Lucie Sharkey

François Medjkane

Renaud Jardri

Axel Bastien

Amandine Cauchie

Flavie Corvoisier

Émilie Dujardin

Marylène Kieken

Karine Mussche

Martine Legrand

Jamila Ogab

Séverine Vanhoove

Guillaume Vaiva

Charles-Edouard Notredame

Lucie Mme

Sharkey-Plantin
email: lucie.sharkeyplantin@chru-lille.fr

Expérience d'un dispositif proactif de soutien aux endeuillés de la Covid-19

2

L'Agence Nationale de Santé Publique dénombre, sur le territoire français, plus de 30 000 décès en lien avec l'épidémie de SARS-CoV-2. Parmi ces décès, plus de 20 000 ont eu lieu au sein d'établissements de santé habilités, qui assurent la prise en charge des patients présentant des formes sévères de l'infection. Si la prise en charge des malades, la sectorisation des services et l'optimisation des ressources font l'objet de stratégies coordonnées par le Ministère des Solidarités et de la Santé, l'accompagnement des proches des défunts semble en revanche laissé à l'initiative des établissements. Or les proches des victimes font l'expérience du deuil dans des conditions d'adversité qui constituent un enjeu de santé publique et justifient la mise en place de dispositifs spécifiques, tel que celui duquel nous faisons ici un retour d'expérience.

1-Complications possibles du deuil

Dans la grande majorité des cas, l'épreuve du deuil est traversée sans complication majeure ni retentissement fonctionnel durable. Cependant, pour une proportion significative de la population, cette perte peut donner lieu à une détresse et une altération des capacités adaptatives inscrites dans le long terme. Le stress intense causé par la mort d'un proche est susceptible d'être à l'origine de décompensations psychiatriques caractérisées sous la forme d'épisodes dépressifs majeurs, de troubles anxieux ou de troubles de stress post-traumatique [START_REF] Stroebe | Health outcomes of bereavement[END_REF]. La littérature est également abondante concernant le retentissement somatique du deuil : celui-ci majorerait, par exemple, les risques de développer ou d'aggraver une affection somatique, d'augmenter sa consommation médicamenteuse ou de nécessiter une hospitalisation [START_REF] Stroebe | Health outcomes of bereavement[END_REF]. De manière plus générale, la perte d'un proche serait associée à une surmortalité non réductible au suicide, plus particulièrement dans les semaines et les mois qui suivent le décès [START_REF] Stroebe | Health outcomes of bereavement[END_REF]. L'ensemble de ces risques, qui appartiennent au deuil en général, sont majorés lorsque la symptomatologie d'un sujet est compatible avec un diagnostic psychiatrique de complication du deuil. La complexité des construits psychiatriques liés au deuil est actuellement en train de s'organiser autour de deux catégories principales. Le trouble du deuil complexe persistant est inclus dans les « troubles liés à des traumatismes ou à des facteurs de stress » de la cinquième édition du Diagnostic and Statistical Manual of Mental Disorders (DSM V). La onzième édition de l'International Classification of Diseases (ICD 11), qui devrait entrer en vigueur en janvier 2022, lui préfère la terminologie de trouble du deuil prolongé, également classifié comme un trouble lié au stress. La validité de ces deux catégories est confirmée [START_REF] Boelen | Evaluation of the factor structure, prevalence, and validity of disturbed grief in DSM-5 and ICD-11[END_REF] et leur proximité en termes de spécificité diagnostique et de validité prédictive a été mise en avant [START_REF] Maciejewski | Prolonged grief disorder" and "persistent complex bereavement disorder", but not "complicated grief", are one and the same diagnostic entity: an analysis of data from the Yale Bereavement Study[END_REF]. Il convient toutefois de noter que le taux d'incidence du trouble du deuil complexe persistant serait significativement plus bas que celui du trouble du deuil prolongé [START_REF] Boelen | Evaluation of the factor structure, prevalence, and validity of disturbed grief in DSM-5 and ICD-11[END_REF]. Cet écart est cependant cohérent, dans la mesure où les travaux concernant le second ont mis l'accent sur une liste plus concise de symptômes et une structure simplifiée, afin de laisser l'appréciation clinique se construire sur des lignes directrices aisément appropriables par les professionnels [START_REF] Killikelly | Prolonged grief disorder for ICD-11: the primacy of clinical utility and international applicability[END_REF].

Ces complications psychiatriques seraient susceptibles de toucher 9,8 % de la population, soit presque une personne sur dix [START_REF] Lundorff | Prevalence of prolonged grief disorder in adult bereavement: A systematic review and meta-analysis[END_REF]. Elles sont structurellement associées à la présence d'idées suicidaires et ont également été corrélées à un surcroit de morbidité psychiatrique, une majoration du retentissement fonctionnel et une dégradation significative de la qualité de vie [START_REF] Maciejewski | Prolonged grief disorder" and "persistent complex bereavement disorder", but not "complicated grief", are one and the same diagnostic entity: an analysis of data from the Yale Bereavement Study[END_REF]. Une augmentation de l'usage de substance et des risques somatiques liés au deuil ont également été rapportées [START_REF] Neimeyer | Grief counselling and therapy: The case for hope[END_REF]. Le risque de développer l'un de ces troubles varie cependant considérablement, en fonction de facteurs tels que la nature du lien avec le défunt, les circonstances du décès, les caractéristiques du sujet, son histoire de vie, la qualité de sa relation au défunt, etc. À ce titre, les spécialistes s'accordent pour considérer que le deuil ne relève pas, en soi, d'une prise en charge professionnelle systématique. Ils recommandent cependant la mise en place de dispositifs de seconde ligne à destination des sujets à risque et de troisième ligne, lorsqu'un diagnostic de pathologie du deuil a été retenu.

2-Facteurs de risque chez les endeuillés de la Covid-19

L'expérience des endeuillés de la Covid-19 doit-elle nous inviter à revisiter ces restrictions et mettre en place des dispositifs de recontact systématique des proches des victimes ? Certains ont pris publiquement la parole pour témoigner des conditions d'adversité dans lesquelles ils font l'expérience du deuil. Des professionnels de la santé mentale se sont joints à eux et les premiers travaux sur le sujet émergent actuellement [START_REF] Pandémie | E Hisch[END_REF]. Ces éléments narratifs et qualitatifs ne suffisent pas toujours à vaincre les résistances que suscitent la mise en place de tels dispositifs, tant la notion qu'une sanitarisation systématique du deuil pourrait être inutile, voire néfaste, est largement intégrée. Pourtant, la littérature internationale est unanime sur la nécessité de repérer les personnes les plus à risques et l'expérience des endeuillés de la Covid-19, aussi inédite soit-elle, concentre une série de facteurs de risques documentés.

La fragilisation du tissu social.

Le manque de soutien social constitue un facteur prédictif de deuil complexe résistant confirmé [8]. Or, le renforcement du tissu social qui s'organise habituellement autour des endeuillés est largement empêché par les mesures de confinement et de distanciation sociale.

La soudaineté du décès et l'absence de « pré-deuil ».

L'incertitude pronostique et le caractère aigu des décompensations liées au virus confèrent aux décès, même lorsqu'il s'agit de celui d'une personne âgée et/ou souffrant d'une condition médicale préexistante, un caractère de soudaineté. Celui-ci est identifié dans la littérature comme potentiel facteur prédictif de deuil complexe résistant [8]. Il peut être associé à l'impression subjective que la mort aurait pu être évitée, qui constitue également un potentiel facteur de risque [8]. Or ce sentiment est particulièrement actif en contexte épidémique, notamment en raison de l'évolution de certaines des mesures de prévention.

2.3.Les difficultés de communication et d'accès à l'information.

La littérature souligne l'importance, pour l'amorce du deuil, d'une compréhension aussi complète que souhaitée des circonstances du décès, à la faveur d'une communication fluide avec les équipes médicales [START_REF] Kentish-Barnes | Complicated grief after death of a relative in the intensive care unit[END_REF]. Or, malgré leurs efforts, le contexte épidémique est de nature à restreindre la communication des équipes soignantes avec les proches des patients et à compliquer la possibilité pour ces derniers de récupérer des informations après la mort ;

L'entrave à la mise en sens.

La possibilité de faire sens du décès est un facteur de résilience reconnu, au point que la mobilisation des croyances spirituelles et philosophiques a été proposée comme levier thérapeutique dans les suites du deuil [START_REF] Park | Religion and spirituality in adjusting to bereavement: Grief as burden, grief as gift. Grief and bereavement in contemporary society: Bridging research and practice[END_REF]. Cependant, l'interdiction de visiter le malade au cours de son hospitalisation, le maintien à distance lorsqu'une visite au mourant est autorisée et la restriction d'accès au corps et au cercueil constituent autant d'entraves à cette dimension humanisante et existentielle du travail de deuil. Ajoutons, de ce point de vue, que l'empêchement des rites concernant le soin du corps ou la tenue des funérailles pourrait constituer un facteur de risque supplémentaire.

2.5.L'effet d'accumulation individuelle.

Certains endeuillés COVID+ sont suspectés d'avoir contaminé leur proche décédé. D'autres développent eux-mêmes une forme sévère du virus et subissent le stress simultané de la maladie, d'une éventuelle hospitalisation et de la perte. Il arrive également que plusieurs membres d'une même famille soient décédés et que les survivants soient confrontés à des deuils consécutifs. Or le sur-risque de complication des deuils en lien avec l'exposition à un risque vital, des décès multiples ou, plus généralement, des conditions particulièrement adverses est largement documenté [START_REF] Heeke | A systematic review and metaanalysis of correlates of prolonged grief disorder in adults exposed to violent loss[END_REF][START_REF] Kristensen | Bereavement and Mental Health after Sudden and Violent Losses: A Review[END_REF]. L'accumulation de ces facteurs de risque est d'autant plus problématique que les endeuillés les plus vulnérables seraient les moins susceptibles de solliciter les dispositifs de soutien. En effet, si certains patients augmentent la fréquence de leurs visites chez le médecin traitant après la perte d'un proche, la littérature suggère au contraire que les personnes présentant les niveaux de détresse les plus élevés réduisent leur usage du système de soin [START_REF] Lichtenthal | Underutilization of Mental Health Services Among Bereaved Caregivers With Prolonged Grief Disorder[END_REF]. Cette dernière hypothèse semble également cohérente avec la baisse de la participation sociale généralement associée au deuil. Dans une perspective de santé publique, l'ensemble de ces considérations nous a encouragé à mettre en place un dispositif de soutien professionnel, qui soit non seulement à disposition des endeuillés mais aille proactivement à leur rencontre. Une des assistantes sociales est mobilisée en cas d'identification de facteurs de fragilités sociales ou de besoins d'accompagnement aux démarches funéraires, financières et administratives. Elle entre en contact avec la personne en deuil afin de la soutenir et de l'orienter dans ses démarches, ainsi que pour l'aider à identifier les ressources à sa disposition. Cette dimension pluridisciplinaire de l'accompagnement permet de soutenir les endeuillés de manière globale, ajustée à leurs besoins psychiques mais aussi pratiques.

3-Mise en place d'un dispositif proactif de soutien aux endeuillés

Le

En cas de vulnérabilité psychique majeure préexistante, de décompensation psychiatrique aiguë ou de crise suicidaire, le psychiatre coordonnateur peut être appelé à conduire une évaluation et une intervention médicale.

4.Bilan d'activité

Le Dasec a débuté son activité le 23 mars 2020. Un mois plus tard, 58 défunts étaient inclus dans le dispositif, soit 87% des morts survenus sur le campus. N'ont pas été inclues les personnes pour lesquelles nous ne disposions d'aucune coordonnée de proche (n=7) ou ayant refusé d'être contactées (n=2). Au total, 101 endeuillés ont été appelés au moins une fois, principalement les enfants (63%) ou conjoint.e.s (23%) des défunts. Un contact a effectivement pu être établi dans près de 74% des cas. La montée en charge progressive de l'activité est illustrée en Lorsque des situations à risque élevé de complications du deuil ou relevant de troubles psychiatriques caractérisés ont été identifiées, une orientation vers des services de soin de plus long cours s'est construite au fil des échanges. Trois situations de crise suicidaire ont requis l'intervention du psychiatre.

5.Discussion

L'évaluation des dispositifs d'accompagnement des endeuillés constitue un champ de recherche dynamique, animé par des échanges vigoureux depuis la publication de l'article Does grief counselling work ? de Jordan et Neimeyer il y'a un peu moins d'une vingtaine d'années [START_REF] Jordan | Does grief counselling work?[END_REF]. La ligne directrice selon laquelle la perte d'un proche ne doit pas entraîner une orientation systématique vers des dispositifs thérapeutiques fait désormais consensus. Il est cependant intéressant de noter que les auteurs affirmaient déjà, à l'époque, l'importance d'identifier les personnes présentant un risque élevé de complication psychiatrique du deuil et de les accompagner vers des dispositifs de soin adaptés. Ce dernier point constitue également un consensus théorico-clinique constamment réaffirmé dans les travaux actuels concernant sur les enjeux psychopathologiques du deuil [START_REF] Waller | Assisting the bereaved: A systematic review of the evidence for grief counselling[END_REF].

Or les proches des victimes de l'infection à SARS-CoV-2 font l'expérience du deuil dans des conditions inédites, certes, mais concentrant une série de facteurs de risques documentés. Il est possible d'opérer un recoupement entre ces facteurs de risques et les caractéristiques communes aux morts dites « violentes ». Les études concernant le deuil suite à une mort violente se sont, à ce jour, concentrées sur les décès consécutifs à des homicides, des suicides, des accidents, mais aussi des désastres naturels ou encore des conflits armés.

Elles mettent l'accent sur la surmorbidité accrue associée à ce type de décès [START_REF] Heeke | A systematic review and metaanalysis of correlates of prolonged grief disorder in adults exposed to violent loss[END_REF]. Sur un plan qualitatif, il est utile de souligner, tant cela est pertinent dans le contexte épidémique, que « le sens donné à la mort violente au niveau individuel peut être enkysté et compliquer le processus de deuil lorsqu'elle est raptée par les enjeux politico-médiatiques » [START_REF] Romano | Accompagner des personnes confrontées à la mort violente d'un proche[END_REF]. La concentration de facteurs de risque, ainsi que la proximité de cette expérience avec celle des morts classiquement considérées comme violentes permettent de donner corps à l'hypothèse que les endeuillés de la Covid-19 sont significativement plus vulnérables à des complications du deuil. Ces éléments justifient de la mise en place de dispositifs de soutien et de repérages spécifiques, Ils semblent justifier, en accord avec les recommandations internationales, un souci particulier pour les proches des victimes, maillon nécessaire d'un effort collectif pour réduire les conséquences de l'épidémie en termes de santé publique.

Dans un contexte de crise sanitaire majeure, où les dispositifs de soin doivent faire coïncider souplesse et simplicité, l'intérêt d'un dispositif de recontact systématique des proches des victimes est de permettre une évaluation individualisée des besoins. La nécessité de connaître les facteurs de risques en lien avec le sexe, l'âge, le niveau socio-économique et le lien avec le défunt est évidente. Mais porter une attention fine à l'histoire du sujet, à sa structure de personnalité, ses mécanismes de coping et les ressources dont il dispose est nécessaire pour cibler les endeuillés qui en ont le plus besoin [START_REF] Jordan | Does grief counselling work?[END_REF] et dont nous savons qu'ils seront les moins en mesure de demander un soutien. Ajoutons qu'une évaluation fine du degré de souffrance, des particularités symptomatologiques et du retentissement fonctionnel est considéré comme essentiel à l'estimation clinique du risque de complications du deuil [START_REF] Waller | Assisting the bereaved: A systematic review of the evidence for grief counselling[END_REF] [14].

Bien que l'existence d'un dispositif tel que le DASEC semble se construire dans le prolongement des recommandations internationales, l'évaluation de son efficacité à moyen et long terme demeure à la fois nécessaire et épineuse. Les études concernant l'efficacité des approches psychothérapeutiques centrées sur le deuil souffrent de l'hétérogénéité de leurs échantillons concernant des facteurs essentiels : le lien au défunt, la qualité de la relation, la possibilité d'anticiper le décès ainsi que le temps écoulé depuis le décès [START_REF] Waller | Assisting the bereaved: A systematic review of the evidence for grief counselling[END_REF]. C'est donc dans le contexte d'une littérature internationale dynamique mais encore en construction que devront se construire l'évaluation des dispositifs construits pendant la crise sanitaire.

Certaines dimensions spécifiques du DASEC, telles que le court délai entre le décès et le premier contact, mais aussi l'accompagnement téléphonique ou encore la transition vers des soins de secteurs non spécialisés dans la prise en charge du deuil devraient être l'objet d'une attention spécifique. Par ailleurs, l'existence d'un outil de mesure spécifique ouvre des perspectives intéressantes, susceptibles de soutenir une évaluation de effets du dispositif sur la prévalence et la sévérité des symptômes de complication du deuil [17].

[17] Boelen PA, Djelantik AAAMJ, de Keijser J, Lenferink LIM, Smid GE. Further validation of the Traumatic Grief Inventory-Self Report (TGI-SR): A measure of persistent complex bereavement disorder and prolonged grief disorder.

  Dispositif d'Accompagnement et de Soutien aux Endeuillés de la Covid-19 (DASEC) est né de la mobilisation du pôle de Psychiatrie, Médecine légale et Médecine en milieu pénitentiaire du Centre Hospitalier Universitaire de Lille, avec le soutien de la direction de l'établissement. Il s'est constitué à moyens constants par le redéploiement d'un psychiatre coordinateur (0,2 ETP), de cinq psychologues (0,2 ETP chacune), de deux assistantes sociales (0,1 ETP chacune), d'une secrétaire (0,1 ETP) et d'une cadre de santé (0,1 ETP). Le dispositif s'organise autour de trois principaux objectifs : (a) porter la représentation d'un souci collectif et sociétal à l'égard des proches des défunts, (b) proposer aux personnes à risque modéré un soutien professionnel dans l'amorce du travail de deuil et (c) identifier les sujets à risque élevé de complication, afin de favoriser leur accès aux dispositifs de soin de droit commun. Chaque jour, le psychiatre coordinateur est informé des décès liés à la COVID lors des 24 dernières heures sur l'ensemble du campus. Il contacte alors les services concernés ou se met à leur disposition afin de se renseigner sur les circonstances de la mort et de recueillir les coordonnées des personnes de confiance. Dans la mesure du possible, celles-ci sont informées de l'existence et du fonctionnement du dispositif par un courrier remis dans les services. Elles sont ensuite contactées par une psychologue référente de la situation et peuvent lui transmettre les coordonnées d'autres proches à contacter. Ce premier contact téléphonique a lieu entre 1 et 4 jours après le décès. Il a pour but de proposer une première écoute, de s'enquérir des besoins de l'entourage, de repérer d'éventuelles situations à risque de complication du deuil et d'initier, chaque fois que pertinent, un accompagnement. Menés par téléphone ou en visiocommunication, les suivis peuvent ensuite se prolonger autant que nécessaire. L'équipe bénéficie également d'un espace d'accueil physique, dans le respect des gestes barrière, mais celui-ci n'a pas été mis à contribution. Lorsque le contact ne peut être établi ou qu'il se clôture, une carte de condoléances personnalisée, rappelant la disponibilité du dispositif et ses coordonnées est envoyée par voie postale.

Figure 1 .

 1 Le 23 avril 2020, les professionnels avaient passé 222 appels, dont 134 étaient aboutis. Le temps cumulé passé au téléphone était de 34 heures et 30 minutes. Quarante-deux cartes de condoléances ont été envoyées. Sur l'ensemble des endeuillés joints, seuls deux ont décliné notre proposition d'un premier temps d'échange. Dans la grande majorité des cas, les personnes se sont montrées touchées de la sollicitude qui leur était témoignée et s'en sont saisi pour faire part de leur vécu, témoigner des conditions difficiles de leur deuil et partager leurs souvenirs du défunt. Les proches se sont fréquemment transmis nos coordonnées, afin d'assurer la possibilité d'un soutien extra-familial à chacun et d'apaiser leurs inquiétudes réciproques. Ces interventions ont également permis d'apporter des réponses concrètes à des difficultés procédurales, matérielles ou financières (ex. demande de récupération de dossier médical, souhait d'adresser un courrier à la maison des usagers, impossibilité d'assumer le coût des funérailles, etc.)

  Death Studies 2019;43:351-64. https://doi.org/10.1080/07481187.2018.1480546. Évolution du nombre et du nombre cumulé d'appels émis ou reçus par le Dispositif d'Accompagnement et de Soutien aux Endeuillés du Covid-19 en fonction du temps, du 23 mars au 23 avril 2020.

	Légende
	Figure 1.


Orianne Domengé

Hélène Ragot

Robin Deloux

Agnès Crépet

Gaëlle Revet

Emmanuelle Solène

Alexandre Boitard

Nathalie Simon

Laurent Mougenot

Thierry David

Delair

Solène Emmanuelle Boitard

Alexandre Simon

Nathalie Mougenot

Laurent David

Thierry Delair

Dr Alexandra Montembault
email: alexandra.montembault@recherche.univ-lyon1.fr

Onnik Agbulut
email: onnik.agbulut@sorbonne-universite.fr

Prof. Onnik Agbulut

Efficacy of epicardial implantation of acellular chitosan hydrogels in ischemic and nonischemic heart failure: impact of the acetylation degree of chitosan

Keywords: Hydrogel, Natural Polymers, Cardiac Patch, Biocompatibility, Epicardial implantation, Heart failure, Dilated cardiomyopathy, Myocardial infarction

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Heart failure is a progressive and life-threatening disease characterized by reduced systolic function as a consequence of cell death and by diastolic dysfunction due to increased fibrosis and reduced ventricular compliance [START_REF] Tomasoni | Highlights in heart failure[END_REF]. Although ischemic cardiomyopathy induced by myocardial infarction (MI) is the most common cause of heart failure worldwide [START_REF] Bui | Epidemiology and risk profile of heart failure[END_REF], nonischemic cardiomyopathy also significantly contributes to the number of patients with heart failure. In contrast to ischemic cardiomyopathies, which are amenable to a variety of pharmacological, electrical, and interventional treatments, the nonischemic forms of heart failure can only be treated symptomatically by drugs.

In the last few decades, engineered cardiac tissue scaffolds or cardiac patches have emerged as therapeutic candidates to substitute for damaged cardiac tissues as well as deliver regenerative factors after their epicardial implantation [START_REF] Feric | Strategies and Challenges to Myocardial Replacement Therapy[END_REF][START_REF] Kitsara | Fibers for hearts: A critical review on electrospinning for cardiac tissue engineering[END_REF][START_REF] Jackman | Engineered cardiac tissue patch maintains structural and electrical properties after epicardial implantation[END_REF]. Cardiac patches were first investigated as a possible solution to cell injection challenges for cell therapy purposes [START_REF] Ye | Patching the heart: cardiac repair from within and outside[END_REF]. Indeed, cell retention in damaged hearts appears to be one of the major roadblocks to successful myocardial regeneration in stem cell therapy.

In this context, many studies demonstrated that the use of biomaterials for delivery and support of transplanted cells can provide a real solution to this obstacle [START_REF] Hamdi | Cell delivery: intramyocardial injections or epicardial deposition? A head-to-head comparison[END_REF][START_REF] Hamdi | Epicardial adipose stem cell sheets results in greater post-infarction survival than intramyocardial injections[END_REF][START_REF] Hamdi | Efficacy of Epicardially-delivered Adipose Stroma Cell Sheets in Dilated Cardiomyopathy[END_REF]. In addition, cardiac patches can provide mechanical support to injured cardiac tissue to reduce adverse post myocardial infarction remodeling. In this perspective, in addition to cellularized cardiac patches, acellular cardiac patches have been developed from a variety of materials to provide structural support for the damaged ventricle, stimulate cellular recruitment into the material, and/or induce adaptive tissue remodeling and endogenous repair mechanisms [START_REF] Burdick | Acellular biomaterials: an evolving alternative to cell-based therapies[END_REF][START_REF] Svystonyuk | Using Acellular Bioactive Extracellular Matrix Scaffolds to Enhance Endogenous Cardiac Repair[END_REF]. Some authors have also demonstrated that acellular cardiac patches minimize inflammatory reactions after implantation by inducing the polarization of proinflammatory M1 macrophages into a pro-reparative M2 phenotype [START_REF] Dziki | Immunomodulation and Mobilization of Progenitor Cells by Extracellular Matrix Bioscaffolds for Volumetric Muscle Loss Treatment[END_REF]. Epicardial implantation of acellular patches has been extensively explored across a number of clinically relevant animal models of ischemic injury, which demonstrated overall that surgically implanted patches can attenuate infarct expansion and left ventricular (LV) remodeling [START_REF] Serpooshan | The effect of bioengineered acellular collagen patch on cardiac remodeling and ventricular function post myocardial infarction[END_REF][START_REF] Mewhort | Epicardial infarct repair with bioinductive extracellular matrix promotes vasculogenesis and myocardial recovery[END_REF][START_REF] Efraim | Biohybrid cardiac ECM-based hydrogels improve long term cardiac function post myocardial infarction[END_REF][START_REF] Park | Heparin Augmentation Enhances Bioactive Properties of Acellular Extracellular Matrix Scaffold[END_REF].

Numerous strategies have been proposed, including 3D-bioprinted scaffolds, electrospun scaffolds, decellularized tissues and various types of hydrogels to optimize the patches for cardiac regeneration [START_REF] Kitsara | Fibers for hearts: A critical review on electrospinning for cardiac tissue engineering[END_REF][START_REF] Kim | Reverse remodelling and myocardial recovery in heart failure[END_REF][START_REF] Han | Development of functional hydrogels for heart failure[END_REF]. Hydrogels are promising candidates in regenerative medicine because they are composed of hydrophilic polymer networks that can mimic specific physical aspects of the tissue microenvironments and extracellular matrix [START_REF] Tibbitt | Hydrogels as extracellular matrix mimics for 3D cell culture[END_REF][START_REF] Geckil | Engineering hydrogels as extracellular matrix mimics[END_REF][START_REF] Saldin | Extracellular matrix hydrogels from decellularized tissues: Structure and function[END_REF]. Chitosan-based hydrogels have been widely studied for biomedical applications, especially for myocardial applications [START_REF] Rufaihah | Hydrogels for therapeutic cardiovascular angiogenesis[END_REF]. Chitosan exhibits many key properties such as biodegradability [START_REF] Chatelet | Influence of the degree of acetylation on some biological properties of chitosan films[END_REF], immune-promoting activities [START_REF] Zhao | Chitosan degradation products facilitate peripheral nerve regeneration by improving macrophage-constructed microenvironments[END_REF][START_REF] Chang | Effect of chitosan molecular weight on anti-inflammatory activity in the RAW 264.7 macrophage model[END_REF][START_REF] Chedly | Physical chitosan microhydrogels as scaffolds for spinal cord injury restoration and axon regeneration[END_REF],

antimicrobial activity [START_REF] Kong | Antimicrobial properties of chitosan and mode of action: a state of the art review[END_REF][START_REF] Khor | Implantable applications of chitin and chitosan[END_REF] and wound-healing acceleration [START_REF] Ishihara | Acceleration of wound contraction and healing with a photocrosslinkable chitosan hydrogel[END_REF][START_REF] Ono | Experimental evaluation of photocrosslinkable chitosan as a biologic adhesive with surgical applications[END_REF]. In this work, it is expected that chitosan promotes the regeneration process of the heart muscle and resorbs concomitantly leaving place to the neo-tissue that forms. This polysaccharide is industrially produced from the Ndeacetylation of chitin in alkaline conditions [START_REF] Lamarque | New route of deacetylation of alpha-and beta-chitins by means of freeze--pump out--thaw cycles[END_REF][START_REF] De | Ultrasound-assisted deacetylation of beta-chitin: influence of processing parameters[END_REF]. Chitin is mainly extracted from the cuticles of arthropods, the endoskeletons of cephalopods and the cell wall of fungi. Chitosan, as chitin, belongs to the family of linear copolymers of 2-amino-2-deoxy-D-glucose residues (glucosamine unit) and 2acetamido-2-deoxy-D-glucose residue (acetyl-glucosamine unit) linked by β (1→4) glycosidic bonds.

The degree of acetylation (DA) of these polymers is defined as the molar fraction of acetylglucosamine units. The name "chitosan" refers to polymers soluble in dilute acidic solutions, and then to DAs below 70% when the structure is that of a statistical copolymer [START_REF] Domard | A perspective on 30 years research on chitin and chitosan[END_REF]. As a general trend, the DA impacts the physico-chemical [START_REF] Schatz | Typical physicochemical behaviors of chitosan in aqueous solution[END_REF][START_REF] Sorlier | Relation between the degree of acetylation and the electrostatic properties of chitin and chitosan[END_REF] and mechanical [START_REF] Montembault | Rheometric study of the gelation of chitosan in aqueous solution without cross-linking agent[END_REF] but also the biological properties of chitosans [START_REF] Rami | Physicochemical modulation of chitosan-based hydrogels induces different biological responses: interest for tissue engineering[END_REF].

The physical gelation of chitosan is widely reported in the literature [START_REF] Montembault | Rheometric study of the gelation of chitosan in aqueous solution without cross-linking agent[END_REF][START_REF] Draget | Smidsrød, Chitosan cross-linked with Mo(VI) polyoxyanions: a new gelling system[END_REF][START_REF] Vachoud | Formation and characterisation of a physical chitin gel[END_REF][START_REF] Montembault | Physico-chemical studies of the gelation of chitosan in a hydroalcoholic medium[END_REF]. Our previous work described the design of physical hydrogel patches composed only of water and chitosan of low DA, and their implantation onto the surface of the cardiac muscle in order to regenerate the infarcted myocardium [START_REF] Fiamingo | Chitosan Hydrogels for the Regeneration of Infarcted Myocardium: Preparation, Physicochemical Characterization, and Biological Evaluation[END_REF]. These physical chitosan hydrogels have the advantage of being cross-linker-free and stable in physiological medium. Such chitosan hydrogels were prepared by varying two parameters: the polymer concentration and the gelling agent (gelation under ammonia vapors [START_REF] Montembault | Rheometric study of the gelation of chitosan in aqueous solution without cross-linking agent[END_REF] or by contact with a NaOH aqueous solution [START_REF] Malaise | Bioresorption mechanisms of chitosan physical hydrogels: a scanning electron microscopy study[END_REF]). This work pointed out that the most adapted chitosan concentration was approximately 3% (w/w (equivalent to g/g)) and that the mechanical properties of chitosan hydrogels prepared with aqueous NaOH solution were more suitable for cardiac implantation than those of hydrogels prepared using ammonia vapors. Biologically, patches produced by neutralization of chitosan solution with NaOH were perfectly incorporated onto the epicardial surface of the heart but the cell invasion into the patches was low [START_REF] Fiamingo | Chitosan Hydrogels for the Regeneration of Infarcted Myocardium: Preparation, Physicochemical Characterization, and Biological Evaluation[END_REF]. Nevertheless, previous works have shown that the cell invasion within chitosan hydrogels obtained from the same gelation process may be promoted with an increase in the DA of chitosan [START_REF] Rami | Physicochemical modulation of chitosan-based hydrogels induces different biological responses: interest for tissue engineering[END_REF][START_REF] Malaise | Bioresorption mechanisms of chitosan physical hydrogels: a scanning electron microscopy study[END_REF].

In this context, the goal of the present study was to develop a hydrogel patch that would be better integrated in contact with the cardiac tissue. For this purpose, the influence of the DA of chitosan on the biological properties of the patches was investigated. Thus, we generated physical chitosan hydrogels by a NaOH induced-gelation process, using chitosan with DAs ranging from 2.5% to 38%, while having the same final polymer concentration (~ 3% (w/w)). The mechanical properties of the hydrogel patches were then studied before their implantation. We chose to work with chitosans with a relatively high molar mass (Mw). The mechanical properties of chitosan hydrogels prepared from polymers with high Mw are generally higher. Finally, the potential benefit of epicardial implantation of chitosan hydrogels with different DAs was evaluated in a murine model of both ischemic and nonischemic cardiomyopathies, focusing particularly on their potential to restore the functional capacity of the heart.

Material and Methods

Purification, acetylation and characterization of chitosan

The chitosan used in this study was produced from the N-deacetylation of chitin extracted from squid pens (calamari endoskeletons) and was purchased from Mahtani Chitosan (batch type 114). Before use, the polymer was purified as follows. The chitosan was dissolved at 0.5% (w/w) in an aqueous solution of acetic acid. The amount of acetic acid was added in stoichiometric amounts with the NH2 sites of the chitosan. The resulting solution was filtered successively through 1.2, 0.8 and 0.45 µm membranes (Millipore) and then the chitosan was precipitated by the addition of ammonium hydroxide solution (29% (w/w)) until a pH of 9 was reached. The chitosan was thoroughly washed with deionized water and freeze-dried.

The N-acetylation of chitosan was obtained in a water/1,2-propanediol mixture with acetic anhydride as the reagent [START_REF] Vachoud | Formation and characterisation of a physical chitin gel[END_REF]. Briefly, an aqueous acetic acid solution of chitosan was prepared at a concentration of 1% (w/w) (chitosan was dissolved in deionized water containing the amount of acid necessary to achieve the protonation of the NH2 sites). Then, 1,2-propanediol was added to obtain a final polymer concentration of 0.5% (w/w). A solution of acetic anhydride was slowly added to this hydroalcoholic solution under strong stirring. The amount of acetic anhydride corresponded to the stoichiometric amount necessary to achieve a given degree of acetylation of chitosan (in this study, 12%, 24% or 38%). The medium was left to stand for 12 h. We afterwards precipitated the final chitosan with aqueous ammonia (29% (w/w)). Finally, the polymer was washed in deionized water and freeze-dried as previously described [START_REF] Montembault | Physico-chemical studies of the gelation of chitosan in a hydroalcoholic medium[END_REF].

After freeze-drying, 1 H NMR analysis was used to precisely determine (±5%) the degree of acetylation (DA) of each chitosan [START_REF] Hirai | Determination of degree of deacetylation of chitosan by 1H NMR spectroscopy[END_REF]. This spectral analysis also allowed us to check the absence of 1,2propanediol in the reacetylated chitosans. We dissolved the polymer (15 mg) in 1 g of D2O containing 5 µL of HCl overnight, and the spectrum was acquired at 25 ± 1°C by using a spectrometer Bruker Avance III 400 MHz 5 mm (ν=400 MHz). The DA was then calculated by the method of Hirai et al. [START_REF] Hirai | Determination of degree of deacetylation of chitosan by 1H NMR spectroscopy[END_REF]. As proposed by these authors in their publication, the DA was deduced from the ratio of the integrated area of the methyl protons peak of the N-acetyl glucosamine residues to that of all the H2 to H6' protons of both glucosamine and N-acetyl glucosamine residues (see an example of 1 H NMR spectrum in Figure S2).

The weight-average molecular weight (Mw) and dispersity (Đ) of the different chitosans were determined using size exclusion chromatography (SEC) coupled online with a differential refractometer (Optilab T-rEX, Wyatt; λ=658 nm) and with a multiangle laser light scattering (MALS) detector (Dawn-HELOES II, Wyatt; λ=664 nm). Thus, the polymer was dissolved in 0.15 mol.L -1 ammonium acetate/0.20 mol.L -1 acetic buffer (pH 4.8); the solution was then filtered through a 0.45 µm Millipore membrane, injected (v=100 µL) and eluted at a flow rate of 0.5 mL.min -1 (1260 Infinity pump, Agilent Technologies) through two columns, TSK G2500 PW and TSK G6000 PW. As the sample passes through the steric exclusion chromatography columns, it is separated into fractions according to different hydrodynamic volumes. The online refraction index measurement allows us to determine the amount of each fraction. The online multi-angle light scattering allows us to determine the molar masses of each fraction. In order to perform measurements without calibrant samples, the refractive index increments (dn / dC) have been determined in a previous study [START_REF] Schatz | Typical physicochemical behaviors of chitosan in aqueous solution[END_REF]. The water content of the freeze-dried chitosan samples was evaluated using a thermogravimetric analyzer (DuPont Instrument 2950 thermogravimetric analyzer) operating at a temperature ramp of 10°C.min -1 , from 25°C to 250°C, under a flow of helium.

The full molecular characteristics of the different prepared chitosan samples are given in Table 1. Degree of acetylation (DA), weight-average molecular weight (Mw), dispersity (Đ), weight-average degree of polymerization (DPw) defined as the ratio between the weight-average molecular weight of a chitosan chain (Mw) and the molar mass of the constitutive unit of the polymer.

Preparation, sterilization and storage of chitosan hydrogels

Chitosan physical hydrogels were formed according to a gelation process named aqueous NaOH induced-gelation [START_REF] Fiamingo | Chitosan Hydrogels for the Regeneration of Infarcted Myocardium: Preparation, Physicochemical Characterization, and Biological Evaluation[END_REF][START_REF] Malaise | Bioresorption mechanisms of chitosan physical hydrogels: a scanning electron microscopy study[END_REF]. Purified chitosan (DA=2.5, 12, 24 or 38%) was dissolved in diluted aqueous acetic acid. The amount of acetic acid corresponded to the stoichiometric protonation of the NH2 sites of chitosan. The resulting chitosan solution (polymer concentrations of 1.5, 1.65, 1.85 or 2.35%, depending on the DA (for DA=2.5, 12, 24 or 38%, respectively)) was centrifuged to remove air bubbles and then poured into a Petri dish with a diameter of 3.5 cm. We obtained chitosan physical hydrogels at a final concentration of approximately 3% (w/w) and with different DAs by putting the polymer solution in contact with concentrated aqueous NaOH (3.0 mol.L-1) for 1 hour. For this, a same volume of NaOH solution was poured over the chitosan solution for the different hydrogels prepared (the amount of chitosan solution was the same for the different hydrogels). Finally, we thoroughly washed the chitosan hydrogels with deionized water until neutral pH and stored them at room temperature in deionized water. Before analysis and implantation into animals, the chitosan physical hydrogels were steam-sterilized under standard conditions (121°C, 1 bar, 20 min). Hereafter, the codification "Ch-X" is used to identify the chitosan hydrogels. The "X" stands for the DA of chitosan (i.e., Ch-2.5, Ch-12, Ch-24 or Ch-38). The hydrogels were ready to use after the sterilization step.

Determination of the final polymer concentration in hydrogels

The final chitosan concentration within the hydrogels was determined as described in a previous work [START_REF] Montembault | Rheometric study of the gelation of chitosan in aqueous solution without cross-linking agent[END_REF]. A known weight of chitosan physical hydrogel was freeze-dried, and further, the water content of the freeze-dried hydrogels was precisely determined by thermogravimetric analysis. We can thus deduce the concentration of the polymer within the hydrogel.

Rheometry analysis

Dynamic-mechanical rheological measurements were carried out by using an ARES rheometer (TA Instruments) operating with a plate-plate geometry (diameter of 25 mm) at room temperature. The strain amplitude was monitored to ensure the measurements were carried out within the linear viscoelastic region, resulting in storage modulus (G') and loss modulus (G'') independent of the strain amplitude. Thus, we carried out angular frequency sweep measurements in the range from 100 rad.s-1 down to 0.1 rad.s-1 (at an applied strain of 0.5%). We repeated these analyses three times for each type of hydrogel ("destructive characterization").

Suturability tests

The suturability test consisted of making a loose suture in the hydrogel with a suture thread and then gradually increasing the force applied to the thread until the material broke/tore [START_REF] Fiamingo | Chitosan Hydrogels for the Regeneration of Infarcted Myocardium: Preparation, Physicochemical Characterization, and Biological Evaluation[END_REF]. Gel samples of 10 mm × 10 mm with similar thicknesses (approximately 0.8±0.1 mm) were analyzed using a dynamic mechanical analyzer (DMA Q800, TA 190 Instruments). We punctured each chitosan physical hydrogel at 3 mm of its down edge with a suture thread (Polyvinylidene Fluoride 6/0, PREMIO Peters Surgical). Afterwards, the hydrogel was attached to the upper clamp of the DMA using a tension gripper, and on the other side, the suture thread to the lower clamp. The tensile suture measurements were performed at room temperature in force-controlled mode with a strength ramp of 0.5 N.min -1 up to 18.0 N and a preloading strength of 0.01 N, allowing the ultimate suture load to be determined. We repeated such analyses five times for each hydrogel.

Animals

Fifty-eight male male Wistar rats (Janvier Labs, Genest Saint Isle, France) weighing an average of 200-225 g and twenty female 3 to 5-month-old mice with conditionally invalidation for Serum response factor gene (SRF HKO mice) [START_REF] Parlakian | Temporally controlled onset of dilated cardiomyopathy through disruption of the SRF gene in adult heart[END_REF] were used in this study. All procedures were approved by our institutional Ethics Committee "Charles Darwin" (Permit number: #4370) and complied with the European legislation (Directive 2010/ 63/EU) on animal care.

Cardiac surgery and experimental groups

Myocardial infarction (MI) was induced as described previously [START_REF] Hamdi | Long-term functional benefits of epicardial patches as cell carriers[END_REF][START_REF] Bellamy | Long-term functional benefits of human embryonic stem cell-derived cardiac progenitors embedded into a fibrin scaffold[END_REF]. Briefly, rats were anaesthetized with isoflurane (Baxter, Maurepas, France), 3% at induction and 2% for maintenance,

and tracheally ventilated at a rate of 45 min -1 and with a 1 mL average insufflate volume for 100 g of body weight (Harvard Apparatus, France). The heart was exposed through a left thoracotomy and the left coronary artery was permanently ligated between the pulmonary artery trunk and the left atrial appendage. Analgesia was performed for 2 days after surgery with a 10 mg.kg -1 intraperitoneal injection of ketoprofen (Merial, Lyon, France).

Four weeks after myocardial infarction, the rats underwent a baseline echocardiographic assessment to evaluate their left ventricular (LV) function. Following a median sternotomy, the animals were then allocated to receive an epicardial implantation of chitosan hydrogels with four different DAs (Ch-2. Additional experiments were conducted for the Ch-24 chitosan hydrogel. Briefly, four weeks after myocardial infarction, the rats underwent a median sternotomy to receive an epicardial implantation of Ch-24 chitosan hydrogels or an open-chest placement of similar sutures, without any implantation.

The animals were sacrificed one week (n=6 per group) after treatment.

Dilated cardiomyopathy was induced by injection of tamoxifen into conditionally invalidated Serum

Response Factor (SRF HKO ) mice [START_REF] Parlakian | Temporally controlled onset of dilated cardiomyopathy through disruption of the SRF gene in adult heart[END_REF]. Briefly, SRF HKO mice were generated by mating mice harboring a homozygous conditional allele of SRF gene (SRFflox) with a cardiac specific tamoxifen-inducible CreER driver mouse line, which express CreER recombinase under the control of cardiomyocyte specific α-myosin heavy chain promoter. The excision of cardiac SRF gene was induced by daily intraperitoneal tamoxifen injections (20 mg/g/day; Sigma Aldrich, St. Quentin Fallavier, France) on 3 consecutive days. Before tamoxifen administration, the animals were first anaesthetized with isoflurane (Baxter, Maurepas, France) for a baseline echocardiographic assessment and then allocated to receive an epicardial implantation of chitosan hydrogels with two different DAs (Ch-2.5, n=10; Ch-24, n=10). Analgesia was performed for 2 days after surgery with a 10 mg.kg -1 subcutaneous injection of ketoprofen (Merial, Lyon, France).

Three weeks after treatment, all animals underwent another echocardiographic assessment of LV function and were sacrificed thereafter. The explanted hearts were separated into two halves by a short-axis section through the midportion of the grafted area. The blocks were immediately fixed in 4% of formaldehyde for 24 h, dehydrated, and embedded in paraffin for histological analysis or immediately fixed in Tissue-Tek (Sakura Finetek, Villeneuve d'Ascq, France) and frozen in liquid nitrogen-cooled isopentane for immunohistochemical analysis.

Evaluation of cardiac function

Pre-and post-transplantation cardiac function were evaluated before transplantation and one month thereafter for the myocardial infarction rats and three weeks thereafter for the SRF HKO mice by transthoracic echocardiography of lightly anesthetized animals under isoflurane (induction with 2%

and maintained with 0.5%) [START_REF] Hamdi | Epicardial adipose stem cell sheets results in greater post-infarction survival than intramyocardial injections[END_REF][START_REF] Hamdi | Efficacy of Epicardially-delivered Adipose Stroma Cell Sheets in Dilated Cardiomyopathy[END_REF]. This noninvasive measurement of left ventricular dimensions and volumes was conducted using echocardiography-Doppler (Vivid 7 Dimension/Vivid7 PRO; GE Medical System Co, Vélizy, France) with an ultrasound probe with a 9 to 14 MHz frequency range.

The bidimensionally guided time-motion recording mode (parasternal long-axis view) of the left ventricle (LV) provided the following measurements: diastolic (IVSd) and systolic interventricular septum (IVSs) and posterior wall thicknesses (LVPWd and LVPWs), LV end-diastolic (LVEDD) and end-systolic diameters (LVESD), and heart rate. Each set of measurements was obtained from the same cardiac cycle. The LV shortening fraction (LVSF) was calculated using the formula: (LVEDD-LVESD)/LVEDD×100. LV myocardial volumes, LV end-diastolic (LVEDV), and end-systolic (LVESV) volumes were calculated using a half-ellipsoid model of the LV. From these volumes, LV ejection fraction (LVEF) was calculated using the formula: (LVEDV-LVESV)/LVEDV×100. The LV thickness/LV radius ratio (h/r) was also assessed in all animals. All measurements were performed on digital loops in triplicate and averaged by an investigator blinded to the treatment group.

Histology and immunolabeling

Ten µm heart sections were made using a microtome (Leica Microsystems, Nanterre, France), stained with hematoxylin and eosin for visualization of general morphology and Sirius red for visualization of fibrosis, mounted in Eukitt resin (CML, France) and examined by transmitted light microscopy.

Images were taken with a microscope (Leica Microsystems, Nanterre, France) equipped with a digital camera.

Immunolabeling was performed on ten µm frozen heart sections. The primary antibodies which we used in this study are: anti-caveolin-1α (rabbit IgG, dilution 1:150, Santa Cruz Biotechnologies, Heidelberg, Germany), anti-CD-206 (rat IgG2a, dilution 1:50, Santa Cruz Biotechnology, Heidelberg, Germany) and anti-vinculin (mouse IgG, dilution 1:200, Sigma Aldrich, St. Quentin Fallavier, France). Binding of primary antibodies was detected by incubating the sections with Alexa-conjugated secondary antibodies (dilution 1:1000; Life Technologies). Images were captured using an inverted microscope (Leica Microsystems, DMi8). All images were acquired using identical settings for exposure for each parameter. All of the measurements were performed blinded to the experimental conditions using ImageJ software (National Institutes of Health [NIH], Bethesda, MD, USA).

The amount of fibrosis was quantified as the Sirius red-positive area/ventricular surface ratio on 6 fields per section on 3 sections per heart, and 5-8 animals per group. Capillary density, defined as the capillary-to-cardiomyocyte ratio, was measured in the same area as the number of caveolin-1-labeled cells (endothelium) over the number of vinculin-positive cells (cardiomyocytes). A minimum of 4 fields per cross-section on 3 cross-sections per heart, and at least 3-5 animals per group were used.

The maximal length and width of the cardiomyocytes was measured using ImageJ following vinculinimmunofluorescence staining. At least 500 cardiomyocytes per group were analyzed. Measurements were performed on samples from at least five individuals in each group.

Relative quantification of gene expression by real-time PCR

Total RNA was extracted from the hearts using TRIzol® (Thermo Fisher Scientific, Saint-Herblain, France) following the manufacturer's instructions. From 500 ng of extracted RNA, the first-strand cDNA was then synthesized using a RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Saint-Herblain, France) with random hexamers according to the manufacturer's instructions.

Using the Light Cycler® 480 system (Roche Diagnostics), the reaction was carried out in duplicate for each sample in a 6-µl reaction volume containing 3 µl of SYBR Green Master Mix, 500 nM of the forward and reverse primers each and 3 µl of diluted (1:25) cDNA. The thermal profile for the SYBR Green qPCR was 95°C for 8 min, followed by 40 cycles at 95°C for 15 s, 60°C for 15 s and 72°C for 30 s. To exclude PCR products amplified from genomic DNA, primers were designed, when possible, to span one exon-exon junction. The mean gene expression stability of 2 genes, Hmbs (hydroxymethylbilane synthase) and B2M (beta-2-microgobulin), were used as the reference transcripts. Data were collected and analyzed using the LightCycler® 480 software release 1.5.0 (Roche Diagnostics).

Cytokine quantification by antibody array

The protein concentration was measured using a bicinchoninic acid kit (Sigma Aldrich, St. Quentin Fallavier, France) with bovine serum albumin as a standard. Cytokine antibody arrays were carried out according to the manufacturer's instructions (Abcam, USA) using 200 µg of protein per sample.

Expression of 38 different cytokines was quantified by measuring the intensity of the different spots using ImageJ software (National Institutes of Health [NIH], Bethesda, MD, USA).

Peptide and protein analysis by liquid chromatography tandem-mass spectrometry (LC-MS/MS)

Total proteins were extracted from the heart samples by homogenization in 400 µL of ice-cold lysis buffer containing: 50 mM Tris (pH 8), 0.1% SDS, 50 mM NaCl, 1 mM EDTA, 1% Triton, and 1 mM DTT with protease and phosphatase inhibitor cocktails (Sigma Aldrich, St. Quentin Fallavier, France).

The supernatant was collected after centrifugation at 12 000 g for 5 min at 4 °C. The protein concentration was measured using a bicinchoninic acid kit (Sigma Aldrich, St. Quentin Fallavier, was limited to two for trypsin digestion. P-values of the peptides were calculated using the percolator algorithm, and a 1% filter was applied as a false-discovery rate threshold. Abundance variations of proteins with a p-value under 0.1 obtained from ANOVA and a maximum fold change of 1.5 were further considered.

Statistical Analysis

Analyses were conducted using GraphPad Prism 7.00 (GraphPad Software Inc., SD, USA). For oneway analysis of variance, the normality was checked using the Shapiro-Wilk normality test. If the normality of distribution assumption was not met, the nonparametric Mann-Whitney test was used instead of ANOVA. If a significant difference was found, then multiple comparison tests were performed to compare the different groups analyzed (Dunn's or Dunnett's multiple comparison test following a nonparametric or parametric test, respectively). For statistical analysis of cardiomyocyte size distribution, Kolmogorov-Smirnov tests were used. Grouped data were analyzed using two-way ANOVA, followed by post hoc Holm-Sidak tests for multiple comparisons when an interaction between the main factors reached significance. A p-value < 0.05 was considered significant. Values are given as the means ± standard error of the mean (SEM).

Results

In this study, we investigated the impact of chitosan hydrogel patches with different DAs on cardiac tissue regeneration using two murine models of cardiomyopathy. First, we described the formation of hydrogel patches composed of only water and pure chitosan with different DAs and then we characterized their mechanical properties before epicardial implantation in animals.

Preparation of chitosan hydrogels with different degrees of acetylation

We investigated the impact of DA on the mechanical and biological properties of hydrogels in order to define an optimal DA for the design of a cardiac patch. It should be noted that we have chosen a wide chitosan DA range, starting from a DA of 2.5% (i.e. the DA of initial deacetylated Mahtani chitosan) up to a maximum DA to obtain stable gels (38%). Indeed, above 40%, chitosans re-acetylated in the solution state remain soluble over a wider pH range and it is not possible to obtain stable gels by neutralization of a chitosan aqueous solution either with ammonia vapors or sodium hydroxide solutions. We first produced gels with different DAs (2.5%, 12%, 24% and 38%) while having the same final polymer concentration (~ 3% (w/w)). To prepare these hydrogels with different DAs, we needed to N-reacetylate the chitosan that we purchased (initial DA of 2.5%). As described previously [START_REF] Schatz | Typical physicochemical behaviors of chitosan in aqueous solution[END_REF][START_REF] Sorlier | Relation between the degree of acetylation and the electrostatic properties of chitin and chitosan[END_REF], the conditions of acetylation were sufficiently mild to avoid any chain degradation. As described in Table 1, these acetylations allowed for the production of a homogeneous series of chitosans of different DAs with similar degrees of polymerization.

Macroscopically, shrinkage was observed for all hydrogels after the gelation step (a slight decrease in diameters in the Petri dishes). This phenomenon was previously reported [START_REF] Fiamingo | Chitosan Hydrogels for the Regeneration of Infarcted Myocardium: Preparation, Physicochemical Characterization, and Biological Evaluation[END_REF] for the same kind of chitosan hydrogels at low DA, and for another kind of physical chitosan hydrogel [START_REF] Montembault | Rheometric study of the gelation of chitosan in aqueous solution without cross-linking agent[END_REF]. Meanwhile, the shrinkage led to significant changes in polymer concentration. Thus, the final polymer concentrations were measured for various sterilized hydrogels of different DAs and are reported in Figure 1. The experimental data demonstrated a linear correlation between the initial polymer concentration in the chitosan solution and the concentration of polymer in the final hydrogels regardless of the DA. The increase in concentration was more important at a lower DA and at a lower polymer concentration. A higher polymer concentration induces a more entangled system less likely to shrink during gelation. Accordingly, from these data, we can deduce the least square fit relationship between the polymer concentration in the solution and in the gel (% (w/w)):

[Chitosan]Gel = (-0.0106 DA + 1,2731) [Chitosan]Sol + 0.0095 DA + 0.636 / eq (1)

Such a relationship is probably impacted by the molar mass distribution of the polymer. Chitosan dissolves in acidic aqueous solution via the protonation of its NH2 sites. In the solution state, before the gelation process, chitosan behaves as a highly entangled polyelectrolyte (polycation bearing -NH3 + groups), and apparently its charge density decreases with the DA. For high DA values, the polysaccharide can be considered as a polymer bearing isolated charges, with a hydrophobicity increasing with the DA [START_REF] Montembault | Rheometric study of the gelation of chitosan in aqueous solution without cross-linking agent[END_REF][START_REF] Popa-Nita | Structure of natural polyelectrolyte solutions: role of the hydrophilic/hydrophobic interaction balance[END_REF].

The gelation process conceptually consists of the modification of the balance between hydrophilic and hydrophobic interactions within the system, favoring interchain hydrophobic interactions: this change could be achieved here thanks to a decrease in the apparent charge density of chitosan induced by neutralization. Then, interchain interactions occur and result in the formation of a network combining hydrophobic interactions, H-bonding and high functionality physical crosslinks constituted by crystallites [START_REF] Malaise | Bioresorption mechanisms of chitosan physical hydrogels: a scanning electron microscopy study[END_REF].

The observed shrinkage phenomenon is less important for chitosan gels with a high DA than chitosan gels with a low DA. At a low DA, chitosan chains present a maximal hydrophilic contrast between the protonated and neutralized forms. Thus, after the gelation process, high DA chitosan is more hydrophilic than low DA chitosan due to the presence of many polar acetyl groups present in the polymer chains. This residual hydrophilicity, together with a lower impact of protonation on the conformation, could explain why chitosan hydrogels with high DA exhibit less shrinkage.

Moreover, it is worth noting that the sterilization process also induced a slight increase in the polymer concentration. This increase was higher at low DA (see the examples in Table 2). These results are in accordance with previous results reported for sterilized chitosan physical hydrogels [START_REF] Montembault | Rheometric study of the gelation of chitosan in aqueous solution without cross-linking agent[END_REF][START_REF] Fiamingo | Chitosan Hydrogels for the Regeneration of Infarcted Myocardium: Preparation, Physicochemical Characterization, and Biological Evaluation[END_REF]. During the sterilization step, the temperature increases up to 121°C lead to a decrease in hydrogen bonds in favor of hydrophobic interactions [START_REF] Fiamingo | Chitosan Hydrogels for the Regeneration of Infarcted Myocardium: Preparation, Physicochemical Characterization, and Biological Evaluation[END_REF], resulting in shrinkage. Again, such effects are limited at high DAs, since the chitosan keeps its residual hydrophilicity. Thus, in this study dealing with the effect of DA on gel properties, each hydrogel was prepared in order to have a final concentration within the hydrogels of approximately 3% (w/w) after sterilization, regardless of the DA. As the shrinkage was different depending on the DA, the concentration of chitosan in the initial solution had to be adapted for each DA (see eq. 1) in order to obtain the same final concentration of approximately 3% (w/w).

Hydrogels formed with chitosans of high DAs seemed to be more flexible than hydrogels formed with chitosans of low DAs (Figure 2C). Macroscopically, the hydrogels prepared from chitosan DA=38%

were softer, more flexible and translucent. In contrast, the use of chitosan DA=2.5% resulted in harder hydrogels that were easy to manipulate and more opaque. This change in opacity might come from the differences of the microstructure of the hydrogels. All hydrogels are composed of interpenetrated submicrometric gel aggregates constituting the material structure [START_REF] Popa-Nita | Continuum of structural organization from chitosan solutions to derived physical forms[END_REF][START_REF] Sereni | Dynamic Structuration of Physical Chitosan Hydrogels[END_REF]. These aggregates may present different diffusion properties depending on the DA of chitosan. The mechanical properties of these hydrogels were then evaluated by carrying out rheological measurements, tensile tests, and suturability tests.

Higher DA results in chitosan hydrogels with lower viscoelastic moduli and suturability

The rheological study allowed us to compare the viscoelasticity of the different hydrogel patches developed in this work. We carried out rheological measurements to study the evolution of storage and loss moduli as a function of DA. For all hydrogels, the moduli were nearly frequency-independent and G' was at least ten times higher than G'' (see an example of the curves of the storage and loss moduli in Figure 2A), in accordance with the definition of a gel [START_REF] Almdal | Towards a phenomenological definition of the term 'gel[END_REF]. Consistent with physical gels [START_REF] Ross-Murphy | Fundamentals of Hydrogels and Gelation[END_REF][START_REF] Clark | Structural and mechanical properties of biopolymer gels[END_REF], a very slight decrease in the G' and a very slight increase in G" at low frequencies are observed for our gels (Figure 2A). The equilibrium storage modulus, noted here as Ge', was estimated corresponding to the value of G' at the plateau for low frequencies. The value of Ge' for the different hydrogels (DA:

2.5, 12, 24, and 38%) was reported in Figure 2B (Figure 2B). As already mentioned, all of the hydrogels were steam-sterilized before mechanical characterizations. In this concentration range, previous works reported that this sterilizing treatment has a low impact on the mechanical properties of chitosan hydrogels through the viscoelastic moduli and ultimate suture strength [START_REF] Fiamingo | Chitosan Hydrogels for the Regeneration of Infarcted Myocardium: Preparation, Physicochemical Characterization, and Biological Evaluation[END_REF].

In the DAs range investigated, we observed a decrease in Ge' with increasing DAs. These results are in line with the semiquantitative experiments shown in Figure 2C and with the literature [START_REF] Rami | Physicochemical modulation of chitosan-based hydrogels induces different biological responses: interest for tissue engineering[END_REF][START_REF] Malaise | Bioresorption mechanisms of chitosan physical hydrogels: a scanning electron microscopy study[END_REF]. The presence of N-acetyl glucosamine residues in chitosan chains contributes to a reduction in the charge density of the polyelectrolyte chains. As a consequence, this leads to the limitation of chain extension in solution and the decrease of entrapped macromolecular entanglements (i.e. "interchain cross-links") in the gel. In addition, Montembault et al. showed that the viscosity of chitosan solutions decreased with increased DA [START_REF] Montembault | Rheometric study of the gelation of chitosan in aqueous solution without cross-linking agent[END_REF].

Conversely, at a low DA, the chain extension due to strong electrostatic repulsions (polyelectrolyte behavior), as a result of the elevated charge density of the chains, induced a high level of entanglement of the chitosan macromolecules. The gelation of more entangled systems leads to hydrogels with higher mechanical properties. In this work, the gelation was fast enough to preserve entanglements in the gel (gelation by neutralization in a NaOH bath at 3.0 mol.L -1 ), resulting in mechanically improved hydrogels at lower DAs. In the context of our application, the design of hydrogel implants requires evaluation of their suture properties. Thus, different hydrogel membranes were prepared to investigate and compare their suture resistance in a quantitative way. The test consists of making a loose suture in the hydrogel and progressively increasing the load applied to the suture until the extraction of the suture (Figure 3A).

This ultimate suture strength can be defined as "suturability".

As we demonstrated in previous works that the suturability increased with the gel thickness [START_REF] Fiamingo | Chitosan Hydrogels for the Regeneration of Infarcted Myocardium: Preparation, Physicochemical Characterization, and Biological Evaluation[END_REF], we normalized the different measured suturability values with respect to sample thickness. Our results demonstrated that the normalized suturability decreased with increasing DA, consistent with the rheological measurements: for the Ch-2.5 hydrogel, the value was 0.35 ± 0.04 N.mm -1 while for the Ch-38 hydrogel value it was 0.03 ± 0.01 N.mm -1 (Figure 3B).

Moreover, regardless of the degree of acetylation, in vivo preliminary tests indicated that no tears were observed after fixation by suture points. These tests allowed us to ensure that these patches were compatible with surgical practice.

Chitosan hydrogels with the highest DAs improved cardiac function after epicardial implantation in myocardial infarction

The potential benefit of epicardial implantation of chitosan hydrogels at different DAs was first evaluated in a rat model of myocardial infarction. One month after the implantation, the chitosan hydrogels were macroscopically visualized on the surface of the hearts (Figure 4). We noticed a visible degradation at higher DAs (Ch-24 and Ch-38 hydrogels). For a closer analysis, serial heart sections were made and some of these sections were stained with hematoxylin and eosin to analyze the general aspect of the hydrogels, as well as foreign body responses with respect to inflammation, granulomatous reactions and epicardial fibrosis (Figure 4A). All implanted hydrogels were visible on the surface of the hearts with hematoxylin and eosin staining at one month after implantation. Low DA hydrogels (Ch-2.5 and Ch-12 hydrogels) showed partial degradation accompanied by mononuclear cell infiltration. In contrast, at high DAs (Ch-24 and Ch-38 hydrogels), the chitosan hydrogels were highly degraded after implantation and only small fragments of hydrogels were visible at one month in the grafted area. No exuberant fibrosis and no multinucleated giant cells were visible in the grafted area, demonstrating the absence of a foreign body reaction and the perfect biocompatibility of these chitosan hydrogels at all of the DAs.

In parallel to morphological assessment, we also evaluated the functional impact of epicardial implantation of the Ch-2.5, Ch-12, Ch-24 and Ch-38 hydrogels using transthoracic echocardiography before (baseline) and one month after implantation (Figure 4B). We first measured two surrogate heart function parameters, left ventricular ejection fraction (LVEF) and left ventricular shortening fraction (LVSF). Neither LVEF nor LVSF differed at baseline between the groups, thereby suggesting initial ischemic injuries of similar extents. However, after implantation of the chitosan hydrogels, the changes were markedly different between the DAs. As demonstrated in Figure 4B, both LVEF and LVSF significantly increased one month after implantation in the presence of the patch in comparison to MI, except for the Ch-2.5 group. For Ch-2.5, the LVEF and LVSF values tended to remain stable in comparison to MI.

This effect was primarily the result of a decrease in LVESV from baseline values in Ch-24 and Ch-38 hydrogels treated hearts whereas in MI these parameters were increased (Figure 4B). Hearts treated with low DA hydrogels featured a smaller increase in LVESV from baseline values compared with MI. In contrast, the LVEDVs did not differ among the groups (Figure 4B). Echocardiographic data were recorded at similar heart rates.

The improvement of systolic function in transplanted rats was further supported by histological analysis. We quantified the capillary density and the extent of interstitial fibrosis in the border zone of the infarcted area. Our results demonstrated no difference in the mean number of capillaries per cardiomyocytes in the border zone between the different treatment groups (Figure 4C). The extent of fibrosis was assessed by Sirius red staining. As demonstrated in Figure 4D, the mean percentage area of fibrosis was significantly lower for the Ch-24 hydrogel compared to MI. The extent of fibrosis was also lower with the Ch-12 hydrogel but the differences failed to reach a significant level. Taken together, our results demonstrated that the optimal hydrogel for cardiac applications was the Ch-24 hydrogel. Following these results demonstrating the absence of differences of mRNA expression profiles of proinflammatory markers, we investigated the inflammatory response at the protein level. The immunostaining for CD206, a marker of activated macrophages [START_REF] Shiraishi | Alternatively activated macrophages determine repair of the infarcted adult murine heart[END_REF], showed no differences between treated and nontreated animals at both 7 and 30 days after epicardial implantation (Figure S1).

Finally, the degree of inflammatory response after treatment with the Ch-24 hydrogel compared to MI was investigated by cytokine array 7 days after implantation. Cytokine arrays are an antibody-pair-

Chitosan hydrogels with DA 24% improve cardiac function and prevent cardiac remodeling in a mouse model of dilated cardiomyopathy

To broaden the spectrum of possible application of this promising therapeutic approach, we also implanted chitosan hydrogels in a nonischemic dilated cardiomyopathy model, the SRF HKO mouse model. Twenty SRF HKO mice received the Ch-2.5 (n=10) and Ch-24 (n=10) hydrogels after tamoxifen injection, before the onset of the dilated cardiomyopathy, to evaluate the impact of chitosan hydrogels on adaptive cardiac remodeling. First, we evaluated the functional impact of epicardial implantation of the Ch-2.5 and Ch-24 hydrogels using transthoracic echocardiography before (baseline, one week after tamoxifen injection) and three weeks after implantation (sacrifice, 4 weeks after tamoxifen injection) (Figure 7). At baseline, the LVSF did not differ among the groups. Three weeks after implantation, the LVSF was significantly decreased from the baseline in both the Ch-2.5 and Ch-24 hydrogel groups, but this decrease was lower in Ch-24 compared to the Ch-2.5 hydrogels (Figure 7A). This effect was primarily the result of a lower increase in LVESD from baseline values in the Ch-24 compared to the Ch-2.5 hydrogels (Figure 7B). LVEDD was increased in both the Ch-2.5 and Ch-24 hydrogels (Figure 7C).

Discussion

The main result of this study is that the epicardial implantation of an acellular chitosan hydrogel, dependent on the DA of the chitosan, improves the functional outcomes of both ischemic and nonischemic hearts and that this benefit is associated with a significant reduction in fibrosis and hypertrophic stress.

In this study, ischemic cardiomyopathy was induced by permanent coronary ligation in rats, which is a commonly used and excellent ischemic model. One of the limits of this model lies in the differences in the observed phenotypes relative to those observed in patients, especially in the early stage, because in patients, ischemic heart disease progresses slowly, while in this model, after the permanent coronary ligation, the death of the cardiomyocytes is immediate and massive. After this early stage, it takes approximately 10 days for the infarcted area to evolve towards a predominantly fibrotic scar, and the cardiac remodeling follows a similar process compared to that observed in humans [START_REF] Jugdutt | Rate of collagen deposition during healing and ventricular remodeling after myocardial infarction in rat and dog models[END_REF]. To overcome this limitation and to use a more clinically relevant animal model, we chose to implant the chitosan hydrogels 4 weeks after coronary ligation. This chronic ischemic model allowed us to explore the impact of chitosan hydrogels at the late stage of cardiac remodeling.

For the nonischemic model, we used a well-characterized and well-documented mouse model of dilated cardiomyopathy based on cardiac-specific and tamoxifen-inducible invalidation of SRF (SRF HKO mice) [START_REF] Hamdi | Efficacy of Epicardially-delivered Adipose Stroma Cell Sheets in Dilated Cardiomyopathy[END_REF][START_REF] Parlakian | Temporally controlled onset of dilated cardiomyopathy through disruption of the SRF gene in adult heart[END_REF][START_REF] Deloux | Voluntary Exercise Improves Cardiac Function and Prevents Cardiac Remodeling in a Mouse Model of Dilated Cardiomyopathy[END_REF][START_REF] Joanne | Nanofibrous clinical-grade collagen scaffolds seeded with human cardiomyocytes induces cardiac remodeling in dilated cardiomyopathy[END_REF]. As previously described, as early as 5 days after tamoxifen injection, there is a marked decrease in SRF accompanied by a progressive impairment of cardiac function, ending in dilated cardiomyopathy, heart failure, and death within 8 weeks. One of the major strengths of this model is the advantage of a timely controlled onset of functional deterioration (once tamoxifen has been given) and the reproducibility of the observed phenotypes between each individual, thereby allowing for a standardized comparison of outcomes between treated and nontreated mutant animals.

Using this model, we evaluated the impact of epicardial implantation of chitosan hydrogels on adaptive cardiac remodeling. For this reason, we implanted chitosan hydrogels just after tamoxifen injection, before the onset of the dilated cardiomyopathy, and we assessed the progression of the disease over time.

Chitosan hydrogels are well-tolerated by the host myocardium after implantation, both in ischemic and nonischemic animal models. They provide structural support for the damaged ventricle and induce adaptive tissue remodeling and endogenous repair mechanisms. Chitosan is a polysaccharide classified by the United States Food and Drug Administration as a "safe" substance [START_REF] Baldrick | The safety of chitosan as a pharmaceutical excipient[END_REF]. Chitosan-based biomaterials are widely used for tissue regeneration purposes [START_REF] Cheung | Chitosan: An Update on Potential Biomedical and Pharmaceutical Applications[END_REF]. The DA of chitosan is a key structural parameter impacting its degradation and biological activity [START_REF] Rami | Physicochemical modulation of chitosan-based hydrogels induces different biological responses: interest for tissue engineering[END_REF][START_REF] Malaise | Bioresorption mechanisms of chitosan physical hydrogels: a scanning electron microscopy study[END_REF][START_REF] Montembault | A material decoy of biological media based on chitosan physical hydrogels: application to cartilage tissue engineering[END_REF][START_REF] Croisier | Chitosan-based biomaterials for tissue engineering[END_REF]. However, limited data are available on the impact of the DA of the chitosan on tissue regeneration. Therefore, in this study, four different DAs ranging from 2.5% to 38% were investigated. Our results showed that epicardial implantation of chitosan hydrogels with a DA of 24% improved the functional outcomes of both ischemic and nonischemic hearts and this benefit was associated with a significant reduction in fibrosis and hypertrophic stress. Although chitosan has been shown to modulate the immune response [START_REF] Zhao | Chitosan degradation products facilitate peripheral nerve regeneration by improving macrophage-constructed microenvironments[END_REF][START_REF] Chang | Effect of chitosan molecular weight on anti-inflammatory activity in the RAW 264.7 macrophage model[END_REF][START_REF] Chedly | Physical chitosan microhydrogels as scaffolds for spinal cord injury restoration and axon regeneration[END_REF], interestingly, in our study, the expression of CD206, a marker of activated macrophages, and the level of cytokines measured by cytokine arrays and quantitative real-time PCR, were not modified by the chitosan treatment, suggesting that the positive effect of chitosan on the cardiac remodeling was independent of inflammation, thus implying a different scenario as described in [START_REF] Hanna | The Role of the TGF-β Superfamily in Myocardial Infarction[END_REF]. One of the explanations for the positive effect of the Ch-24 hydrogel on cardiac function could be linked to the capacity of the chitosan patch to provide mechanical support to the injured cardiac tissue [START_REF] Serpooshan | The effect of bioengineered acellular collagen patch on cardiac remodeling and ventricular function post myocardial infarction[END_REF][START_REF] Mewhort | Epicardial infarct repair with bioinductive extracellular matrix promotes vasculogenesis and myocardial recovery[END_REF][START_REF] Efraim | Biohybrid cardiac ECM-based hydrogels improve long term cardiac function post myocardial infarction[END_REF][START_REF] Park | Heparin Augmentation Enhances Bioactive Properties of Acellular Extracellular Matrix Scaffold[END_REF].

Although all hydrogel patches used here offer mechanical support to injured tissue regardless of the degree of acetylation, the Ch-24 hydrogel is perhaps the most suitable patch in terms of its mechanical properties (this kind of patch was not too rigid, but not too flexible either). In other hand, label-free proteomic analysis performed in the Ch-24 hydrogel treated hearts highlighted an increased expression of mitochondrial proteins and cytosolic proteins involved in the glycolysis process. These results could indicate that the treatment with the Ch-24 hydrogel was inducing a metabolic switch towards glycolysis to the detriment of the tricarboxylic acid cycle and fatty acid oxidation, which is in relation with energy preservation in the context of heart failure. These results are consistent with previous works that have shown that chitosan and their derivatives could have anti-oxidant effects [START_REF] Varlamov | Chitin/Chitosan and Its Derivatives: Fundamental Problems and Practical Approaches[END_REF]. It is possible that the hydrogel via the delivery of gel degradation products, depending on its degradation kinetics, protects surrounding host cardiomyocytes via induction of adaptive tissue remodeling.

However, the role of chitosan gel "fragments" with different DAs in the implanted region still needs to be elucidated. Indeed, chitosan oligosaccharides exhibited a beneficial effect on cardiac cells in response to the apoptosis and oxidative stress induced by doxorubicin [START_REF] Zhang | Chitosan oligosaccharides prevent doxorubicin-induced oxidative stress and cardiac apoptosis through activating p38 and JNK MAPK mediated Nrf2/ARE pathway[END_REF].

In accordance with the efficacy of chitosan hydrogels implantation, we observed a reduced LV wall stress in the animals treated with the Ch-24 hydrogel. Our results demonstrated the reduced expression of pro-hypertrophic stress and fibrosis markers and higher expression of cytoskeletal proteins, such as troponin I and desmin, with Ch-24 hydrogel treatment. Moreover, using a nonischemic SRF HKO mouse model, we have demonstrated that the cardiomyocyte length to width ratio was significantly lower in Ch-24 hydrogel treated mice compared to Ch-2.5 hydrogel treated mice, indicating that Ch-24 hydrogel treatment prevents cardiac remodeling.

Conclusions

Taken together, in this study, we showed that the epicardial implantation of acellular chitosan hydrogels with a DA of 24% is able to reverse the deleterious cardiac remodeling in the setting of both ischemic and nonischemic cardiomyopathies. The extent of fibrosis, the cardiomyocyte length-towidth ratio, as well as the genes involved in fibrosis and hypertrophic stress were repressed after implantation. Our findings indicate they have great potential as a reliable therapeutic approach to heart failure. The possibility of enriching these hydrogel patches with molecules of beneficial interest for cardiac regeneration and functional recovery of the heart is currently under study.

France) with bovine

  serum albumin as a standard. For LC-MS/MS acquisition, 50 µg of protein for each sample was precipitated with cold acetone overnight at -20 °C. Proteins were digested overnight at 37°C in 20 μL of 25 mM NH4HCO3 with 1.25 µl sequencing grade trypsin (0.2 µg/µL, Promega Corporation, WI, USA). Peptide mixtures were desalted and concentrated on ZipTip µ-C18 pipette tips (Millipore). Peptides were separated by chromatography with the following parameters: Acclaim PepMap100 C18 precolumn (2 cm, 75 μm i.d., 3 μm, 100 Å), Pepmap-RSLC Proxeon C18 column (50 cm, 75 μm i.d., 2 μm, 100 Å), 300 nL/min flow rate, a 98 min gradient from 95% solvent A (water, 0.1% formic acid) to 35% solvent B (100% acetonitrile, 0.1% formic acid) followed by column regeneration, giving a total time of 120 minutes. Peptides were analyzed in the Orbitrap cell at a resolution of 70,000, with a mass range of m/z 375-1500. MS/MS data were acquired in the Orbitrap cell in a Top20 mode, with an AGC target of 3.106 for full MS. Fragments were obtained by Higherenergy C-trap Dissociation (HCD) activation with a collisional energy of 27% and a quadrupole isolation window of 1.4 Da, with an AGC target of 2.105. MS/MS data were acquired by an Orbitrap in a data-dependent mode with a dynamic exclusion of 30 seconds. Peptides with unassigned charge states or monocharged were excluded from the MS/MS acquisition. The maximum ion accumulation times were set to 50 ms for MS acquisition and 45 ms for MS/MS acquisition.For the peptide and protein identification step, all MS and MS/MS data were processed with Proteome Discoverer software (Thermo Scientific, version 2.2) and with the Mascot search engine (Matrix Science, version 5.1). The mass tolerance was set to 7 ppm for precursor ions and 0.5 Da for fragments. The following variable modifications (maximum two per peptide) were allowed: oxidation (M), phosphorylation (ST), and acetylation (N-term of protein). The maximum number of missed cleavages was limited to two for the trypsin protease. The SwissProt database (02/17) with the Rattus norvegicus taxonomy was used for the MS/MS identification step. Peptide identifications were validated using a 1% FDR (false discovery rate) threshold calculated with the Percolator algorithm.Label Free quantitation was performed using Progenesis QI for proteomics software (Waters). Data were processed with Proteome Discoverer 2.4 software (Thermo Fisher Scientific) using the HI-3 method for quantification of proteins. Identification was performed on a Mascot search server (Matrix Science; version 2.4) used in Proteome Discoverer 2.4. The mass tolerance of the fragment ions was set to 6 ppm for precursor ions and 0.02 Da for fragments. Identification of tryptic peptides related to the proteins were performed using the Mus musculus or Rattus Norvegicus database from Uniprot RefSeq. Post translation modifications were searched in the dynamics parameters: oxidation (M), phosphorylation (S/T/Y), and acetylation (K/N-terminal). The maximum number of missed cleavages

Figure 1 .

 1 Figure 1. Final hydrogel concentration ([Chitosan]Gel) after neutralization with a NaOH solution (3 M, 1 h) and autoclave sterilization, with different degrees of acetylation (Ch-2.5, Ch-12, Ch-24 and Ch-38 hydrogels) with different polymer solution concentrations ([Chitosan]Sol).

Figure 2 .

 2 Figure 2. Mechanical characterization of chitosan hydrogels. (A) Evolution of the storage and loss moduli for a Ch-12 hydrogel. (B) Influence of the DA on the equilibrium elastic modulus (Ge') of the chitosan hydrogels. (C) Hydrogels obtained from chitosans of different degrees of acetylation (2.5, 12, 24, and 38%) and with the same final polymer concentration ([Chitosan]Gel~3% (w/w)).

Figure 3 .

 3 Figure 3. Suturability characterization of the chitosan hydrogels. (A) Normalized static strength diagram of chitosan hydrogels as a function of the DA of chitosan. (B) Ultimate suture normalized strength diagram of chitosan hydrogels as a function of the DA of chitosan.

Figure 4 .

 4 Figure 4. Evaluation of chitosan hydrogels after one month post epicardial implantation in the infarcted hearts. (A) Hematoxylin and eosin staining of the rat heart implanted with different DA of chitosan (2.5%, 12%, 24%, 38%). White asterisks (*) in bottom Ch-12 panel shows remaining chitosan hydrogel fragments; Black dotted lines represent the border between the implanted hydrogels (red) and the heart; White dotted lines (upper panel) show a higher magnification area (of the lower panel). (B) Functional evaluation of chitosan hydrogels after one month post epicardial implantation (n=9-14 per group). (C) Capillary density per cardiomyocyte was evaluated in the left ventricle by caveolin-1α (endothelial cells) and vinculin (cardiomyocyte) immunostaining (n=3-5 per groups). (D) Assessment of cardiac fibrosis by Sirius red staining in the border zone (n=6-10 per group). Note that fibrosis was expressed as the ratio (%) of the area of fibrosis to the whole area of the left ventricle. MI, myocardial infarction. Values are given as the means ± standard error of the mean. *p<0.05; **p<0.01; ***p<0.001.

Figure 5 .

 5 Figure 5. Evaluation of chitosan hydrogels after 7 days post epicardial implantation in the infarcted hearts. Relative gene expression of selected cardiac stress and remodeling markers (A) and proinflammatory cytokines markers (B) were evaluated after 7 days of implantation in a rat model of chronic myocardial infarction (n=6 per group). MI, myocardial infarction. Values are given as the means ± standard error of the mean. *p<0.05; **p<0.01.

Figure 7 .

 7 Figure 7. Evaluation of epicardial-implanted chitosan hydrogels in a mouse model of dilated cardiomyopathy. Impact on cardiac function of the epicardial implantation of Ch-24 versus Ch-2.5 hydrogels was evaluated before (BL, baseline) and three weeks after implantation (Sacrifice) in a mouse model of dilated cardiomyopathy based on cardiac-specific and tamoxifen-inducible invalidation of serum response factor. (A) Left ventricular shortening fraction (LVSF); (B) Left ventricular end-systolic diameter; (C) Left ventricular end-diastolic diameter; (D) Interventricular septum thickness in systole (IVSs); (E) Left ventricular posterior wall thickness in systole (LVPWs); (F) Left ventricular thickness to left ventricular radius ratio (h/r). Data are represented in the box plot. The boxes represent the lower to upper quartile range, the error bars represent the entire range, the horizontal bar represents the median value and the dot points represent mean values. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

Figure 8 .

 8 Figure 8. Impact of the Ch-24 hydrogel on cardiac remodeling after epicardial implantation in a mouse model of dilated cardiomyopathy. (A-C) Relative quantification of mRNA levels by realtime PCR for some stress-induced genes (n=7-10 per group). Nppa, atrial natriuretic factor (A); Nppb, brain natriuretic peptide (B); Myh7, beta-myosin heavy chain genes (C). (D-G) Morphometric analysis of cardiomyocytes after vinculin immunostaining. Distribution of cardiomyocyte lengths (D) and widths (E) in Ch-2.5 and Ch-24 hydrogels treated hearts (n>500 cardiomyocytes for each group, including five to eight mice per group). (F) Length-to-width ratio of Ch-2.5 and Ch-24 hydrogels. (G) Representative images of vinculin immunostaining of a heart section three weeks after implantation. Scale bar: 50 µm. (H-J) Assessment of cardiac fibrosis by Sirius red staining (n=5-8 per group) and by real-time PCR (n=7-10 per group). (H) Representative images of Sirius red staining of a heart section three weeks after implantation. Scale bar: 150 µm. (I) Fibrosis is expressed as the ratio (%) of the area of fibrosis to the whole area of the left ventricle. The boxes represent the lower to upper quartile range, the error bars represent the entire range, the horizontal bar represents the median value and the dot points represent mean values. (J) Relative quantification by real-time PCR of the mRNA level of a surrogate fibrosis marker, connective tissue growth factor (Ctgf). *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

Table 1 . Characteristics of the different chitosan samples used in this work. DA (%) Water content (%) (w/w) Mw (g.mol -1 ) Đ DPw

 1

	2.5 ± 0.1	7.6 ± 0.8	580400 ± 29000	1.6 ± 0.2	3600 ± 400
	12 ± 0.6	5.5 ± 0.6	557400 ± 28000	1.7 ± 0.2	3400 ± 300
	25 ± 1.3	11.6 ± 1.2	599000 ± 30000	1.5 ± 0.2	3500 ± 400
	38 ± 1.9	10.7 ± 1.1	575700 ± 29000	1.6 ± 0.2	3200 ± 300

Table 2 . Concentration of polymers in chitosan gels before and after sterilization.

 2

	Degree of acetylation (%)	Concentration of the initial solution (%) (w/w)	Concentration of the corresponding final gel before sterilization (%) (w/w)	Concentration of the corresponding final gel after sterilization (%) (w/w)	Percentage of concentration increase induced by the sterilization (%)
	2.5%	5	5.83 ± 0.05	6.88 ± 0.93	18
	12%	5	5.66 ± 0.44	6.54 ± 0.06	15
	24%	5	5.55 ± 0.26	5.97 ± 0.22	8
	38%	5	5.15 ± 0.09	5.28 ± 0.11	3

Acknowledgments

The authors thank Lea Deltourbe, Christina Fissoun, Zakaria Mougin, Lucas Rougier, Ceren Yazici (IBPS, Paris-France) and Maria Mihoc (UMS28, Paris-France) for punctual technical assistance. The authors also thank all of the personnel of the Animal Facility of the Sorbonne Université (UMS28, Paris-France); the Microscopy Platform (IBPS, Paris-France) for helpful advice and technical assistance during microscopy image acquisition and analysis; and the Proteomics facility (Jacques Monod Institute, Paris-France) for performing the mass spectrometry experiments and for their support in data analysis.

Funding

This work was supported by funds from Sorbonne Université, Claude Bernard Lyon 1, the CNRS, the INSERM, the Agence Nationale de la Recherche (ANR-16-CE29-0021-MYOCHITO), the Fondation de l'Avenir (grant number AP-RM-17-032), the Fédération Française de Cardiologie, Fondation France. Orianne Domengé was supported by a Ph.D. fellowship from the Agence Nationale de la Recherche (ANR-16-CE29-0021-MYOCHITO).

based assay that allows for the screening of multiple mediators, including activin A, agrin, CD86, cytokine-induced neutrophil chemoattractants (CINC), ciliary neurotrophic factor (CNTF), Fas ligand, fractalkine, granulocyte macrophage-colony stimulating factor (GMSF), intercellular adhesion molecule 1 (ICAM-1), interferon-γ (IFN-γ), interleukins, leptin, LPS-induced CXC chemokine (LIX), L-selectin, monocyte chemotactic protein 1 (MCP-1), macrophage inflammatory protein 3α (MIP-3α), metalloproteinase 8 (MMP-8), neural growth factor β (NGF-β), platelet-derived growth factor AA (PDGF-AA), prolactin R, RAGE, chemokine-1, tissue inhibitor of metalloproteinase 1 (TIMP-1), tumor necrosis factor α (TNFα) and vascular endothelial growth factor (VEGF). As shown in Table 3, no differences were observed after 7 days of epicardial implantation of the Ch-24 hydrogel compared to MI, demonstrating that the positive impact of chitosan on cardiac remodeling and function is probably independent of its role as a modulator of the inflammatory response. Additionally, we analyzed the global protein expression in response to chitosan hydrogel implantation using label-free proteomic analysis to better understand the signaling pathways underlying the benefits of chitosan on cardiac function. Protein extracts from the left ventricle were submitted to mass spectrometry analysis and all identified proteins (n=2598) are presented in For a closer analysis of systolic function, we also evaluated systolic interventricular septum (IVSs) and posterior wall thickness (LVPWs) in the treated animals. The IVSs thickness was maintained over the three week period in Ch-24 treated SRF HKO mice but was thinner in Ch-2.5 treated SRF HKO mice at the time of sacrifice (Figure 7D). Both the Ch-2.5 and Ch-24 groups exhibited a significant decrease in LVPWs at the time of sacrifice compared to baseline (Figure 7E), but the thinning was significantly less pronounced in Ch-24 treated mice compared to Ch-2.5 treated mice at the time of sacrifice. Finally, we calculated the LV thickness to LV radius ratio (h/r), which has a constant relationship with left ventricular systolic wall stress [START_REF] Gaasch | Left ventricular radius to wall thickness ratio[END_REF]. We demonstrated that the LV h/r ratio was significantly decreased in Ch-2.5 compared to Ch-24 treated animals, indicating that Ch-24 limits the process of LV chamber dilatation (Figure 7F). The functional efficiency of Ch-24 compared to Ch-2.5

during the establishment of heart failure was reinforced by the demonstration of a reduction in the expression of the stress-induced Nppa (p<0.05), Nppb (p<0.05) and Myh7 (p=0.067) in Ch-24 treated SRF HKO mice compared to the Ch-2.5 treated ones (Figure 8A-C).

To evaluate the impact of the chitosan hydrogels on cardiac remodeling, we also analyzed cardiomyocyte size (Figure 8D-G) and cardiac fibrosis (Figure 8H-J) in Ch-2.5 and Ch-24 hydrogels treated SRF HKO mice. Fibrosis was measured by Sirius red staining (Figure 8H). At the time of sacrifice, all of the SRF HKO mice exhibited interstitial fibrosis (Figure 8H). Nevertheless, the mean percentage area of fibrosis was lower in the Ch-24 hydrogel treated mice compared to the Ch-2.5 hydrogel treated group (Figure 8I). The reduction in fibrosis was also supported by real-time PCR, which demonstrated significantly less induction of the expression of Ctgf in Ch-24 hydrogel treated animals in comparison to Ch-2.5 hydrogel treated ones (Figure 8J).

Finally, we conducted morphometrical analysis after vinculin immunostaining to measure the maximal length and width of the cardiomyocytes three weeks after treatment. In SRF HKO mice treated with Ch-2.5 compared to Ch-24 hydrogels, the cardiomyocyte length distribution was shifted toward the higher values whereas the cardiomyocyte width distribution was shifted toward the lower values (Figure 8D-E). Moreover, the cardiomyocyte length to width ratio was significantly lower in Ch-24 hydrogel compared to Ch-2.5 hydrogel treated mice, indicating that Ch-24 hydrogel treatment prevents cardiac remodeling in a mouse model of dilated cardiomyopathy (Figure 8F).

Conflicts of interest