Marie-Josèphe Leroux-Sostenes

Prof. Dr Emmanuel Bayle
email: emmanuel.bayle@unil.ch

Martin Kopp

The crowdfunding of sport -paving the way to shared sponsorship?

Keywords:

The rise of crowdfunding as a way of funding sport projects has prompted numerous companies to become involved in these campaigns. This paper explores a model of crowdfunding in which a sponsor company supports individual projects. A qualitative study based on interviews with crowdfunding executives showed that campaigns are more likely to be successful if they include a sponsor company, and that crowdfunding platforms and the projects they support have become a new interactive, online communication tool for sponsors. Our data revealed four modes of corporate involvement in crowdfunding and fourteen objectives companies hope to achieve through this involvement. Three of these objectives can be attained exclusively via this communication tool. In addition, companies focus on five "success factors" when deciding whether or not to sponsor a crowdfunding campaign. This model of corporate-supported crowdfunding is creating a new paradigm of "shared" (by a community) sponsorship that will complement the current system of "confined" (to specific companies) sponsorship.

Introduction

According to the annual KPMG report, French crowdfunding websites raised € 336 million in the first half of 2018, double the amount they had raised during the same period the previous year (Odent & Ptachek 2018). This spectacular growth, which is largely due to the development of loan-based crowdfunding, is forecast to continue, thereby consolidating crowdfunding's position as an important alternative mode of finance in almost every area of human activity, including sport. Crowdfunding's success quickly attracted the interest of the research community. Many early studies of crowdfunding were conducted by economists, who investigated consumer behavior and the criteria consumers use when choosing products and services online [START_REF] Belleflamme | Crowdfunding: Tapping the right crowd[END_REF]. At the same time, management scientists noted crowdfunding's ability to eliminate the distance between project creators and funders [START_REF] Agrawal | Offline relationships, distance and the Internet: the geography of crowdfunding[END_REF], and the importance of both personal and situational factors in motivating crowdfunders (Agrawal & al., 2013, Gerber & Hui, 2013). To date, most crowdfunded projects have followed the original model, in which project creators directly solicit donations from contributors (target community) by posting their projects on the Internet, usually on specialist crowdfunding platforms such as Sponsorise.me (France's leading crowdfunding platform). However, recent years have seen the emergence of a new crowdfunding model involving a fourth actor, in the form of a sponsor/company, who provides creators and/or platforms with public support in order to help them reach/extend their communities of individual contributors. Although this approach is still quite rare, it is an interesting way of increasing a project's/platform's chances of success in what has become a crowded market. The present paper describes what is, to the best of our knowledge, the first academic study to examine this new approach to crowdfunding. Focusing on crowdfunding in the field of sport, our aim was to determine the objectives of companies that support the crowdfunding process. We begin by presenting the concept of crowdfunding and the ways it is being applied in the field of sport. Section two uses these observations to examine crowdfunding as a possible new field of sponsorship. We then present the results of our study in terms of the modes of corporate involvement in crowdfunding and sponsor companies' objectives. Finally, we discuss the notion that corporate support for crowdfunding is creating a new paradigm in which the sponsorship of athletes, clubs, teams, events, etc. is shared by an entire community, rather than being restricted to a limited number of sponsors. We call this new paradigm "shared sponsorship".

How is sport embracing crowdfunding?

Crowdfunding, appealing to the masses to obtain the funds needed to realize a project, first developed as a way of financing start-ups in the arts. In recent years, this concept has been embraced by the sports sector, mostly through specialist platforms.

The roots of crowdfunding

Crowdfunding, which arose out of the more general concept of crowdsourcing (obtaining input to a project from a large number of people), is the act of funding a project by using the Internet to solicit monetary donations, sometimes in exchange for a future product, service, or reward. Donations are collected by using web technologies, including online payment systems, to facilitate contacts and transactions between creators (the people soliciting funds) and contributors (the people who donate money). In a study including all categories of creators and contributors, [START_REF] Gerber | Crowdfunding: why people are motivated to post and fund projects on crowdfunding platforms[END_REF] found that people are motivated to participate in crowdfunding communities by a desire to obtain recognition, improve their social standing, build relations with peers, and improve society. Crowdfunding exploits the fact that contributors are motivated to contribute to projects by the social interactions they experience via crowdfunding platforms (e.g., reinforcement of their commitment to an idea or a cause) and which create a strong sense of connectedness to a community with similar ideals.

What is crowdfunding?

According to Onnée and Renault (2013, p.55), for a project creator (whatever that creator's status: private individual, commercial company, non-commercial body, etc.), crowdfunding consists of "having recourse to a funding platform (generalist or specialist) in order to propose a project (finalized or not) to a community (general or targeted) of potential contributors (backers), possibly in exchange for an agreed reward". These same authors differentiated between four crowdfunding models on the basis of the rewards offered to contributors: • A model based on donations, akin to a sort of patronage.

(For example, donations to fund a sports association.); • A model based on compensation in the form of a tangible or intangible reward (thanks, invitations, etc.). (For example, support given to an athlete in exchange for invitations to sports events.); • A model based on investment, in which contributors become shareholders in the company requesting funds.

(For example, buying shares in a sports club.); • A model based on loans, in which contributions are reimbursed according to a pre-defined schedule of payments.

Three-quarters of successful campaigns use the compensation model [START_REF] Onnée | Le financement participatif: atouts, risques et conditions de succès[END_REF], which is the most widely used model overall. It is also the predominant model in the sports sector; however, very few studies have looked at crowdfunding in this sector [START_REF] Abdourazakou | Crowd Funding: the New Frontier of Sports Sponsorship[END_REF].

Crowdfunding in sport

Sponsors in the field of sport have long been aware of the importance of social ties and the ability of sports events to generate powerful relational and emotional experiences. Sponsoring sports events enables companies to become associated with these experiences and thereby create close ties with their target audience. Sponsorship also allows companies to communicate a social message that touches the spectator's or listener's desire to feel part of a social or sporting community.

Crowdfunding provides an alternative/complementary way for sport-related projects to obtain funding. In addition to general crowdfunding sites that include sports projects (e.g., KKBB), a large number of specialist sports crowdfunding platforms (Sponsorise.me, Fosburit, Makeachamp, WePlaySport, I Believe in You, etc.) have been created in recent years. These platforms list a wide range of personal (e.g., take part in a competition) and social (e.g., climb Russia's Mount Elbrus in order to raise funds for the charity Handi'Chiens) projects. Sports crowdfunding via these specialist platforms meets the needs of both the creators of projects directly related to sport or physical activity, and of funders, whether individuals or companies. teams, sport administrators, clubs, federations, or companies that organize sports events, etc. In most cases, if the entire sum requested is raised, the project is financed; if not, contributors receive a full refund. [START_REF] Hemer | A Snapshot on Crowdfunding[END_REF] refers to this as an "all-or-nothing model". Another model, albeit one that is used more rarely, allows for the project creator to be given the amount collected by the platform, event if this sum is less than the amount requested. Moreover, Loïc Yviquel, one of Sponsorise.me's co-founders, notes that 80% of projects ask for amounts of less than €2,000, but the range of sums requested is extremely wide. Although most contributions are provided by private individuals who believe in the project, sports crowdfunding platforms have begun asking sponsor companies to help finance certain projects.

Sports crowdfunding, a new approach to sports sponsorship?

Project creators solicit donations via a crowdfunding platform when they are unable or unwilling [START_REF] Hemer | A Snapshot on Crowdfunding[END_REF] to seek funding from classic sources such as personal funds, loans, subsidies, or individual sponsors. Many crowdfunding platforms now encourage support companies to contribute to projects alongside the crowd in order to help creators achieve their funding targets. So, is crowdfunding becoming a medium for a new form of "shared sponsorship" in which companies and individuals jointly support projects?

A model of crowdfunding incorporating sponsor companies

Before examining the objectives underlying an eventual sponsor's/company's decision to become involved in crowdfunding campaigns, it is necessary to differentiate between two coexisting crowdfunding models. Both models are founded on the notion of social value, defined by Mucchielli (2001, p 13) as "a reference principle shared by a group of individuals; this value is the root of a behavior that is considered ideal and commendable by the group and that guides the conduct of the group's members". In the first model (primary form of crowdfunding), a project creator (individual, athlete, club, federation, organization, etc.) posts a project on a crowdfunding platform in order to bring it to the attention of a target community, that is, "a group of individuals with shared objectives and values" (Bessière & Stéphany, 2014, p 155), which is asked to finance the project. An example of this primary form of crowdfunding is provided by the way Racing Club de Toulon (RCT), a leading French rugby club, raised money to renovate an historic part of its premises. In 2014 RCT posted its "Avenue of Legends" project on the Sponsorise.me website, asking rugby fans to help the club refurbish its training center by contributing to the project. In return, contributors would have their names inscribed in the pavement alongside the names of RCT's most illustrious players. The project raised 106% of its funding objective.

In the second model (secondary form of crowdfunding), a project's fundraising efforts are supported by the active involvement of a sponsor company. The "Ladji Doucoure: Back to the Top" project (supported by a chain of sports stores) provides a very successful illustration of this model, as the athlete obtained the €8,000 he asked for within 24 hours. In addition to the rewards offered to contributors (spend a day at a training session, race against the athlete, etc.), each person who donated at least €30 received a gift voucher for the Intersport chain of sports shops. This example is typical of one way in which companies become involved in crowdfunding, that is, by sponsoring a crowdfunding platform. Figure 1 illustrates these two crowdfunding models. The present study examines only the second model. [START_REF] Derbaix | Essai de conceptualisation d'une activité éminemment pratique : le parrainage[END_REF][START_REF] Derbaix | Essai de conceptualisation d'une activité éminemment pratique : le parrainage[END_REF][START_REF] Didellon | Mode de persuasion et mesure d'efficacité du parrainage: une application au modèle sportif[END_REF] to produce a consensual definition, they defined sponsorship as "an advertising technique that, for all organizations, brands, or products, involves creating or directly supporting a socio-culturally independent body and developing a widely publicized association with that body in order to achieve marketing promotion objectives". Contributors in the first form of crowdfunding (see figure 1) are uniquely individuals, whereas in the second form of crowdfunding support is provided by sponsor companies, alongside individual contributors. By providing this support, sponsor companies hope to do much more than increase their name exposure, as it enables them to pursue another important marketing objective-building ties with members of a community. In fact, according to Walliser (2006a, p.31), sponsorship should be considered in terms of networks of actors, rather than as individual links between sponsors and sponsees. chose here was particularly suited to our study's exploratory nature. Our first step was to collect data from the very sparse literature on this subject, from semi-structured interviews with executives from crowdfunding platforms that have signed partnership agreements with sponsor companies, and from an exploratory interview with a sponsor company. The crowdfunding platforms included in our study were either specialist sports platforms or general platforms with a section for sports projects. We then supplemented this initial data set by carrying out further interviews with the founders of platforms that had not yet set up partnerships with companies.

In total, we carried out in-depth interviews with eleven platform executives plus an exploratory interview with La Banque Postale, as a sponsor company (Table 1).

The small number of interviews was the result of two factors:

• A saturation effect due to the small number of platforms that have set up partnerships with sponsor companies; • The "exemplary" [START_REF] David | Des rapports entre généralisation et actionnabilité: le statut des connaissances dans les études de cas[END_REF] and "revealing" [START_REF] Yin | Case study research: design and methods[END_REF] nature of our case study • corporate support for crowdfunding platforms is both an innovative practice and a domain in which information is strategic and confidential. Eight platforms refused our request for an interview, generally citing a lack of time and confidentiality issues.

Each of the eleven semi-structured interviews lasted between 30 minutes and 1 hour, and was conducted according to an interview guide organized into three main themes:

• The practicalities of setting up the platform/support company relationship (history of the partnership, objectives, choice of projects, etc.); the fact that the parties involved in a sponsorship arrangement are members of a network of interacting stakeholders. Viewed from this perspective, corporate involvement in crowdfunding could be considered a new approach to sponsorship, a sort of sponsorship 2.0 in which a wide variety of stakeholders, including individual contributors, come together to support a project. Both crowdfunding and sponsorship are based on interactions between actors. This commonality has allowed the emergence of a new model of crowdfunding incorporating support companies. The present study examined the objectives of these support companies, as described by crowdfunding platform executives.

Research methodology

Given the lack of previous studies into sponsor-company involvement in crowdfunding projects, for the present study we decided to use the qualitative and exploratory approach described by Evrard, Bras, and Roux (1997). Our aims were to:

• Identify the different modes of corporate involvement in the crowdfunding process. • Determine the objectives of companies that sponsor projects.

Our methodology was based on qualitative interviews with members of the crowdfunding industry, which we then used as the basis for a sort of collective case study. By adopting a casestudy methodology, we were able to bring together multiple empirical sources in order to investigate a contemporary phenomenon (the involvement of sponsor companies in crowdfunding) in its real context [START_REF] Yin | Case study research: Design and methods[END_REF]). The collective case study methodology (Stake, 1994, cited in David, 2005) we

Person

Results and discussion

We begin by describing the different modes of corporate involvement in the crowdfunding process. We then use this framework to describe companies' objectives in sponsoring a project and define the success factors impacting companies' decisions on whether or not to support a project. As a final stage, we draw on these results to define a new concept for sponsoring: shared sponsoring.

Modes of corporate involvement in crowdfunding

Many platform executives have begun implementing a new idea: associate a sponsor with certain, carefully selected crowdfunding campaigns. In fact, numerous platforms have formed or intend to form partnerships with companies. For example, KKBB has created partnerships with several companies, including La Banque Postale and, in 2016, the Swiss platform I Believe in You signed partnership agreements with three large Swiss companies: Postfinance, Helsana, and Aldi.

Postfinance gives up to € 200 to projects that have attracted at least 50% of the finance requested; Helsana donates € 200 to sport health projects; and Aldi gives € 400 to projects targeted at the under-20s. In addition, Helsana and Aldi donate € 1000 and € 3000, respectively, to the best projects every month. We used the data obtained during the interviews to draw up a typology of these different partnerships (see table 2, below):

• A partner company supports an entire platform because it adheres to values with which the partner company would like to be associated. This allows a platform to, for example, "display the company's logo on projects" (EB, head of Ecobole). • A partner company supports certain categories of project, such as sports activities. DK, the co-founder of Fédérêves, explained why she chose this type of partnership: "We take a proactive approach. Our platform includes three themes: Women, Disability, and Insertion, so, we are looking for one or two companies for each theme. We don't feel it is necessary to have a company for every project.

Communication is oriented more towards a theme than towards a project". • A company contributes to projects in the same way as other contributors (members of the community). Our interviews with platform executives suggest that this is currently the most widely sought type of involvement. We identified three distinct cases within this category.

First, for AC, the co-founder of Weplaysport, it is frequently companies belonging to the project creator's network that contribute to a project. Consequently, companies are solicited directly by project creators, not by the platform. Second, some platforms combine this first approach with a more proactive approach in which the platform directly solicits potential partner companies to contribute to projects. For example, specialist sports platform Fosburit mostly targets local SMEs/SMIs: "First and foremost, we work with project creators. Then we contact companies who may be interested in supporting projects in their local area". Third, platforms such as Sponsorise.me have set up a system through which sponsor companies can look at projects posted on the platform and choose which, if any, they would like to contribute to financially.

• A company creates a platform specifically for campaigns initiated by its staff. With this in mind, BB, co-founder of myannona.com, explained: "I want to build products with internal communities (that is, the company's staff ). For example, EDF contributes to projects supported by its staff and to community projects instigated by its staff".

So, corporate communication includes both an external dimension, targeting stakeholders within the company's orbit, and an internal dimension, focusing on the company's own staff.

There are, of course, numerous interactions between these two dimensions. Of all these modes of corporate involvement, the present study focused on the "Contribution to individual projects as a sponsor" approach, as this is the only mode involving a strategic approach to sports marketing by sponsor companies. Even if it is a rapidly expanding way of obtaining funding, crowdfunding has by no means reached maturity. Consequently, little is known about the objectives of sponsor companies or the success factors impacting potential sponsors' decisions about whether to support a crowdfunding campaign. Analyzing the content of the interviews carried out for our study was a first step in remedying this situation.

Sponsor companies' objectives and the processes through which they become involved in crowdfunding

A qualitative analysis of the interviews led us to classify sponsor companies' objectives into three categories: civic,

Makeachamp

There is an image objective, but it is also about benefiting from the image, values, and identity of our platform, and those of the type of project supported.

KKBB

Above all, it's about image, about communication.

Sponsorise.me

Raise the company's profile Companies choose the best project that will get the most media coverage.

Makeachamp

Companies look at the project's online attractiveness.

Fosburit

Benefit from the buzz produced by other actors

Given two projects, a company will choose the athlete with the most fans, the one that will generate the most buzz.

Fosburit

Increase goodwill Generate goodwill.

Sponsorise.me

Activate rights as a sponsor Share the experience with fans and be a producer of meaning.

Sponsorise.me

Centralize and manage individual requests for sponsorship

This allows a company that receives a lot of requests to centralize requests for sponsorship.

Sponsorise.me

Target a community La Banque Postale would like to move into a more modern, younger world than that of traditional banking.

Aldi wants to create better connections with the youngest through a strong emotionnal basis KKBB I Believe in You

Internal communication

Companies want to help their staff. These companies are looking for a tool that meets their staff's needs when they are looking for funding for a sport project.

Weplaysport

Support a community initiative by staff Myannona

Commercial

Increase turnover

For Intersport, it is about getting people into their stores. go beyond the classic model of "confined" sponsorship and embrace a new model of shared sponsorship. We believe the development of shared sponsorship marks the beginning of a new phase in the evolution of sponsorship.

Sponsorise.me

Create a database Myannona

An interactive channel

As an intrinsically interactive process, shared sponsorship allows companies to highlight the existence of strength-giving popular support for a cause or project. For Makeachamp founder HR, "the crowd is the best judge of projects. Instead of having one person judging on the basis of criteria that are often highly subjective, the job is done by hundreds, if not thousands, of contributors". At the same time, it enables companies/brands to demonstrate their attachment to a project. CM (Fosburit) feels that "this sponsorship of values creates a relationship between the sponsor and the project creator's community".

An online dimension

A company that supports a project in the same way as other contributors does not, in theory, divert crowdfunding from its original purpose. According to VR, head of the generalist crowdfunding platform KKBB, it is "a new angle of attack in terms of communication, distinctive with respect to the competition, to be tied to a target, especially young people, as is the case for La Banque Postale". For VR "the two concepts are complementary; deep down, the objectives are the same (improved image, stories to tell, unifying values to share), but the levers for attaining them are different". CM (Fosburit) took this idea further: "the strategies used by leading advertisers are 360°. They continue communicating through traditional channels and would like to include online channels. In fact, it is in the online realm that audiences are increasing the most.

Crowdfunding allows them to create ties with the project creator's community. They capitalize on this community, which consists of their target group". Nevertheless, this complementarity does not exclude the need to take into account the size of the sponsor company. On this issue, HR (Makeachamp) noted: "sponsors that support crowdfunding projects are essentially large companies that invest in complementary promotional channels or expanding companies who need to make themselves known quickly". In contrast, CM (Fosburit) believes that crowdfunding is an interesting promotional tool even for small companies with small advertising budgets. According to CM, local sponsorship does not achieve much for SMEs/SMIs, whereas sponsorship through crowdfunding gives an extra, online dimension to a company's strategy. Hence, there is a certain complementarity between online and physical tools. CM reinforced this point when stressing the potential of platforms as a new medium based on the power of crowdfunding and on the shared excitement sport produces. He describes the relationship between sponsorship and crowdfunding as one in which crowdfunding requests. Objectives were categorized as commercial when their aim was to increase a sponsor's turnover.

Of the fourteen sponsor or support company objectives cited by crowdfunding executives, three-"be a stakeholder in a project's success", "become part of a community", and "benefit from the buzz generated by other actors"-can be achieved only by financing crowdfunding projects.

Theoretical contribution: The concept of shared sponsorship

The results of our research led us to define a new concept in the field of sponsorship, which we call shared sponsorship. Shared sponsorship occurs when a company provides support (financial or in-kind) to a project that will be largely funded by donations from a wide community of contributors. This support is provided as part of a larger promotional strategy and is designed to build associations in the minds of a community between the company and the project's success. Sponsorship has evolved considerably over the years. From the 1970s to the 1990s, sponsorship tended to consist of providing one-off support, whether financial, in-kind, or skills, to a sports event or other aspect of sport (athlete, team, club, etc.) in exchange for advertising. This was followed in the 1990s and 2000s by the emergence of a more strategic approach to sponsorship, combined with ex-ante and ex-post monitoring and measurement of its efficacy [START_REF] Tribou | Sponsoring sportif[END_REF]. The 2000s saw the beginning of a third phase in this evolution, when sponsors began looking beyond effective promotion and commercial efficacy (inclusion in the marketing mix) in order to pursue an eco-civic and socially responsible form of sponsorship [START_REF] Maltese | Parrainage sportif éco-citoyen[END_REF][START_REF] Pope | The making of special sponsorship[END_REF] that allows them to display their credentials as socially and environmentally responsible organizations. However, a constant feature throughout all these phases has been the "confined" nature of sponsorship, with events, clubs, teams, etc. being sponsored by a very small number of entities. As a result, sponsorship was "imposed" on fans, spectators, and television viewers, without them having the opportunity to contribute. In contrast, crowdfunding opens the way for the shared sponsorship of projects, with a large community of individuals and corporations coming together to promote and share in a project's success. The term "shared sponsorship" was suggested to us by PQ (Sponsorise.me), who believes that corporate involvement in crowdfunding will herald a move "from imposed sponsorship to shared sponsorship". He used the example of Olympique de Marseille football club to illustrate what he meant: "the sponsor imposes itself on the fans of Olympique de Marseille, whereas in the case of crowdfunding, the sponsorship is shared with the fans". "enables (companies) to complement and revive a traditional form of communication".

Conclusion

The present study used data from interviews with crowdfunding executives to explore a model of crowdfunding including a sponsor company. To the best of our knowledge, this model

has not yet been analyzed in the literature, even though it is being adopted ever more widely. In fact, companies have come to view crowdfunding as a new communication channel, which they use either intermittently, providing support to carefully chosen projects, or in a more structured way, as part of a communication strategy. Our data revealed four different modes of corporate involvement in the crowdfunding process, three of which (supporting a platform, supporting a category of projects, and supporting a project) are used as part of an external communication strategy. The fourth mode (supporting a project created by a member of staff ) is used for internal communication (see table 2). According to the platform executives we interviewed, this involvement is motivated by the desire to achieve one or more of fourteen objectives (see table 3), three of which-becoming a stakeholder in the success of a project, becoming part of a community, benefiting from the buzz generated by other actors-can be achieved only by supporting crowdfunding. Finally, potential sponsors take into account five success factors when deciding whether or not to support a crowdfunding campaign.

In addition to these empirical findings, the final contribution of our research is to suggest a new sponsorship concept in which sponsorship is shared between the crowd, fans, supporters, and companies. This new concept, which we call "shared sponsorship", provides an interesting complement to the classic model of "confined" sponsorship. As our study shows, developing a new model of crowdfunding based around the concept of shared sponsorship will provide value for both platforms and support companies. One of the most interesting aspects of shared sponsorship, as engendered by this model, is to give sponsorship a much more modern visage.

Although our interviews provided a wealth of information, our study has a number of limitations. First, the interviews did not reveal any negative effects for companies involved in crowdfunding platforms. Future studies should investigate this issue.

Second, concerns about confidentiality led some crowdfunding platforms to refuse our request for an interview, so the only way we could expand our data was to include interviews with executives from platforms that do not specialize in sport and/or that have not (yet) created partnerships with companies. Nevertheless, collecting data from such a wide range of interviewees should not fundamentally affect our results because, whatever their market position, all platforms have to take into account the issue of profitability. In fact, it would be worthwhile expanding the data collection net to obtain the views of sponsor companies. As the subject of our research becomes less innovative, and therefore les confidential, the difficulties we faced in obtaining information should disappear. Third, our study focused on a single mode of corporate involvement in the crowdfunding process (support for a project); the other three modes (supporting a project created by company staff, supporting sections of a platform, supporting entire platforms) remain to be explored. Finally, another interesting research avenue would be to measure the impact of a shared sponsorship action on individuals, whether or not they are contributors to the project being sponsored [START_REF] Cornwell | Sponsorship-linked marketing: opening the black box[END_REF].

At the end of this exploratory study, three fundamental questions about shared sponsorship remain to be answered. First, how does shared sponsorship fit into companies' online communication strategies? Second, will the development of this model be to the detriment of the crowd? For example, if sponsor companies start becoming important elements in the crowdfunding process, is there a risk of platforms accepting only projects likely to attract sponsorship and refusing those that will be supported only by the crowd? If this occurred it would destroy the very essence of crowdfunding. Third, could the emergence of support companies be seen as a new form of ambush marketing in the form of competition between a traditional sponsor and a project sponsor? For example, what would happen if, via a crowdfunding site, Adidas supported a project created by a team sponsored by Nike?

Figure 1 :

 1 Figure 1: Two forms of sport crowdfunding

  J. Leroux-Sostenes & E. BayleThe crowdfunding of sport -paving the way to shared sponsorship?

interviewed Position Organization Type

	AB	Head of the crowdfunding project La Banque Postale	Company that supports crowdfunding
				operations
	HR	Founder	Makeachamp	Platform specializing in sport
	VR	Founder	KissKissBankBank	Generalist platform
	PQ	Project manager	Sponsorise.me	Platform specializing in sport
	CM	Co-founder	Fosburit	Platform specializing in sport
	EB	Founder	Ecobole	Platform specializing in environmental
				projects
	AC	Co-founder	Weplaysport	Platform specializing in sport
	DK	Co-founder	Federeves.com	Platform specializing in sport
	BB	Co-founder	Myannona.com	Platform specializing in projects for women
	ED	Founder	Credofunding	Platform specializing in Christian projects
	FK	Co-founder	I Believe in You	Platform specializing in sport (Switzerland)

Table 1 :

 1 Organizations and executives interviewed

	CISS 4 (2019)

Table 2 :

 2 Sponsor companies' objectives for crowdfunding projects

	Dimensions	Sponsor's objectives	Extracts from the semi-structured interviews	Platform
	Civic	Be a stakeholder in a project's success	Aldi helps young to realize their projects	I Believe in You
		(spirit of solidarity)	Be seen to promote the realization of creative	KKBB
		Become part of a community	and innovative projects alongside individuals.
		Promote values	Companies can show their attachment to values	Fédérêves
			connected with the platform's different project cat-
			egories.
		Raise awareness of the company's	Demonstrate its local credentials.	Sponsorise.me
		attachment/importance to an area (feeling of belonging)	that express their values, such as team spirit. Local companies are interested in local projects	Fosburit
		Make a donation	They help because they know the project creator,	Weplaysport
			to lend a helping hand. Companies claim contribu-
			tions against tax, but nothing more.
			For companies, it is not a commercial transaction;	Credofunding
			it is a pure donation.
	Communication	Improve the company's image	Above all, companies invest to improve their im-
			age, so it is marketing or communication manag-
			ers who decide whether to support crowdfunding
			projects.

Funding

The authors have no funding or support to report.

Data Availability Statement

All relevant data are within the paper.

Competing Interests

The authors have declared that no competing interests exist.


Fares Gouzi

| Marine Blaquière

Matthias Catteau

| François Bughin

Jonathan Maury

Emilie Passerieux

| Bronia Ayoub

Jacques Mercier

Maurice Hayot

Pascal Pomiès
email: pascal.pomies@inserm.fr

Oxidative stress regulates autophagy in cultured muscle cells of patients with chronic obstructive pulmonary disease

The proteolytic autophagy pathway is enhanced in the lower limb muscles of patients with chronic obstructive pulmonary disease (COPD). Reactive oxygen species (ROS) have been shown to regulate autophagy in the skeletal muscles, but the role of oxidative stress in the muscle autophagy of patients with COPD is unknown. We used cultured myoblasts and myotubes from the quadriceps of eight healthy subjects and twelve patients with COPD (FEV1% predicted: 102.0% and 32.0%, respectively; p < 0.0001). We compared the autophagosome formation, the expression of autophagy markers, and the autophagic flux in healthy subjects and the patients with COPD, and we evaluated the effects of the 3-methyladenine (3-MA) autophagy inhibitor on the atrophy of COPD myotubes. Autophagy was also assessed in COPD myotubes treated with an antioxidant molecule, ascorbic acid. Autophagosome formation was increased in COPD myoblasts and myotubes (p = 0.011; p < 0.001), and the LC3 2/LC3 1 ratio (p = 0.002), SQSTM1 mRNA and protein expression (p = 0.023; p = 0.007), BNIP3 expression (p = 0.031), and autophagic flux (p = 0.002) were higher in COPD myoblasts. Inhibition of autophagy with 3-MA increased the COPD myotube diameter (p < 0.001) to a level similar to the diameter of healthy subject myotubes.

Treatment of COPD myotubes with ascorbic acid decreased ROS concentration (p < 0.001), ROS-induced protein carbonylation (p = 0.019), the LC3 2/LC3 1 ratio (p = 0.037), the expression of SQSTM1 (p < 0.001) and BNIP3 (p < 0.001), and increased the COPD myotube diameter (p < 0.001). Thus, autophagy signaling is enhanced in cultured COPD muscle cells. Furthermore, the oxidative stress level contributes to the regulation of autophagy, which is involved in the atrophy of COPD myotubes in vitro.

K E Y W O R D S
atrophy, autophagy, chronic obstructive pulmonary disease (COPD), oxidative stress, satellite cells

| INTRODUCTION

Chronic obstructive pulmonary disease (COPD) is characterized by airflow obstruction resulting from the inflammatory response of the lungs to inhaled toxic particles. Among the comorbidities associated with COPD, muscle dysfunction has a crucial impact on the quality of life and mortality rate of patients. Muscle dysfunction is associated with reduced muscle strength and endurance and is often accompanied by muscle atrophy of the lower limbs, which has been attributed in great part to increased protein degradation due to activation of the ubiquitin/proteasome pathway [START_REF] Doucet | Muscle atrophy and hypertrophy signaling in patients with chronic obstructive pulmonary disease[END_REF][START_REF] Plant | Cellular markers of muscle atrophy in chronic obstructive pulmonary disease[END_REF].

The autophagy-lysosome pathway is a proteolytic system that contributes to muscle homeostasis through the elimination of protein aggregates and damaged organelles. Autophagy has been shown to be induced in skeletal muscles in response to various pro-atrophic stimuli such as starvation [START_REF] Mizushima | In vivo analysis of autophagy in response to nutrient starvation using transgenic mice expressing a fluorescent autophagosome marker[END_REF], denervation-induced muscle disuse (O'Leary, Vainshtein, Carter, Zhang, & Hood, 2012), hypoxia [START_REF] Bellot | Hypoxia-induced autophagy is mediated through hypoxia-inducible factor induction of BNIP3 and BNIP3L via their BH3 domains[END_REF], and oxidative stress [START_REF] Dobrowolny | Skeletal muscle is a primary target of SOD1G93A-mediated toxicity[END_REF]. Furthermore, use of the antioxidant molecule N-acetylcysteine (NAC) has shown that reactive oxygen species (ROS) regulate autophagy in in vitro myogenic C2C12 cells, as well as in the skeletal muscles of mice [START_REF] Qi | Antioxidant supplement inhibits skeletal muscle constitutive autophagy rather than fasting-induced autophagy in mice[END_REF][START_REF] Rahman | Reactive oxygen species regulation of autophagy in skeletal muscles[END_REF]. In COPD, autophagy signaling is enhanced in the tibialis anterior and vastus lateralis of stable patients [START_REF] Guo | Autophagy in locomotor muscles of patients with chronic obstructive pulmonary disease[END_REF][START_REF] Puig-Vilanova | Oxidative stress, redox signaling pathways, and autophagy in cachectic muscles of male patients with advanced COPD and lung cancer[END_REF].

Moreover, indirect measurements suggest that oxidative stress might contribute to autophagy, and autophagy markers are inversely correlated with thigh atrophy and lung function impairment in patients with COPD [START_REF] Guo | Autophagy in locomotor muscles of patients with chronic obstructive pulmonary disease[END_REF]. Nevertheless, while ROS have been shown to regulate autophagy in skeletal muscles, the involvement of oxidative stress in COPD muscle autophagy has yet to be clearly demonstrated.

We recently developed a cellular model based on purified satellite cells derived from the quadriceps of patients with COPD and showed that oxidative stress is involved in the atrophy of cultured COPD myotubes via the ubiquitin/proteasome signaling pathway [START_REF] Pomiès | Reduced myotube diameter, atrophic signalling and elevated oxidative stress in cultured satellite cells from COPD patients[END_REF][START_REF] Pomiès | Involvement of the FoxO1/MuRF1/atrogin-1 signaling pathway in the oxidative stress-induced atrophy of cultured chronic obstructive pulmonary disease myotubes[END_REF]. Here, we use this cellular model to assess the role of autophagy in the atrophy of COPD muscle cells in vitro and to investigate whether oxidative stress is involved in COPD muscle autophagy. Autophagy signaling was evaluated with various autophagy markers in in vitro muscle cells of patients with COPD and healthy subjects, while the effect of the autophagy inhibitor 3-methyladenine (3-MA) was tested on COPD myotube atrophy.

Autophagy signaling was also assessed in cultured COPD myotubes subjected to antioxidant treatment.

| MATERIALS AND METHODS

| Study population

The two study groups, sedentary healthy subjects (n = 8) and patients with COPD (n = 12), are identical to the groups we have previously studied [START_REF] Pomiès | Involvement of the FoxO1/MuRF1/atrogin-1 signaling pathway in the oxidative stress-induced atrophy of cultured chronic obstructive pulmonary disease myotubes[END_REF], and their characteristics are detailed in Table 1. Anthropometry, pulmonary function, exercise level and peripheral muscle function were assessed, as previously described [START_REF] Pomiès | Involvement of the FoxO1/MuRF1/atrogin-1 signaling pathway in the oxidative stress-induced atrophy of cultured chronic obstructive pulmonary disease myotubes[END_REF]. All subjects gave an informed written consent, and the research protocol was approved by the institutional ethics committee of the Montpellier University Hospitals (no. 2009-04-BPCO-V2 and no. 2011-A00842-39) and conducted in accordance with the Helsinki Declaration and the European guidelines for "good clinical practice."

| Muscle biopsy, myoblast purification, and cell culture

Muscle biopsies were obtained from the vastus lateralis using our needle method [START_REF] Gouzi | Fiber atrophy, oxidative stress, and oxidative fiber reduction are the attributes of different phenotypes in chronic obstructive pulmonary disease patients[END_REF]. One piece of the biopsy was flash frozen in a liquid nitrogen precooled beaker of isopentane and then conserved at -80°C, until total RNA extraction using the NucleoSpin RNA/protein purification kit (Macherey-Nagel, Düren, Germany).

Another piece of the biopsy was progressively frozen for later satellite cell purification, as described previously [START_REF] Pomiès | Reduced myotube diameter, atrophic signalling and elevated oxidative stress in cultured satellite cells from COPD patients[END_REF]. Myoblast purification was performed using an immunomagnetic method, as previously described in detail [START_REF] Pomiès | Reduced myotube diameter, atrophic signalling and elevated oxidative stress in cultured satellite cells from COPD patients[END_REF].

The myoblasts were grown in collagen-coated petri dishes in proliferation medium: Dulbecco modified Eagle medium (DMEM)/20% fetal bovine serum (FBS)/0.5% Ultroser. Myoblasts were always cultured at a passage below P4. Differentiation was induced by replacing the proliferation medium with the differentiation medium: DMEM/2% FBS. Myotubes were obtained after 5 days in differentiation medium.

| Cell treatments

Healthy and COPD myoblasts and myotubes were grown in the presence of 200 nM bafilomycin A1 or phosphate-buffered saline (PBS) for 90 min before harvesting the cells. The LC3 2 autophagic flux was determined by calculating the difference between the LC3 2/tubulin ratio in the presence and in the absence of bafilomycin A1. The BODE index takes into account the body mass index, the airflow obstruction, the functional dyspnea, and the exercise capacity [START_REF] Celli | The body-mass index, airflow obstruction, dyspnea, and exercise capacity index in chronic obstructive pulmonary disease[END_REF]. Values are presented as median [25th percentile-75th percentile].

The autophagy inhibitor 3-MA was added to myotubes after 4 days of differentiation at a concentration of 500 µM for 18 hr. These conditions were chosen because they can induce a significant decrease in autophagy marker expression associated with an increase in the COPD myotube diameter, while higher 3-MA concentrations do not improve these effects (data not shown).

Ascorbic acid was added at a concentration of 0.14 mM to the culture medium of myoblasts, 1 day before induction of differentiation. Ascorbic acid was then renewed every day until the end of the differentiation, as previously described [START_REF] Pomiès | Involvement of the FoxO1/MuRF1/atrogin-1 signaling pathway in the oxidative stress-induced atrophy of cultured chronic obstructive pulmonary disease myotubes[END_REF].

| Fluorescence microscopy and image analysis

Myoblasts or myotubes were fixed for 15 min in PBS/3.7% formaldehyde, permeabilized in PBS/0.1% Triton X-100 for 5 min, Images were anonymized and the LC3 staining density for each image was analyzed using ImageJ (National Institutes of Health, Bethesda, MD), as previously described [START_REF] Zhang | Apelin inhibits the proliferation and migration of rat PASMCs via the activation of PI3K/Akt/mTOR signal and the inhibition of autophagy under hypoxia[END_REF]. For assessment of the myotube diameter, myotubes were first stained with an antitroponin T antibody and Hoechst 33258. Images were captured in five random fields at 5× magnification and anonymized. The diameter was then assessed by measuring three diameters along each myotube, for 100 myotubes per condition.

| Electron microscopy

Myotubes were immersed in a solution of 2.5% glutaraldehyde in PHEM (PIPES, HEPES, EGTA and MgCl 2 ) buffer pH 7.4 overnight at 4°C. They were then rinsed in PHEM buffer and fixed in 0.5% osmic acid for 2 hr in the dark at room temperature. After two rinses in PHEM buffer, the cells were dehydrated in a graded series of ethanol solutions 30%-100%). The cells were then embedded in EMbed 812 (Electron Microscopy Sciences, Hatfield, PA) using an EM AMW automatic microwave tissue processor (Leica Microsystems, Wetzlar, Germany). Thin sections of 70 nm were collected with an Ultracut E microtome (Leica) at different levels of each block. These sections were counterstained with 1.5% uranyl acetate in 70% ethanol and lead citrate and observed using a H7100 transmission electron microscope (Hitachi, Tokyo, Japan).

| SDS-PAGE and immunoblotting

Proteins were extracted from myoblasts and myotubes with hypotonic buffer: 20 mM Tris pH 7.5/10 mM NaCl/1 mM DTT/ protease inhibitor cocktail, separated by SDS-PAGE and transferred to Immobilon-P PVDF membranes (Millipore, Bedford, MA). LC3 1 and LC3 2 were detected with a specific anti-LC3A/B antibody. The membranes were analyzed on an Odyssey imaging system (LI-COR Biotechnology, Lincoln, NE).

| Quantitative polymerase chain reaction

Total RNA extraction from cultured cells, first strand complementary DNA synthesis, and quantitative polymerase chain reaction protocol and analysis have been described [START_REF] Pomiès | Involvement of the FoxO1/MuRF1/atrogin-1 signaling pathway in the oxidative stress-induced atrophy of cultured chronic obstructive pulmonary disease myotubes[END_REF]. Specific values were normalized to the values of the housekeeping gene GAPDH. The primer sequences are shown in Table 2.

| Reagents and antibodies

Mouse monoclonal anti-troponin T was purchased from Sigma-Aldrich (Saint Louis, MO), and rabbit polyclonal anti-LC3A/B and rabbit monoclonal anti-SQSTM1/p62 from Cell Signaling Technology (Danvers, MA). Hoechst 33258, 3-MA, bafilomycin A1 and ascorbic acid were purchased from Sigma-Aldrich.

| Statistical analysis

The variables of the healthy control and COPD patient groups were compared with the Student t-test. The percentage of variation between the "with" and "without" treatment was compared with a reference value of 100 using the Student t-test. Spearman's coefficient was used to assess correlations between variables. The variations in the myotube diameter of the study groups "with" or "without" 3-MA treatment were compared using the Wilcoxon signed rank test. Statistical analysis was performed using GraphPad Prism 5

(GraphPad Software, San Diego, CA). [START_REF] Celli | The body-mass index, airflow obstruction, dyspnea, and exercise capacity index in chronic obstructive pulmonary disease[END_REF].

| RESULTS

| Characteristics of the study groups

| Elevated autophagy signaling in COPD myoblasts and myotubes

To evaluate the autophagosome formation level in the cultured COPD myotubes, autophagosomes were detected using transmission electron microscopy. Representative images of myotube cultures from two healthy subjects and two patients with COPD showed the abundance of autophagosomes in COPD myotubes compared with control myotubes (Figure 1). To quantify autophagosome formation, the expression level of the autophagosome marker, LC3, was assessed using immunofluorescence micro- To confirm the elevated autophagy signaling in COPD myoblasts and myotubes, several autophagy-related markers were assessed. The LC3 2/LC3 1 ratio was significantly higher in COPD myoblasts compared with muscle cells from healthy subjects (p = 0.002; Figure 3a). In addition, SQSTM1 mRNA and protein as well as BNIP3 mRNA expression in COPD myoblasts (Figure 3b,c) was significantly more elevated than in myoblasts from healthy subjects (p = 0.023, p = 0.007, p = 0.031, respectively). No significant difference in GABARAPL1 expression was detected in the COPD muscle cells (Figure 3d).

To determine whether the enhanced expression of the autophagic markers in COPD myoblasts was due to an enhanced induction of autophagy or a reduced autophagic degradation, the autophagic flux was assessed using the lysosomal inhibitor bafilomycin A1. The LC3 2 autophagic flux was significantly more elevated in COPD myoblasts than in healthy myoblasts (p = 0.002; Figure 4a), whereas no difference was observed between COPD and healthy myotubes (Figure 4b).

To assess the link between the enhanced autophagy signaling we observed in vitro and the increased autophagy described in the lower limb muscles of patients with COPD by others [START_REF] Guo | Autophagy in locomotor muscles of patients with chronic obstructive pulmonary disease[END_REF][START_REF] Puig-Vilanova | Oxidative stress, redox signaling pathways, and autophagy in cachectic muscles of male patients with advanced COPD and lung cancer[END_REF], we determined the expression of the autophagy marker BNIP3 in quadriceps biopsies obtained from five healthy subjects and six patients with COPD of our study groups. Although the BNIP3 expression in the patients with COPD was not significantly higher than that in the healthy subjects (p = 0.219; Figure 5a), BNIP3 expression in quadriceps biopsies was positively correlated with the BNIP3 expression in the cultured myoblasts (r = 0.663; p = 0.031; Figure 5b). Interestingly, the BNIP3 expression in muscle biopsies was inversely correlated with both FEV1 (% predicted) (r = -0.645; p = 0.037; Figure 5c) and

| Atrophy is decreased in acid ascorbic-treated COPD myotubes

We previously reported that antioxidant treatment of COPD muscle cells with ascorbic acid significantly decreased ROS concentration and oxidative stress-induced cellular damage, and increased myotube diameter [START_REF] Pomiès | Involvement of the FoxO1/MuRF1/atrogin-1 signaling pathway in the oxidative stress-induced atrophy of cultured chronic obstructive pulmonary disease myotubes[END_REF] (p < 0.001, p = 0.019, p < 0.001, respectively; Figure 7a). To determine whether oxidative stress is also involved in the elevated autophagy signaling of COPD muscle cells, we assessed the effect of ascorbic acid treatment of COPD muscle cells on the expression of autophagy-related markers. As shown in Figure 7b, the antioxidant treatment induced a significant decrease in the LC3 2/LC3 1 ratio and the SQSTM1 and BNIP3 expression levels (p = 0.037, p < 0.001, p < 0.001, respectively). No variation was observed for GABARAPL1 expression (p = 0.998; Figure 7b).

| DISCUSSION

Although oxidative stress has been shown to regulate autophagy signaling in skeletal muscles, its involvement in the skeletal muscle autophagy of patients with COPD has never been demonstrated. In the current study, we used a cellular model to show that autophagosome formation was increased in COPD myoblasts and myotubes compared with healthy control cells (Figures 1 and2), along with increased expression of autophagy markers and enhanced autophagic flux in COPD myoblasts (Figures 3 and4). We also showed that in vitro autophagy signaling was correlated with the autophagy signaling observed in the quadriceps of the study subjects (Figure 5). Use of the 3-MA autophagy inhibitor confirmed the involvement of the autophagy pathway in the atrophy of cultured COPD myotubes (Figure 6). Most interestingly, the use of an antioxidant molecule on COPD myotubes, which resulted in a induced a significant decrease in autophagy signaling (Figure 7).

Taken together, our findings showed that autophagy signaling was increased in cultured COPD muscle cells, where it played a role in the atrophy of COPD myotubes in vitro, and that oxidative stress was involved in COPD muscle autophagy.

Muscle dysfunction and atrophy are common processes that have been associated with a decreased survival rate in patients with COPD independent of the pulmonary obstruction [START_REF] Marquis | Midthigh muscle cross-sectional area is a better predictor of mortality than body mass index in patients with chronic obstructive pulmonary disease[END_REF]. Atrophy of the lower limb muscles of patients has been shown

to result mostly from elevated protein degradation due to increased ubiquitin/proteasome signaling [START_REF] Doucet | Muscle atrophy and hypertrophy signaling in patients with chronic obstructive pulmonary disease[END_REF][START_REF] Plant | Cellular markers of muscle atrophy in chronic obstructive pulmonary disease[END_REF] Recently, autophagosome formation and autophagy marker expression were shown to be increased in the quadriceps of both, patients with COPD with mild to severe airflow obstruction [START_REF] Guo | Autophagy in locomotor muscles of patients with chronic obstructive pulmonary disease[END_REF] and patients with cachectic COPD [START_REF] Puig-Vilanova | Oxidative stress, redox signaling pathways, and autophagy in cachectic muscles of male patients with advanced COPD and lung cancer[END_REF], compared with healthy subjects. Furthermore, autophagosome formation was inversely correlated with FEV1 (% predicted), whereas the LC3 2/LC3 1 ratio was negatively correlated with the thigh cross-sectional area, suggesting a link between autophagyassociated muscle degradation and COPD muscle atrophy, with a protective role of autophagy toward muscle function [START_REF] Guo | Autophagy in locomotor muscles of patients with chronic obstructive pulmonary disease[END_REF]. Our in vitro studies showed that autophagosome formation, autophagy-related marker expression, and autophagic flux were enhanced in COPD myoblasts and/or myotubes compared with muscle cells derived from sedentary healthy subjects (Figures 1234).

However, since SQSTM1 is an adapter protein of the autophagic degradation process, one could expect a decrease in expression of SQSTM1 in COPD muscle cells. Nevertheless, our result showing increased expression of SQSTM1 in COPD myoblasts (Figure 3) is in agreement with the enhanced expression of mRNA and protein SQSTM1 observed in the vastus lateralis of patients with COPD [START_REF] Guo | Autophagy in locomotor muscles of patients with chronic obstructive pulmonary disease[END_REF]. Furthermore, it has been shown that an enhanced expression of SQSTM1 can occur in parallel with activated autophagic flux in some situations, including autophagy activation in cardiac proteinopathy [START_REF] Zheng | Autophagy and p62 in cardiac proteinopathy[END_REF] and autophagy induction, contributing to muscle wasting in mouse models of cancer cachexia [START_REF] Penna | Autophagic degradation contributes to muscle wasting in cancer cachexia[END_REF]. It is, therefore, reasonable to think that in COPD muscle cells, an increased expression of SQSTM1 is associated with an enhanced autophagic flux.

Unlike a previous report [START_REF] Guo | Autophagy in locomotor muscles of patients with chronic obstructive pulmonary disease[END_REF], we did not detect a significant difference in the quadriceps expression of BNIP3 in healthy subjects and patients with COPD, but this discrepancy might be due to the relatively low number of quadriceps biopsies available for our study. In their study, [START_REF] Guo | Autophagy in locomotor muscles of patients with chronic obstructive pulmonary disease[END_REF] found an inverse (% predicted) and 6MWD (% predicted; Figure 5), although our experimental study would have benefited from a larger population of patients with COPD with various severities. We also observed a correlation between BNIP3 expression in the quadriceps of the study subjects and BNIP3 expression in the cultured myoblasts (Figure 5).

Together, these observations indicate that the increased autophagy signaling we identified in the cultured COPD muscle cells derived from human satellite cells reflects the enhanced autophagy observed in the quadriceps muscle of the stable patients with COPD [START_REF] Guo | Autophagy in locomotor muscles of patients with chronic obstructive pulmonary disease[END_REF][START_REF] Puig-Vilanova | Oxidative stress, redox signaling pathways, and autophagy in cachectic muscles of male patients with advanced COPD and lung cancer[END_REF].

Autophagy, a major catabolic process in the elimination of damaged molecules and organelles, plays a crucial role in muscle cell homeostasis. A relationship between autophagy and atrophy has been demonstrated in skeletal muscles subjected to various atrophic stimuli such as starvation and denervation [START_REF] Mizushima | In vivo analysis of autophagy in response to nutrient starvation using transgenic mice expressing a fluorescent autophagosome marker[END_REF]O'Leary et al., 2012). In the skeletal muscles of patients with COPD, it was proposed that enhanced autophagy participates in the signaling [START_REF] Qi | Antioxidant supplement inhibits skeletal muscle constitutive autophagy rather than fasting-induced autophagy in mice[END_REF][START_REF] Rahman | Reactive oxygen species regulation of autophagy in skeletal muscles[END_REF], suggesting that oxidative stress regulates skeletal muscle autophagy. Direct evidence of the involvement of oxidative stress in skeletal muscle autophagy was reported in a study using a transgenic mouse model expressing a mutant of the antioxidant enzyme superoxide dismutase 1 selectively in skeletal muscle [START_REF] Dobrowolny | Skeletal muscle is a primary target of SOD1G93A-mediated toxicity[END_REF]. Nevertheless, the involvement of oxidative stress in COPD muscle autophagy has never been studied. Here, we showed that antioxidant treatment of COPD myotubes with ascorbic acid significantly decreased ROS concentration, oxidative stress-induced cellular damage, as well as expression of various autophagy markers, and increased the myotube diameter (Figure 7). Therefore, lowering the oxidative burden in COPD muscle cells reduced the autophagy signaling, showing for the first time that the oxidative stress level regulates autophagy in COPD skeletal muscle cells. This result suggests that reduction of autophagy by antioxidant molecules might be a potential therapeutic strategy to counteract oxidative stress-induced atrophy in patients with COPD.

Mitophagy is an autophagic process dedicated to the selective elimination of damaged and dysfunctional mitochondria to avoid excessive ROS generation and preserve muscle homeostasis [START_REF] Youle | Mechanisms of mitophagy[END_REF]. In COPD skeletal muscles, it was shown that the altered functioning of mitochondria led to enhanced mitochondrial ROS production linked to skeletal muscle oxidative stress [START_REF] Puente-Maestu | Abnormal mitochondrial function in locomotor and respiratory muscles of COPD patients[END_REF], 2012). It would, therefore, be of interest to determine whether the increased ROS concentration we have observed in cultured COPD myoblasts and myotubes is coming from altered mitochondria. Furthermore, our results showed that the expression levels of SQSTM1 and BNIP3, two molecular adapters targeting mitochondria to autophagosomes for mitophagy, were increased in cultured COPD muscle cells compared with healthy control cells (Figure 3), and that ascorbic acid reduced their expression (Figure 7). These results suggest that oxidative stress is involved in the autophagy-associated remodeling of the mitochondrial network by the removal of dysfunctional mitochondria in the atrophic COPD muscle cells, a process that will be addressed in a forthcoming study.

In summary, we assessed autophagosome formation, autophagy

  Autophagosomes in healthy and COPD myotubes. Representative electron micrographs of healthy subject myotubes (a-d) and COPD patient myotubes (e-h). b, d, f and h are blow-up images (×10,000) of a, c, e and g images (x5,000), respectively. Arrows point towards autophagosomes, while N indicates a nucleus. Bar = 1 µm and then stained for 1 hr at 37°C with an anti-LC3 antibody and Hoechst 33258. Images were taken in five random fields using an AxioCam MRm CCD camera (Carl Zeiss, Oberkochen, Germany) on an AxioImager M1 microscope (Carl Zeiss) at 10× magnification.

  scopy in myoblasts and myotubes derived from a limited number of patients with COPD and healthy subjects from our study groups. Representative images of one control and one COPD myoblast culture, as well as one control and one COPD myotube culture, are presented in Figure2a,b,d,e, respectively. Figure2b,eshowed typical dot-like staining of autophagosomes in COPD myoblasts and myotubes, respectively. Analysis of the LC3 expression level showed significantly increased autophagosome formation in COPD myoblasts (p = 0.011; Figure2c) and COPD myotubes (p < 0.001; Figure2f) compared with healthy subject cells.

  6MWD (% predicted) (r = -0.736; p = 0.013; Figure 5d) of the study subjects. A statistical significance at p < 0.05 is still observed for the three presented correlations when the outer COPD value is removed. F I G U R E 2 LC3 expression levels in healthy and COPD myoblasts and myotubes. (a) Representative images of control healthy subject (CONT) and COPD (COPD) myoblasts, showing fluorescence double-labeling using an anti-LC3 antibody (red) and Hoechst (blue). Bar = 200 µm. (b) Higher magnification showing dot-like staining of autophagosomes in COPD myoblasts. Bar = 50 µm. (c) Densitometric analysis of the LC3 staining (arbitrary unit) in myoblasts from five healthy subjects and five patients with COPD. (d) Representative images of control healthy subject (CONT) and COPD (COPD) myotubes, showing fluorescence double-labeling using an anti-LC3 antibody (red) and Hoechst (blue). Bar = 200 µm. (e) Higher magnification showing dot-like staining of autophagosomes in COPD myotubes. Bar = 50 µm. (f) Densitometric analysis of the LC3 staining (arbitrary unit) in myotubes from five healthy subjects and five patients with COPD. (*) and (***) indicate statistical significance at p < 0.05 and p < 0.001, respectively. The medians are indicated. COPD, chronic obstructive pulmonary disease [Color figure can be viewed at wileyonlinelibrary.com]3.3 | Atrophy is reduced in 3-MA-treated COPD myotubes To investigate the involvement of autophagy in the atrophy of the cultured COPD myotubes, the autophagy inhibitor 3-MA was added to the culture medium of the myotubes derived from twelve patients with COPD. Treatment induced the inhibition of autophagy signaling as depicted by the significant decrease in SQSTM1 and BNIP3 (p = 0.001, p = 0.017, respectively; Figure 6a), and a tendency to a decreased expression of GABARAPL1 (p = 0.060; Figure 6a). Representative images of COPD myotubes cultured in the absence and the presence of the autophagy inhibitor are shown in Figure 6b. Analysis of the COPD cultures indicated that 3-MA significantly increased the myotube diameter (p < 0.001; Figure 6b). However, 3-MA treatment F I G U R E 3 Expression of autophagyrelated markers in healthy and COPD myoblasts and myotubes. (a) Analysis of the LC3 2/LC3 1 ratio, from the quantification of two independent western blots, in eight control healthy (CONT) and twelve COPD (COPD) myoblasts and myotubes. The mean values are presented in arbitrary units. Expression levels (arbitrary unit) of SQSTM1 mRNA and protein (b), BNIP3 mRNA (c) and GABARAPL1 mRNA (d), in myoblasts and myotubes derived from healthy subjects and patients with COPD. (*) and (**) indicate statistical significance at p < 0.05 and p < 0.01, respectively. (n.s.) indicates statistically nonsignificant. The medians are indicated. COPD, chronic obstructive pulmonary disease of cultured myotubes derived from healthy subjects did not increase the myotube diameter (p = 0.641; Figure 6c). Interestingly, treatment of the COPD myotubes with the autophagy inhibitor 3-MA could increase their diameter to a level similar to the diameter of healthy myotubes (Figure 6b,c, lower panels).

F

  I G U R E 4 LC3 2 autophagic flux in healthy and COPD myoblasts and myotubes. (a) Representative western blots showing the expression levels of LC3 1, LC3 2 and tubulin in cultured myoblasts derived from eight control healthy subjects (CONT 1-8) and eight patients with COPD (COPD 1-8) in absence or in presence of bafilomycin A1, and analysis of the LC3 2 autophagic flux in the eight control healthy (CONT) and eight COPD (COPD) myoblasts. (b) Representative western blots showing the expression levels of LC3 1, LC3 2 and tubulin in cultured myotubes derived from six control healthy subjects (CONT 1-6) and six patients with COPD (COPD 1-6) in absence or in presence of bafilomycin A1, and analysis of the LC3 2 autophagic flux in the six control healthy (CONT) and six COPD (COPD) myoblasts. (**) indicates statistical significance at p < 0.01. (n.s.) indicates statistically nonsignificant. COPD, chronic obstructive pulmonary disease decrease of ROS concentration and an increase in myotube diameter,

F

  I G U R E 5 BNIP3 expression levels in the quadriceps of healthy subjects and patients with COPD. (a) Expression levels of BNIP3 in the quadriceps biopsies obtained from five healthy subjects and six patients with COPD of our study groups. (n.s.) indicates statistically nonsignificant. The medians are indicated. (b) Statistical analysis showing the correlation between BNIP3 quadriceps expression and BNIP3 expression in cultured myoblasts derived from the same healthy subjects and patients with COPD. Statistical analysis showing correlations between BNIP expression in the quadriceps and (c) FEV1 (% predicted) and (d) 6MWD (% predicted) of the healthy subjects and patients with COPD. Healthy subjects: open circles; patients with COPD: filled circles. COPD, chronic obstructive pulmonary disease; FEV1, forced expiratory volume in 1 s; 6MWD, 6-min walking distance correlation between markers of autophagosome formation (autophagosomes/field, LC3 intensity, LC3 2/LC3 1 ratio) in the lower limb muscles and lung and muscle function (FEV1 [% predicted], FEV1/ forced vital capacity [%], thigh cross-sectional area). In our study, we observed similar significant correlations between autophagy signaling (BNIP3 expression) in the quadriceps and FEV1

F

  I G U R E 6 Expression levels of autophagy-related markers and myotube diameter after 3-MA treatment of COPD and healthy myotubes. (a) SQSTM1, BNIP3, and GABARAPL1 expression levels in myotubes derived from twelve patients with COPD with 3-MA treatment, relative to the expression levels of the same markers without treatment (dotted line). The means ± SEM are indicated. (b) Representative images of COPD myotubes without or with 3-MA treatment, and analysis of the variation of the diameter of myotubes derived from twelve patients with COPD after 3-MA treatment. (c) Representative images of healthy myotubes without or with 3-MA treatment, and analysis of the variation of the diameter of myotubes derived from eight healthy subjects after 3-MA treatment. The medians are indicated. (*), (**) and (***) indicate statistical significance at p < 0.05, p < 0.01 and p < 0.001, respectively. (n.s.) indicates statistically nonsignificant. Bar = 200 µm. COPD, chronic obstructive pulmonary disease; 3-MA, 3-methyladenine; SEM, standard error of the mean [Color figure can be viewed at wileyonlinelibrary.com] F I G U R E 7 Expression levels of autophagy-related markers in COPD myotubes after ascorbic acid treatment. (a) Analysis of ROS concentration, protein carbonylation level and myotube diameter in cultured myotubes derived from twelve patients with COPD after ascorbic acid treatment, relative to the detection of the same markers without treatment (dotted line). (b) Analysis of the LC3 2/LC3 1 ratio, and SQSTM1, BNIP3 and GABARAPL1 expression levels in cultured myotubes derived from twelve patients with COPD with ascorbic acid treatment, relative to the expression levels of the same markers without treatment (dotted line). (*) and (***) indicate statistical significance at p < 0.05 and p < 0.001, respectively. (n.s.) indicates statistically nonsignificant. The means ± SEM are indicated. COPD, chronic obstructive pulmonary disease; ROS, reactive oxygen species; SEM, standard error of the mean remodeling of the muscle, contributing to muscle atrophy (Hussain & Sandri, 2013). In our study, treatment of atrophic COPD myotubes with the autophagy inhibitor 3-MA induced a drastic increase in the myotube diameter, showing that autophagy was involved in the atrophy of COPD muscle cells in vitro (Figure 6). Although the pro-oxidant molecule, hydrogen peroxide, has been shown to activate autophagy in cultured C2C12 myotubes (McClung, Judge, Powers, & Yan, 2010; Rahman et al., 2014), administration of the antioxidant molecule NAC to mice reduced the autophagy

  marker expression, and autophagic flux and showed that autophagy signaling was enhanced in cultured muscle cells derived from patients with COPD compared with those from healthy subjects, reflecting the increased autophagy signaling observed in the quadriceps of patients with COPD. Furthermore, treatment of COPD myotubes with an autophagy inhibitor significantly decreased their atrophic phenotype, confirming the involvement of autophagy in the atrophy of COPD muscle cells. Last, antioxidant treatment diminished the ROS concentration and the autophagy signaling and increased the COPD myotube diameter. These data are therefore the first evidence that the oxidative stress level contributes to the regulation of autophagy in COPD muscle cells and that autophagy may be involved in the atrophy of COPD skeletal muscles. While little information is available on how oxidative stress levels affect autophagy in muscle, such in vitro approaches should help us to decipher this signaling pathway in COPD muscles.

  T A B L E 1 Characteristics of the study groups

		Healthy subjects	COPD patients	P-value
	n	8	1 2	-
	Gender (M/F)	7/1	9/3	-
	Age (year)	62.0 [57.0-67.5]	59.0 [54.0-62.8]	0.393
	BMI (kg/m 2 )	25.9 [24.3-27.8]	21.6 [19.0-26.0]	0.071
	FFMI (kg/m 2 )	19.4 [18.4-20.5]	17.1 [15.0-18.3]	0.025
	FEV1 (% pred.)	102.0 [93.0-105.0] 32.0 [26.8-47.5] <0.0001
	FEV1/FVC (%)	75.0 [68.8-81.5]	39.0 [32.0-41.0]	0.002
	6MWD (% pred.)	88.0 [83.5-97.0]	68.0 [55.5-71.5] <0.0001
	qMVC (kg)	27.7 [25.1-31.6]	17.6 [12.9-32.2]	0.071
	BODE index	-	5.0 [4.3-8.5]	-
	Note. BMI, body mass index; COPD, chronic obstructive pulmonary
	disease; FFMI, fat-free mass index; FEV1, forced expiratory volume in 1 s;
	FVC, forced vital capacity; 6MWD, 6-min walking distance; qMVC,
	quadriceps muscle voluntary contraction.

Table 1

 1

illustrates the main clinical and functional characteristics of the healthy subjects (n = 8) and patients with COPD (n = 12). The following values reflected the moderate to severe clinical state of the COPD group: forced expiratory volume in 1 s (FEV1) lower than 50% predicted, moderately reduced 6-min walking distance (6MWD) and quadriceps muscle voluntary contraction (qMVC) values, and a relatively high BODE index

ACKNOWLEDGMENTS

We thank Nicolas Molinari (IMAG, Montpellier, France) for his advice on statistical analysis, and the Fontalvie/5-Santé Group for the use of their facilities during the clinical evaluation and biopsies of the patients with COPD. We also acknowledge Chantal Cazevielle for her expertize with electron microscopy (Centre de Ressources en Imagerie Cellulaire de Montpellier, France).

CONFLICTS OF INTEREST

The authors declare that they have no conflict of interests.