protein synthesis and breakdown [START_REF] Chopard | Molecular events and signalling pathways involved in skeletal muscle disuse-induced atrophy and the impact of countermeasures[END_REF]. These alterations of protein turnover are generally characterized by inhibition of the anabolic AKT/mammalian target of rapamycin (mTOR) signaling pathways and increased activity of the catabolic ubiquitin-proteasome pathway [START_REF] Chopard | Molecular events and signalling pathways involved in skeletal muscle disuse-induced atrophy and the impact of countermeasures[END_REF]. In agreement, western blot analysis and quantification of the relative phosphorylation levels of key markers of these signaling pathways showed that activation of AKT was decreased whereas that of 4E-BP1 (mTOR target) was increased in soleus protein lysates from HU rats compared with controls (-28%, p=0.054 and +29%, p<0.001, respectively, Fig. 1C-D). This was associated with higher protein ubiquitination levels after HU (+21% compared with controls, p=0.012, Fig. 1C-D). IR prevented HU effect on AKT activation and protein ubiquitination (values comparable in HU-IR animals and controls) (Fig 1C -D), but not on 4E-PB1 phosphorylation (+15% compared with controls, p<0.001, Fig. 1C-D).

Intermittent reloading prevents the slow-to-fast MHC phenotype transition in soleus muscle.

Analysis of the relative contents of the fast and slow MHC isoforms in the three experimental groups (representative SDS-PAGE in Fig. 1E) showed that expression of type I slow MHC (MHC I), the predominant form in control soleus muscles (90%), was significantly reduced in the HU group (76%, p=0.002, Fig. 1E andF). Conversely, MHC IIx (from 1 to 7%, p<0.001) and MHC IIb (from 1 to 6%, p<0.001) (Fig. 1E andF) significantly increased. MHC IIa did not significantly differ between control and HU animals (p=0.32, Fig. 1E andF). MHC I expression was similarly decreased in the HU-IR group compared with controls (90 vs. 78%, p=0.004, Fig. 1E andF), whereas only MHC IIa was significantly increased in these animals .

(from 7 to 17%, p=0.003, Fig. 1E andF). IR efficiently attenuated the MHC IIx and MHC IIb increase observed in the HU group (7% versus 2%, p<0.001 and 6% versus 2% in the HU and HU-IR group, respectively; p<0.001 for both, Fig. 1E andF).

Effects of 14 days of hindlimb unloading and intermittent reloading on iron distribution. We then determined the iron metabolism changes induced by HU, and whether they can be influenced by IR. After 14 days of HU, serum iron concentration p=0.019,Table. 4) and transferrin saturation (-18%, p=0.041, Table. 4) were significantly reduced in the HU group compared with controls. This reduction of the available iron was not associated with changes in hemoglobin, hematocrit, and serum EPO levels (Table 4). To determine where iron was stored during HU, we measured iron concentration in liver and spleen, the main organs responsible for iron storage. We found no difference between control and HU rats in liver and spleen iron content. As muscle atrophy could affect iron metabolism, we also measured iron concentration in gastrocnemius muscles, but did not find any difference between control and HU rats. Despite the positive effect on soleus muscle atrophy, HU+IR group failed to significantly prevent decreases of serum iron concentration and transferrin saturation observed in HU group (Table. 4). Furthermore, serum iron concentration, liver, spleen and skeletal muscle iron concentrations, as well as hemoglobin, hematocrit and serum EPO levels were not altered in HU+IR group vs CTL and HU groups (Table. 4).

Effects of 14 days of hindlimb unloading and intermittent reloading on hepcidin regulation.

To investigate the mechanism by which HU affected iron availability, we quantified the liver expression of hepcidin, the master regulator of iron metabolism. Hepcidin mRNA level (Hamp) was increased in the HU group compared with control (+77%, p=0.036, Fig. 2A), suggesting a role for this hormone in the iron homeostasis alteration during HU. In agreement with the lack of preventive effects of HU-IR on iron availability reduction, Hamp level was also increased in the HU-IR group compared with control (+95%, p=0.008, Fig. 2A).

Hepcidin expression is upregulated by different signaling pathways, including BMP6/SMAD, ERK1/2 and IL-6/STAT3 (52). Therefore, we investigated whether these three signaling pathways were activated in the liver of HU and HU-IR animals. However, we did not detect any change in STAT3, SMAD1-5-8 and ERK activation (Fig. 2B-C). Furthermore, mRNA expression of C-reactive protein (Crp) and haptoglobin (Hp) in liver was not significantly different in the three groups.

.

DISCUSSION

The present study was designed to evaluate the medium-term effect of simulated microgravity on iron metabolism, and to determine the role of skeletal muscle in iron metabolism regulation in this specific condition. Using a HU rat model, we previously demonstrated that 7 days of exposure leads to hepcidin upregulation and concomitantly to iron distribution changes [START_REF] Cavey | Simulated microgravity decreases circulating iron in rats: role of inflammation-induced hepcidin upregulation[END_REF]. In the present study, we found that iron availability remains low after 14 days of HU, probably due to persistent hepcidin upregulation. Moreover, we observed that IR efficiently prevents HU-induced soleus atrophy and slow-to fast twitch MHC isoform transition. However, it cannot prevent the reduction of iron availability and hepcidin upregulation.

During space missions, microgravity reduces serum iron concentration and increases iron storage in humans [START_REF] Smith | The nutritional status of astronauts is altered after long-term space flight aboard the International Space Station[END_REF]. The HU model has been widely used for decades to mimic the effects of microgravity on the organism during spaceflight. This model presents the advantage of exploring the organ-specific responses to microgravity, a key characteristic of the spaceflight environment, but without the concomitant effects of the exposure to radiation or hypercapnia. In a previous study, we found that 7 days of HU reduces circulating iron concentration and transferrin saturation in rats [START_REF] Cavey | Simulated microgravity decreases circulating iron in rats: role of inflammation-induced hepcidin upregulation[END_REF]. Here, we obtained similar results after 14 days of HU, suggesting that the available iron is reduced also upon prolonged exposure to simulated microgravity. The decreased iron availability for erythropoiesis could play an important role in spaceflight anemia [START_REF] Smith | Red blood cell and iron metabolism during space flight[END_REF]. In our previous study, we found that 7 days of HU reduces also serum EPO concentrations in rats (unpublished data), as reported in humans after short-term exposure to real and simulated microgravity [START_REF] Leach | The endocrine system in space flight[END_REF][START_REF] Gunga | Erythropoietin under real and simulated microgravity conditions in humans[END_REF]. Conversely, here we found that serum EPO concentrations did not differ between controls and rats exposed to HU for 14 days. This suggests that erythropoiesis decrease might play a role in anemia only during the first days of exposure to microgravity.

To understand whether iron metabolism is dysregulated in conditions of simulated microgravity, we investigated hepcidin synthesis in the liver of HU rats. We found that hepcidin transcription was increased after 14 days of HU in the same order of magnitude we previously observed after 7 days of HU [START_REF] Cavey | Simulated microgravity decreases circulating iron in rats: role of inflammation-induced hepcidin upregulation[END_REF]. High hepcidin levels induce the decrease of serum iron concentration and the increase of iron storage in cells that express ferroportin, especially macrophages [START_REF] Ganz | Hepcidin and iron regulation, 10 years later[END_REF]. After 7 days of HU, we previously . observed that iron accumulates in spleen, which is particularly rich in macrophages [START_REF] Cavey | Simulated microgravity decreases circulating iron in rats: role of inflammation-induced hepcidin upregulation[END_REF], but not in liver. Here, we found that iron concentrations in spleen and liver were not significantly different in HU rats (14 days) and controls. On the other hand, Xu et al. reported that 21 days of HU increases iron concentration in liver [START_REF] Xu | The regulation of iron metabolism by hepcidin contributes to unloading-induced bone loss[END_REF]. These results suggest that splenic iron storage during the first days of HU is transient, possibly to protect other organs from iron overload. However, this mechanism might not be effective when exposure to mechanical unloading is prolonged. The fact that serum iron concentration (and thus availability) remained low after 14 days of HU, despite the return to normal iron concentration in spleen, suggests that iron started to accumulate in other organs. Therefore, we also measured iron concentration in atrophied gastrocnemius muscles, but levels were comparable in controls and rats that experienced HU for 14 days. It would be interesting to explore the effects of prolonged real and simulated microgravity on iron storage in other organs that are functionally affected by this condition (e.g., brain, kidney, heart).

We previously showed that in rats, HU for 7 days increases STAT3 activation in liver [START_REF] Cavey | Simulated microgravity decreases circulating iron in rats: role of inflammation-induced hepcidin upregulation[END_REF]. Conversely, after 14 days of HU, activation of STAT3 and of the BMP/SMAD and ERK1/2 signaling pathways was comparable in control and HU rats. Recently, [START_REF] Xu | The regulation of iron metabolism by hepcidin contributes to unloading-induced bone loss[END_REF] observed in mice that after 21 days of HU, Bmp6 mRNA levels are increased and hepcidin upregulated. Altogether, these data support the hypothesis that hepcidin upregulation is not a transient adaptation associated with mechanical unloading, and that the underlying mechanisms differ depending on the exposure duration. Thus, low-grade inflammation might play a role during the first days of simulated microgravity; however, upon prolonged exposure other mechanisms might be involved, including iron overload in liver.

In our previous study we observed that 7 days of HU also increases Il6 mRNA levels in atrophied skeletal muscle, concomitantly with STAT3 activation in liver [START_REF] Cavey | Simulated microgravity decreases circulating iron in rats: role of inflammation-induced hepcidin upregulation[END_REF]. Therefore, we proposed the existence of a muscle-to-liver crosstalk to regulate iron metabolism under microgravity. As mechanical unloading increases both circulating and muscle IL-6 levels in humans and rodents [START_REF] Felix | Altered cytokine expression in tissues of mice subjected to simulated microgravity[END_REF][START_REF] Bosutti | Calorie restriction modulates inactivity-induced changes in the inflammatory markers C-reactive protein and pentraxin-3[END_REF][START_REF] Mutin-Carnino | Effect of muscle unloading, reloading and exercise on inflammation during a head-down bed rest[END_REF][START_REF] Kwon | MyD88 regulates physical inactivityinduced skeletal muscle inflammation, ceramide biosynthesis signaling, and glucose intolerance[END_REF][START_REF] Cavey | Simulated microgravity decreases circulating iron in rats: role of inflammation-induced hepcidin upregulation[END_REF][START_REF] Yakabe | Inhibition of interleukin-6 decreases atrogene expression and ameliorates tail suspension-induced skeletal muscle atrophy[END_REF], we hypothesized that IR would prevent hepcidin upregulation and the related iron distribution changes by limiting muscle atrophy and the associated inflammatory state. Here, we did not find any preventive effect of IR on iron metabolism, although it limited HU-induced soleus . muscle atrophy and the transition from the slow to fast muscle contractile phenotype. These results refute the hypothesis of a skeletal muscle-liver dialogue during mechanical unloading.

However, IR prevented HU-induced atrophy of the soleus muscle, but less efficiently the atrophy of the gastrocnemius muscle. In rats, this muscle represents a mass approximately 20 times bigger than that of the soleus muscle. Based on this data, we cannot exclude that the IR preventive effect on whole-body skeletal muscle mass was too small to significantly influence iron metabolism regulation. Moreover, after 14 days of HU, the IL-6/STAT3 signaling pathway was no longer activated in liver, both in the HU and HU-IR groups.

Therefore, we cannot exclude that IR might exert a preventive effect on hepcidin synthesis during the first days of mechanical unloading that are characterized by an intense catabolic and inflammatory state of skeletal muscle. This effect might then decrease after 14 days, following the progressive reduction of the systemic and muscle inflammatory state.

To conclude, the present study demonstrates in rats that iron availability is durably reduced during simulated microgravity, but that iron distribution changes differ after short and medium-term exposure to HU. It also confirms that hepcidin plays a key role in this process, although the underlying mechanisms are still unclear. Finally, IR effectively prevented HUinduced soleus wasting, but did not have any effect on iron distribution changes, suggesting that skeletal muscle might not be a key player in iron balance regulation in conditions of microgravity.

Zwart SR, Morgan JLL & Smith SM (2013). Iron status and its relations with oxidative damage and bone loss during long-duration space flight on the International Space Station. Am J Clin Nutr 98, 217-223.

TABLES

Table 1 :

 1 List of primary antibodies used for western blotting

	Antibody target	Molecular weight (kDa)	Reference	Dilution
	Phosphorylated 4E-BP1 Thr37/46	15 to 20	Cell Signaling, 2855	1:1000
	4E-BP1	15 to 20	Cell Signaling, 9644	1:1000
	Phosphorylated AKT Ser473	60	Cell Signaling, 9271	1:1000
	AKT	60	Cell Signaling, 9272	1:1000
	Phosphorylated ERK1/2 Thr202/Tyr204	42/44	Cell Signaling, 9101	1:1000
	ERK1/2	42/44	Santa Cruz, sc-514302	1:200
	HSC70	70	Cell Signaling, 9271	1:5000
	Phosphorylated SMAD1/5/8 Ser463/465	60	Abcam, ab92698	1:1000
	SMAD5	60	Cell Signaling, 12534	1:1000
	Phosphorylated STAT3 Tyr705	90	Cell Signaling, 9131	1:1000
	STAT3	86	Cell Signaling, 4904	1:1000
	Ubiquitin		Cell Signaling, 3933	1:1000

Table 2 .

 2 Primer sequences (5'-3')

	Gene	Reference	Forward	Reverse
	Crp	NM_017096.3	AGGCTTTTGGTCATGAAGACATG	CTTCGGCTCACATCAGCGT
	Gapdh	NM_017008.4	GGTCGGTGTGAACGGATTTG	CGTGGGTAGAGTCATACTGGAACA
	Hp	NM_012582.2	AACACTTGGTTCGTTATCGCTG	CCACACACTGCCTCACACTTG
	Hamp	NM_053469.1	CTGCAGCCTTGGCATGG	CAGCAGCGCA CTGTCATCA -3
	Hprt1	XM_008773659.2 CTGATTATGGACAGGACTGAAAGAC CCAGCAGGTCAGCAAAGAACT
	Rpl4	NM_022510.1	CGAGCACCACGCAAGAAGAT	ATTCCTAGCCTGGCGGAGAA

ACKNOWLEDGMENTS

The authors thank Pascale Bellaud (H2P2 facility, University Rennes 1, Rennes) for technical help with histological analyses, and Guillemette Gauquelin-Koch (Centre National d'Etudes Spatiales, Paris) for scientific support. The authors thank Elisabetta Andermarcher for expert manuscript editing.

AUTHOR CONTRIBUTIONS

All authors approved the final version of the manuscript and agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part . of the work are appropriately investigated and resolved. All persons designated as authors qualify for authorship, and all those who qualify for authorship are listed.

GRANTS

This study was supported by grants from the French "Centre National d'Etudes Spatiales" (CNES), the Brittany Research Council, and INRA.

DISCLOSURES

No conflict of interest, financial or otherwise, is declared by the author(s).

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