Michael Holzinger

Henrique M Paulo

Serge Buzzetti

Cosnier

Polymers and nano-objects, a rational combination for developing health monitoring biosensors

Biosensors are essential tools in the health and the environmental sectors since they allow fast and reliable diagnostics and analysis. Biosensors are defined by the biological sensing element, which contains biomolecules or synthetic, bioinspired entities with unique specificity towards the analyte. The biological or bioinspired recognition even can be transduced into an electric, electrochemical, or optical signal. A constant challenge for the development of biosensors is the confinement of these selective entities in devices without altering the biological activity. Polymers are great candidates to serve as substrates for the immobilization or entrapment of bioreceptors related to well established synthesis routes with highly modulable chemical functions to create an almost ideal environment for the biomolecules. Nanomaterials are of constant increasing interest since they allow not only to increase drastically the specific surface for higher amounts of receptor units, but also provide electric or optical properties leading to enhanced signal capture. As for bioreceptors, such nanomaterials can be integrated polymer matrices for reliable processability. This review aims to summarize selected original examples about biosensors for health monitoring using beneficial combinations of nanomaterials and polymers.

Introduction

Monitoring risk factors of patients or medical screening of the population is essential for early detection and control of diseases. The strong evolution in this field led to portable, miniaturized devices with wireless data transmission systems to improve comfort and treatments [START_REF] Gonzalez | Chapter 10 -Applications to Improve the Assistance of First Aiders in Outdoor Scenarios[END_REF]. Very prominent became point of care tests where patients can monitor the pathogen at home without the need of qualified staff or clinical laboratories [START_REF] Vashist | An Overview of Point-of-Care Technologies Enabling Next-Generation Healthcare Monitoring and Management, Point-of-Care Technologies Enabling Next-Generation Healthcare Monitoring and Management[END_REF]. To analyse biological parameters like blood sugar, cancer or cardiovascular markers, biosensors are the most efficient devices related to their capacity to capture one specific analyte in complex body fluids [START_REF] Bhattarai | Basics of Biosensors and Nanobiosensors[END_REF][START_REF] Mohankumar | Recent developments in biosensors for healthcare and biomedical applications: A review[END_REF]. The sensing entities are generally enzymes [START_REF] Bollella | Enzyme based amperometric biosensors[END_REF], proteins of immune systems [START_REF] Zhang | Chapter 5 -Immuno-Biosensor[END_REF], DNAs [START_REF] Kowalczyk | Trends and perspectives in DNA biosensors as diagnostic devices[END_REF], or synthetic nucleotides like aptamers [START_REF] Stanciu | Recent Advances in Aptamer-Based Biosensors for Global Health Applications[END_REF] or peptides like DARPins [START_REF] Stumpp | DARPins: a true alternative to antibodies[END_REF] depending on the type of disease.

One important factor is the choice of the substrate for optimal immobilization of the bioreceptors and efficient transduction of the recognition event. Historically, the first biosensors for health monitoring was an electrochemical glucose sensor, where the enzyme glucose oxidase was trapped in a polyacrylamide gel [START_REF] Updike | The Enzyme Electrode[END_REF]. Polymers became the material of choice for the development of biosensors since properties and chemistry can easily be tuned and customized for each receptor type [START_REF] Long | Polymers in Biosensors[END_REF]. Particular interesting are conductive polymers which can facilitate electron transfers with enzymes, change the electronic signature and thus serve as transducer element [START_REF] Ramanavicius | Conducting Polymers in the Design of Biosensors and Biofuel Cells[END_REF]. Such polymers can directly be generated on electrodes via electropolymerization which allows the control of morphology, thickness and spatial addressing of polymer whatever size and the form of conducting surface [START_REF] Cosnier | Electrosynthesized polymers for biosensing[END_REF].

More recently, nanomaterials emerged in the field of biosensors developments [START_REF] Pirzada | Nanomaterials for Healthcare Biosensing Applications[END_REF][START_REF] Holzinger | Nanomaterials for biosensing applications: A Review[END_REF]. The global advantage of nanomaterials is the high specific surface that allows the immobilization of more bioreceptor units leading consequently to improve performances in terms of sensitivity and limit of detection. When reduced to the nanoscale, some materials change their properties with the appearance of intrinsic quantum effects which lead to particular optical, electrical and mechanic features [START_REF] Asha | Chapter 15 -Nanomaterials properties[END_REF]. Due to their unique physicochemical properties, nanoparticles, in particular metal and metal oxide nanoparticles, have been widely exploited for health and medical applications [START_REF] Boisselier | Gold nanoparticles in nanomedicine: preparations, imaging, diagnostics, therapies and toxicity[END_REF][START_REF] Tayo | Stimuli-responsive nanocarriers for intracellular delivery[END_REF][START_REF] Yaqoob | Recent Advances in Metal Decorated Nanomaterials and Their Various Biological Applications: A Review[END_REF][START_REF] Akintelu | A review on synthesis, optimization, characterization and antibacterial application of gold nanoparticles synthesized from plants[END_REF]. Since nanomaterials are in the same size range as biomolecules used for biosensors, the immobilization or confinement is as important. Polymers play again a crucial role for the processing of nanomaterials and this led to the vast research topic of nanocomposites [START_REF] Hassan | Functional nanocomposites and their potential applications: A review[END_REF][START_REF] Fu | Some basic aspects of polymer nanocomposites: A critical review[END_REF].

Nanocomposites can be defined as a material formed from at least two components and where at least one is a nanosized material. Such nanofillers can be of 0 (spheres), 1 (tubes, fibres) and 2 (layered sheets) dimensions and be of any imaginable chemical nature. In this context, biomacromolecules used as receptor units in biosensors can thus be considered as nanomaterial where the first bio-nanocomposite was formed in 1967. However, the researchers Roy and Komarneni used firstly term nanocomposite describing sol-gel processes in the period 1982-1983 [START_REF] Komarneni | Feature article. Nanocomposites[END_REF][START_REF] Hoffman | Diphasic Xerogels, A New Class of Materials: Phases in the System Al2o3-Sio2[END_REF].

Nanocomposites revolutionized the vast topic of health care research and are present in various applications concerning gene therapy, drug delivery, tissue engineering, food preservation, bioimaging, and biosensing [START_REF] Kumar | Recent advances and remaining challenges for polymeric nanocomposites in healthcare applications[END_REF]. Tremendous advances were reported and reviewed in the biomedical diagnostic sector about high performing sensors and biosensors with detection limits down to individual molecules using polymer nanocomposites [START_REF] Shrivastava | Next-generation polymer nanocomposite-based electrochemical sensors and biosensors: A review[END_REF][START_REF] Wang | Nanomaterial-doped conducting polymers for electrochemical sensors and biosensors[END_REF].

In the following section, a selection of original approaches for the combination of nanomaterials and polymers for healthcare biosensors is presented.

Imprinted Polymer -Quantum dots (QDs):

Quantum dots (QDs) are semiconducting nanoparticles with particular optical properties. Their narrow emission spectrum depends on the particle size influencing the band gap and thus the emission wavelength [START_REF] Weller | Colloidal Semiconductor Q-Particles: Chemistry in the Transition Region Between Solid State and Molecules[END_REF]. Quantum dots are therefore of interest for optoelectronics [START_REF] Litvin | Colloidal quantum dots for optoelectronics[END_REF] but also play an important role in the biomedical field like bioimaging, drug delivery, or sensing [START_REF] Wagner | Quantum dots in biomedical applications[END_REF]. However, the most studied semiconductor material is based on cadmium chalcogenides which represents toxicological issues [START_REF] Reshma | Quantum dots: Applications and safety consequences[END_REF]. Zn based QDs [START_REF] Labiadh | ZnS quantum dots and their derivatives: Overview on identity, synthesis and challenge into surface modifications for restricted applications[END_REF]and carbon QDs [START_REF] Wang | A Mini Review on Carbon Quantum Dots: Preparation, Properties, and Electrocatalytic Application[END_REF] represent less toxic alternatives but the obtained QDs and, in particular, carbon QDs, still lack of homogeneity for defined narrow emission spectra. The quantum efficiency and synthesis yields are also still to improve [START_REF] Zheng | Glowing Graphene Quantum Dots and Carbon Dots: Properties[END_REF]. For optical biosensing, the transduction principle is generally based on non-radiative energy transfer between the excited QDs and an acceptor such as gold nanoparticles [START_REF] Dyadyusha | Quenching of CdSe quantum dot emission, a new approach for biosensing[END_REF] or graphene [START_REF] Dong | Fluorescence Resonance Energy Transfer between Quantum Dots and Graphene Oxide for Sensing Biomolecules[END_REF]. QDs and acceptors are therefore used as labels and need relative complex preparation and synthesis procedures to construct the sensing element. An elegant solution is when the analyte itself can act as acceptor or quenching unit. One quite original strategy is to coat the QDs with molecular imprinted polymers (MIPs) which serves as specific receptor. MIPs are formed by polymerization of appropriate monomers like silicates, silanes, or organic monomers in presence of the target analyte. After the polymerization reaction, the target molecule is extracted leaving a specific imprint on the polymer. With the resulting MIP-QD composite, the measured quenching effect during the recognition events in the evaluated target solutions allows to quantify the analyte concentration [START_REF] Diaz-Alvarez | Molecularly Imprinted Polymer-Quantum Dot Materials in Optical Sensors: An Overview of Their Synthesis and Applications[END_REF]. The different quenching mechanisms and a series of examples are nicely reviewed in references [START_REF] Li | Advances in Molecularly Imprinting Technology for Bioanalytical Applications[END_REF][START_REF] Yang | Strategies of molecular imprinting-based fluorescence sensors for chemical and biological analysis[END_REF]. This detection principle is very efficient for small molecules like drugs or biomarkers but can also be applied to biomacromolecules like proteins. For instance, Wang et al. used phycocyanin as model protein to imprint polydopamine on thioglycollic acid and glutathione capped QDs [START_REF] Wang | Quantum dots based imprinting fluorescent nanosensor for the selective and sensitive detection of phycocyanin: A general imprinting strategy toward proteins[END_REF]. This biosourced polymer is presented as very appropriate polymer for protein imprints. The phycocyanin concentration dependent quenching provided a nicely linear profile and detection limits in the sub micromolar range could be obtained and was validated with phycocyanin spiked sea and lake waters. To demonstrate the suitability of this setup, bovine haemoglobin was used as template leading to similar biosensor performances. Related to the electroactivity of dopamine, a similar setup with electrochemical transduction was reported. Mo et al. electropolymerized dopamine on glassy carbon electrodes in presence of squamous cell carcinoma antigen (SCCA), the target analyte. SCCA is a 48 kDa glycoprotein and a useful tumor marker for the diagnosis of lung cancer. ZnSe quantum dots, attached to a metal organic framework (MOF) and modified with anti-SCCA antibodies. After rebinding of SCCA on the polymer in the analyte solution, the antibody MOF QDs are immobilized on the electrode surface leading to an electrochemiluminescent signal. The MOF allowed an improved efficiency to reduce the sacrificial electron acceptor S 2 O 8 2-and thus an improved electrochemiluminescent signal. With this configuration, a stable and reproducible SCCA biosensor was obtained [START_REF] Mo | A sensitive electrochemiluminescence biosensor based on metal-organic framework and imprinted polymer for squamous cell carcinoma antigen detection[END_REF].

Another elegant strategy was firstly reported by Li et al. This team used the epitope phenylphosphonic acid as printing template for the detection of tyrosine phosphopeptide [START_REF] Li | A "turn-on" fluorescent receptor for detecting tyrosine phosphopeptide using the surface imprinting procedure and the epitope approach[END_REF]. In this work, a "turn on" concept was developed which is based on a passivation of "trap-states" in the MIP-QD composite after capture of phenylphosphonic acid or tyrosine phosphopeptide leading to enhanced fluorescence. A more sophisticated setup for the same analyte was proposed by Saeedzadeh Amiri and colleagues [START_REF] Saeedzadeh Amiri | Application of ratiometric fluorescence sensorbased microwave-assisted synthesized CdTe quantum dots and mesoporous structured epitopeimprinted polymers for highly efficient determination of tyrosine phosphopeptide[END_REF]. Here, two optical probes with different emission wavelengths were used to detect phenylphosphonic acid or tyrosine phosphopeptide via colour changes since the target quenched the emission of the QDs with red emissions but not the one with green emission. Furthermore, a mesoporous structure was formed on the outer shell of the MIP using the amphiphile ionic liquid 1tetradecyl-3-methylimidazolium chloride. The microwave assisted polymerization of the silane in presence of these ionic liquid micelles and the epitope led to a porous structure with enhanced receptor sites (Figure 1). This biosensor design had excellent regeneration capacities and led to a 3 orders of magnitude improvement for the detection limit compared to the precedent setup.

Polymer Gold NP

Gold nanoparticles are by the mostly used material for analytics and diagnosis related to the combination of excellent conductivity and optical properties [START_REF] Li | Gold nanoparticle-based biosensors[END_REF][START_REF] Elahi | Recent biomedical applications of gold nanoparticles: A review[END_REF][START_REF] Qin | Gold rush" in modern science: Fabrication strategies and typical advanced applications of gold nanoparticles in sensing[END_REF]. In particular, the plasmonic behaviour of nanosized gold particles made this material the component of choice for analytical SPR and Raman setups. The localized resonant plasmons are extremely sensitive to their environment and can interact with surface plasmons on macroscopic surfaces leading to an amplification of the signals [START_REF] Amendola | Surface plasmon resonance in gold nanoparticles: a review[END_REF]. By eliminating bulk effects from classic SPR setups, localized plasmon resonance allows higher selectivity and does not need sophisticated equipment [START_REF] Dutta | Smartphone based LSPR sensing platform for bio-conjugation detection and quantification[END_REF]. However, the sensitivity and analytical performances are generally lower. This issue can be improved by controlling the spatial distribution and spacing between the gold nanoparticles to optimize the plasmonic properties [START_REF] Jain | On the Universal Scaling Behavior of the Distance Decay of Plasmon Coupling in Metal Nanoparticle Pairs: A Plasmon Ruler Equation[END_REF]. One interesting approach is the use of block copolymers forming self-organized nanopatterns to obtain an array of homogeneously distributed gold nanoparticles. Lu et al. used poly(styrene-b-4vinylpyridine) block copolymer to form a brush layer on glass fibres [START_REF] Lu | Polymer-Templated Gold Nanoparticles on Optical Fibers for Enhanced-Sensitivity Localized Surface Plasmon Resonance Biosensors[END_REF]. The exposed vinylpyridine patterns allowed to form dense gold nanoparticle array with controlled distances and particle sizes without aggregates and vacancies. The authors then formed an optical immunosensor for the detection of human IgG with immobilized goat anti-human IgG on the glass fibre AuNP array (Figure 2). The immune response was monitored in real time by measuring changes in resonance wavelength and intensity with a standard fibre-coupled spectrophotometer. The obtained detection limit was in the same order of magnitude as the detection limit using a prism-based SPR instrument. Less sophisticated but as efficient patterns are necessary for electrochemical transduction. Avelino et al. formed a flexible DNA sensor using an electropolymerized polypyrrole layer on ITO modified polyethylene terephthalate [START_REF] Avelino | Flexible sensor based on conducting polymer and gold nanoparticles for electrochemical screening of HPV families in cervical specimens[END_REF]. Polydisperse gold nanoparticles were electro synthesized on the polypyrrole film, functionalized with a designed oligonucleotide for the recognition of families of human papillomavirus in cervical specimens. In this setup, the gold nanoparticles served as amplifier for the cyclic voltammetric and impedimetric signals due to their good conductivity and enhanced surface area. The detection limit covers the realistic range of infected persons. This setup could therefore be validated with clinical samples, which also confirmed the excellent specificity of the designed oligonucleotide.

One important point in health monitoring is the environmental impact of the devices since they have to be littered after direct contact with patients. Toxic or polluting components are therefore to avoid. Vural et al. developed a disposable and cost-effective sensing element based on pencil graphite electrodes which were modified with synthesized gold nanoparticle / peptide nanotube composites [START_REF] Vural | Electrochemical immunoassay for detection of prostate specific antigen based on peptide nanotube-gold nanoparticle-polyaniline immobilized pencil graphite electrode[END_REF]. The conductive polymer polyaniline was then electropolymerized as thin film around the deposited composite. This setup served to detect prostate specific antigen (PSA) via amperometry. The electrochemical signal was obtained by formation of sandwich structures with anti-PSA antibody as bioreceptor unit, which capture the target PSA. The final layer was a secondary anti-PSA antibody labeled with horseradish peroxidase, which reduced hydrogen peroxide to water via hydroquinone mediated electron transfer with the electrode. The obtained detection limit is suitable for medical applications. Table 1 summarises the performances of the discussed biosensors setups based on the combination of polymers and nanomaterials. Considering the different analytes and transduction techniques, it is difficult to compare the reported values. Nonetheless, a clear tendency to higher performances with more sophisticated systems can be observed. ).

[ ) [START_REF] Vural | Electrochemical immunoassay for detection of prostate specific antigen based on peptide nanotube-gold nanoparticle-polyaniline immobilized pencil graphite electrode[END_REF] For repeatable production of such analytical tools, however, such complex assemblies are less suitable for industrial application where simplified protocols are generally preferred to reduce costs and to assure reliability. The examples using low cost and environmental friendly materials combined with original but abridged fabrication procedures take into account such industrial needs. But, this should not prevent academic research to explore new principles despite the complexity at short term.

Polymer based functional organic nanoparticles.

Beside metallic nanoparticles, considerable efforts have been made in the development of organic nanoparticles based on self-assembly such as micelles, liposomes or polymersomes [START_REF] Fang | Different Strategies for Organic Nanoparticle Preparation in Biomedicine[END_REF][START_REF] Zhang | Self-Assembled Organic Nanomaterials for Drug Delivery, Bioimaging, and Cancer Therapy[END_REF]. Among then, biocompatible carbohydrate-coated nanoparticles have recently attracted a wide attention for biosensing and bioconversion applications. In particular, the synthesis of amphiphilic glycopolymers based on the coupling of hydrophilic poly-saccharides and hydrophobic synthetic polymers such as polystyrene has enabled the design of various structures (micelle, vesicle, worm-like micelle, nanoparticle) by self-assembly of these block copolymers [START_REF] Miura | Glycopolymer Nanobiotechnology[END_REF][START_REF] Pramudya | Recent progress of glycopolymer synthesis for biomedical applications[END_REF]. Among the oligosaccharides, cyclodextrins as a hydrophilic block with a hydrophobic internal cavity can form inclusion complexes with a wide range of poorly water-soluble compounds. Thanks to these host-guest interaction properties of the nanoparticle shell, these nanoparticles provide a versatile platform that has been exploited for medical applications such as drug delivery [START_REF] Gadade | Cyclodextrin Based Nanoparticles for Drug Delivery and Theranostics[END_REF].

In particular, the self-assembly of a β-cyclodextrin-polystyrene copolymer leads to spherical and stable glyconanoparticles (NP-βCD) in aqueous medium, which present an outer layer composed of β-cyclodextrin for post-functionalization by host-guest interactions. NP-βCD have thus permit entrapment of hydrophobic fluorescent molecules in aqueous solution such as tetrazinenaphthalimide or redox mediators such as quinone and thiazoline, for the electrical wiring of enzymes [START_REF] Gross | Redox-Active Glyconanoparticles as Electron Shuttles for Mediated Electron Transfer with Bilirubin Oxidase in Solution[END_REF][START_REF] Gross | Redox-Active Carbohydrate-Coated Nanoparticles: Self-Assembly of a Cyclodextrin-Polystyrene Glycopolymer with Tetrazine-Naphthalimide[END_REF][START_REF] Hammond | Solubilized Enzymatic Fuel Cell (SEFC) for Quasi-Continuous Operation Exploiting Carbohydrate Block Copolymer Glyconanoparticle Mediators[END_REF] (Figure 3). As previously reported for β-cyclodextrin-modified gold nanoparticles [START_REF] Holzinger | Adamantane/-cyclodextrin affinity biosensors based on single-walled carbon nanotubes[END_REF], the possibility of immobilizing the glyconanoparticles on a surface was studied via the electrogeneration of an anchoring polymer on an electrode. Thus, the immobilization of NP-βCD was carried out on an electropolymerized film of poly (pyrrole-adamantane) thanks to the affinity interactions between βcyclodextrin and adamantane. Owing to the high affinity of biotin to form inclusion complexes with β-cyclodextrin [START_REF] Holzinger | Biotin --cyclodextrin: a new host-guest system for the immobilization of biomolecules[END_REF], the immobilized NP-βCD layer was applied to the anchoring of biotinylated enzymes to develop amperometric biosensor. Biotinylated glucose oxidase was thus anchored onto the NP-βCD-poly (pyrrole-adamantane) film for the detection of glucose via the electro-oxidation of H 2 O 2 at the underlying platinum electrode. It should be noted the amperometric detection performance was improved by electropolymerizing a less hydrophobic polymer via the copolymerization of pyrrole-adamantane and 11-(1-pyrrolyl)undecanol increasing the permeation of H 2 0 2 to the underlying platinum surface.

With the aim of developing new enzymatic labels for immunosensors or aptasensors, nanoparticles can be functionalized by enzymes and antibodies or antigens previously modified by adamantane or biotin groups. In this context, the density of the host-guest interaction sites of the nanoparticle shell must be reduced to avoid generating too many bonds between the nanoparticle and a protein.

Indeed, the latter could induce the blocking of the conformational mobility of the enzyme and the creation of steric constraints, which would decrease the enzymatic activity. In order to modulate the density of β-cyclodextrin at the surface of the nanoparticles, hybrid nanoparticles have been produced by self-assembly of maltoheptaose-polystyrene and β-cyclodextrin-polystyrene in different proportions. Taking into account that maltoheptaose-polystyrene provides regular nanoparticles [START_REF] Otsuka | Self-Assembly of Maltoheptaose-block-Polystyrene into Micellar Nanoparticles and Encapsulation of Gold Nanoparticles[END_REF], as expected, its association with β-cyclodextrin-polystyrene also leads to stable and spherical nanoparticles with diameters ranging from 30 to 40 nm [START_REF] Carriere | Functionalizable Glyconanoparticles for a Versatile Redox Platform[END_REF]. The host-guest interaction capacity of the hybrid nanoparticles was evaluated by cyclic voltammetry using the inclusion of a water-soluble redox probe: anthraquinone sulfonate. It appears that the intensity of the electroactivity of the hybrid nanoparticles is inversely proportional to the percentage of maltoheptaose-polystyrene present in the nanoparticle composition. This effect corroborates the possibility to modulate the anchoring properties of nanoparticles Another type of nanoparticles based on a polymer shell surrounding a protein was described by M. Yan et al in 2010 for the intracellular delivery of proteins [START_REF] Yan | A novel intracellular protein delivery platform based on single-protein nanocapsules[END_REF]. Three enzymes, HRP, Caspase 3 and superoxide dismutase were thus trapped in a polymeric shell, the diameter of the resulting nanocapsules being three times greater than the diameter of the native enzymes. The concept of manufacturing nanoparticles based on single protein consists in chemically grafting acrylic acid (5 to 10 units per protein) onto a protein and then using this functionalized protein as a template for the polymerization of vinyl monomers. This polymerization process leads to a crosslinked polymer cage anchored on the protein, described by the authors as a nanocapsule.

Following a similar approach, Zhu, Lu and coworkers reported the elaboration of polyzwitterionic nanoparticles containing urate oxidase or L-asparaginase in order to improve the "in vivo" circulation time and to prevent their uptake by macrophages [START_REF] Zhang | Encapsulating Therapeutic Proteins with Polyzwitterions for Lower Macrophage Nonspecific Uptake and Longer Circulation Time[END_REF]. After the step of vinylation of the amino groups of the two enzymes, the in-situ polymerization of two vinyl derivatives, a zwitterionic 2methacryloyloxyethyl phosphorylcholine and a bifunctional N, N'-methylenebisacrylamide leads to a polyzwitterionic polymer cage containing the enzyme. It appears that the urate oxidase, which is encapsulated in a crosslinked polymer shell 5 nm thick, retains its activity, the permeation of the urate not being hampered by steric or electrostatic constraints.

Thanks to the polyzwitterionic polymer shell, the stability of the urate oxidase nanoparticle was highly improved compared to the native enzyme. In addition, these enzyme-based nanoparticles resist the process of cell uptake in aqueous solution containing 10% serum unlike native enzymes, which are rapidly internalized by macrophages. In view of these properties, the urate oxidase-based nanoparticles injected into the blood vessels of mice exhibit markedly improved stability in vivo. It appears that a partial activity is still present after 127 hours of circulation time whereas the native enzymes completely lose their activity after 5 hours. These results highlight the key role of the nature of the polymer matrix that envelops the unique enzyme and the promising potential of these nanoparticles for therapeutic applications.

A second generation of nanocapsules has been made by introducing a specific polymerizable group into the backbone of the polyacrylamide polymer surrounding a protein leading to redox and fluorescent nanoparticles [START_REF] Chen | Fabrication of Activity-Reporting Glucose Oxidase Nanocapsules with Oxygen-Independent Fluorescence Variation[END_REF]. Following previous work, the enzyme glucose oxidase was first chemically modified by acryloyl groups and then an in situ polymerization was carried out with a derivative of riboflavin (2'-diallyamino-ethyl flavin) and N,N′-methylene bis-acrylamide. The formation of the polymeric redox shell around the glucose oxidase induces an increase in diameter size from 8nm for the native enzyme to 30 nm for the resulting nanoparticle. As previously reported, the presence of the polymer via its anchoring on the protein structure reinforces the enzyme conformation and hence its activity at higher temperatures.

More importantly, the polymerized flavin groups allow efficient electron transfer between the prosthetic glucose oxidase FAD and the electrode. In addition, the flavin groups confer fluorescence on the nanocapsules. The fluorescence intensity depending on the oxidized or reduced state of the flavin groups, the latter has been elegantly used to monitor the activity of the enzyme located in the core of the nanoparticle.

Recently, a redox nanoparticle based on glucose oxidase was designed by using a more hydrophilic riboflavin derivative functionalized with a vinyl group. Its polymerization around the enzyme carrying on average 12 acryloyl groups in presence of acrylamide derivatives provides a redox polymer shell around a single enzyme [START_REF] Huang | Rational design of electroactive redox enzyme nanocapsules for high-performance biosensors and enzymatic biofuel cell[END_REF]. The resulting redox nanocapsule provides a catalytic oxidation peak at -0.4 V vs Ag/AgCl in presence of glucose indicating an excellent wiring of the enzyme by the flavinbased polymer. The adsorption of these redox nanoparticles on a glassy carbon electrode modified by a deposit of multi-walled carbon nanotubes leads to an amperometric glucose biosensor without interference but with a low detection limit of 0.13 mmolL -1 . Its application in the design of biofuel cells in combination with a biocathode based on bilirubin oxidase and ABTS as redox mediator leads to interesting performances in terms of stability and power, a maximum power density of 1mW cm -2 being recorded at 0.378 V. However, it should be noted that the operation of this biofuel cell requires an anode compartment saturated with N 2 while the cathodic compartment contains an oxygen saturated solution. Table 1 shows the performance of some amperometric biosensors and biofuel cells prepared by immobilization of redox nanoparticles based on glucose oxidase or regular glucose oxidase in different electrode materials. The comparison of the performances obtained in term of glucose sensitivity, power density and open circuit voltage for identical quantities of glucose oxidase clearly shows the advantage of using redox nanoparticles. The immobilization of nanoparticles on a surface with preservation of their spherical form could be exploited to develop multifunctional systems associating redox particles with particle-enzymes. These systems could lead to more efficient bioelectrochemical interfaces due to the porosity of the structure compared to layer-by-layer systems. The development of redox and enzymatic nanoparticles should make it possible to create a new generation of biofuel cells based on enzymatic cascade reactions. In addition, replacing the hydrophobic polystyrene part with a polycaprolactone in the structure of the copolymer would lead to totally bio-based and therefore biodegradable nanoparticles, thus opening the way to single-use biosensors and biofuel cells.

Polymer-nanocarbon materials

Since the discovery of C 60 in 1985 [75], carbon nanomaterials are intensively studied for any type of applications and therein biosensors since their particular optical and electronic properties provide more than just an enhanced specific surface [START_REF] Pasinszki | Carbon Nanomaterial Based Biosensors for Non-Invasive Detection of Cancer and Disease Biomarkers for Clinical Diagnosis[END_REF][START_REF] Wang | Carbon nanomaterial-based electrochemical biosensors: an overview[END_REF]. The most investigated structures in this research topic are fullerenes [START_REF] Pilehvar | Recent Advances in Electrochemical Biosensors Based on Fullerene-C 60 Nano-Structured Platforms[END_REF] and carbon quantum dots [START_REF] Sahin | Affinity biosensors developed with quantum dots in microfluidic systems[END_REF], carbon nanotubes [START_REF] Sireesha | A review on carbon nanotubes in biosensor devices and their applications in medicine[END_REF], and graphene [START_REF] Peña-Bahamonde | Recent advances in graphenebased biosensor technology with applications in life sciences[END_REF] .

Despite the electrochemical properties, C 60 fullerenes did not become a privileged material in biosensing applications related to the inappropriate redox potential, little solubility and the need of sophisticated processing [START_REF] Bianco | Carbon science perspective in 2020: Current research and future challenges[END_REF]. Relatively new and promising members of the 0D nanocarbon family are carbon quantum dots. As their inorganic counterpart, these carbon dots have specific optical properties and are therefore used for optical transductions. As already discussed in the corresponding section, imprinted polymers are more and more used in combination with carbon quantum dots for biosensing applications [START_REF] Haupt | Molecularly Imprinted Polymers and Their Use in Biomimetic Sensors[END_REF][START_REF] Demir | Tracking Hyaluronan: Molecularly Imprinted Polymer Coated Carbon Dots for Cancer Cell Targeting and Imaging[END_REF]. Demir et al. synthesized N-doped carbon quantum dots and used the light emission to photopolymerize the functional monomer 4acrylamidophenyl(amino)methan-iminium acetate in presence of glucuronic acid, an epitope of hyaluronan which is a biomarker for certain cancers [START_REF] Demir | Tracking Hyaluronan: Molecularly Imprinted Polymer Coated Carbon Dots for Cancer Cell Targeting and Imaging[END_REF]. This setup allowed quantitative imaging of cancer cells and could distinguish between healthy and tumor cells. The authors could further demonstrate no cytotoxicity of the N-doped quantum dots when applied to HaCaT cells.

Carbon nanotubes (CNTs) were considered as the carbon nanomaterial of choice for biosensors until graphene became more fashioned [START_REF] Yang | Carbon nanotube based biosensors[END_REF]. Related to the excellent electronic properties, CNTs are mostly used for electrochemical biosensing but examples of CNT based optical and gravimetric biosensors rely on other properties than conductivity or specific surface [START_REF] Sireesha | A review on carbon nanotubes in biosensor devices and their applications in medicine[END_REF]. In combination with polymers, very efficient is the electrogeneration of thin polymer films out of functional monomers on CNTs for efficient immobilization of the receptor unit [START_REF] Cosnier | Conductive Polymers, Immobilization of Macromolecular Bio-Entities[END_REF]. Such functions can be activated esters for covalent immobilization or host-guest units for non-covalent grafting [START_REF] Holzinger | Supramolecular immobilization of bio-entities for bioelectrochemical applications[END_REF]. For enzymatic biosensors, the analyte is in most cases a small organic molecule like glucose or a pesticide. An efficient method to immobilize enzymes can be the entrapment in an electrogenerated polymer matrix which remains permeable for the analyte [START_REF] Scouten | Enzyme or protein immobilization techniques for applications in biosensor design[END_REF]. Beside the conductivity enhancement of carbon nanotube doped polymers [START_REF] Cosnier | Aqueous dispersions of SWCNTs using pyrrolic surfactants for the electro-generation of homogeneous nanotube-composites. Application to the design of amperometric biosensor[END_REF], electrochemical biosensors using the molecular imprint technique are more and more developed [START_REF] Pandey | Carbon nanomaterials integrated molecularly imprinted polymers for biological sample analysis: A critical review[END_REF]. As an example, Kewarin et al. developed an electrochemical biosensor toward cardiac troponin T by imprinting this protein biomarker during electropolymerization of aniline [START_REF] Phonklam | A novel molecularly imprinted polymer PMB/MWCNTs sensor for highly-sensitive cardiac troponin T detection[END_REF]. Methylene blue was electrodeposited as redox probe on multiwalled carbon nanotubes before the formation of imprinted polyaniline (Figure 4). The differential pulse voltammetric signal of polymethylene blue decreased linearly with increasing cardiac troponin T concentrations. This phenomenon is explained by the passivation of the electrode attenuating the polymethylene blue signal during rebinding of cardiac troponin T. The reliability of this approach was confirmed with measurement in human blood plasma where the detection limit and linear range are in a realistic range. Parallel experiments with non-imprinted polyaniline further demonstrated the efficiency of this setup. The scientific interest in graphene, related 2D carbon and composites widely overpassed all other carbon nanomaterials for biosensing applications [START_REF] Celik | Graphene-based biosensors: methods, analysis and future perspectives[END_REF][START_REF] Krishnan | A review on graphene-based nanocomposites for electrochemical and fluorescent biosensors[END_REF][START_REF] Muñoz | Characterization protocol to improve the electroanalytical response of graphene-polymer nanocomposite sensors[END_REF] due to their electronic and optical properties, and inexpensive availability [START_REF] Suvarnaphaet | Graphene-Based Materials for Biosensors: A Review[END_REF]. Graphene and related materials are commonly used with other nanomaterials to form biosensors [START_REF] Krishnan | A review on graphene-based nanocomposites for electrochemical and fluorescent biosensors[END_REF]. Within the vast number of examples about graphene composites [START_REF] Chee | Nanocomposites of graphene/polymers: a review[END_REF] An original approach was applied to construct an impedimetric biosensors for the detection, and classification of the dengue virus [START_REF] Navakul | A novel method for dengue virus detection and antibody screening using a graphene-polymer based electrochemical biosensor[END_REF]. A copolymer was formed using acrylamide, methacrylic acid, methylmethacrylate, and N-vinylpyrrolidone with N,N-(1,2dihydroxyethylene) bisacrylamide as cross linker in presence of graphene oxide and a specific dengue virus serotype. The conductive imprinted polymer allowed to discriminate the subtypes of the dengue virus.

Conclusion

Polymer nanocomposites became essential components in biosensor research. The advantage is that relatively simple but reliable processes can be applied to form highly performing tools for health monitoring. The nanosized components with their specific properties mostly confer to the device higher performances but can also lead to new and efficient transduction, which cannot be obtained with macroscopic compounds. The high scientific activities around this material can be expressed with around 2000 research articles in the last twenty years (source: web of science, keywords: nanocomposites and biosensor). This technology even became of interest for commercialised devices where a couple of nanobiosensors are already in clinical use [START_REF] Dedeoglu | Chapter Twelve -Nanotechnological approaches and materials in commercial biosensors[END_REF] Here, some recent original examples are summarized since there are already excellent exhaustive review articles about the different nanocomposites for health care biosensing [START_REF] Omidfar | Point-of-care biosensors in medicine: a brief overview of our achievements in this field based on the conducted research in EMRI (endocrinology and metabolism research Institute of Tehran University of medical sciences) over the past fourteen years[END_REF][START_REF] Lu | Recent developments in bio-monitoring via advanced polymer nanocomposite-based wearable strain sensors[END_REF][START_REF] D'souza | Nanocomposite biosensors for point-of-care-evaluation of food quality and safety[END_REF].

Figure 1 :

 1 Figure 1: Illustration of a phenylphosphonic acid imprinted nanoparticle with a green light emitting QD core and a porous quenchable red light emitting QD shell during recognition of tyrosine phosphopeptide or phenylphosphonic acid.

Figure 2 :

 2 Figure 2: Schematic presentation of the self-assembled patterning of a glass fiber using poly(styrene-b-4vinylpyridine) block copolymer for spatial controlled growth of gold nanoparticles and the formation of a human IgG biosensors using localized plasmon resonance.

Figure 3 :

 3 Figure 3: Formation of a biosourced block copolymer nanoparticle with β-cyclodextrin shell and its capacity to form inclusion complexes with a variety of redox molecules for the wiring of enzymes.

Figure 4 :

 4 Figure 4: Sketch of polymethylene blue modified carbon nanotube electrodes with a cardiac troponin T imprinted polyaniline coating. The recognition of the analyte provokes a passivation of the electrode surface and lowers linearly with the cardiac troponin T concentration the polymethylene blue signal.

Table 1 :

 1 biosensor performances using polymer nanoparticle assemblies

	Polymer	Template / Analyte	Nanomaterial	Limit of		Linear	ref
				detection		range
			CdTe QDs capped
	Polydopamine	Phycocyanin	with thioglycollic acid and	0.069 µmol L	-1	0.3-5.0 (µmol L -1
			glutathione

Table 2 :

 2 Comparison of immobilized redox enzymatic nanocapsules and regular glucose oxidase (GOX) immobilized in different materials in terms of biosensor sensitivity to glucose and power density and open circuit voltage (OCV) of biofuel cell (BFC).

	Material/system	Immobilized	Biosensor sensitivity	BFC power	OCV	ref
		GOX amount	(mA -1 molL -1 cm -2 )	density	(V)
				(mWcm -2 )
	Nanocapsule	100 g	63.69	1.01	0.9	[68]
	MWCNT	50 mg	-	1.3	0.93	[69]
	compression/cellulose
	MWCNT-naphthoquinone	15 mg	-	1.54	0.76	[70]
	compression/polymers
	MWCNT-naphthoquinone	15 mg	-	1.03	0.66	[71]
	compression (flow-through)
	Spray-freezing reduced	200 g	-	0.38	0.85	[72]
	graphene oxide
	SWCNT-pyrene-biotin/avidin	20 g	1.6	-	-	[73]
	Polypyrrole-	0.5 g	0.175	-	-	[61]
	adamantane/cyclodextrin
	Au/MXene (metal carbide)	320 g	4.2	-	-	[74]
	nanocomposite

Acknowledgments

The authors thank the French National Research Agency in the framework of the ANR-18-CE09-0022-01 and CBH-EUR-GS (ANR-17-EURE-0003).


Didier F Pisani

Lilian Leclerc

Gisèle Jarretou

Jean-François Marini

Claude A Dechesne
email: dechesne@unice.fr

SMHS1 is involved in oxidative/glycolytic-energy metabolism balance of muscle fibers

Keywords: skeletal muscle, atrophy, hypertrophy, muscle fiber type, RTP801, HIF-1

With the aim of finding important mediators of muscle atrophy, we cloned SMHS1, a novel gene that was found to be upregulated in rat soleus muscle atrophied by restriction of activity.

The SMHS1 amino-acid sequence shares 65% similarity with RTP801 -which is a cellular stress response protein regulated by HIF-1 -but SMHS1 expression was demonstrated to be independent of HIF-1. SMHS1 was found to be mainly expressed in skeletal muscle, and comparisons of its expression in atrophied versus hypertrophied muscles and in oxidative versus glycolytic muscles suggested that SMHS1 contributes to the muscle energy metabolism phenotypes.

Introduction

The well-documented plasticity of skeletal muscle is largely based on the heterogeneity of fiber types which vary in metabolic equipment, contraction speed, and fatigability [START_REF] Pette | Cellular and molecular diversities of mammalian skeletal muscle fibers[END_REF][START_REF] Schiaffino | Molecular diversity of myofibrillar proteins: gene regulation and functional significance[END_REF][START_REF] Zhang | Skeletal muscle fibre types: detection methods and embryonic determinants[END_REF].

Briefly, at one end of the classification, slow-twitch oxidative fibers (type I) have a slow contraction time and are fatigue resistantthey are responsible for both maintaining posture and carrying out slow repetitive movements. Then, we found fast-twitch oxidative fibers (type IIa) with a rapid contraction time associated to fatigue resistance. The other end of the classification includes fast-twitch glycolytic fibers (types IIx and IIb), which have a fast contraction time and low fatigue resistance. They are adapted for high-power output. In addition of these principal fibers, it exists several intermediary and transitory fibers (types I/IIa, IIa/IIx and IIx/IIb). Chronic skeletal muscle activity variations cause changes in muscle mass and metabolism, which are associated with shifts in fiber types [START_REF] Fitts | Physiology of a microgravity environment invited review: microgravity and skeletal muscle[END_REF][START_REF] Edgerton | Human fiber size and enzymatic properties after 5 and 11 days of spaceflight[END_REF][START_REF] Booth | Changes in skeletal muscle gene expression consequent to altered weight bearing[END_REF]. These well established changes ultimately modify muscle performance, e.g. oxidative slow-to glycolytic fast-twitch transitions in the case of atrophy of slow-twitch muscles (soleus muscle). Many cellular components are involved in this switch, including myofibril protein isoforms, oxidative enzymes, glycolytic enzymes, calcium homeostasis players, transcription factors, and signaling molecules [START_REF] Thomason | Atrophy of the soleus muscle by hindlimb unweighting[END_REF][START_REF] Talmadge | Distribution of myosin heavy chain isoforms in non-weight-bearing rat soleus muscle fibers[END_REF][START_REF] Price | Increased transcription of ubiquitin-proteasome system components: molecular responses associated with muscle atrophy[END_REF][START_REF] Glass | Molecular mechanisms modulating muscle mass[END_REF]. Several previous studies, including those conducted by our laboratory, focused on large-scale gene profiling or proteomic analysis in experimental rat and mouse models of muscle atrophy induced by hindlimb unweighting or immobilization [START_REF] Cros | Upregulation of M-creatine kinase and glyceraldehyde3-phosphate dehydrogenase: two markers of muscle disuse[END_REF][START_REF] Cros | Analysis of altered gene expression in rat soleus muscle atrophied by disuse[END_REF][START_REF] Bodine | Identification of ubiquitin ligases required for skeletal muscle atrophy[END_REF][START_REF] St-Amand | Characterization of control and immobilized skeletal muscle: an overview from genetic engineering[END_REF][START_REF] Wittwer | Prolonged unloading of rat soleus muscle causes distinct adaptations of the gene profile[END_REF][START_REF] Chen | Response of rat muscle to acute resistance exercise defined by transcriptional and translational profiling[END_REF]. In order to identify genes involved in skeletal muscle adaptive responses to chronic activity modifications, we characterized a novel gene that we previously demonstrated to be two-fold upregulated in rat soleus muscles atrophied by restriction of activity imposed by hindlimb suspension (03A12 clone) [START_REF] Cros | Analysis of altered gene expression in rat soleus muscle atrophied by disuse[END_REF]. This gene, named SMHS1 (Skeletal Muscle Hindlimb Suspension 1), encodes a protein with 65% similarity (35% identity) to the RTP801 protein [START_REF] Shoshani | Identification of a novel hypoxia-inducible factor 1responsive gene, RTP801, involved in apoptosis[END_REF]. RTP801 is a novel recently identified protein under the control of hypoxiainducible factor 1 (HIF-1). After we submitted the full-length SMHS1 cDNA sequence to GenBank the same cDNA sequence was found through in silico EST contigization and named RTP801-like [START_REF] Shoshani | Identification of a novel hypoxia-inducible factor 1responsive gene, RTP801, involved in apoptosis[END_REF].

In this study, we cloned SMHS1 full-length cDNA and characterized the SMHS1 sequence and expression, especially in different muscle mass and energy metabolism conditions. Our data suggest that SMHS1 is a novel gene potentially important for the oxidative versus glycolytic specificities of muscle fibers.

Materials and Methods

Animals and tissues. All procedures were approved by the local Centre National de la Recherche Scientifique ethics committee. Ten-week-old Ico: OF1 (Caw) animals were used for mouse tissue RNA preparations. Atrophied soleus and gastrocnemius muscles were derived from a previous study [START_REF] Cros | Analysis of altered gene expression in rat soleus muscle atrophied by disuse[END_REF]. Ten week-old female Sprague Dawley rats (n = 9 / experimental group) were used for soleus muscle compensatory hypertrophies and glycolytic versus oxidative muscle analysis. For hypertrophy experiments, rats were anesthetized and separated in two groups. The first group was sham-operated for control, and the second group was submitted to synergist gastrocnemius muscles ablations to induce a compensatory hypertrophy of plantaris and soleus muscles by overload. Six weeks later, rats were sacrificed and soleus muscles were taken, weight and immediately frozen in liquid nitrogen.

Cell culture. C2C12 mouse skeletal muscle cells (ATCC n° CRL-1772) were grown in Dulbecco's modified Eagle's medium with 4.5 g/L glucose and 10% fetal calf serum.

Myotube C2C12 differentiation was induced by withdrawing fetal calf serum and adding 10 µg/ml insulin, 5 µg/ml transferrin and 2% horse serum.

cDNA library screening and cloning of rat SMHS1 full-length cDNA. An oligo(dT)random primed cDNA library of rat skeletal muscle (Clontech) was screened with the 03A12 cDNA fragment [START_REF] Cros | Analysis of altered gene expression in rat soleus muscle atrophied by disuse[END_REF]. Approximately 1.2 10 6 gt11 phage clones were transfected into host strain Y1090r-and then transferred onto nylon membranes (Macherey-Nagel) for hybridization, as previously described [START_REF] Cros | Analysis of altered gene expression in rat soleus muscle atrophied by disuse[END_REF]. Positive clones were plaque-purified and the cDNA phage inserts were PCR-purified using gt11 specific vector amplimers. RNA analysis. Multiple rat tissue RNA blots were purchased from Clontech Laboratories Inc.

Total RNA was prepared with RNAInstaPure kit (Eurogentec). Northern blot analyses were performed as previously described, with 32 P-labeled mouse partial SMHS1 or actin cDNA and 18S rRNA probes [START_REF] Cros | Analysis of altered gene expression in rat soleus muscle atrophied by disuse[END_REF]. Reverse transcription reactions were carried out with M-MLV-RT (Invitrogen) and oligo(dT). Advantage Klen Taq polymerase (Clontech) was used for PCR amplification of partial SMHS1 cDNA with the forward primer 5'-GAAACAGAGCCGTTGACCAT-3' and the reverse primer 5'-ATTAGCCACTCATTAGG GAC-3'. The 18S rRNA cDNA fragment was amplified with the forward primer 5'-AGTTG GTGGAGCGATTTGTC-3' and the reverse primer 5'-GGCCTCACTAAACCATCCAA-3'.

PCR samples were taken in exponential amplification phase, i.e. 25 cycles for SMHS1 and 20 cycles for 18S rRNA. Agarose electrophoretic bands and Northern blot specific signals were quantified using the PCBas software package.

In situ hybridizations were performed on 12 µm serial sections of mouse total embryos, at 75°C overnight, with 239 bp anti-sense and 400 bp sense SMHS1 digoxigenin-UTP-labeled riboprobes. Riboprobes were synthesized with the DIG RNA labeling kit (Roche), from SMHS1 cDNA cloned in pGEM-T Easy (Promega) using SP6 and T7 transcription starts after plasmid linearization with NheI. Visualization of digoxigenin (DIG)-nucleic acids with alkaline-phosphatase-conjugated anti-DIG antibody was carried out according to Roche's procedure.

Protein analysis.

Western blotting analyses were performed on protein whole extracts of wild-type C2C12 cells as previously described [START_REF] Pisani | The topoisomerase 1interacting protein BTBD1 is essential for muscle cell differentiation[END_REF]. Anti-HIF-1 monoclonal antibody (AbCam, clone hif 67 ) and anti-FLAG monoclonal antibody (Sigma) were used at 1:200 and 1:2000 dilution, respectively. Statistical analysis. t-test was performed using SigmaStat software in order to compare the differences between two groups of values. Statistical differences were noted * (p<0.05).

Results

Molecular cloning and sequence analysis of SMHS1

We previously cloned the 03A12 novel partial cDNA on the basis of its upregulation in rat soleus muscle atrophied after activity restriction induced by hindlimb suspension [START_REF] Cros | Analysis of altered gene expression in rat soleus muscle atrophied by disuse[END_REF]. This cDNA was used to screen a rat skeletal muscle cDNA library, and a putative 1.1 kb fulllength cDNA was cloned and named SMHS1 for "Skeletal Muscle Hindlimb Suspension 1".

The nucleotide sequence (GenBank accession no. AF327511) contained a 579 bp open reading frame compatible with the size of the ~1.2 kb SMHS1 mRNA. The mouse ortholog cDNA (GenBank accession no. AF327512) was cloned through RT-PCR experiments using rat SMHS1 oligonucleotides as primers and mouse muscle RNAs as templates. The open reading frame accuracy was confirmed by expression in C2C12 muscle cells (not shown). The highest SMHS1 sequence homologies were found with a few vertebrate proteins, including an unknown zebrafish protein and an hypothetical Xenopus tropicalis protein (Fig. 1). The only meaningful sequence homology concerned the 65% amino acid residue similarity (35% identity) with a recently identified protein named RTP801 [START_REF] Shoshani | Identification of a novel hypoxia-inducible factor 1responsive gene, RTP801, involved in apoptosis[END_REF], or REDD1 [START_REF] Ellisen | REDD1, a developmentally regulated transcriptional target of p63 and p53, links p63 to regulation of reactive oxygen species[END_REF], or dig2 [START_REF] Wang | Dexamethasone-induced gene 2 (dig2) is a novel pro-survival stress gene induced rapidly by diverse apoptotic signals[END_REF].

This protein is involved in cellular stress responses and its RNA transcription is regulated by hypoxia-inducible factor 1 (HIF-1). Although no known functional domains were found within the sequences reported in Fig. 1, it was interesting to note the presence of several residue stretches highly identical in the different proteins, especially in the C terminal portion, suggesting that these domains have been conserved throughout evolution.

SMHS1 mRNA expression

Hybridization of rat RNAs with the rat SMHS1 cDNA revealed an abundant ~1.2 kb mRNA with high expression in skeletal muscle, in addition to expression in heart and testis (Fig. 2A).

In fact, as previously noted with the initial 03A12 clone, two less abundant and longer mRNAs also hybridized with SMHS1 probes, but were not investigated in the present study [START_REF] Cros | Analysis of altered gene expression in rat soleus muscle atrophied by disuse[END_REF]. The rat expression profile was roughly confirmed in mouse by RT-PCR performed with rat/mouse SMHS1 specific primers and mouse tissue RNA templates. Expression was mainly detected in mouse skeletal muscle and eye, and lower expression was detected in heart and brain (Fig. 2B). Eye RNA was not tested on the above-mentioned rat Northern blots. SMHS1 mRNA tissue distribution was further confirmed by in situ hybridizations on sections of 18 day-old mouse embryos (Fig. 2C). This revealed expression in skeletal muscle, heart, and brain similar to that in adult mouse/rat (testis and eye expression was not investigated).

SMHS1 expression is independent of HIF-1 variations

Because of the sequence similarities between SMHS1 and RTP801, which is an HIF-1 target gene, we wondered whether HIF-1 could also regulate SMHS1 expression. HIF-1 comprises two subunits, HIF-1α and HIF-1β, and HIF-1α is the oxygen-dependent regulatory subunit.

We induced CoCl2 cellular stress, which is known to inhibit proteasome-induced HIF-1α degradation, thus allowing formation of the active HIF-1α/HIF-1β complex [START_REF] Poellinger | HIF-1 and hypoxic response: the plot thickens[END_REF]. Murine muscle C2C12 cells were differentiated in myotubes and stressed with 200 µM CoCl2 treatments. Changes in SMHS1 mRNA expression were assessed by semi-quantitative RT-PCR in relation with variations in HIF-1α protein quantities, followed by Western blot analysis. As expected [START_REF] Wang | Hypoxia-inducible factor 1 is a basic-helix-loop-helix-PAS heterodimer regulated by cellular O2 tension[END_REF], CoCl2 stress induced HIF-1α protein accumulation, which increased over the 3 h treatment (Fig. 3A). However, no significant variations in SMHS1 RNA were noted (Fig. 3B-C), suggesting that the HIF-1 transcription factor does not directly control SMHS1 expression.

SMHS1 expression and muscle fiber oxidative/glycolytic-energy metabolism status

In soleus muscles, activity restriction induces atrophy associated with the transition from a large majority of oxidative fibers to a mixture of oxidative and glycolytic fibers [START_REF] Cros | Upregulation of M-creatine kinase and glyceraldehyde3-phosphate dehydrogenase: two markers of muscle disuse[END_REF]. Figure 4A shows that a 40% atrophy was seen in soleus muscles after 2 weeks of activity restriction imposed by hindlimb suspension. SMHS1 was found to be two-fold upregulated in such muscles. This upregulation, which was the starting point of the SMHS1 cloning, may be thus related to the muscle mass variation or/and to the energy metabolism shift. To further clarify this point, SMHS1 mRNA expression was analyzed in 3 other situations by semi-quantitative RT-PCR and Northern blots.

First, we checked SMHS1 mRNA expression in the same rat muscle atrophy model but in the predominantly glycolytic gastrocnemius muscle (Fig. 4B). After 2 weeks of activity restriction, this muscle presented a 25% atrophy and it has been previously demonstrated that under these experimental conditions the gastrocnemius muscle undergoes no variations in myosin isoform, and consequently no variations in energy metabolism type [START_REF] Jiang | Adaptation of fibers in fast-twitch muscles of rats to spaceflight and hindlimb suspension[END_REF][START_REF] Ohira | Metabolic adaptation of skeletal muscles to gravitational unloading[END_REF]. Interestingly, no significant variation was found in SMHS1 mRNA expression.

Secondly, approximately 50% hypertrophy of soleus muscle was obtained as a compensatory effect 6 weeks after gastrocnemius muscle ablation (Fig. 4C). Such soleus muscle hypertrophy does not involve changes in the oxidative/glycolytic fiber ratio [START_REF] Almurshed | The effects of dietary energy restriction on overloaded skeletal muscle in rats[END_REF][START_REF] Almurshed | Dietary protein does not affect overloaded skeletal muscle in rats[END_REF] and no alteration of SMHS1 expression was found in the hypertrophied soleus muscles.

Thirdly, we compared SMHS1 mRNA levels in the predominantly glycolytic quadriceps muscle versus the predominantly oxidative soleus muscle, independently of any muscle mass variations. SMHS1 mRNA expression was found to be three-fold higher in the glycolytic muscle than in the oxidative muscle (Fig. 5).

Discussion

We report on cloning of a complete cDNA that we named SMHS1 for "Skeletal Muscle Hindlimb Suspension 1". The tissue origins of sequence-matching EST and cDNA clones as well as our Northern blot and RT-PCR data show that SMHS1 expression is restricted to a few tissues, mainly skeletal muscle, and to a lesser extent heart, testis, eye and brain.

As SMHS1 function is unknown, we assessed it in skeletal muscle. Our first line of investigation was based on sequence homologies with RTP801 (Fig. 1), a recently described HIF-1-regulated gene. Since SMHS1 expression is regulated in skeletal muscle, as discussed below, HIF-1 regulation of SMHS1 mRNA was considered. This actually seemed unlikely since the upregulation of HIF-1 proteinwhich allows the formation of an active HIF-1α/HIF-1β complexin the CoCl2 treatment of C2C12 cells had no impact on the SMHS1 mRNA level (Fig. 3). The non-control of SMHS1 transcription is further supported by the fact that we did not find any cis-acting nucleotide sequence binding HIF-1 in the SMHS1 gene. This motif is named the hypoxia-responsive element and its consensus sequence is either 5'-(G/C/T)ACGTGC(G/C)-3' or 5'-RCGTG-3' [START_REF] Wang | Hypoxia-inducible factor 1 is a basic-helix-loop-helix-PAS heterodimer regulated by cellular O2 tension[END_REF]. The SMHS1 8 kb-region upstream from the ATG translation initiation start codon was unsuccessfully screened for the presence of the consensus sequence. These observations strongly suggest that SMHS1 is not an HIF-1 responsive gene like RTP801.

To gain further insights in SMHS1 function, we based our in vivo experiments on the initial finding that SMHS1 expression was altered in soleus muscle atrophy due to hindlimb suspension; this was the basis for SMHS1 cloning [START_REF] Cros | Analysis of altered gene expression in rat soleus muscle atrophied by disuse[END_REF]. In this model, two modifications are primordial, muscle mass decrease and energy metabolism shift [START_REF] Cros | Upregulation of M-creatine kinase and glyceraldehyde3-phosphate dehydrogenase: two markers of muscle disuse[END_REF][START_REF] Cros | Analysis of altered gene expression in rat soleus muscle atrophied by disuse[END_REF][START_REF] Bodine | Identification of ubiquitin ligases required for skeletal muscle atrophy[END_REF][START_REF] St-Amand | Characterization of control and immobilized skeletal muscle: an overview from genetic engineering[END_REF][START_REF] Wittwer | Prolonged unloading of rat soleus muscle causes distinct adaptations of the gene profile[END_REF][START_REF] Chen | Response of rat muscle to acute resistance exercise defined by transcriptional and translational profiling[END_REF]. To assess the role of SMHS1 in each of these modifications, we analyzed SMHS1 expression in different in vivo situations. The situations associated or dissociated muscle mass variations and energy metabolism variations (Fig. 3 and4). In total, (i) a significant upregulation of SMHS1 mRNA was found in soleus muscle atrophy. The 2-week hindlimb suspension is characterized by a 40% decrease of muscle mass, and a large oxidative-to glycolytic-energy transition associated to a variation in fiber type content. The amount of type I/IIa oxidative fibers decreases from 100 to 85% and the amount of type IIx/IIb glycolytic fibers increases from 0 to 15%, according to the myosin heavy chain isoform contents [START_REF] Cros | Upregulation of M-creatine kinase and glyceraldehyde3-phosphate dehydrogenase: two markers of muscle disuse[END_REF]. (ii) Conversely, no significant SMHS1 mRNA alteration was detected during gastrocnemius muscle atrophy due to a 2-week hindlimb suspension. This muscle was 25% atrophied and no variation in energy metabolism / fiber type composition was found in this situation [START_REF] Jiang | Adaptation of fibers in fast-twitch muscles of rats to spaceflight and hindlimb suspension[END_REF][START_REF] Ohira | Metabolic adaptation of skeletal muscles to gravitational unloading[END_REF]. (iii) Our analysis showed a stable SMHS1 mRNA expression in soleus muscle hypertrophy due to a 6-weeks synergists muscles ablation. Soleus muscle was 50% hypertrophied and it has been shown that this does not imply any significant energy-metabolism switch [START_REF] Almurshed | The effects of dietary energy restriction on overloaded skeletal muscle in rats[END_REF][START_REF] Almurshed | Dietary protein does not affect overloaded skeletal muscle in rats[END_REF]. (iv) SMHS1 mRNA expression was finally checked in the predominantly glycolytic gastrocnemius muscle (70% type IIb/x fibers and 30% type I/IIa fibers) compared to the predominantly oxidative soleus muscle (100% type I/IIa fibers) [START_REF] Cros | Upregulation of M-creatine kinase and glyceraldehyde3-phosphate dehydrogenase: two markers of muscle disuse[END_REF][START_REF] Jiang | Adaptation of fibers in fast-twitch muscles of rats to spaceflight and hindlimb suspension[END_REF], and found to be three-fold higher in the more glycolytic muscle. These results taken together strongly suggest that an higher SMHS1 mRNA expression is associated with muscle glycolytic energy metabolism; SMHS1 expression is related to oxidative/glycolytic metabolism balance and varies with skeletal muscle energy metabolism transitions, independently of muscle mass variations per se.

Many molecules are involved in the development and in the differences between oxidative and glycolytic muscle fibers, and the plasticity between these fibers according to muscle functional constraints [START_REF] Pette | Cellular and molecular diversities of mammalian skeletal muscle fibers[END_REF][START_REF] Schiaffino | Molecular diversity of myofibrillar proteins: gene regulation and functional significance[END_REF][START_REF] Fitts | Physiology of a microgravity environment invited review: microgravity and skeletal muscle[END_REF][START_REF] Edgerton | Human fiber size and enzymatic properties after 5 and 11 days of spaceflight[END_REF][START_REF] Booth | Changes in skeletal muscle gene expression consequent to altered weight bearing[END_REF][START_REF] Thomason | Atrophy of the soleus muscle by hindlimb unweighting[END_REF][START_REF] Schiaffino | Calcineurin signaling and neural control of skeletal muscle fiber type and size[END_REF]. SMHS1 is another good candidate molecule that could be involved in such mechanisms, and this could be further supported by the fact that SMHS1 has sequence homologies with RTP801/REDD1/dig2 [START_REF] Shoshani | Identification of a novel hypoxia-inducible factor 1responsive gene, RTP801, involved in apoptosis[END_REF][START_REF] Ellisen | REDD1, a developmentally regulated transcriptional target of p63 and p53, links p63 to regulation of reactive oxygen species[END_REF][START_REF] Wang | Dexamethasone-induced gene 2 (dig2) is a novel pro-survival stress gene induced rapidly by diverse apoptotic signals[END_REF], which is claimed to be involved in oxidative stress and hypoxia response. Indeed, muscle fiber energy metabolism is strongly linked to oxygen homeostasis.

Finally, it seems likely that the novel SMHS1 gene, whose exact function still remains to be elucidated, is not a muscle mass variation marker, nor a cellular stress response gene (unlike RTP801/REDD1/dig2), but is involved in the energy metabolism status within skeletal muscle fibers. It will be interesting to find out what is the exact SMHS1 contribution to the skeletal muscle fiber phenotypes.

Figure 1 .

 1 Figure 1. Multiple alignment of the SMHS1 protein sequence with vertebrate homologous

Figure 2 .

 2 Figure 2. SMHS1 tissue expression analysis. A) Tissue distribution in rat. Multiple tissue

Figure 3 .

 3 Figure 3. SMHS1 expression in differentiated C2C12 myotubes treated with 200 µM CoCl2.

Figure 4 .

 4 Figure 4. SMHS1 mRNA amounts and muscle mass variations. Soleus (A) and

Figure 5 .

 5 Figure 5. Comparison of SMHS1 expression in predominantly oxidative soleus muscle (S)

Figure 5 .

 5 Figure 5.

Acknowledgments:

We are grateful to David Manley for the English revision of the manuscript. D. F. Pisani is a recipient of grants from the Association Française contre les Myopathies and the Fondation pour la Recherche Médicale.


Didier F Pisani

Claude A Dechesne²
email: dechesne@unice.fr

Skeletal muscle HIF-1α expression is dependent on muscle fiber type

Keywords: HIF-1, skeletal muscle, oxidative fiber, glycolytic fiber, atrophy

Oxygen homeostasis is an essential regulation system for cell energy production and survival.

The oxygen-sensitive subunit α of the HIF-1 complex is a key protein of this system. In this work, we analyzed rodent HIF-1α protein and mRNA expression in parallel to energetic metabolism variations within skeletal muscle. Two physiological situations were studied.

First, we compared HIF-1α expression between the predominantly oxidative soleus muscle and three predominantly glycolytic muscles. Second, HIF-1α expression was assessed in an energy-metabolism switch model which was based on muscle disuse. Our results gave rise to the hypothesis that the oxygen homeostasis regulation system depends on the fiber type, with higher constitutive HIF-1α expression in glycolytic muscle fibers.

INTRODUCTION

Eukaryotic cells sense oxygen and adapt to hypoxia by regulating a certain number of genes. This was shown many years ago for various mammalian cell types, including C 2 C 12 myoblasts [START_REF] Webster | Regulation of glycolytic enzyme RNA transcriptional rates by oxygen availability in skeletal muscle cells[END_REF]. The HIF-1 (hypoxia inducible factor-1) transcription factor is a key element of this pleiotropic response. Wang and Semenza observed, more than a decade ago, that HIF-1 DNA binding activity is specifically present under low oxygen conditions for different cell lines, including muscle cells [START_REF] Wang | General involvement of hypoxia-inducible factor 1 in transcriptional response to hypoxia[END_REF]. Activated HIF-1 comprises two subunits, i.e. HIF-1α and HIF-1β. HIF-1α plays a central role as it is subjected to drastic O 2dependent proteasomal control. At normoxia, HIF-1α and HIF-1β are constitutively expressed but HIF-1α is mainly ubiquitinated and degraded by the proteasome [START_REF] Jaakkola | Targeting of HIF-alpha to the von Hippel-Lindau ubiquitylation complex by O2-regulated prolyl hydroxylation[END_REF]. In severe hypoxia, the HIF-1α protein level increases by relaxing its degradation, and HIF-1α can form an active complex with HIF-1β. Activated HIF-1 induces the expression of genes involved, for example, in angiogenesis, erythropoiesis, glucose uptake and energy metabolism [START_REF] Semenza | Oxygen-regulated transcription factors and their role in pulmonary disease[END_REF][START_REF] Wenger | Mammalian oxygen sensing, signalling and gene regulation[END_REF]. At the RNA level, no HIF-1α upregulation has been described in response to short-term hypoxia or other stimuli, e.g. CoCl 2 , CO or insulin [START_REF] Ladoux | Cardiac expressions of HIF-1 alpha and HLF/EPAS, two basic loop helix/PAS domain transcription factors involved in adaptative responses to hypoxic stresses[END_REF]. However, several studies have reported an increase of HIF-1α mRNA in chronic hypoxia conditions, such as adaptation to altitude (Hoppeler and Vogt, 2001a, b) or high glucose concentration situations [START_REF] Marfella | Myocardial infarction in diabetic rats: role of hyperglycaemia on infarct size and early expression of hypoxia-inducible factor 1[END_REF], without any clear explanations. Moreover, a recent study involving a global analysis of DNA arrays of genes altered in chronic critical and acute-on-chronic human skeletal muscle ischemia conditions, demonstrated upregulation of genes involved in the HIF-1 system, including HIF1 itself [START_REF] Tuomisto | HIF-VEGF-VEGFR-2, TNF-alpha and IGF pathways are upregulated in critical human skeletal muscle ischemia as studied with DNA array[END_REF]. These observations show that HIF-1α could also be regulated at the RNA level in several conditions. HIF-1 studies in skeletal muscle are difficult due to the presence of a number of energy metabolism systems which include different O 2 supplies and homeostasis, with different proportions of oxidative, glycolytic, and intermediate fibers. Indeed, it has been shown that skeletal muscle presents a continuum of mechanical and metabolic properties from the slow contractile speed type I fibers, which have a low fatigability, to the fast contractile speed type IIb fibers, which have a high fatigability. Type I fiber energy is mainly sustained by an oxidative metabolism, while type IIb energy is mainly generated by a glycolytic metabolism [START_REF] Booth | Muscle plasticity: energy demand and supply processes[END_REF]. Moreover, this metabolic/mechanical status is not fixed, but adapts to environmental changes such as chronic activity modifications. For instance, slowtwitch muscles modify their phenotype from a predominantly oxidative slow-to a glycolytic fast-type metabolism under different physiological or experimental conditions of muscle atrophy induced by restriction of muscle activity [START_REF] Diffee | Contractile and biochemical properties of rat soleus and plantaris after hindlimb suspension[END_REF][START_REF] Ohira | Rat soleus muscle fiber responses to 14 days of spaceflight and hindlimb suspension[END_REF][START_REF] Cros | Analysis of altered gene expression in rat soleus muscle atrophied by disuse[END_REF].

In this study, we investigated HIF-1α mRNA and protein expression in relation with muscular energy metabolism types. Our observations strongly suggest that HIF-1α expression is fibertype dependent.

MATERIALS AND METHODS

Animals and tissues. All procedures were approved by the local Centre National de la Recherche Scientifique ethics committee. Skeletal muscles were quickly excised from anesthetized (intra-peritoneal injection of pentobarbital, 1 µL/g of body mass) 10 week-old Ico: OF1 (Caw) mice (Charles Rivers Laboratories), then cleaned and immediately frozen in liquid nitrogen for RNA and protein immunoblotting analysis. Rat gastrocnemius and soleus muscles atrophied by 2 weeks of hindlimb suspension and control muscles were derived from a previous study [START_REF] Cros | Analysis of altered gene expression in rat soleus muscle atrophied by disuse[END_REF].

Cell culture. C 2 C 12 mouse skeletal muscle cells were obtained from the American Type Culture Collection (ATCC n° CRL-1772), and grown in Dulbecco's modified Eagle's medium with 4.5 g/L glucose and 10% fetal calf serum. Myotube C 2 C 12 differentiation was induced by withdrawing fetal calf serum and adding 10 µg/ml insulin, 5 µg/ml transferrin and 2% horse serum. Cells were stressed by adding 200 µM CoCl 2 to the culture medium.

RNA analysis. Skeletal muscle RNAs were prepared from nine-rat pool muscles to eliminate individual variations or from three cell culture dishes. Semi-quantitative RT-PCR (reverse transcriptase and polymerase chain reaction) cDNA amplification and quantification were performed as described previously [START_REF] Pisani | Myodulin is a novel potential angiogenic factor in skeletal muscle[END_REF]. HIF-1α cDNA fragments were amplified as described by Marfella et al. [START_REF] Marfella | Myocardial infarction in diabetic rats: role of hyperglycaemia on infarct size and early expression of hypoxia-inducible factor 1[END_REF] and 18S rRNA fragments were amplified as described by [START_REF] Pisani | Myodulin is a novel potential angiogenic factor in skeletal muscle[END_REF].

Protein analysis. For immunoblotting analysis, total soluble proteins were prepared from fresh cell cultures or frozen skeletal muscles as previously described [START_REF] Yeow | Increased MAPK signaling during in vitro muscle wounding[END_REF].

Equal amounts of proteins were separated by SDS-PAGE on 6% acrylamide gels. Proteins were transferred to Hybond-C extra membranes (Amersham) and stained with ponceau red to check whether the transfers were uniform. Blots were then incubated for 4 h at room temperature in TBS with 0.1% Tween-20 and 5% nonfat powdered milk. Membranes were probed overnight at 4°C with anti-HIF-1α polyclonal antibody (kindly provided by Dr.

Christiane Brahimini-Horn and Dr. Jacques Pouysségur), at 1:2000 dilution in TBS, 0.1% Tween-20, 1% nonfat powdered milk, and then for 1 h at room temperature with an HRPconjugated swine anti-rabbit IgG (Dako) used at 1:2000 dilution in TBS, 0.1% Tween-20 and 5% nonfat powdered milk. After each incubation, membranes were washed three times in TBS, 0.1% Tween-20, for 10 min per wash. Proteins were then revealed using the Amersham ECL system. Statistical analysis. t-tests were performed using SigmaStat software to compare differences between two groups of values. Statistical differences were noted * (p<0.05).

RESULTS

Difference in HIF-1α expression between oxidative-and glycolytic-energy metabolism muscle fibers. We compared HIF-1α expression between a predominantly oxidative muscle (soleus) and three predominantly glycolytic muscles (gastrocnemius, tibialis anterior, and quadriceps). An analysis was performed on mouse muscles, which were immediately snapfrozen in liquid nitrogen after excision to avoid any modifications induced by tissue hypoxia.

With the same goal, the first step of protein or RNA preparations were carefully carried out directly using frozen tissues. First, we investigated the HIF-1α protein level by Western blotting (Fig. 1A). A specific 120 kDa signal was detected with a monoclonal anti-HIF-1α antibody on nuclear protein extracts. HIF-1α quantities were reproducibly found to be 8-to 10-fold higher in glycolytic muscles than in the oxidative soleus muscle. Despite the care taken in tissue storage and protein preparation for all muscles, we could not rule out that the drastic difference noted between glycolytic muscles and the oxidative muscle was caused by some more hypoxic conditions for glycolytic muscle sample preparations. Therefore, we compared HIF-1α mRNA quantities in the oxidative soleus muscle with gastrocnemius and quadriceps glycolytic muscles by semi-quantitative RT-PCR, since no mRNA variations were expected under short-term hypoxia. HIF-1α mRNA expression was found to be three-fold higher in glycolytic muscles than in the oxidative muscle (Fig. 1B). The higher HIF-1α protein quantity observed in glycolytic muscles was thus found to be associated with a higher HIF-1α mRNA content.

CoCl 2 stress induces an HIF-1α protein increase but no mRNA modifications in differentiated C 2 C 12 muscle cells. We wondered whether HIF-1α regulation by hypoxia would also involve mRNA regulation in skeletal muscle, unlike other tissues. CoCl 2 is known to mimic acute hypoxia upon in vitro HIF-1 activity induction [START_REF] Wang | Hypoxia-inducible factor 1 is a basic-helix-loophelix-PAS heterodimer regulated by cellular O2 tension[END_REF]. It inhibits HIF-1α ubiquitination and consequently its degradation by proteasomes, thus allowing formation of the active HIF-1α/HIF-1β complex. Myotube differentiated C 2 C 12 cells were stressed for 3 h with 200 µM CoCl 2 treatments. As expected [START_REF] Wang | Hypoxia-inducible factor 1 is a basic-helix-loophelix-PAS heterodimer regulated by cellular O2 tension[END_REF], Western blot analysis revealed an increase in HIF-1α protein (Fig. 2A), but no variations in mRNA contents were detected by semi-quantitative RT-PCR (Fig. 2B-C). This was in agreement with the hypothesis that the increase in HIF-1α protein was only due to a decrease in HIF-1α degradation, as very well documented in other tissues [START_REF] Wenger | Mammalian oxygen sensing, signalling and gene regulation[END_REF].

Variations in HIF-1α mRNA in a model of muscle energy metabolism switch. To further test the hypothesis that the HIF-1α protein level differences reported above between oxidative and glycolytic muscles was due to constitutive differences in HIF-1α mRNA quantities between the two kinds of muscles and not to artefactual hypoxic conditions, we analysed another physiological situation. Instead of comparing different muscles, we used a procedure which induced an energy-metabolism switch within the same muscle. Thus, HIF-1α mRNA expression was analysed by semi-quantitative RT-PCR in rat soleus and gastrocnemius muscles submitted to 2 weeks of activity restriction by hindlimb suspension [START_REF] Diffee | Contractile and biochemical properties of rat soleus and plantaris after hindlimb suspension[END_REF]. The anti-gravity oxidative soleus muscles were more than 40% atrophied and approximately 20% of the oxidative fibers were replaced by glycolytic fibers, as assessed via the myosin isoform contents [START_REF] Cros | Upregulation of M-creatine kinase and glyceraldehyde3-phosphate dehydrogenase: two markers of muscle disuse[END_REF]. In this situation of oxidative-to glycolyticenergy metabolism transition within soleus muscle, HIF-1α mRNA was found to be threefold upregulated (Fig. 3). Conversely, the glycolytic gastrocnemius muscles, which underwent only 25% atrophy, showed no variation in energy metabolism [START_REF] Jiang | Adaptation of fibers in fast-twitch muscles of rats to spaceflight and hindlimb suspension[END_REF][START_REF] Ohira | Metabolic adaptation of skeletal muscles to gravitational unloading[END_REF] and the HIF-1α mRNA content was not significantly modified (Fig. 3).

DISCUSSION

Energy for muscle contraction is provided by oxidative and glycolytic metabolisms. Skeletal muscles are made of fibers that differ according to the relative involvement of each of these energy metabolisms. These metabolism differences are associated with different mechanical properties that allow muscles to meet various functional demands. As HIF-1 is a critical sensor of cellular oxygen, we investigated expression of the regulatory HIF-1α subunit in accordance with the oxidative/glycolytic energy balance in skeletal muscle. Variations in this balance naturally occur in different muscles or can be experimentally forced by modifying the mechanical constraints. In this hypoxia-free study, we have shown: (i) higher HIF-1α expression in predominantly glycolytic muscles than in the predominantly oxidative soleus muscle, and (ii) an HIF-1α expression increase in the oxidative-to glycolytic-energy transition noted in soleus muscle activity reduction imposed by hindlimb suspension. These variations were noted at the protein level but also at the mRNA level. The existence of HIF-1α mRNA variations ruled out the possibility of simple hypoxia artifacts in RNA preparations from glycolytic muscles. Indeed, as confirmed by the experimental CoCl 2 treatment of C 2 C 12 myoblasts, muscle cells sense oxygen like other cells by regulating HIF-1α protein and not HIF-1α mRNA.

It was not surprising to detect HIF-1α expression in normoxic skeletal muscle. Indeed, contrary to most tissues, it has been shown that HIF-1α is present under normoxic conditions in a few mouse tissues, including brain and skeletal muscle, where its level increases in response to systemic hypoxia [START_REF] Stroka | HIF-1 is expressed in normoxic tissue and displays an organ-specific regulation under systemic hypoxia[END_REF]. In the present work, we confirmed these observations, suggesting that HIF-1 could have a potential function in muscle oxygen homeostasis under normoxia. In addition, our results highlighted that HIF-1 expression was constitutively higher in glycolytic fibers than in oxidative fibers. This would explain previous observations regarding HIF-1α mRNA increases in human adaptation to altitude (Hoppeler and Vogt, 2001a, b), or under high glucose concentrations [START_REF] Marfella | Myocardial infarction in diabetic rats: role of hyperglycaemia on infarct size and early expression of hypoxia-inducible factor 1[END_REF].

Variations in HIF-1α content according to skeletal muscle fiber types in normoxia conditions should be compared to the myoglobin content. According to our results, oxidative fibers have a low HIF-1α content and are known to express a high level of myoglobin (Mb) [START_REF] Lin | Transcriptional co-activator PGC-1 alpha drives the formation of slow-twitch muscle fibres[END_REF], which is responsible for their red color. Mb is a cytoplasmic hemoprotein able to link O 2 in the interstitial compartment to carry it inside the fiber [START_REF] Brunori | Nitric oxide, cytochrome-c oxidase and myoglobin[END_REF]. Mb forms an intracellular oxygen reserve which to a certain extent enables muscular fibers to buffer variations in blood O 2 supply (i.e. hypoxia), or to meet fiber metabolism needs. In contrast, glycolytic fibers were shown here to have a high HIF-1α content but they express a low level of Mb [START_REF] Vogt | Molecular adaptations in human skeletal muscle to endurance training under simulated hypoxic conditions[END_REF]. In this case, we could speculate that the Mb level is not efficient enough to compensate for alterations in O 2 homeostasis (i.e. hypoxia), which implies a higher fiber sensitivity to O 2 variations-the higher HIF-1α content in glycolytic fibers would be necessary to provide satisfactory hypoxia protection for these fibers. Moreover, a balance between HIF-1α and Mb was recently documented in a study of Mb -/-knocked-out mice [START_REF] Grange | Functional and molecular adaptations in skeletal muscle of myoglobin-mutant mice[END_REF]. Indeed, these mice were viable and showed significant phenotypic modifications to compensate for the Mb absence, including HIF-1α upregulation and metabolism transitions from oxidative to glycolytic muscle fibers [START_REF] Grange | Functional and molecular adaptations in skeletal muscle of myoglobin-mutant mice[END_REF].

Conversely, a recent study of partially HIF-1α -/-knocked-out mice showed no variation in fiber type composition in soleus and gastrocnemius muscles [START_REF] Mason | Loss of Skeletal Muscle HIF-1alpha Results in Altered Exercise Endurance[END_REF]. However, a clear shift from glycolytic to oxidative muscle metabolism was detected during exercise of these modified mice in comparison to wild-type mice. Taken together, these points could be explained by the presence of an O 2 regulation system based mainly on Mb in oxidative fibers and on an HIF-1 complex in glycolytic fibers.

In conclusion, our data confirmed differences in the HIF-1-mediated oxygen regulation system between normoxic oxidative and glycolytic skeletal muscle fibers. The higher HIF-1α expression observed under the lower O 2 supply conditions in glycolytic fibers underlines the importance of HIF-1 for maintaining muscle homeostasis and HIF-1 involvement in muscle plasticity to tailor the metabolism, and therefore the mechanical properties, to meet the functional demand.

FIGURE LEGENDS Figure 1 .

 LEGENDS1 FIGURE LEGENDS

Figure 2 .

 2 Figure 2. HIF-1α expression in differentiated C 2 C 12 myotubes treated with 200 µM

Figure 3 .

 3 Figure 3. Comparison of HIF-1α mRNA expression in soleus and gastrocnemius muscles

Figure 3 .

 3 Figure 3.

Acknowledgements: We thank Dr. Christiane Brahimini-Horn and Dr. Jacques Pouysségur for providing anti-HIF-1α polyclonal antibody, Dr. Annie Ladoux for constructive discussions on the HIF-1 system, Anne-Sophie Coldefy for the statistical analysis and David Manley for the English revision of the manuscript. D.F. Pisani is a recipient of grants from the Association Française contre les Myopathies and the Fondation pour la Recherche Médicale.