Mathilde Lacôte

Šárka Jelínková

Yvonne Sleimane

Nourdine Chakouri

Jean-Marie Launay

Pr Jacques

Gilles Renault

Carmen Marchiol

Nada Joe Melhem

Mouna Chajadine

Ingrid Gomez

Kiave-Yune Howangyin¹

Marion Bouvet

Camille Knosp

Yanyi Sun

Marie Rouanet

Ludivine Laurans

Olivier Cazorla²

Mathilde Lemitre

José Vilar

Ziad Mallat

Alain Tedgui¹

Hafid Ait-Oufella

Jean- Sébastien Hulot

Jacques Callebert

Jeremy Fauconnier²

Jean- Sébastien Silvestre

Soraya Taleb

VSMC: Vascular smooth muscle cell WHO: World Health Organization

Keywords: Indoleamine 2, 3-dioxygenase 1, Kynurenine, acute myocardial infarction, cardiac remodeling, Kynurenine, aryl hydrocarbon receptor, apoptosis, reactive oxygen species am ...50 b. Endothelial cells ……………………………………………………...…53 c. Fibroblasts …………………………………………………...………….54 d. Vascular smooth muscle cells and pericytes …………………………....56 e. Immune cells ……………………………………………………………57 ii. Cellular cross-talk following myocardial infarction ………………………….58 .....66 Abdominal aortic aneurysm AngII: Angiotensin-II ACE: Angiotensin converting enzyme ACh: Acetylcholine ACS: Acute coronary syndrome AF: Atrial fibrillation AHR: Aryl Hydrocarbon receptor Akt Coronary artery bypass grafting CAD: Coronary artery disease cAMP: Cyclic adenosine monophosphate Central nervous system CO: Carbon monoxide CO2: Carbon dioxide COOH: Carboxyl COVID-19: Coronavirus disease 2019 COX2: Cyclo-oxygenase 2 CpG Cytochrome P450 1B1 DAMP: Danger-associated molecular patterns DBP: Diastolic blood pressure Ejection fraction EGFR: Epidermal growth factor receptor EndoMT: endothelial-to-mesenchymal transition eNOS: Endothelial nitric oxide synthase Fibroblast growth factor FOXO3: Forkhead Heme oxygenase 1 H2O2: Hydrogen peroxide HSP: Heat shock protein 5-HT: 5-Hydroxytryptamin HMGB1: High mobility group protein 1 HSPC: Hematopoietic stem and progenitor cell IC: Immune cell IC50: Inhibitory concentration ICAM1: Intercellular adhesion molecule 1 IDO: Indoleamine 2, 3-dioxygenase 1 IDO2: Indoleamine 2, 3-dioxygenase 2 Kynurenine monooxygenase ventricular ejection fraction Ly6C: lymphocyte antigen 6C Arterial oxygen saturation SBP: Systolic blood pressure segment elevation myocardial infarction TCDD: 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin TDO

Acute myocardial infarction (MI) is mainly caused by the rupture of an atherosclerotic plaque leading to a thrombus forming within the lumen of a coronary artery resulting in its occlusion. Cardiomyocytes (CM) start to dye because of the lack of oxygen and nutrients. A reparative response consisting on an inflammation followed by a fibrosis takes place resulting in cardiac remodeling. Indoleamine 2, 3 dioxygenase 1 (IDO), induced by pro-inflammatory stimuli such as interferon-gamma (IFN)-γ, catalyzes the degradation of L-Tryptophan (Trp) into kynurenine (Kyn) and other metabolites implicated in different pathologies like atherosclerosis. Different studies showed that plasmatic Kyn/Trp ratio, which is a surrogate marker of IDO activity, is associated with a bad cardiovascular prognosis in patients suffering from coronary artery disease. Our hypothesis is that IDO is induced following MI and its local activity participates in cardiac remodeling and function. Our mouse model consists on left anterior descending coronary artery ligation in order to induce MI. Our results show that Kyn/Trp ratio is increased in the heart and plasma of wild-type mice (WT) starting from the first day following MI. IDO genetic invalidation as well as pharmacologic inhibition of its activity preserve cardiac function and reduce cardiac remodeling following MI. In order to study the role of inflammatory cells, WT or Ido-1 -/- chimeric mice irradiated and transplanted with bone-marrow derived cells were generated. Our results show that IDO expression in non-bone marrow derived cells has deleterious effects following MI. We found that CM express IDO and its activity increases following MI. Moreover, CM isolated from Ido-1 -/-mice had accelerated contraction and relaxation velocities compared to Ido-1 +/+ . In order to study the role of IDO in CM, we generated mice displaying IDO deficiency in these cells. However, this deletion has no effect neither on cardiac function nor on cardiac remodeling following MI. In order to study the role of vascular cells expressing IDO in the observed effects, Ido-1 flox/flox SM22-Cre et Ido-1 flox/flox VECad-CreERT2 + IDOdeficient respectively in smooth muscle cells (SMC) and endothelial cells (EC) were generated. We did not observe any significant effect in mouse model displaying IDO invalidation in SMC. Nevertheless, a significant amelioration of cardiac function and a beneficial remodeling were observed in EC IDO-deficient mice. A complete analysis of metabolites: Kyn and Kyn-derived metabolites by high performance liquid chromatography (HPLC) showed an increase only in Kyn but not in the other metabolites such as Kynurenic acid, quinolinic acid, hydroxylkynurenine, and anthranilic acid. Corroborating these results, supplementation with Kyn of IDO-deficient mice abolishes protective effect observed in the absence of IDO, showing that this metabolite is directly implicated in the phenotype. The study of the mechanisms implicated in the phenotype showed that Kyn produced by EC exerts a pro-apoptotic effect on CM through the AHR (Aryl Hydrocarbon receptor) as well as generation of reactive oxygen species. In summary, we demonstrated a new role of local IDO activity in the heart through the paracrine generation of Kyn by EC on CM, thus impacting cardiac remodeling and function.

This doctoral project was realized in team 5 headed by Pr. Hafid It could be achieved thanks to "Lebanese Association for Scientific Research (LASeR)" fellowship and "Medical Research Foundation (FRM)" end-of-thesis fellowship.

Humanity has been given a precious donation.

LIFE!

It is a big treasure.

In it, dreams have no limit.

My dream is to fly so high En revanche, une amélioration significative de la fonction cardiaque et un remodelage bénéfique ont été observés dans les souris déficientes en IDO dans les CE. L'analyse complète des métabolites : Kyn ainsi que des métabolites dérivés de la Kyn par chromatographie liquide de haute performance (HPLC) a montré une augmentation uniquement de la Kyn et pas des autres métabolites comme l'acide kynurénique, l'acide quinolinique, l'hydroxy-kynurénine et l'acide anthranilique. Corroborant ces résultats, la supplémentation des souris déficientes en IDO avec la Kyn abolit l'effet protecteur observé en absence d'IDO, montrant que ce métabolite est directement impliqué dans le phénotype. L'étude des mécanismes impliqués dans le phénotype a montré que la Kyn produite par les CE exerce un effet pro-apoptotique sur les CM via l'AHR (Aryl hydrocarbon receptor) et la génération de dérivés réactifs de l'oxygène. En résumé, nous avons mis en évidence un nouveau rôle de l'activité d'IDO localement dans le coeur à travers la génération paracrine de Kyn par les CE sur les CM impactant ainsi le remodelage et la fonction cardiaque.

Mots-clés: Indoléamine 2,3-dioxygenase 1, infarctus du myocarde, remodelage cardiaque, Kynurénine, AHR, apoptose, dérivés réactifs de l'oxygène YOU are my daily strength and support. YOU are making my wellbeing, happiness and hope.

YES HOPE for a successful future. And why the dream cannot be a reality?

THANK YOU ALL for your unique and valuable contributions.

I would like to express my gratitude to the jury members Dr. Ebba BRAKENHIELM, Dr.

Mathias MERICSKAY, Dr. Jean-Sébastien SILVESTRE and Dr. Jeremy FAUCONNIER for accepting to evaluate my work. I am really convinced that your expertise in the field will help me to gain new ideas and broaden my knowledge.

I would also thank Dr. Chantal BOULANGER, the head of the PARCC, for her prudence, care, benevolence and good management.

Thank you so much Dr. Alain TEDGUI, Pr. Ziad MALLAT and Pr. Hafid AIT-OUFELLA for accepting me as a member in your team which is for me my second family. Your wisdom, motivation and love for science inspired me a lot. It has been an honor to know you and learn from you. Thank you for ensuring all the conditions needed to realize my project. Your support and confidence were essential for my work, and progress.

Dr. Soraya TALEB, by occupying decision-making positions, women can do miracles. Their weapons are struggle and empowerment. You are one of them. I knew you as a courageous adventurer known for her unlimited love for research. A captain whom boat never sink off even when there is a thunderstorm. I really admire the strong and ambitious person in you. During these 4 years, you have been always by my side, seeing in you the intelligent leader and the cautious mom. Thank you for the endless help, support and confidence. Thanks for giving me the opportunity to supervise students, and participate to national and international congresses.

With you and with every challenge, I learnt that everything is possible and nothing does not exist. Thanks to your encouragement, I was able to overcome many obstacles which I encountered during this journey. Over these 4 years, you gave me a wonderful assistance and numerous ideas and advices. I have greatly appreciated discussions with you. By sharing your knowledge and wisdom you made my success a reality. Thank you for spending considerable time receiving this manuscript, reading it, making corrections and providing me with feedback, encouraging comments and helpful suggestions. Many thanks for all your efforts. Finally, I will miss every moment I passed working with you, the discussions and the calls. Now, I leave with the only hope that the end is a new beginning and maybe one day our paths will cross again.

A special thought for Dr. Stéphane POTTEAUX. I would like to thank her for her kindness, the valuable comments during the lab meetings and the discussions.

Many thanks to all my friends and colleagues from team 5:

Bruno ESPOSITO, for your help and the valuable advices.

Ludivine LAURANS, for your help and for sharing your computer. I really appreciated your kindness and the morning discussions.

Mouna CHAJADINE thank you for your help. Now it is your turn! Best of luck for your PhD journey.

Marie VANDESTIENNE (l'ancêtre), Yujiao ZHANG (ma petite Cici), and Icia SANTOS ZAS (It's nine to nine), I will never forget the wonderful moments we spent together in the BDE working, discussing, kidding, shouting, singing and dancing. Thanks for all the positive vibes.

Rida AL-RIFAI (Ridz), I am so grateful to know you as a colleague, friend and sister. I really appreciated discussing with you not only about work but also about politics in our beloved country, Lebanon. Thanks for your generosity and for being by my side, giving me advices, help and support.

Dima KHARRAT (Damdoum), the trail for success is long. Every day and with every experience we learn and ameliorate ourselves. I am so glad to know you as a friend and a sister too. You were always supportive and helpful. I really appreciated your kindness, generosity, sincerity and courage. Best of luck for your future plans.

With Nadine SUFFEE, I learnt that the other version of "Salut" or "Bonjour" is when will be your wedding? Nadine, I am a big fan of your funny jokes, smile and good humor.

Rhita LAMTAHRI, the TOP chef! I really admire your talent. Thank you for your delicious cakes.

Jean-Rémi LAVILLEGRAND, thank you for your kindness and best of luck for your PhD journey.

With Florent, I learnt that I have two names Nada and Jojo. For him the choice between them depends on the degree of my seriousness. Thank you so much Florent for adding some fun and best of luck for your next plan.

Thank you for the kindest Natalie FOURNIER. I am so glad to know you. Wish you the best in your career.

I would also thank all my trainees Marie, Ilana, Arthur and Karen for their help and contribution to the progress of my project. We learnt a lot together. This was, for me, a wonderful enriching experience. I cannot forget all the other students who did their internship in our team during these four years.

A special thought for Alice PELHAITRE and Mélanie MONTABORD. Thank you so much for the help and the wonderful time we spent together.

I am glad to know my colleagues who already left the team: Wared NOUR-ELDINE, Hajer HICHRI, Yacine HADDAD, Sarvenaz METGHALCHI, Linda ZEBOUDJ, Andreas GIRAUD, and Jeremy JOFFRE.

Be sure that all my accomplishments were possible thanks to your help and encouragement.

TOGETHER we made the difference. I wish you all a brilliant future. I hope that every one of you can realize his/her ambitions.

My warmest thanks to all the people who shared their ideas and experience with me: I would also thank all my friends and colleagues who are actually at PARCC or already left:

Stephan MAZLAN (Steph), we were the best "BDE" team. I will always remember your smile and funny jokes. Thank you so much for adding positive vibes. I am also glad to know

Michael and would thank him for the valuable discussions. Thank you both for the welcome, generosity, and the beautiful gatherings at your place.

Shruti CHATTERJEE, the "BDE" team is missing without you. Thank you for your kindness and best wishes for your future plans.

A special thank you for the best Camille KNOSP for the valuable help in FACS and a thought for Camille BRUNAUD.

I would also thank Aurélie Morin, Pierre-Michaël COLY, Cécile DEVUE, Marion TANGUI, and a special thank you for Johanne POISSON and Marouane KHELOUFI.

I am also so glad to know my friends and colleagues from the first, second and third floors at PARCC, Rami EL ZEIN, Alaa ABDELLATIF, Mengyao Yu, Lu LIU, Charline GUERY, Fatima ZEMALI, Adel HAMMOUTENE, Michael ROBILLARD, Fariza MEZINE, Anja Nitzsche, Hira Niazi, Ammar BENARAB, and Radia BRIOLLE.

To my friends Ala'a El-JAMAL, Toni EL-KHAWAND, Naila ABOU HAIDAR and Rima KAMEL, I will never forget the beautiful moments we spent together in Lebanon and France.

Agathe, Edith, Bertille and all the family VERMERSCHE, I am so happy and proud to know you and I am so grateful for your kindness, goodness, and endless care and welcome.

Charlotte VOLGERS, what a beautiful and special coincidence! I found a friend and a sister too from other horizons. Charlotte, I really admire your kindness, goodness, sincerity and courage. I am so happy to know you and will always remember the beautiful time we spent together and the long interesting discussions. Thanks for your care and for sharing my good and bad moments. Wish you a lot of happiness, success and best of luck in your new adventure.

Looking forward to see you again.

A special thought for Dr. Fadwa El-MERHABI. I am so proud to be your student. Thank you so much for the many advices since I was in Lebanon. I am also grateful for your encouragement, availability and the interesting discussions. Your motivation inspires me a lot.

You deserve all the best Dr. Fadwa. Best of luck in your career.

I offer all my love and sincere appreciation to my family in Lebanon, Australia and USA.

Special thanks to my uncle, Dr. Edmond MELHEM, and the great support from Melbourne and the many advices. I am proud of you uncle! A special thought for my uncle Raymond MELHEM who already left us.

My parents and sisters, Joane and Ange-Marie, let me please express my endless love to you. No thank you is enough to express how much I am grateful for everything you made for me. You are my strength, support and the reason for my success. Thank you so much for your love, generosity and confidence. Thanks for helping me, providing beneficial advices and encouraging me in every challenge. Thanks for being always by my side.

I hope that I deserve all what you are doing for me. Be sure that I will always live up to your expectations.

To my friend Chris, life teaches us valuable lessons. Every day and with every experience, we add to our knowledge new concepts. We learn that we should be strong, believe in ourselves whatever the circumstances are, and that we should never give up. We should always remember that life is a precious donation and that ambitions and dreams have no limits. Chris, thank you so much for your priceless care and support. Acute Myocardial Infarction: when the heart kingdom is the victim.

List of Figures

A. Epidemiology of cardiovascular diseases

The heart and the circulatory system, together, form the cardiovascular system. The heart has been at the center of cardiovascular research investigation because of its important role in physiology in addition to the fact that cardiac diseases are the major cause of death globally.

Since its capacity of highly complex roles, orchestrated function and interaction between its components, more precisely cells, arteries and veins blood vessels and valves, are substantial to maintain it healthy and their disruption leads to pathological conditions.

The heart has the role to pump the blood to the entire body in order to supply the different organs with oxygen and nutrients. It is situated within the chest cavity and surrounded by a fill sac called the pericardium. The heart wall is composed of three layers: The epicardium is the outer layer, the myocardium is the muscular middle layer and the endocardium is the inner layer. Heart is composed of 4 chambers: two upper chambers called atria and two lower chambers called ventricles. The right atrium (RA) receives the deoxygenated blood from the body. Blood passes then through the tricuspid valve to the right ventricle (RV) which propels it through pulmonic valve to the lungs for re-oxygenation. After that, oxygenated blood enters the left atrium (LA) then passes through the mitral valve known as bicuspid valve to the left ventricle (LV) before it is pumped to the aorta through the aortic valve then to the rest of the body [START_REF] Swirski | Cardioimmunology: the immune system in cardiac homeostasis and disease[END_REF]. The cardiac cycle consists of two phases: diastole and systole which occurs when the heart beats. During the diastole, the heart ventricles are relaxed and the heart fills with blood. During the systole, the ventricles contract and pump blood to the aorta which in turn delivers it to the other arteries. The coronary arteries are the first blood vessels which extend from the aorta to the heart in order to supply it with oxygenated blood.

There are two main coronary arteries: the right and the left coronary arteries (RCA, LCA). The RCA branches into the posterior descending artery and the right marginal artery. It irrigates the right atrium and ventricle, a part of the LV and the sinoatrial nodal artery. The posterior descending artery supplies the posterior third of the interventricular septum. The right marginal artery supplies blood to both surfaces of the RV. In turn, the main LCA, which is one or two centimeters long, is divided into two major trunks: the left anterior descending (LAD) coronary artery and the left circumflex coronary artery. The LAD artery supplies most of interventricular septum, a small margin of the free wall of the RV, and a large portion of the adjacent free wall of the LV including the apex cordis [START_REF] James | Coronary circulation in acute myocardial infarction[END_REF]. The left circumflex coronary artery supplies the LA, the LV and the sinoatrial nodal artery. Since blood supply is crucial for all the regions of the heart to ensure its needs in oxygen and nutrients, coronary artery disease (CAD), also known as coronary heart disease (CHD) or ischemic heart disease (IHD), involving reduction of blood flow to the heart muscle, could result in cardiac damage known as acute myocardial infarction (MI). Of note, these diseases are the most common cause of the cardiovascular diseases (CVDs) [START_REF] Ibanez | ESC Guidelines for the management of acute myocardial infarction in patients presenting with ST-segment elevation: The Task Force for the management of acute myocardial infarction in patients presenting with ST-segment elevation of the European Society of Cardiology (ESC)[END_REF].

Nowadays, CVDs constitute a global tsunami. According to the World Health Organization (WHO), CVDs are the primary cause of death globally accounting for ~1 of every 3 deaths in the United States (US) and 45% of all deaths in Europe, equating to > 4 million deaths per year [START_REF] Nitz | Amino Acids and Their Metabolism in Atherosclerosis[END_REF][START_REF] Timmis | European Society of Cardiology: Cardiovascular Disease Statistics[END_REF][START_REF] Virani | Heart Disease and Stroke Statistics-2020 Update: A Report From the American Heart Association[END_REF]. They are followed by cancer (No.

2), unintentional injuries (No. [START_REF] Timmis | European Society of Cardiology: Cardiovascular Disease Statistics[END_REF]. Here are some unraveled new cases; 34.9 million living with IHD, 20.4 million with stroke, 25.8 million of peripheral vascular disease (PVD) and 10.0 million of atrial fibrillation (AF).

The burden of CVDs is considered to be not only a health issue, but also an economic challenge to the healthcare systems that is expected to grow exponentially in the future years [START_REF] Anand | Stemming the global tsunami of cardiovascular disease[END_REF]. According to the World Heart Federation, the global cost of CVDs will increase from $863 billion in 2010 to $1044 billion in 2030. In the US, costs of CVDs were evaluated to $213.8 billion in 2014 to 2015. In a European Heart Network study, CVDs costs were estimated to 210 billion euros a year in 2015 [START_REF] Wilkins | European Cardiovascular Disease Statistics[END_REF]. In France, CVDs cost was estimated to 15.1 billion euros. Of note, premature CVDs death is more common in low and middle-income countries which are under-resourced in terms of cardiological person-power and technological infrastructure and are less able to meet the costs of healthcare compared to high-income countries. Thus, prevention is the best solution being independent from financial muscle. Based on INTERHEART study [START_REF] Yusuf | Effect of potentially modifiable risk factors associated with myocardial infarction in 52 countries (the INTERHEART study): case-control study[END_REF]) some risk factors and core health behaviors were identified as important risks predisposing to the development of CVDs such as atherosclerosis and acute MI, notably blood pressure (BP), cholesterol, overweight and obesity, smoking, alcohol, nutrition, physical inactivity, diabetes, age, kidney disease [START_REF] Virani | Heart Disease and Stroke Statistics-2020 Update: A Report From the American Heart Association[END_REF], and coronavirus disease-19.

B. Risk factors of cardiovascular diseases i. Blood pressure

BP is independently and continuously related to the incidence of cardiovascular events such as MI, heart failure (HF), stroke as well as end-stage renal disease [START_REF] Timmis | European Society of Cardiology: Cardiovascular Disease Statistics[END_REF][START_REF] Williams | ESC/ESH Guidelines for the management of arterial hypertension[END_REF] [START_REF] Timmis | European Society of Cardiology: Cardiovascular Disease Statistics[END_REF].

In 2004, the INTERHEART study estimated that hypertension was responsible for 22% of MI in Europe [START_REF] Yusuf | Effect of potentially modifiable risk factors associated with myocardial infarction in 52 countries (the INTERHEART study): case-control study[END_REF]. In 2015, hypertension resulted in approximately 10 million deaths among them 4.9 million deaths due to IHD, 2 million due to hemorrhagic stroke and 1.5 million due to ischemic stroke. According to the authors, 1.13 billion persons suffered from hypertension in 2015, among them more than 150 million were in central and Eastern Europe.

Notably, its prevalence in adults was about 30-45% with 24% men and 20% women, and was > 60% in people aged > 60 years [START_REF] Williams | ESC/ESH Guidelines for the management of arterial hypertension[END_REF]. Of note, the estimated number of hypertensive persons will importantly increase to reach 1.5 billion by 2025, thus increasing the risk of cardiovascular events incidence as well as mortality [START_REF] Williams | ESC/ESH Guidelines for the management of arterial hypertension[END_REF]. Therefore, more effort should be done in order to ameliorate high BP diagnosis and further treatment which is known to be beneficial.

ii. Cholesterol

Cholesterol levels variability, including low-density lipoprotein (LDL) cholesterol and highdensity lipoprotein (HDL) cholesterol, constitutes an important risk factor of major adverse cardiovascular events. A nationwide population-based study involving 3 656 648 Koreans without a history of CVD showed that cholesterol variability is an independent predictor for developing MI, stroke, and mortality [START_REF] Kim | Cholesterol variability and the risk of mortality, myocardial infarction, and stroke: a nationwide population-based study[END_REF]. CVD risk increases linearly when blood concentrations increase [START_REF] Ference | Effect of long-term exposure to lower low-density lipoprotein cholesterol beginning early in life on the risk of coronary heart disease: a Mendelian randomization analysis[END_REF]. Hypercholesterolemia occurs when cholesterol level exceeds 6.2 mmol/L [START_REF] Timmis | European Society of Cardiology: Cardiovascular Disease Statistics[END_REF]. According to 2019 ESC statistics, the prevalence of hypercholesterolemia was 18.8% in females and 18.1% in males from highincome countries compared to 11.8% and 8.7%, respectively in middle-income countries [START_REF] Timmis | European Society of Cardiology: Cardiovascular Disease Statistics[END_REF].

Lipid accumulation in the arterial wall plays an important role in the development of atherosclerosis which is the first underlying pathology contributing to MI. Of note, given the relationship between atherosclerosis and circulating LDL, statin therapy aiming to lower LDL cholesterol prevents atherosclerosis progression and cardiac events incidence [START_REF] Palasubramaniam | Myocardial Infarction-From Atherosclerosis to Thrombosis[END_REF]. Another LDL-lowering therapy consists on the use of proprotein convertase subtilisin-kexin type 9 (PCSK9) inhibitors which have the role to stop LDL receptors destruction mediated by the hepatic protease PCSK9. Notably, PCSK9

inhibitors, evolocumab and alirocumab, used in clinical trials resulted in 40% to 60% reductions in LDL levels thus decreasing the risk of major adverse cardiovascular events (MACE) [START_REF] Ms | Evolocumab and Clinical Outcomes in Patients with Cardiovascular Disease[END_REF][START_REF] Szarek | Alirocumab Reduces Total Nonfatal Cardiovascular and Fatal Events: The ODYSSEY OUTCOMES Trial[END_REF]. Moreover, a long-acting RNA interference agent having the role of inhibiting the synthesis of PCSK9 was responsible to reduce LDL cholesterol levels in phase I trial [START_REF] Fitzgerald | A Highly Durable RNAi Therapeutic Inhibitor of PCSK9[END_REF]. In addition, clustered regularly interspaced short palindromic repeat-associated 9 (CRISPR-Cas9) gene editing of the Pcsk9 gene in liver tissue of adult mice was responsible for plasma PCSK9 reduction by more than 50% accompanied with 30% reduction of plasma cholesterol levels by 30% [START_REF] Chadwick | In Vivo Base Editing of PCSK9 (Proprotein Convertase Subtilisin/Kexin Type 9) as a Therapeutic Alternative to Genome Editing[END_REF].

Although previous recommendations consisted in reducing dietary cholesterol, no contemporary guidance has been issued about it. Probably, because there is no appreciable relationship between consumption of dietary cholesterol and serum cholesterol, nor a significant positive association between dietary cholesterol and CVD risk [START_REF] Carson | Thrombosis and Vascular Biology; Council on Cardiovascular and Stroke Nursing; Council on Clinical Cardiology; Council on Peripheral Vascular Disease; and Stroke Council[END_REF].

iii. Overweight and Obesity

While a healthy weight is defined as a body mass index (BMI: weight kg/height m 2 ratio) between 20 Kg/m 2 and < 25 Kg/m 2 [START_REF] Ibanez | ESC Guidelines for the management of acute myocardial infarction in patients presenting with ST-segment elevation: The Task Force for the management of acute myocardial infarction in patients presenting with ST-segment elevation of the European Society of Cardiology (ESC)[END_REF], overweight and obesity are defined by BMI of ≥ 25 Kg/m 2 and ≥ 30 Kg/m 2 , respectively [START_REF] Timmis | European Society of Cardiology: Cardiovascular Disease Statistics[END_REF].

Overweight and obesity are associated with earlier development of CVD and greater mortality.

Their prevalence is increasing in both developed and developing countries. Based on what has been reported in the Heart Disease and Stroke Statistics-2020, the prevalence of obesity among youth in the US was 18.5% and 38.3% among adults [START_REF] Virani | Heart Disease and Stroke Statistics-2020 Update: A Report From the American Heart Association[END_REF]. In turn, ESC:

Cardiovascular Disease Statistics 2019 showed that one in five adult females and males were obese (≥ 30Kg/m²). Prevalence of obesity in females and males among ESC member countries was 21.4% and 23.7%, respectively in high income countries compared to 24.1% and 21% in middle-income countries. It remarkably increased from 9.6% to 22.6% between 19809.6% to 22.6% between and 20169.6% to 22.6% between (Timmis et al., 2020)). This could be due to global trade liberalization, economic growth, and rapid urbanization fueling dramatic changes in lifestyle and food intake with a greater tendency to consume animal fat and added sugar [START_REF] Malik | Global obesity: trends, risk factors and policy implications[END_REF].

iv. Smoking

In 2017, smoking caused 8.1 million deaths worldwide [START_REF] Virani | Heart Disease and Stroke Statistics-2020 Update: A Report From the American Heart Association[END_REF]. According to Timmis and colleagues, it was responsible for many forms of cancer and CVDs [START_REF] Timmis | European Society of Cardiology: Cardiovascular Disease Statistics[END_REF]. Smoking prevalence in the ESC member countries was higher in males (26.5%) than in females (15%), and more males and less females smoked in middle income-countries compared to high-income countries [START_REF] Timmis | European Society of Cardiology: Cardiovascular Disease Statistics[END_REF]. Based on the Heart Disease and Stroke

Statistics-2020, the use of electronic cigarettes among adolescents in the US has been sharply increased between 2011 (1.5%) and 2018 (20.8%) [START_REF] Virani | Heart Disease and Stroke Statistics-2020 Update: A Report From the American Heart Association[END_REF]. A recent study conducted by Alzahrani and colleagues showed that daily electronic cigarette use is associated with increased risk of MI [START_REF] Alzahrani | Association Between Electronic Cigarette Use and Myocardial Infarction[END_REF]. This could be explained by increased oxidative stress, endothelial dysfunction, inflammation, oxidation of LDL cholesterol, platelet activation, and thrombosis [START_REF] Ambrose | The pathophysiology of cigarette smoking and cardiovascular disease: an update[END_REF].

v. Alcohol

Alcohol consumption is defined as the recorded amount (in liters) of pure alcohol consumed per adult (≥ 15 years) over a calendar year. Whether alcohol is beneficial or not depends on the amount consumed and drinking frequency which predict the cardiovascular risk. In their study, Mostofsky and colleagues found that both moderate alcohol consumption (2-4 drinks) and heavy one (6-9 drinks) are associated with a higher cardiovascular risk which was only attenuated for moderate intake after 24 hours and was even protective for MI and ischemic stroke within one week (6 drinks) [START_REF] Mostofsky | Alcohol and Immediate Risk of Cardiovascular Events: A Systematic Review and Dose-Response Meta-Analysis[END_REF].

In the US, 1 in 10 adults at working-age dies because of excessive alcohol consumption [START_REF] Stahre | Contribution of excessive alcohol consumption to deaths and years of potential life lost in the United States[END_REF]. In the European Union (EU), harmful alcohol drinking is the third cause of premature death after tobacco and hypertension. Based on ESC Cardiovascular Disease Statistics 2019, the prevalence of alcohol consumption was higher in high-income countries (72.7%) than in middle-income countries (37.5%). In France, for example, the prevalence of alcohol consumption per calendar year by persons aged ≥15 years was estimated to 12.6%, in

2016. The highest one was in Republic of Moldova (15.2%) and the lowest one was in Syrian Arab republic (0.3%) [START_REF] Timmis | European Society of Cardiology: Cardiovascular Disease Statistics[END_REF].

vi. Nutrition

According to the Global Burden of Disease 2017 Study, around 11 million people die because of dietary risks the most important are high sodium (Na + ) intake and low whole grain and fruit intakes. Of note, low fruit and vegetable intakes are associated with increased risk of cancer and CHD [START_REF] Aune | Fruit and vegetable intake and the risk of cardiovascular disease, total cancer and all-cause mortality-a systematic review and doseresponse meta-analysis of prospective studies[END_REF]. Therefore, promoting cardiovascular health implies a good diet quality which should be based on fruits, vegetables, whole grains, low-fat or fat-free dairy products, lean protein sources, nuts, seeds, and liquid vegetable oils [START_REF] Carson | Thrombosis and Vascular Biology; Council on Cardiovascular and Stroke Nursing; Council on Clinical Cardiology; Council on Peripheral Vascular Disease; and Stroke Council[END_REF].

vii. Physical inactivity

Physical activity can be calculated by dividing the daily total energy expenditure of an individual by the daily basal metabolic rate (kJ/min). Three cases exist: a physical inactivity when the physical activity ratio (PAR) is between 1.40 and 1.69, a moderate activity when PAR is between 1.70 and 1.99, and vigorous activity when PAR ≥ 2.00 [START_REF] Fiuza-Luces | Exercise benefits in cardiovascular disease: beyond attenuation of traditional risk factors[END_REF]. More simply, the minimum of physical activity recommended for an adult is about 150 min per week. Less than 150 min of moderate-intensity physical activity per week or 75 min of vigorous-intensity physical activity is considered insufficient and responsible for increasing the risk of many diseases including IHD. Based on ESC: Cardiovascular Disease Statistics 2019, 31% of the adults aged ≥ 18 years across the ESC member countries had an insufficient physical activity with a higher tendency in females than in males and a higher prevalence in high-income countries compared to middle income-countries [START_REF] Timmis | European Society of Cardiology: Cardiovascular Disease Statistics[END_REF].

As the prevalence of physical inactivity is increasing across the world, thus augmenting the risk of CVDs, it is necessary to remember some of the beneficial effects exerted by a regular

viii. Diabetes Mellitus

ix. Age

Despite the fact that ageing is not a disease in itself, it may be another risk factor for heart disease. Ageing is accompanied with an increased interstitial fibrosis, hypertrophy and stiffness associated to a lower capacity of the heart to pump blood. In addition, as we age immune system undergo remodeling. This process is known as "inflamm-ageing" during which inflammation increases, tolerance decreases, cytokines levels are deregulated, and mitochondria function is impaired. Hence, age accompanied with changes in immune system results in a higher risk of CVDs [START_REF] Swirski | Cardioimmunology: the immune system in cardiac homeostasis and disease[END_REF].

x. Kidney disease

Chronic kidney disease constitutes a risk factor for incident and recurrent coronary events, stroke, HF, venous thromboembolism, and AF. It is estimated by glomerular filtration rate and albuminuria categories. Chronic kidney disease occurs when glomerular filtration rate < 60 mL.min -1 .1.73 m -2 or albumin/creatinine ratio ≥ 30 mg/g. Its prevalence in 2013 to 2016 was 14.8% [START_REF] Virani | Heart Disease and Stroke Statistics-2020 Update: A Report From the American Heart Association[END_REF].

xi. Coronavirus disease 2019

The recent coronavirus disease 2019 (COVD-19) is rapidly spreading around the world. The most recent statistics describe 650 805 deaths and 16 341 920 confirmed cases with Americas at the top of the list, followed by Europe, and South-East Asia, then Eastern Mediterranean, and Africa, and the last one was Western Pacific. COVID-19 is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). While 80% of infected patients present mild symptoms with good prognosis, about 20% display respiratory distress and need urgent intervention consisting of immediate oxygen therapy and mechanical ventilation. Some clinical manifestations concern a respiratory rate of ≥ 30 times/min, mean oxygen saturation ≤ 93% in the resting state, or arterial blood oxygen partial pressure/oxygen concentration ≤ 300 mmHg [START_REF] Shen | Proteomic and Metabolomic Characterization of COVID-19 Patient Sera[END_REF].

In the clinical study based on data from the Kaiser Permanente Northern California health system, Solomon and colleagues found that the weekly rates of hospitalization for acute MI decreased by up to 48% during the COVID-19 period and compared to same weekly periods in 2019 (Figure 1) [START_REF] Solomon | The Covid-19 Pandemic and the Incidence of Acute Myocardial Infarction[END_REF]. De Filippo and colleagues found similar results in northern Italy where a significant decrease in acute coronary syndrome (ACS)-related hospitalization rates were noticed during the early days of COVID-19 outbreak compared to an earlier period from 2020 and the same period in 2019 [START_REF] De Filippo | Reduced Rate of Hospital Admissions for ACS during Covid-19 Outbreak in Northern Italy[END_REF]. At the same time, a significant increase in mortality was observed during this period which was not only explained by COVID-19 cases but could be also due to ACS with the absence of medical attention for these cases during COVID-19 pandemic. pneumonitis promotes both local inflammation in the lungs and systemic one leading to myocardial ischemia even due to pre-existing plaque rupture or an impaired balance between myocardial oxygen supply and demand. In the first case, inflammation is accentuated and is characterized by cytokines production such as interleukin-1 (IL-1) and -6 (IL-6) and tumor necrosis factor (TNF). These cytokines stimulate macrophages within the plaque and contribute to tissue factor expression participating to thrombogenesis as well as reduced fibrinolysis. They also stimulate leukocyte adhesion molecule expression on the endothelial cells (ECs) enhancing recruitment of inflammatory cells (ICs) thus promoting pre-existing plaque rupture or leading to coronary vessels dysfunction in the absence of pre-existing vessel disease. In the second case, myocardial ischemia, due to COVID-19 infection, is explained by increased oxygen demands of the myocardium due to fever and tachycardia, and decreased oxygen supply because of hypoxemia associated to pneumonitis and hypotension in sepsis resulting in impaired coronary perfusion. Given this information, it is important to note that the increase in cTn biomarker does not always reflect the presence of CAD that needs an invasive intervention.

Instead, more attention should be given to anti-inflammatory therapies including glucocorticoids and cytokine antagonists as well as antiviral agents [START_REF] Libby | The Heart in COVID19: Primary Target or Secondary Bystander?[END_REF].

C. Consequences of risk factors on cardiovascular diseases

These risk factors are known to accelerate CVDs such as atherosclerosis and acute MI incidence.

i. Atherosclerosis: an underlying pathology of myocardial infarction

Atherosclerosis is the underlying pathology of the majority of CVDs [START_REF] Nitz | Amino Acids and Their Metabolism in Atherosclerosis[END_REF][START_REF] Theodorou | Endothelial Cell Metabolism in Atherosclerosis[END_REF]. Its development and progression are due to increased blood lipids levels, namely hypercholesterolemia. Hence, it is known as lipoprotein driven disease.

Atherosclerosis is also considered as a chronic inflammatory condition of the medium-and large-sized arteries including the coronary arteries [START_REF] Nitz | Amino Acids and Their Metabolism in Atherosclerosis[END_REF][START_REF] Palasubramaniam | Myocardial Infarction-From Atherosclerosis to Thrombosis[END_REF]. Many changes participate to the development of atherosclerosis including EC activation and dysfunction. This latter is explained by phenotype modification of EC becoming proinflammatory and pro-thrombotic with a reduced barrier function leading to a disturbed blood flow dynamics at lesion-areas of the arterial vasculature. This is also accompanied with retention and modification of LDL at the sub-endothelial level. These occurring events initiate an inflammatory response characterized by monocytes recruitment to the intima and differentiation into macrophages which have the role of engulfing modified lipoproteins before they switch into foam cells.

Over years, atherosclerosis progression is marked by plaque formation in the intimal space of the coronary artery in addition to lesions with a fibrous cap covering a lipid rich necrotic core as well as leukocytes accumulation in the lateral edges leading to plaque instability which is associated with a high probability of rupture. Either plaque rupture or coronary artery endothelium erosion caused by EC apoptosis may result in an atherothrombotic event. Plaque disruption consists in the release of its thrombogenic contents, platelet activation, and initiation of the coagulation cascade, mural thrombus formation, and embolization of atherosclerotic debris downstream. Therefore, obstruction of the artery that cannot anymore supply blood, more precisely oxygen to the heart leads to ischemic injury or acute MI [START_REF] Ibanez | ESC Guidelines for the management of acute myocardial infarction in patients presenting with ST-segment elevation: The Task Force for the management of acute myocardial infarction in patients presenting with ST-segment elevation of the European Society of Cardiology (ESC)[END_REF][START_REF] Swirski | Cardioimmunology: the immune system in cardiac homeostasis and disease[END_REF].

ii. Acute myocardial infarction: an example of cardiovascular diseases

a. Definition and classification

Acute MI, also known as heart attack, is causally related to CAD [START_REF] James | Coronary circulation in acute myocardial infarction[END_REF]. It is the first and most severe manifestation of this disease. MI is defined as an event of myocardial death due to prolonged ischemia. This is explained by ultra-structural changes including mitochondrial abnormalities, diminished cellular glycogen, relaxed myofibrils and sarcolemmal disruption which occur as early as 10 to 15 minutes following the onset of ischemia. Moreover, necrosis starts in the subendocardium and then it evolves towards the subepicardium over several hours [START_REF] Thygesen | and Executive Group on behalf of the Joint European Society of Cardiology (ESC)/American College of Cardiology (ACC)/American Heart Association (AHA)/World Heart Federation (WHF) Task Force for the Universal Definition of Myocardial Infarction (2018). Fourth Universal Definition of Myocardial Infarction[END_REF]. Of note, acute myocardial injury is defined as MI with typical signs or symptoms of myocardial ischemia. In turn, chronic myocardial injury is characterized by the absence of MI symptoms or electrocardiogram (ECG) changes indicating myocardial ischemia [START_REF] Kadesjö | Acute versus chronic myocardial injury and long-term outcomes[END_REF][START_REF] Thygesen | and Executive Group on behalf of the Joint European Society of Cardiology (ESC)/American College of Cardiology (ACC)/American Heart Association (AHA)/World Heart Federation (WHF) Task Force for the Universal Definition of Myocardial Infarction (2018). Fourth Universal Definition of Myocardial Infarction[END_REF]. MI is usually caused by plaque rupture and thrombus forming within the lumen of one or more coronary arteries resulting in blood blockage, thus preventing the perfusion of a part of the heart [START_REF] Anderson | Acute Myocardial Infarction[END_REF]. However, it can be the result of non-obstructive coronary arteries, in this case it represents 1 to 14% of total MIs [START_REF] Ibanez | ESC Guidelines for the management of acute myocardial infarction in patients presenting with ST-segment elevation: The Task Force for the management of acute myocardial infarction in patients presenting with ST-segment elevation of the European Society of Cardiology (ESC)[END_REF].

MI patients are divided into two major categories: patients who develop an ST-segment elevation MI (STEMI), and those without ST-segment elevation or non-ST-elevation MI (NSTEMI). Moreover, unstable angina, which is an imminent precursor to MI, has a similar pathophysiology to NSTEMI. In addition to these categories, MI may be classified into various types. For instance, type 1 MI includes coronary artery disorders like atherosclerotic plaque rupture, ulceration, fissuring or erosion with resulting intraluminal thrombus in one or more of the coronary arteries. MI can also be with non-obstructive coronary artery. For instance, type 2 MI implies imbalance between oxygen supply and demand such as coronary artery spasm and coronary embolism as well as coronary microvascular dysfunction in addition to myocardial disorders which are not the result of acute atherothrombotic plaque disruption (Figure 2) [START_REF] Thygesen | and Executive Group on behalf of the Joint European Society of Cardiology (ESC)/American College of Cardiology (ACC)/American Heart Association (AHA)/World Heart Federation (WHF) Task Force for the Universal Definition of Myocardial Infarction (2018). Fourth Universal Definition of Myocardial Infarction[END_REF]. Of note, MI most commonly occurs in the LV usually after left anterior descending (LAD) coronary artery occlusion. But since 1974, it has also been known to exist in the RV due to the occlusion of the right ventricular branch or acute marginal branch of the RCA or left circumflex artery in the left dominant coronary circulation. However, its occurrence in the RV is seldom probably because RV resists to infarction. It has also been reported that even after prolonged occlusion RV is able to recover, and its performance improves spontaneously even in the absence of reperfusion [START_REF] Namana | Right ventricular infarction[END_REF]. In both cases, MI is responsible for high morbidity and mortality worldwide [START_REF] Thygesen | Joint ESC/ACCF/AHA/WHF Task Force for Universal Definition of Myocardial Infarction[END_REF].

In this chapter, I will focus on LV MI.

b. Epidemiology

Acute MI has become the leading contributor to the burden of disease as assessed on the basis of disability-adjusted life-years. Each year, MI affects more than 7 million individuals and causes more than 2.4 million deaths in the US, and more than 4 million deaths in Europe and northern Asia [START_REF] Reed | Acute myocardial infarction[END_REF]. Approximately 550 000 first episodes and 200 000 recurrent episodes of acute MI occur annually in the US [START_REF] Anderson | Acute Myocardial Infarction[END_REF] Of note, MI occurs more frequently in men than in women below the age of 60 years [START_REF] Reed | Acute myocardial infarction[END_REF]. Hence, CVDs including MI are often considered as male diseases. However, it is important to note that MI remains the number one cause of death in women worldwide. For instance, in the US, statistics show that since 1984, annual mortality was more elevated in women than in men (The [START_REF] Lancet | Acute myocardial infarction in women[END_REF]. According to 2017 ESC Guidelines, women, after the age of 75, represent the majority of patients suffering from ACS. Moreover, older women suffering from MI have a poorer outcome related to more comorbidities [START_REF] Reed | Acute myocardial infarction[END_REF].

Nowadays, lifestyle changes and advances in pharmacological and preventive therapies, as well as reperfusion strategy result in a significant decrease in mortality. However, direct costs due to high rate of hospitalizations are huge suggesting an urgent need for a better way to diagnose, stratify risk, and manage patients with suspected CVD in order to prevent and avoid worse outcome [START_REF] Reed | Acute myocardial infarction[END_REF][START_REF] Wang | The Biomarkers for Acute Myocardial Infarction and Heart Failure[END_REF].

c. Diagnosis

Acute MI is diagnosed based on many clinical features including symptoms, ECG abnormalities, elevated values of biomarkers of myocardial necrosis, and imaging such as echocardiography.

Symptoms include persistent chest pain, radiation of pain to the neck, lower jaw, or left arm.

Some other less-typical symptoms also exist like dyspnea, nausea/vomiting, fatigue, palpitations, diaphoresis, syncope or cardiac arrest. It is important to note that MI can also occur without symptoms, for example in women, the elderly, diabetics, or post-operative and critically ill patients [START_REF] Ibanez | ESC Guidelines for the management of acute myocardial infarction in patients presenting with ST-segment elevation: The Task Force for the management of acute myocardial infarction in patients presenting with ST-segment elevation of the European Society of Cardiology (ESC)[END_REF][START_REF] Thygesen | Joint ESC/ACCF/AHA/WHF Task Force for Universal Definition of Myocardial Infarction[END_REF][START_REF] Thygesen | and Executive Group on behalf of the Joint European Society of Cardiology (ESC)/American College of Cardiology (ACC)/American Heart Association (AHA)/World Heart Federation (WHF) Task Force for the Universal Definition of Myocardial Infarction (2018). Fourth Universal Definition of Myocardial Infarction[END_REF]. In order to relieve pain responsible for sympathetic activation, vasoconstriction and increased workload of the heart, intravenous opioids like morphine are used. Moreover, anxiety is treated with tranquillizer, as benzodiazepine. In hypoxic patients, oxygen is administered when arterial oxygen saturation (SaO2) is less than 90% [START_REF] Ibanez | ESC Guidelines for the management of acute myocardial infarction in patients presenting with ST-segment elevation: The Task Force for the management of acute myocardial infarction in patients presenting with ST-segment elevation of the European Society of Cardiology (ESC)[END_REF].

Patients presenting these symptoms develop STEMI and are subject to ECG monitoring (Figure 3) [START_REF] Thygesen | and Executive Group on behalf of the Joint European Society of Cardiology (ESC)/American College of Cardiology (ACC)/American Heart Association (AHA)/World Heart Federation (WHF) Task Force for the Universal Definition of Myocardial Infarction (2018). Fourth Universal Definition of Myocardial Infarction[END_REF]. Suspicion of myocardial ischemia needs an urgent reperfusion therapy. However, some patients like those with acute occlusion of circumflex coronary artery may not present a ST-segment elevation and do not undergo reperfusion therapy, this leads to a larger infarction and worse outcomes.

In addition, blood sampling for serum markers is routinely carried out in the acute phase. This is what happens for example in patients with main LCA obstruction. cTn isoforms I and T, which are components of the contractile apparatus of myocardial cells exclusively expressed in the heart, are highly sensitive and specific biomarkers for myocardial injury leading to myocytes necrosis. They are detected in the blood within 2-3 hours following the injury and peak within 24-28 hours. Detection of a rise and/or fall of the measurements is essential for the diagnosis of acute MI. Of note, elevated cTn levels can also help diagnose other non-ischemic diseases associated with myocardial injury, like myocarditis, aortic dissection, pulmonary embolism, HF or renal failure. Creatine kinase myocardial band (CK-MB) is another marker for myocardial injury but it is less sensitive than cTn. It is used when cTn assay is not available [START_REF] Ibanez | ESC Guidelines for the management of acute myocardial infarction in patients presenting with ST-segment elevation: The Task Force for the management of acute myocardial infarction in patients presenting with ST-segment elevation of the European Society of Cardiology (ESC)[END_REF][START_REF] Thygesen | Joint ESC/ACCF/AHA/WHF Task Force for Universal Definition of Myocardial Infarction[END_REF][START_REF] Thygesen | and Executive Group on behalf of the Joint European Society of Cardiology (ESC)/American College of Cardiology (ACC)/American Heart Association (AHA)/World Heart Federation (WHF) Task Force for the Universal Definition of Myocardial Infarction (2018). Fourth Universal Definition of Myocardial Infarction[END_REF].

Finally, it is important to note that many patients die very early after STEMI onset usually before arrival to the hospital because of VF which is an abnormal heart rhythm in which the ventricles do not pump normally.

d. Pathophysiology

Acute MI is defined as a myocardial injury with necrosis as a result of decreased perfusion and lack of oxygen and nutrients in a clinical setting consistent with myocardial ischemia [START_REF] Ibanez | ESC Guidelines for the management of acute myocardial infarction in patients presenting with ST-segment elevation: The Task Force for the management of acute myocardial infarction in patients presenting with ST-segment elevation of the European Society of Cardiology (ESC)[END_REF]. Cell death occurs around 20 minutes after the onset of ischemia and takes several hours. Complete myocardial cells necrosis takes at least 2-4 hours [START_REF] Thygesen | Joint ESC/ACCF/AHA/WHF Task Force for Universal Definition of Myocardial Infarction[END_REF]. This is accompanied with subendocardial disruption of collagen fibers [START_REF] Jugdutt | Ventricular remodeling after infarction and the extracellular collagen matrix: when is enough enough?[END_REF]. Of note, partial or complete vessel occlusion as well as its duration, the amount of myocardium supplied, the sensitivity of the myocytes to ischemia, presence of collaterals, the adequacy of reperfusion following treatment, and individual demand for oxygen and nutrients are different factors indicating the severity of ischemia [START_REF] Reed | Acute myocardial infarction[END_REF][START_REF] Wang | The Biomarkers for Acute Myocardial Infarction and Heart Failure[END_REF].

After the onset of ischemia, a process of healing consisting on inflammation and fibrosis takes place in order to preserve structure, shape and function of the LV. The main goal is to actively clean the damaged site from dead cells and repair it by forming a firm scar in the ischemic zone. This process takes several weeks (at least 5-6 weeks) to months depending on the infarct size [START_REF] Jugdutt | Ventricular remodeling after infarction and the extracellular collagen matrix: when is enough enough?[END_REF][START_REF] Thygesen | Joint ESC/ACCF/AHA/WHF Task Force for Universal Definition of Myocardial Infarction[END_REF]. However, an impaired healing could be deleterious and could lead to many complications. It results in structural, functional and geometric alterations involving both the infarcted and non-infarcted myocardial segments of the LV all known as LV remodeling. Adverse LV remodeling involves infarct expansion, thinning, LV dilatation, aneurysm formation, LV eccentric hypertrophy, lengthening of non-infarctedrelated segments, increase sphericity of the ventricle and cardiac dysfunction (Figure 4) [START_REF] Curley | Molecular imaging of cardiac remodelling after myocardial infarction[END_REF]. It is dependent on the infarct size and quality of cardiac repair. LV remodeling may be accompanied with an increased incidence of arrhythmias and poor prognosis in patients surviving a MI. It may also lead to the ventricular rupture of the free wall or septum, or the progression of HF known as the incapacity of the heart to deliver sufficient oxygen to ensure the demands of peripheral tissues ending with death [START_REF] Frangogiannis | The inflammatory response in myocardial injury, repair, and remodelling[END_REF][START_REF] Jugdutt | Ventricular remodeling after infarction and the extracellular collagen matrix: when is enough enough?[END_REF].

e. Myocardial infarction complications

Based on 2017 ESC Guidelines [START_REF] Ibanez | ESC Guidelines for the management of acute myocardial infarction in patients presenting with ST-segment elevation: The Task Force for the management of acute myocardial infarction in patients presenting with ST-segment elevation of the European Society of Cardiology (ESC)[END_REF], acute MI complications include:

1. Myocardial dysfunction

Left ventricular dysfunction

Morphologic changes affecting the LV including loss of cardiomyocytes (CMs) in addition to inflammatory changes are responsible for LV functional impairment participating to myocardial dysfunction. These changes increase the systolic work load in the remote myocardium thus stimulating hypertrophy of the non-infarcted-related myocardium [START_REF] Bogaert | Remote myocardial dysfunction after acute anterior myocardial infarction: impact of left ventricular shape on regional function: a magnetic resonance myocardial tagging study[END_REF]. Notably, LV systolic dysfunction is the most frequent consequence of MI. This latter may be clinically silent or contribute to progressive ventricular failure thus predicting mortality. According to Bogaert and colleagues, patients suffering from MI had a severe decrease in global endocardial LVEF (46.7% vs 65.5% control subjects), and endocardial regional LVEF in the infarct-related myocardium (25.6% vs 65.1% control subjects) [START_REF] Bogaert | Remote myocardial dysfunction after acute anterior myocardial infarction: impact of left ventricular shape on regional function: a magnetic resonance myocardial tagging study[END_REF]. Moreover, LV dysfunction could also be associated to LV aneurysm occurring in patients with a large transmural MI undergoing adverse remodeling, as well as LV thrombus formation happening in patients with anterior MI. In this case, oral anticoagulant therapy is administered for up to 6 months. Another complication concerns secondary mitral valve regurgitation due to LV remodeling with lateral and apical displacement of the papillary muscles, leaflet tethering, and annular dilatation. It is treated with diuretics and arterial vasodilators, or even with mitral valve replacement.

Right ventricular involvement

It most frequently occurs in the form of an acute infero-posterior MI. It is rare, and when it happens, it is often silent. In addition to the typical manifestations listed above, some more specific signs and symptoms for right ventricular MI (RVMI) include hypotension, elevated jugular venous pressure and clear lungs [START_REF] Ibanez | ESC Guidelines for the management of acute myocardial infarction in patients presenting with ST-segment elevation: The Task Force for the management of acute myocardial infarction in patients presenting with ST-segment elevation of the European Society of Cardiology (ESC)[END_REF][START_REF] Namana | Right ventricular infarction[END_REF]. According

to Namana and colleagues, not all RCA occlusions result in RVMI, most probably because RV has a smaller muscle mass compared to LV, a coronary perfusion occurring both in systole and diastole, lesser oxygen demand, in addition to the presence of more extensive collateral vessels from left to right. When it exists, the RV undergoes some morphologic and functional changes.

For example, it becomes stiff, dilated, dyskinetic, with reduced compliance, filling, and stroke volume. These changes in the RV affect the function of the LV explained by diastolic filling abnormalities, changes in compliance, a significant impairment of contractile function, ending by a reduced cardiac output [START_REF] Namana | Right ventricular infarction[END_REF]. Treatment of RVMI consists on early reperfusion, dual antiplatelet therapy including aspirin and thienopyridine agents in addition to oxygen if the patient is hypoxic, and correction of AF and/or atrioventricular block with sequential pacing if needed. Of note, therapies which reduce pre-load such as nitrates, opioids and diuretics and medications with negative inotropic and chronotropic effect should be avoided. Instead, dobutamine which is an inotropic agent should be administered in order to enhance RV performance. In turn, dopamine is used to restore an adequate coronary perfusion pressure. Moreover, intra-aortic balloon pumps are used in patients suffering from persisting hypotension in order to improve RV function by enhancing coronary perfusion [START_REF] Namana | Right ventricular infarction[END_REF]. In some situations, complications of RV dysfunction are unavoidable such as arrhythmias, atrioventricular block, bradychardia hypotension, ventricular tachyarrhythmias, and ventricular tachycardia/fibrillation, in addition to mechanical complications such as tricuspid regurgitation, ventricular septal rupture, as well pulmonary edema, elevating pulmonary pressures and resistance.

Heart failure

HF, which is the most frequent complication of MI, is defined as the incapacity of the heart to pump blood and deliver sufficient oxygen to ensure the demands of peripheral tissues ending sometimes with death. HF occurs in 1-2% of the adult population in developed countries, and ≥ 10% of people aged 70 years old [START_REF] Ponikowski | ESC Guidelines for the diagnosis and treatment of acute and chronic heart failure: The Task Force for the diagnosis and treatment of acute and chronic heart failure of the European Society of Cardiology (ESC)Developed with the special contribution of the Heart Failure Association (HFA) of the ESC[END_REF] [START_REF] Metra | Heart failure[END_REF]. Further classification, based on bedside physical examination, helps differentiate between four groups warm and wet (well perfused and congested), cold and wet (hypo perfused and congested), cold and dry (hypo perfused without congestion), and warm and dry (compensated, well perfused without congestion). Another classification concerning patients with HF complicating MI divides them into four classes according to Killip and Kimball [START_REF] Mello | Validation of the Killip-Kimball classification and late mortality after acute myocardial infarction[END_REF]: class I without any clinical sign of HF, class II: HF with rales and S3 gallop, class III: with frank acute pulmonary edema, and class IV: with cardiogenic shock, hypotension, and evidence of peripheral vasoconstriction such as oliguria, cyanosis and diaphoresis [START_REF] Ponikowski | ESC Guidelines for the diagnosis and treatment of acute and chronic heart failure: The Task Force for the diagnosis and treatment of acute and chronic heart failure of the European Society of Cardiology (ESC)Developed with the special contribution of the Heart Failure Association (HFA) of the ESC[END_REF].

The most recommended treatments for HF consists of ACE, beta-blockers, mineralocorticoid/aldosterone receptor antagonists, diuretics, angiotensin receptor neprilysin inhibitor, If-channel inhibitor as an inhibitor of heart rate, angiotensin II type I receptor blockers, and combination of hydralazine and isosorbide dinitrate acting as vasodilators [START_REF] Metra | Heart failure[END_REF][START_REF] Ponikowski | ESC Guidelines for the diagnosis and treatment of acute and chronic heart failure: The Task Force for the diagnosis and treatment of acute and chronic heart failure of the European Society of Cardiology (ESC)Developed with the special contribution of the Heart Failure Association (HFA) of the ESC[END_REF].

Arrhythmias

They occur during the first hours following MI and they are responsible for sudden cardiac death. Some treatment options such as electrical cardioversion, correction of electrolyte imbalances, and early treatment with beta-blockers, ACE inhibitors/angiotensin receptor blockers, and statins are recommended. However, antiarrhythmic drugs have negative effects and should be avoided.

Mechanical complications

They may occur in the first days following MI. Some signs like hypotension, recurrent chest pain, cardiac murmurs and pulmonary congestion may be alarming.

Here some examples of dangerous mechanical complications which can take place in the ischemic heart [START_REF] Ibanez | ESC Guidelines for the management of acute myocardial infarction in patients presenting with ST-segment elevation: The Task Force for the management of acute myocardial infarction in patients presenting with ST-segment elevation of the European Society of Cardiology (ESC)[END_REF].

Free wall rupture

Based on 2017 ESC guidelines, ˂ 1% of patients may be the victims of a free wall rupture which occurs during the first week following MI. Some conditions such as, older age, lack of reperfusion, or late fibrinolysis, facilitate its occurrence. Of note, there is a possibility of wall rupture diagnosis by echocardiography. Sometimes, it is accompanied with a thrombus formation helping to seal the damaged site and delay the rupture. In this case, an immediate surgery via the use of pericardial patch is recommended. Despite the effort made to avoid harmful consequences, mortality rates remain high (20-75%).

Ventricular septal rupture

It may occur within 24 hours to several days following MI and presents with HF or cardiogenic shock. One major alarming sign is a loud systolic murmur. Of interest, ventricular septal rupture can be diagnosed with echocardiography and Doppler. When it is confirmed, surgery should be performed. To this end, patients are divided into two categories: those with severe HF can be directly operated and the others who respond well to the surgery are asked to wait in order to allow a septal repair in scarring tissue. Unfortunately, surgery is accompanied with high mortality rate (20-40%), in addition to an elevated risk of rupture recurrence.

Papillary muscle rupture

It results in acute mitral regurgitation which may occur 2-7 days following MI. Symptoms include hemodynamic deterioration, dyspnea, pulmonary edema, and/or cardiogenic shock.

Diagnosis is possible through echocardiography. Based on 2017 ESC guidelines, patients suffering from papillary muscle rupture are treated with intravenous diuretic and vasodilator with inotropic support. Moreover, surgery consisting on valve replacement or papillary muscle suture are also performed. However, surgery is accompanied with high risk of mortality (20-25%).

Pericarditis

It is an inflammation of the pericardium which may occur following MI. Complications are early pericarditis, late pericarditis, or post-cardiac injury (Dressler syndrome) and pericardial effusion explained by a buildup of excess fluid in the pericardium.

Both early and late pericarditis are rare. They may occur as a result of a late or failed reperfusion and when it concerns larger infarct size. Early pericarditis occurs soon after MI and is transient.

However late pericarditis takes place 1-2 weeks following MI as a result of immune-mediated pathogenesis caused by myocardial necrosis. Most of patients suffer from pleuritic chest pain, others display new widespread ST-segment elevation, many present pericardial effusion, and some have pericardial friction rub. Treatment consists on anti-inflammatory therapy. Notably, aspirin is the first choice and is recommended at a dose of 500-1000 mg every 6-8 hours for 1-2 weeks, then at a daily dose of 250-500 mg. Moreover, colchicine, an anti-inflammatory drug, is administered as an adjunct to aspirin for 3 months and 6 months for the recurrent forms [START_REF] Ibanez | ESC Guidelines for the management of acute myocardial infarction in patients presenting with ST-segment elevation: The Task Force for the management of acute myocardial infarction in patients presenting with ST-segment elevation of the European Society of Cardiology (ESC)[END_REF].

f. Treatment of myocardial infarction 1. Short-term reperfusion therapy

It includes primary PCI, fibrinolysis, and coronary artery bypass graft surgery. According to 2017 ESC guidelines, PCI is the preferred reperfusion strategy performed within 120 minutes from STEMI diagnosis and can be executed within 12 hours of symptom onset. Different procedures can be used during PCI. For instance, coronary stenting, which is the technique of choice, is associated with a lower risk of reinfarction and target vessel revascularization. Other techniques are thrombus aspiration, multivessel coronary revascularization and intra-aortic balloon pump. Periprocedural pharmacotherapy consists on platelet inhibition by administrating a combination of aspirin and a P2Y12 inhibitor. Of note, P2Y12 is a protein expressed on platelets promoting their aggregation. Prasugrel, clopidogrel, ticagrelor and cangrelor, and a parenteral anticoagulant like enoxaparin and bivalirudin are some examples of P2Y12 inhibitors. In turn, fibrinolysis therapy, by using fibrinolytic agents like tenecteplase tissue plasminogen activator, is recommended within 12 hours of symptom onset when primary PCI cannot be done within 120 min. In this case, angiography is recommended following fibrinolysis. Moreover, coronary artery bypass graft surgery is considered in patients with unsuitable anatomy for PCI and having a large myocardial area [START_REF] Ibanez | ESC Guidelines for the management of acute myocardial infarction in patients presenting with ST-segment elevation: The Task Force for the management of acute myocardial infarction in patients presenting with ST-segment elevation of the European Society of Cardiology (ESC)[END_REF].

Despite the beneficial effects of many used strategies, there is still no therapy capable of reducing I/R injury or MI size responsible for improved clinical outcomes. Therefore, more effort should be done for this aim given the importance of reducing MI size in improving longterm ventricular function.

Long-term therapies

During the follow-up period, patients with MI should have healthy lifestyle interventions by controlling risk factors. Smoking should be stopped and diet and alcohol controlled. For instance, a Mediterranean diet rich in polyunsaturated fatty acids is highly recommended.

Patients are allowed to less than 5 g daily salt intake, 30-45 g fibers, and 30 g unsalted nuts.

They are encouraged to consume 200g fruits and 200g vegetables per day and 1 to 2 times fish meals per week. Sugar-sweetened drinks should be restricted and daily alcohol consumption should be limited to 1 or 2 glasses for women and men, respectively. Moreover, weight should be maintained healthy (BMI between 20Kg/m 2 and <25Kg/m 2 ) and SBP of less than 140 mmHg. In addition, physical activity particularly exercise-based cardiac rehabilitation is very beneficial [START_REF] Ibanez | ESC Guidelines for the management of acute myocardial infarction in patients presenting with ST-segment elevation: The Task Force for the management of acute myocardial infarction in patients presenting with ST-segment elevation of the European Society of Cardiology (ESC)[END_REF].

According to 2017 ESC guidelines, antithrombotic medication is also recommended as a longterm therapy. Aspirin, for example, is prescribed alone in low doses (75-100 mg) or in combination with anti-platelet therapy for up to 12 months. Other beneficial therapies include administration of beta-blockers like metoprolol which reduces the incidence of ventricular arrhythmias as well as mortality, early lipid-lowering therapy through statins like atorvastatin and simvastatin in order to reach an LDL cholesterol concentration less than 1.8 mmol/L (< 70mg/dL) or at least 50% reduction in LDL cholesterol if the baseline LDL cholesterol level is 1.8-3.5mmol/L, and PCSK9 inhibitors as evolocumab responsible for LDL cholesterol decrease to up to 60% when used alone or with a statin dose. Another example concerns nitrates which can be administered intravenously during the acute phase in patients with hypertension or HF or later to control residual angina symptoms. Furthermore, a calcium antagonist like verapamil is another option in the chronic phase in patients suffering from hypertension or residual angina with contraindications to beta-blockers and without HF or impaired LV function. In addition, ACE inhibitor like captopril is administered in patients with an impaired LVEF (≤ 40%), HF, hypertension or diabetes and is replaced by an angiotensin II receptor blocker such as valsartan when it is not tolerated. Finally, a mineralocorticoid/aldosterone receptor antagonist like eplerenone is recommended in patients with a LVEF ≤ 40% and HF or diabetes and without renal failure [creatinine concentration > 221 mmol/L (2.5 mg/dL) in men and > 177mmol/L (2.0 mg/dL) in women] or hyperkalemia, and who are already under ACE inhibitor and a beta-blocker treatment [START_REF] Ibanez | ESC Guidelines for the management of acute myocardial infarction in patients presenting with ST-segment elevation: The Task Force for the management of acute myocardial infarction in patients presenting with ST-segment elevation of the European Society of Cardiology (ESC)[END_REF].

Despite all the progress made in MI management, some gaps persist and some areas of uncertainty, such as reduction of ischemic injury and myocardial repair, should be solved in the future. Notably, we strongly need to find an efficient and safe novel therapy, either a cell therapy or gene therapy, able to replace dead myocardium or prevent long-term poor remodeling by limiting infarct size. To this end, more effort should be done in order to better understand the biological and cellular processes implicated in cardiac repair following MI.

D. Experimental models of myocardial infarction

Large and small animals such as monkeys, pigs, rabbits, dogs, rats and mice are used to induce MI in order to mimic this pathology happening in human heart. Different methods exist like chemical agent, open surgery or closed-chest catheter methods.

i. Chemical agent

Isoprenaline or isoproterenol is an agonist of adrenergic receptors responsible for positive inotropic, chronotropic and dromotropic effects. It results in infarct-like lesion similar to human MI by exerting a depressor effect on circulation, thus creating an imbalance between cardiac stimulation and decreased coronary blood flow in addition to producing oxidation products responsible for myocardial damage. It can be administered subcutaneously, intraperitoneally (IP) or intravenously. Lesion size depends on the dose administered and whether it is a single dose or repeated doses [START_REF] Aisyah | Induction of Myocardial Infarction in Experimental Animals: A Review[END_REF].

Zhang and colleagues tested different doses of isoproterenol through subcutaneous administration to rats. They saw that isoproterenol used at 125-500 µg/kg was responsible for severe and extensive lesions in the LV explained by CMs necrosis and interstitial fibrosis [START_REF] Zhang | Isoproterenol-induced Cardiotoxicity in Sprague-Dawley Rats: Correlation of Reversible and Irreversible Myocardial Injury with Release of Cardiac Troponin T and Roles of iNOS in Myocardial Injury[END_REF]. In turn, Filho et al., induced acute MI in rats by subcutaneous isoproterenol injection at a dose of 150 mg/Kg/day diluted in 2 mL of saline, on two consecutive days with an interval of 24 hours between applications [START_REF] Lobo Filho | Experimental model of myocardial infarction induced by isoproterenol in rats[END_REF].

ii.

Open-chest surgery a. Permanent ligation

The first illustration is the mouse because it is an experimental animal of choice for the study of human diseases such as acute MI thanks to its low cost, existence of genetically modified mouse, in addition to physiologic similarities to human and its capacity to reproduce the same pathologic features, thus letting us to better understand the pathology and its complications, explore pathophysiological pathways, and find new therapeutic targets.

Mouse model of lasting ischemia consists on a permanent ligation of the LAD coronary artery.

LAD is the major vessel that supplies blood to the LV as described above and it is a predominant site for occurrence of atherosclerosis in humans [START_REF] Wasilewski | Predominant location of coronary artery atherosclerosis in the left anterior descending artery. The impact of septal perforators and the myocardial bridging effect[END_REF]. As a result of LAD coronary artery occlusion, MI takes place in the antero-apical and lateral wall of the LV and the anterior portion of the interventricular septum. This is accompanied with an impairment of the LV function, and the infarct size is estimated to approximately 40% [START_REF] Aisyah | Induction of Myocardial Infarction in Experimental Animals: A Review[END_REF][START_REF] Wang | A simple and fast experimental model of myocardial infarction in the mouse[END_REF].

First, mouse is anesthetized using ketamine (100 mg/kg body weight) and xylazine (10 mg/kg body weight) via IP injection. Then, it is intubated, and ventilated using a small-animal respirator (Figure 5). After that, the chest wall is shaved and a thoracotomy is performed in the fourth left intercostal space. The heart is visualized. The pericardial sac is removed, and the LAD coronary artery, in the LV, is permanently ligated with 7-0 monofilament suture at the site of its emergence close to the LA. The thoracotomy is then closed with 6-0 monofilament sutures. Finally, the endotracheal tube is removed once spontaneous respiration resumed, and the mouse is placed on a warm pad at 37°C until awakened [START_REF] Zlatanova | Iron Regulator Hepcidin Impairs Macrophage-Dependent Cardiac Repair After Injury[END_REF]. Of note, permanent ischemia, by ligating the LCA 1 to 2 mm from its origin, can be also induced in other experimental animals like rats where occlusion is easy to perform and quantification is fast due to their small heart compared to bigger animals, notably dogs and pigs [START_REF] Fishbein | Experimental myocardial infarction in the rat: qualitative and quantitative changes during pathologic evolution[END_REF].

b. Ischemia/reperfusion

In order to mimic the reperfusion performed in human either by PCI or fibrinolytic therapy, an experimental model consisting on a temporary occlusion of LAD is produced. For instance, Peterss and coworkers induced MI in rats by ligating LAD coronary artery after tightening a suture placed around it for 30 minutes followed by 60 minutes of reperfusion by releasing the suture [START_REF] Peterss | An experimental model of myocardial infarction and controlled reperfusion using a miniaturized cardiopulmonary bypass in rats[END_REF]. Mouse and pig models for I/R also exist. For example, Nossuli and colleagues performed I/R in mouse by doing 1 hour of ischemia followed by 2 hours of reperfusion [START_REF] Nossuli | A chronic mouse model of myocardial ischemiareperfusion: essential in cytokine studies[END_REF]. In turn, Esposito et al., performed I/R in adult male swine by ligating LAD artery for 90 minutes followed by 120 minutes reperfusion [START_REF] Esposito | Left Ventricular Unloading Before Reperfusion Promotes Functional Recovery After Acute Myocardial Infarction[END_REF].

iii. Coronary artery embolization

This method is non-invasive and is more suitable for large animals. It consists on a catheterization of the coronary artery in order to insert a material such as sponge foam, coils, polystyrene microspheres, and alcohol injection to form a thrombus resulting in MI. During this procedure, blood clot formation, arrhythmia, and VF should be avoided by using an anticoagulant therapy and an anti-arrhythmic protocol, respectively [START_REF] Aisyah | Induction of Myocardial Infarction in Experimental Animals: A Review[END_REF].

Hanes and colleagues performed MI in pigs by injecting 90 µm polystyrene micro-spheres in the LAD using percutaneous embolization method. Researchers found a decrease in the EF starting immediately after the procedure marking that this latter was done successfully [START_REF] Hanes | Embolization of the first diagonal branch of the left anterior descending coronary artery as a porcine model of chronic trans-mural myocardial infarction[END_REF]. In turn, Reffelmann and colleagues performed MI in pigs using foam sponge inserted into the distal coronary artery [START_REF] Reffelmann | A novel minimal-invasive model of chronic myocardial infarction in swine[END_REF].

iv. Balloon catheter

This method consists in a balloon catheter insertion into the coronary artery. The first step is an inflation of the balloon to produce coronary occlusion followed by its deflation to initiate the reperfusion. Hu and colleagues performed this open-chest technique in mice where they induced ischemia with inflated balloon during 30 minutes followed by reperfusion after deflating it [START_REF] Hu | Outer Balloon Ligation Increases Success Rate of Ischemia-Reperfusion Injury Model in Mice[END_REF].

v. Ameroid constrictor

It is a ring-like structure made of stainless steel, titanium or plastic containing casein material which is hygroscopic. It is placed around the coronary artery by an open-chest method or closechest one using a catheter. Casein has the capacity to absorb the fluid surrounding the artery resulting in a narrowing of its lumen. One example concerns the work of Keeran and coworkers who induced cardiac ischemia in pigs using this device [START_REF] Keeran | A Chronic Cardiac Ischemia Model in Swine Using an Ameroid Constrictor[END_REF].

E. Effectors of post-infarct reparative phase

Multicellular organisms required millions of years of evolution. For instance, human body is composed of more than 200 different cell types [START_REF] Perbellini | Heterocellularity and Cellular Cross-Talk in the Cardiovascular System[END_REF]. Cell generation and turnover, in addition to communication between different cell populations, is the essence of complex multicellular systems. For example, the heart is an organ within the cardiovascular system with a central function of a pump. Cells constituting it are CMs, ECs, fibroblasts (FBs), pericytes (PCs) and vascular smooth muscle cells (VSMCs) and ICs [START_REF] Wagner | Cellular cross-talks in the diseased and aging heart[END_REF]. Each of these cells has specific functions and regulatory roles. Communication between them is of great importance to perform the essential function of the heart. However, loss of cells occurring in some conditions such as acute MI is largely irreversible depending on cell type and dysregulation of cell communications constitute a big health burden [START_REF] Bergmann | Dynamics of Cell Generation and Turnover in the Human Heart[END_REF].

Hence, better understanding of cardiac homeostasis and pathology, particularly acute MI, relies on a greater understanding of the complex cellular milieu of the heart as well as cell communications.

i. Cardiac cell populations a. Cardiomyocytes

CMs are the most physically energetic cells known to be the muscle cells of the heart. Their synchronized contraction is essential to pump blood throughout the circulatory system.

CMs are very large cylindrical cells about 100-150 µm in length and 10-35 µm in width. They occupy two-thirds of total cardiac tissue volume. While CMs in humans are mononucleated (73.6%), binucleated (25.5%) or trinucleated (1.2%), they are multinucleated in rodents [START_REF] Bergmann | Dynamics of Cell Generation and Turnover in the Human Heart[END_REF]. In order to satisfy their high energy needs essential for contraction, CMs are equipped with a large cytoplasm full of mitochondria and contractile units called sarcomeres. Cardiac contraction is initiated by an action potential generated in the sino-atrial node, then conducted to the apex by Purkinje fibers and spread throughout the ventricles. This link between membrane excitation and CM contraction is known as excitation-contraction coupling (Figure 6). Depolarization is induced by the opening of voltage-gated Na + -channels accompanied with calcium ions (Ca 2+ ) entry to the cytosol via activated L-type voltage-gated Ca 2+ channels. Increased Ca 2+ concentration induces Ca 2+ release from sarcoplasmic reticulum (SR) via ryanodine receptor 2 (RyR2). Ca 2+ binds to the contractile apparatus and stimulates contraction. Cytosolic Ca 2+ returns to resting levels by re-entering the SR through sarco/endoplasmic reticulum Ca 2+ (SERCA) or by being extruded from the cell by sodiumcalcium exchanger (NCX) marking the end of systole and the beginning of diastole [START_REF] Drawnel | The role of the paracrine/autocrine mediator endothelin-1 in regulation of cardiac contractility and growth[END_REF]. Of interest, cytosolic Ca 2+ overload is considered as an important pathophysiological mechanism implicated in injuries during I/R. It has been reported that a defect of RyR2 is responsible for this Ca 2+ overload during I/R. Hence, modulation of RyR2 is of great importance since it could prevent a worse I/R outcome [START_REF] Fauconnier | Type 2 ryanodine receptor: a novel therapeutic target in myocardial ischemia/reperfusion[END_REF]. (2). Increased Ca 2+ concentration induces calcium release from SR via RyR2, thus increasing Ca 2+ concentration in the cytosol [START_REF] Schwarcz | Differential vulnerability of central neurons of the rat to quinolinic acid[END_REF]. Ca 2+ binds to the contractile filaments ( 4) and stimulates contraction [START_REF] Wang | Tryptophanderived 3-hydroxyanthranilic acid contributes to angiotensin ii-induced abdominal aortic aneurysm formation in mice in vivo[END_REF]. Cytosolic Ca 2+ returns to resting levels by re-entering the SR through SERCA or by being extruded from the cell by NCX [START_REF] Metghalchi | Indoleamine 2 3-dioxygenase knockout limits angiotensin ii-induced aneurysm in low density lipoprotein receptor-deficient mice fed with high fat diet[END_REF]. Ca 2+ : calcium ions, SR: sarcoplasmic reticulum, RyR2: ryanodine receptor 2, SERCA: sarco/endoplasmic reticulum Ca 2+ , NCX: sodiumcalcium exchanger.

Among markers to identify CMs, α-actinin and α-myosin heavy chain (α-MHC) are widely used [START_REF] Banerjee | Determination of cell types and numbers during cardiac development in the neonatal and adult rat and mouse[END_REF][START_REF] Bergmann | Dynamics of Cell Generation and Turnover in the Human Heart[END_REF][START_REF] Perbellini | Heterocellularity and Cellular Cross-Talk in the Cardiovascular System[END_REF]. Their counts show that in number, they account only for around 25-35% of all the cells in the heart [START_REF] Camelliti | Structural and functional characterisation of cardiac fibroblasts[END_REF][START_REF] Perbellini | Heterocellularity and Cellular Cross-Talk in the Cardiovascular System[END_REF]Pinto et al., 2016). According to Bergman and colleagues, CM nuclei constitute the majority of cell nuclei in the human LV, during the postnatal period (66%).

While the number of CM nuclei and CMs is found to be constant during heart growth, the number of ECs and mesenchymal cells increases. For instance, CMs number was estimated to 18%, ECs 24%, and mesenchymal cells 58% in young adults [START_REF] Bergmann | Dynamics of Cell Generation and Turnover in the Human Heart[END_REF]. Based on fluorescence activated cell sorting (FACS) analysis, Banerjee and coworkers found that the adult murine heart consists of 56% CMs and the LV consists of around 52% CMs, and rat adult heart consists of 62% [START_REF] Banerjee | Determination of cell types and numbers during cardiac development in the neonatal and adult rat and mouse[END_REF].

Of note, CMs turnover is under debate with no possible full deduction yet about that. According

to Banerjee and colleagues, the proliferative capacity of the heart decreases with time. Notably, they found that Bromodeoxyuridine (BrdU)-positive cells decrease from 37% at neonatal day 3 to 5% at neonatal day 13 [START_REF] Banerjee | Determination of cell types and numbers during cardiac development in the neonatal and adult rat and mouse[END_REF]. It has also been shown by FACS that CMs number in the rat heart decreases from 62% at day 1 to 30% at day 15 [START_REF] Banerjee | Determination of cell types and numbers during cardiac development in the neonatal and adult rat and mouse[END_REF].

Similarly, an important decrease in CM nuclei density from around 430 000/mm 3 of the LV shortly after birth to 28 000/mm 3 has been observed in subjects aged 20 to 73 without any history or sign of heart pathology [START_REF] Bergmann | Dynamics of Cell Generation and Turnover in the Human Heart[END_REF]. The same study showed that the final CMs number was reached at 1 month after birth and was stabilized over lifetime which is controversial with what has been reported in humans that CMs number increases by 3.4-fold between 1 to 20 years of age [START_REF] Mollova | Cardiomyocyte proliferation contributes to heart growth in young humans[END_REF][START_REF] Tucker | Transcriptional and Cellular Diversity of the Human Heart[END_REF].

CMs are connected to each other thanks to intercalated-disks containing intercellular channels called gap junctions made of connexins (Cx). Ions, small molecules and small peptides are able to cross these junctions thus ensuring mechanical and electromechanical coupling. These connections are also important to stabilize cells positions and maintain the 3D structural integrity [START_REF] Perbellini | Heterocellularity and Cellular Cross-Talk in the Cardiovascular System[END_REF]. CMs also communicate with other neighbor cells via secretion of factors which may be of considerable importance in the development, growth, and maintenance of the coronary vasculature [START_REF] Giordano | A cardiac myocyte vascular endothelial growth factor paracrine pathway is required to maintain cardiac function[END_REF]. In the heart, CMs are the major source of vascular endothelial growth factor (VEGF). In their study published in 2001 Giordano et al., the first to create a mouse strain with CM-VEGF specific deletion, demonstrated that mice with CM-VEGF specific deletion had 6-fold reduction of cardiac VEGF expression. This indicates that cardiac non-CM, despite their dominant presence, cannot compensate for the loss of CM derived VEGF in basal conditions. This means that in pathological conditions like acute MI, the loss of CMs may significantly alter myocardial paracrine signaling and negatively influence post-MI ventricular remodeling or contribute to myocardial dysfunction. Moreover, CM-derived VEGF-A, by controlling the formation of capillaries, is considered as an important mediator for cardiac development. Remarkably, VEGF-A deficient-mice have reduced body weights, thin-walled, dilated and hypo vascular hearts as well as a basal contractile dysfunction [START_REF] Giordano | A cardiac myocyte vascular endothelial growth factor paracrine pathway is required to maintain cardiac function[END_REF]. CMs also produce and secrete acetylcholine (ACh) which is a parasympathetic neurotransmitter. This latter has the role to counterbalance sympathetic activity and is crucial to maintain normal heart activity.

Remarkably, mice lacking CM-vesicular ACh transporter had an increased heart rate following exercise, more oxidative stress, altered Ca 2+ , remodeling and hypertrophy (Roy et al., 2013).

b. Endothelial cells

ECs role is to control vasomotor tone, and regulate angiogenesis [START_REF] Talman | Cardiomyocyte-Endothelial Cell Interactions in Cardiac Remodeling and Regeneration[END_REF].

In the heart, ECs form the inner surface of the chambers as well as macro-and microvasculature having the role to distribute oxygen and metabolic substrates to CMs. In order to insure their high oxygen demands, CMs are situated in close proximity to capillaries and each one is associated to at least one capillary. It has been reported that ECs not only represent the major non-CM population [START_REF] Talman | Cardiomyocyte-Endothelial Cell Interactions in Cardiac Remodeling and Regeneration[END_REF], but also the most abundant cell type in the heart and their number is three times more elevated than CMs [START_REF] Perbellini | Heterocellularity and Cellular Cross-Talk in the Cardiovascular System[END_REF][START_REF] Talman | Cardiomyocyte-Endothelial Cell Interactions in Cardiac Remodeling and Regeneration[END_REF][START_REF] Wagner | Cellular cross-talks in the diseased and aging heart[END_REF]. Based on immune fluorescence staining, it has been shown that ECs in the murine heart, account for 60% of all non-CMs (Pinto et al., 2016). It has also been reported that ECs constitute 52% of the totality of the cardiac cells [START_REF] Perbellini | Heterocellularity and Cellular Cross-Talk in the Cardiovascular System[END_REF]. According to Wagner and colleagues, ECs constitute 48% of all non-CMs [START_REF] Wagner | Cellular cross-talks in the diseased and aging heart[END_REF]. Whereas, Banerjee et al., reported that ECs content, based on FACS analysis in the total mouse heart, is only 7% [START_REF] Banerjee | Determination of cell types and numbers during cardiac development in the neonatal and adult rat and mouse[END_REF]. In humans, it has been reported that ECs number increases 6.5-fold during heart growth to reach an average of 24% of all the cardiac cells in young adults and it declines during aging [START_REF] Bergmann | Dynamics of Cell Generation and Turnover in the Human Heart[END_REF]. It is clear that ECs number varies depending on the method used. This huge variation, especially in FACS studies, could be due to tissue dissection which could damage EC membrane surface proteins such as cluster of differentiation 144 (CD144) and platelet endothelial cell adhesion molecule-1 (PECAM-1 also known as CD31), thus preventing their recognition by flow cytometry. In addition to ECs, CD31, in the heart, is also found on leukocytes like neutrophils. Therefore, a CD31 + CD45 -staining is used in order to distinguish ECs from the leukocytes when performing FACS analysis. Another EC marker is Lectin Ulex Europaeus Agglutinin I (UEA I) which is a specific marker of human ECs and more specifically the nucleus of cardiac ECs [START_REF] Bergmann | Dynamics of Cell Generation and Turnover in the Human Heart[END_REF].

CMs and ECs neighborhood results in a continuous communication that is fundamental for cardiac development and function regulation by both autocrine and paracrine mechanisms [START_REF] Perbellini | Heterocellularity and Cellular Cross-Talk in the Cardiovascular System[END_REF]. On the one hand, CMs communicate with ECs by secreting factors known as cardiokines. In addition to VEGF-A which mediates EC activation and proliferation by binding to its receptor vascular endothelial growth factor receptor 2 (VEGFR2) in these cells, VEGF-B, VEGF-C, placental growth factor (PlGF), fibroblast growth factor (FGF), hepatocyte growth factor (HGF), and angiopoietin-1 are other cardiokines produced by CMs which induce cardiac angiogenesis [START_REF] Talman | Cardiomyocyte-Endothelial Cell Interactions in Cardiac Remodeling and Regeneration[END_REF]. On the other hand, Narmoneva and colleagues found that cardiac ECs promote CMs viability and synchronized contraction (Narmoneva [START_REF] Daria | Endothelial Cells Promote Cardiac Myocyte Survival and Spatial Reorganization[END_REF]. Moreover, ECs' interaction with CMs is mediated by different factors they release known as angiocrines. NO, for example, induces VSMC relaxation and blood vessels vasodilation, regulates CM contractility [START_REF] Mohan | Myocardial contractile response to nitric oxide and cGMP[END_REF] and inhibits CM hypertrophy [START_REF] Li | Premature death and age-related cardiac dysfunction in male eNOS-knockout mice[END_REF]. Also, NO has been found to have an anti-apoptotic effect on CM by suppressing caspase activity via S-nitrosylation [START_REF] Maejima | Nitric oxide inhibits myocardial apoptosis by preventing caspase-3 activity via S-nitrosylation[END_REF]. Of note, caspases are regulators of apoptotic signaling pathways. They are separated into two groups: the initiators (caspase-8,-9) and the executioners (caspase-3, -6, -7). Under normal conditions, they exist under their inactive forms until they are cleaved into their active forms able to initiate apoptotic process. Neuregulin-1 (NRG-1) is another protein secreted by ECs that binds and activates erbB receptors, members of EGFR family, in CMs which are structurally related to the epidermal growth factor receptor (EGFR). Notably, NRG-1 regulates ROS-induced CM apoptosis through NRG-1β/erbB4 signaling [START_REF] Kuramochi | Cardiac endothelial cells regulate reactive oxygen species-induced cardiomyocyte apoptosis through neuregulin-1beta/erbB4 signaling[END_REF].

Moreover, endothelin-1 (ET-1) which is produced by ECs, CMs, and FBs, regulates cardiac contractility and growth [START_REF] Drawnel | The role of the paracrine/autocrine mediator endothelin-1 in regulation of cardiac contractility and growth[END_REF].

c. Fibroblasts

FBs have a mesenchymal origin from two principal sources: the pro-epicardial organ, and the epithelial-mesenchymal transformation during the formation of cardiac valves. They are spread in the cardiac tissue. They surround CMs and fill the voids between myocardial tissue layers.

According to Jugdutt BI, FBs account for 90% to 95% of non-myocytes cell mass in the healthy heart [START_REF] Jugdutt | Ventricular remodeling after infarction and the extracellular collagen matrix: when is enough enough?[END_REF]. Based on FACS results, Banerjee and coworkers found that cardiac FBs population constitutes 30% of the whole rat heart at neonatal day 1 and it increases to 64% at day 15. In murine heart, they are about 10% of the cardiac cell population at neonatal day 1 and increase to 18% at neonatal day 15 [START_REF] Banerjee | Determination of cell types and numbers during cardiac development in the neonatal and adult rat and mouse[END_REF]. Using morphometric and FACS analysis, the same group showed that murine LV consists of 37% FBs. Of note, FBs do not have a specific marker because many of the proteins highly expressed on these cells are also expressed on other cell types. For instance, vimentin is expressed in intermediate filaments of

FBs, ECs and neurons. Although its non-specificity, it is useful for FBs identification when cyto-morphological differences of these cells are taken into account. FB-specific protein-1 (FSP1) is another example. Other markers include platelet-derived growth factor receptor-beta (PDGFR-β) and the Discoidin domain receptor 2 (DDR2). DDR2 is a collagen specific receptor tyrosine kinase that mediates growth, migration, morphology and differentiation. It is found on mesenchymal cells, leukocytes and tumors but not on CMs or cardiac ECs and SMCs [START_REF] Banerjee | Determination of cell types and numbers during cardiac development in the neonatal and adult rat and mouse[END_REF][START_REF] Bergmann | Dynamics of Cell Generation and Turnover in the Human Heart[END_REF][START_REF] Camelliti | Structural and functional characterisation of cardiac fibroblasts[END_REF][START_REF] Wagner | Cellular cross-talks in the diseased and aging heart[END_REF].

FBs produce interstitial collagen types I which is the most abundant, III and VI. They are essential components of the extra-cellular matrix (ECM) [START_REF] Camelliti | Structural and functional characterisation of cardiac fibroblasts[END_REF]. Instead of having a basement membrane, they have sheet-like extensions. They also have an oval nucleus, extensive rough endoplasmic reticulum, a prominent Golgi apparatus, and an abundant cytoplasmic granular material [START_REF] Camelliti | Structural and functional characterisation of cardiac fibroblasts[END_REF]. In order to maintain myocardial tissue structure, FBs participate to ECM homeostasis and production of factors like cytokines, growth factors and matrix metalloproteinases (MMPs) responsible for preserving a balance between synthesis and degradation of connective tissue components. In addition, FBs contribute to mechanical properties of the myocardium. When mechanical changes occur, ECM, which is stretch-sensitive, detects the signal and transduces it to the cardiac cells like FBs via integrins, ion channels and second messenger responses leading to proliferation, enhanced deposition of ECM protein and release of growth factors and MMPs [START_REF] Camelliti | Structural and functional characterisation of cardiac fibroblasts[END_REF].

Cardiac FBs are interconnected by gap junctions such as Cx40, Cx43 and Cx45. They are electrically "non-excitable cells" but mechano-sensitive. When they detect mechanical stimuli, they respond with changes in their membrane potential attributed to stretch-activated ion channels permeable to Na⁺, potassium ions (K⁺), and Ca 2+ [START_REF] Camelliti | Structural and functional characterisation of cardiac fibroblasts[END_REF]. FBs are also connected to CMs by gap junctions as Cx43 and Cx45 which enable them to ensure a longdistance connection between distant CMs. Despite the fact that FBs are considered as obstacles to the spread of electrical excitation by slowing down or interrupting conduction, they could serve as conductors of electrical excitation and thus affect CMs electrophysiology. Given the close location of CMs and FBs as well as their interconnection, the stimulation of the ones induces the response of the others. Notably, FBs release transforming growth factor-β (TGFβ), interleukin-6 (IL-6), ET-1, angiotensin-II (AngII) as well as miRNA-loaded exosomes (miR-21) thus mediating the hypertrophic response of CMs and their growth [START_REF] Camelliti | Structural and functional characterisation of cardiac fibroblasts[END_REF][START_REF] Wagner | Cellular cross-talks in the diseased and aging heart[END_REF]. In turn, CMs release growth factors resulting in FBs proliferation, collagen synthesis, ECM turnover and changes in gene expression [START_REF] Camelliti | Structural and functional characterisation of cardiac fibroblasts[END_REF].

d. Vascular smooth muscle cells and pericytes

VSMCs are stromal cells forming the vessel wall of small and large vessels. By contracting and relaxing, they control vessel luminal diameter and thus, regulate blood pressure and blood flow [START_REF] Perbellini | Heterocellularity and Cellular Cross-Talk in the Cardiovascular System[END_REF][START_REF] Talman | Cardiomyocyte-Endothelial Cell Interactions in Cardiac Remodeling and Regeneration[END_REF]. [START_REF] Perbellini | Heterocellularity and Cellular Cross-Talk in the Cardiovascular System[END_REF].

VSMCs are able to detect environmental changes and respond adequately. These changes can directly affect VSMCs or indirectly through interactions with other cell types. For instance, when VSMCs sense tensile stress and shear stress induced by blood pressure and blood flow, they initiate vessel wall remodeling. Moreover, given the interaction between ECs and VSMCs, the alteration of the ones participate to the alteration of the others via altered mediators responsible for the communication between both cells such as NO, ET-1, serotonin and prostacyclin. In addition, a part of VSMCs communication with the other neighbor cells including other VSMCs, ECs, and perivascular ICs is done through PDGF molecules secreted by these cells. Changes in these molecules, under some pathologic conditions like pulmonary arterial hypertension, induce synthetic VSMCs proliferation and migration thus participating to arterial remodeling. Furthermore, crosstalk between VSMCs and macrophages, during atherosclerotic plaque formation, results in an alteration of ECM synthesis and deposition, an increase in production of MMPs and angiogenic chemokines such as VEGF and IL-1 [START_REF] Perbellini | Heterocellularity and Cellular Cross-Talk in the Cardiovascular System[END_REF].

In turn, PCs are contractile cells having a thin and elongated cytoplasm, numerous finger-like projections and a rounded nucleus. They are found in the basal membrane in close contact with ECs within the microvasculature more specifically, pre-capillary arterioles, capillaries, and post-capillary venules. In the heart, they are also found in larger vessels forming a layer separating the endothelium from the VSMCs and the elastic structures of the vessel. Different surface markers like PDGFRβ, CD146, CD13 and neural/glial antigen 2 (NG2) proteoglycan, as well as cytoplasmic markers like α-SMA, desmin, vimentin, and nestin are used in order to identify PCs [START_REF] Perbellini | Heterocellularity and Cellular Cross-Talk in the Cardiovascular System[END_REF].While Nees and colleagues found that PCs are the second most abundant cells in the LV with a ratio of ECs to PCs about 2:1 to 3:1 [START_REF] Nees | Isolation, bulk cultivation, and characterization of coronary microvascular pericytes: the second most frequent myocardial cell type in vitro[END_REF],

Pinto and coworkers found that they only constitute around 6% of all the cells in the LV (Pinto et al., 2016). PC function is to regulate homeostasis and permeability of the vasculature, control the blood flow, remove cell debris and participate to the maturation of ECs to whom they are connected by Cx43 and N-Cadherin adherence junctions. Thanks to this cross-talk, endothelial barrier is maintained. It has been reported that PCs secrete TGF-β and angiopoietin 1, and contribute to collagen I, IV and fibronectin formation and deposition in the basal membrane [START_REF] He | Isolation of rat heart endothelial cells and pericytes: evaluation of their role in the formation of extracellular matrix components[END_REF]. PCs also participate to new vessel formation by either inducing maturation of activated ECs or filling the gaps between sprouting ECs. In addition, PCs crosstalks with resident and ICs play an important role in angiogenesis, reparative fibrosis and tissue remodeling following ischemia. For instance, as a result of galectin-3 secretion by macrophages, PCs undergo proliferation and participate to collagen formation and accumulation leading to cardiac fibrosis. Moreover, under PDGF stimulation, PCs migrate to the interstitium, transform into myofibroblasts and release ECM [START_REF] Perbellini | Heterocellularity and Cellular Cross-Talk in the Cardiovascular System[END_REF].

e. Immune cells

Immune system interferes in case of injury and infection such as by bacteria. It is a sentinel for all the body including myocardium when it is infected. However, immune system is crucial for organ development and normal tissue function in the absence of infection or injury. Although present in minor populations, all major leukocyte classes are represented in the heart including monocytes/macrophages, mast cells, dendritic cells (DCs), neutrophils, T-and B-cells [START_REF] Nag | Study of non-muscle cells of the adult mammalian heart: a fine structural analysis and distribution[END_REF][START_REF] Swirski | Cardioimmunology: the immune system in cardiac homeostasis and disease[END_REF][START_REF] Wagner | Cellular cross-talks in the diseased and aging heart[END_REF]. According to Pinto and colleagues, they represent around 8% of the total cells in the LV (Pinto et al., 2016).

Macrophages constitute the first line of defense against invading pathogens. It has been reported that they are among the most abundant cells in the infarcted heart [START_REF] Perbellini | Heterocellularity and Cellular Cross-Talk in the Cardiovascular System[END_REF][START_REF] Zlatanova | Immune Modulation of Cardiac Repair and Regeneration: The Art of Mending Broken Hearts[END_REF]. They are large, round or spindle-like cells with a central nucleus and vacuolated cytoplasm with long protrusions. Macrophages are interspersed between CMs, FBs and ECs [START_REF] Hulsmans | Monocyte and macrophage contributions to cardiac remodeling[END_REF][START_REF] Perbellini | Heterocellularity and Cellular Cross-Talk in the Cardiovascular System[END_REF]. Macrophages are classified into two groups: M1 macrophages which participate to inflammation and M2 ones which decrease inflammation and facilitate tissue repair [START_REF] Perbellini | Heterocellularity and Cellular Cross-Talk in the Cardiovascular System[END_REF]. Despite this oversimplified classification, cardiac macrophages can be divided into 4 groups based on their surface markers. Generally, they are all CD45 + CD11b + F4/80 + CD64 + MERTK + . The difference exists in terms of expression of major histocompatibility complex (MHC) class II molecules, CCchemokine receptor 2 (CCR2) and lymphocyte antigen 6C (Ly6C). The 4 subsets are ranked based on their dominance: MHC class II hi CCR2 -and MHC class II low CCR2 -, CCR2 + expressing MHC class II and Ly6C + subsets [START_REF] Swirski | Cardioimmunology: the immune system in cardiac homeostasis and disease[END_REF]. Cardiac macrophages origin differs depending on macrophage subset, more precisely expression of surface markers. For instance, studies have shown that CCR2 -and Ly6C + within the heart have an embryonic origin. After birth, some cardiac macrophages self-renew without monocyte contribution and some others may arise from monocytes which are considered to play a minimal role in healthy heart tissue in mice and human [START_REF] Bajpai | The human heart contains distinct macrophage subsets with divergent origins and functions[END_REF]. and IL-10 derived from macrophages result in collagen production, neoangiogenesis and resolution of inflammation [START_REF] Swirski | Cardioimmunology: the immune system in cardiac homeostasis and disease[END_REF].

ii. Cellular cross-talk following myocardial infarction

Following acute MI, cells undergo adaptive mechanisms, and phenotypic changes.

Maladaptive responses in different cell types is associated with an altered cross-talk resulting in adverse cardiac remodeling and deteriorated cardiac function.

Dying CMs communicate with neighboring cells specifically ICs and FBs through proinflammatory factors secretion such as ROS, TNFα, IL-6, and IL-1β and via danger-associated molecular patterns (DAMPs)-toll-like receptor (TLR) signaling [START_REF] Wagner | Cellular cross-talks in the diseased and aging heart[END_REF]. In turn, ICs number increases in the injured heart. These pre-existing and newly recruited cells interact with the non-leukocyte cells through a variety of cytokines they secrete.

For instance, macrophages produce TGF-β which promotes CM death and hypertrophy as well Following acute MI, ECs number initially decreases due to cell death. This is then followed by vascular network restoration explained by blood vessels growth and formation between day 7 and 14 to provide oxygen to the ischemic tissue. In addition to their role in controlling ICs invasion, ECs closely interact with CMs. On the one hand, hypertrophic CMs secrete proangiogenic factors such as VEGF or angiopoietin-1, thus promoting adaptive vessel growth.

On the other hand, ECs secrete ET-1, thereby inducing CMs hypertrophy in a paracrine manner [START_REF] Wagner | Cellular cross-talks in the diseased and aging heart[END_REF].

F. Molecular and cellular mechanisms of cardiac repair following MI

Cardiac repair following MI consists in an inflammatory phase followed by a reparative phase marked by the resolution of inflammation and myofibroblasts proliferation, in addition to scar formation, maturation and neovascularization. Major events governing the reparative response are cellular debris elimination, modification of the vascular network and fibrotic scar formation.

i. Necrosis and inflammatory response

The inflammatory phase has the role to clean the infarct zone from damaged cells and ECM debris. Notably, both innate and adaptive immunity play an important role in cardiac repair starting from an early time point following the injury [START_REF] Zlatanova | Immune Modulation of Cardiac Repair and Regeneration: The Art of Mending Broken Hearts[END_REF].

Shortly after the onset of ischemia, CMs start to die (Figure 7). It has been reported that a patient with MI loses on average around 1 billion CMs [START_REF] Sahara | Programming and reprogramming a human heart cell[END_REF]. Dying CMs and damaged ECM generate endogenous signals known as "alarmins" or DAMPs to warn the body of injury and activate innate ICs. One example of alarmins is the high mobility group protein B1 (HMGB1) involving TLRs as TLR2 and TLR4, which are transmembrane receptors that activate downstream pro-inflammatory cascades, and the receptor for advanced glycation end products (RAGE). Moreover, intracellular constituents like heat shock proteins (HSP), adenosine triphosphate (ATP) and deoxyribonucleic acid (DNA) secreted by necrotic cells, in addition to low molecular weight hyaluronan and fibronectin fragments, as well as the complement system, ROS, and free radicals are also involved in the inflammatory response [START_REF] Frangogiannis | The inflammatory response in myocardial injury, repair, and remodelling[END_REF]. As a result, immune and non-immune effector cells are activated.

Immune effector cells are the cells normally found in the heart, in addition to the newly recruited ones. Resident mast cells release their granular content containing TNF, resident macrophages and CMs produce pro-inflammatory cytokines notably, TNF, IL-1β and IL-6 and pro-inflammatory chemokines mostly, C-C and C-X-C chemokines, and FBs acquire a proinflammatory and matrix-degrading phenotype, and release hematopoietic growth factors like GM-CSF. Aggravated inflammation is accompanied with ECs activation and production of adhesion molecules such as vascular cell adhesion molecule 1 (VCAM1) and selectins [START_REF] Swirski | Cardioimmunology: the immune system in cardiac homeostasis and disease[END_REF][START_REF] Wagner | Cellular cross-talks in the diseased and aging heart[END_REF]. Within one day following the ischemia, CD4+ and CD3+ T lymphocytes promptly colonize the infarcted heart. It has been shown that activation of these cells ameliorates wound healing and mice survival after MI [START_REF] Hofmann | Activation of CD4+ T lymphocytes improves wound healing and survival after experimental myocardial infarction in mice[END_REF]. Moreover, leukocytes including Ly6C high CCR2 + CX3CR1 low monocytes (Ly6C hi ), implicated in secretion of large amounts of proteases, and neutrophils, are mainly recruited from hematopoietic stem and progenitor cells (HSPCs) in the bone-marrow (BM) and few from the spleen [START_REF] Swirski | Cardioimmunology: the immune system in cardiac homeostasis and disease[END_REF]. In turn, mesenchymal stem cells and peripheral B cells participate to monocytes and neutrophils mobilization to the blood by releasing monocyte and neutrophil-attracting chemokines. Interestingly, Zouggari and colleagues found that Blymphocytes, by producing CCL7, trigger Ly6C hi monocyte mobilization and recruitment to the myocardium leading to an enhanced tissue injury and impaired heart function following MI (Zouggari et al., 2013).These newly recruited cells interact with activated ECs. They roll along the endothelial surface involving interactions with the selectins, as well as chemokines bound to proteoglycans. Thanks to the integrins they express, recruited cells adhere to ECs, and transmigrate into the infarcted area. By releasing proteases and ROS, these cells clear the wound from dead cells and debris [START_REF] Frangogiannis | The inflammatory response in myocardial injury, repair, and remodelling[END_REF]. Monocytes and macrophages have the role to clean ischemic zone from dead and dying cells. This is possible thanks to scavenger receptors such as tyrosine-protein kinase MER (MERTK), interferon regulatory factor 3 (IRF3)

that helps them to scavenge CM-derived DNA, and proteolytic enzymes for digestion.

Recruited cells also secrete matrix MMPs, proteases and inflammatory cytokines resulting in a more powerful inflammation. Repression of pro-inflammatory signaling, explained by reduced IL-6, TNF and MMPs, marks the end of this phase. This is then followed by the proliferative phase of healing. Starting from day 3 following the ischemia, neutrophils start to decline and entirely disappear at day 7. Of note, Horckmans and colleagues showed that neutrophils are important effectors in cardiac healing response after MI. They found that neutrophil-depleted mice had worse cardiac function and HF, associated to an increased fibrosis. Moreover, they identified a role of neutrophils in polarizing macrophages towards a reparative phenotype through the release of neutrophil gelatinase-associated lipocalin [START_REF] Horckmans | Neutrophils orchestrate post-myocardial infarction healing by polarizing macrophages towards a reparative phenotype[END_REF]. In turn, monocytes continue accumulating in the injured myocardium and differentiate into Ly6C low CCR2 + CX3CR1 high monocytes (Ly6C lo ) implicated in a strong VEGF secretion, as well as reparative macrophages. These macrophages produce IL-10 which results in inflammation attenuation. Moreover, they secrete TGF-β leading to collagen production by FBs. Macrophages also release VEGF. Indeed, during this phase, there is new vessel formation and/or remodeling of pre-existing vasculature, mainly under the control of local proangiogenic program, immuno-inflammatory balance and circulating/local vascular progenitor cells (Silvestre et al., 2013). Furthermore, there is an accumulation of mast cells reaching a peak at day 7 after MI. Notably, mast cells have a beneficial role against cardiac dysfunction through the modulation of CM contractility by regulating myofilament Ca 2+ sensitization via alteration of protein kinase A activity after MI [START_REF] Ngkelo | Mast cells regulate myofilament calcium sensitization and heart function after myocardial infarction[END_REF]. Moreover, regulatory T (Treg) cells actively participate in the cardiac reparative response. Remarkably, it has been demonstrated

that Treg cell-deficient mice had an aggravated cardiac inflammation and deteriorated cardiac function following MI. Namely, Treg-cell activation induced an M2-like macrophage differentiation, thus participating to wound healing post-MI [START_REF] Weirather | Foxp3+ CD4+ T cells improve healing after myocardial infarction by modulating monocyte/macrophage differentiation[END_REF].

Furthermore, it has been reported that LB peaked at around day 5 after the onset of ischemia [START_REF] Zlatanova | Immune Modulation of Cardiac Repair and Regeneration: The Art of Mending Broken Hearts[END_REF]. Interestingly, Wu and coworkers found that these cells improve the outcome of MI by producing IL-10. Notably, Il-10 specific deletion in LB worsened cardiac function and delayed the resolution of inflammation following MI [START_REF] Wu | IL-10-producing B cells are enriched in murine pericardial adipose tissues and ameliorate the outcome of acute myocardial infarction[END_REF]. The end of this phase is marked by infarct maturation and scar formation consisting of cross-linked collagen which takes place within weeks following the onset of ischemia.

As a result of TGF-β production, FBs migrate and transdifferentiate into myofibroblasts secreting large amounts of ECM proteins, leading to a scar tissue formation in order to replace necrotic cells. In addition to resident cardiac FBs, BM-derived FB progenitors, ECs undergoing transdifferentiation into mesenchymal cells, SMCs and PCs might contribute to the infarct myofibroblasts population. Of note, an imbalance between MMPs having the role of degrading ECM and their endogenous tissue inhibitors (TIMPs) can result in adverse remodeling and outcome. Moreover, an abnormal decrease or increase in myocardial collagen leads to LV dilatation and rupture and an increased stiffness, respectively [START_REF] Frangogiannis | The inflammatory response in myocardial injury, repair, and remodelling[END_REF].

Here, it is important to note that a deregulated inflammation and/or an excessive fibrosis may reduce ventricular compliance and contributes to an adverse ventricular remodeling contributing to an overall decline in cardiac function and increased likelihood of HF and mortality.

ii. Revascularization

When tissue's need for oxygen augments, blood flow in the capillaries increases to satisfy its demand. In some conditions, oxygen supply remains insufficient. In order, to ensure an equilibrium between supply and demand, new vessels are formed or a remodeling of preexisting vasculature occurs. Following ischemia, for example, different processes of revascularization including vasculogenesis, angiogenesis, arteriogenesis and collateral growth take place in order to establish a functional vascular network in ischemic zones.

Vasculogenesis is the process by which endothelial stem/progenitor cells (EPCs) proliferate and differentiate to form tubules of ECs which then join together and form a plexus where blood flow remains chaotic and nonfunctional. The differentiation of this plexus as well as its organization into a network of capillaries or in other words, the formation of capillaries from pre-existing primitive tubules is known as angiogenesis. This is then followed by arteriogenesis which is defined as the formation of new arterioles from existing capillaries. During this phase, VSMCs and PCs are produced in addition to ECM having the role to consolidate the new formed vessels. In addition, development and remodeling of preexisting arterioles form the collateral growth which can later give rise to a functional vascular structure. In ischemic conditions, collateral growth has the role to perfuse the region affected by the ischemia in order to protect it against necrosis (Silvestre et al., 2013).

Hypoxia, explained by a lack of oxygen due to a reduced tissue perfusion following ischemia, activates the hypoxia inducible factor (HIF) which is implicated in all steps of the postischemic revascularization. It has been shown that intramyocardial injection of a plasmid encoding HIF-1α was responsible for infarct size reduction and neovascularization increase following MI in the rat [START_REF] Shyu | Intramyocardial injection of naked DNA encoding HIF-1alpha/VP16 hybrid to enhance angiogenesis in an acute myocardial infarction model in the rat[END_REF]. Moreover, HIF-1α regulates the expression of VEGF-A and its receptor VEGFR1, stroma-derived factor 1 or SDF-1 (CXCL12), angiopoietin 2, PDGF-BB, and stem cell factor or c-Kit ligand (SCF), all implicated in neogenesis and vascular remodeling (Silvestre et al., 2013). For instance, NO and ROS participate in the effects of VEGF in the angiogenic response. It has been shown that VEGF-mediated angiogenesis was dependent on eNOS in mouse following hindlimb ischemia [START_REF] Murohara | Nitric oxide synthase modulates angiogenesis in response to tissue ischemia[END_REF].

Furthermore, it has been demonstrated that in both cases, when nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 2 (Nox2), source of ROS, was depleted and when ROS are overproduced, post-ischemic neovascularization was impaired [START_REF] Ebrahimian | NADPH oxidase-derived overproduction of reactive oxygen species impairs postischemic neovascularization in mice with type 1 diabetes[END_REF][START_REF] Tojo | Role of gp91phox (Nox2)-containing NAD(P)H oxidase in angiogenesis in response to hindlimb ischemia[END_REF]. In turn, immune system also plays a central role in angiogenesis in a paracrine way through production of VEGF and interleukins as IL-1β, IL-2, IL-20, as well as proangiogenic proteases such as MMP-9, and by releasing small extracellular vesicles derived

iii. Lymphangiogenesis

In the heart, lymphatic vessels form an extensive network. They accompany blood vessels and play an essential role in myocardial fluid homeostasis by ensuring the return of extravasated fluid and solutes from the tissue interstitium into the blood circulation. According to Henri and colleagues, while the blood vessel-to-CM ratio is ~1:1, the lymphatic-to-CM ratio is 1:10 [START_REF] Henri | Selective Stimulation of Cardiac Lymphangiogenesis Reduces Myocardial Edema and Fibrosis Leading to Improved Cardiac Function Following Myocardial Infarction[END_REF]. Lymphatic vessels also contribute to immuno-modulation by draining tissue antigens and cytokines released during inflammation, and reuptaking infiltrating cells such as DCs, macrophages, and lymphocytes, and transporting them to the lymph nodes where they participate to immune response. In addition, they participate to lipid absorption from the gut [START_REF] Brakenhielm | Cardiac lymphatics in health and disease[END_REF][START_REF] Klotz | Cardiac lymphatics are heterogeneous in origin and respond to injury[END_REF]. Given the essential role accomplished by the lymphatic compartment, it is important to note that changes affecting the heart and its components could highly impact cardiac lymphatic vessels structure and function resulting in deleterious events. Notably, lymph propulsion from the heart highly depends on its contractile force. It has been shown that a decrease in cardiac contractility results in a reduced cardiac lymphatic flow [START_REF] Taira | Flow velocity of cardiac lymph and contractility of the heart: an experimental study[END_REF]. Moreover, some dysregulations occurring in the blood vessels such as an excessive blood capillary ultrafiltration, and an increased permeability, or in the lymphatic vessels explained by an insufficient uptake of tissue fluid in the presence or absence of a poor drainage could result in an edema which occurs in many CVDs such as MI and HF [START_REF] Brakenhielm | Cardiac lymphatics in health and disease[END_REF]. It has been shown that excessive myocardial edema potentiates interstitial fibrosis and compromises cardiac function in dog models of chronic pulmonary hypertension with right HF and chronic arterial hypertension [START_REF] Laine | Left ventricular myocardial edema. Lymph flow, interstitial fibrosis, and cardiac function[END_REF].

(
Studies have shown that lymphatic vessels play crucial role in the heart following MI where they undergo lymphangiogenesis, essentially generating small capillary lymphatics and some large vessels [START_REF] Henri | Selective Stimulation of Cardiac Lymphangiogenesis Reduces Myocardial Edema and Fibrosis Leading to Improved Cardiac Function Following Myocardial Infarction[END_REF]. Remarkably, Klotz and colleagues observed a significant increase in VEGF-C protein levels in mice hearts from 24 hours up to 21 days post-MI, and this was accompanied with increased number and diameter of lymphatic vessels localized in the border zone of the infarcted region at day 7 and persisted to day 35. This response was enhanced following adeno-associated viral gene delivery of VEGF-CC156S treatment responsible for cardiac function improvement [START_REF] Klotz | Cardiac lymphatics are heterogeneous in origin and respond to injury[END_REF]. Moreover, Henri and coworkers found that lymphatic density was increased in the infarcted region of rats' heart by 12 weeks post-MI with a preferential growth of small lymphatic capillaries. This was associated with elevated levels of VEGF-C and VEGFR3 from 4 to 12 weeks. However, larger precollector vessels decreased in number, and other epicardial vessels undergo adverse remodeling, thus affecting the lymphatic drainage capacity of the heart resulting in myocardial edema and inflammation and aggravating the fibrosis and cardiac function. Interestingly, the researchers found that VEGF-CC156S-therapy was beneficial as it improved lymphatic vessels remodeling, reduced edema and inflammation, and prevented fibrosis and cardiac dysfunction [START_REF] Henri | Selective Stimulation of Cardiac Lymphangiogenesis Reduces Myocardial Edema and Fibrosis Leading to Improved Cardiac Function Following Myocardial Infarction[END_REF]. Recently, Houssari et al., discovered that both CD4 + and CD8 + T cells suppress, through interferon-γ (IFN-γ), cardiac lymphangiogenesis post-MI [START_REF] Houssari | Lymphatic and Immune Cell Cross-Talk Regulates Cardiac Recovery After Experimental Myocardial Infarction[END_REF].

iv. Aryl Hydrocarbon Receptor: a potent biomarker for coronary artery disease

Human aryl hydrocarbon receptor (ahr) gene, located on chromosome 7p15, is around 50 kb in size. It contains at least 11 exons and 10 introns [START_REF] Huang | AhR expression and polymorphisms are associated with risk of coronary arterial disease in Chinese population[END_REF]. ahr mRNA expression is found in the placenta, lung, heart, pancreas and liver with a lower expression in the kidney, brain and skeletal muscle. It is also detected in pulmonary microvasculature, aortic arch and umbilical vein [START_REF] Zhang | The role of endogenous aryl hydrocarbon receptor signaling in cardiovascular physiology[END_REF]. AHR is a transcription factor belonging to the basic helix-loophelix PerArnt-Sim family of DNA-binding proteins. It controls the toxicity of environmental compounds. Toxic environmental polluants include halogenated aromatic hydrocarbons such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and polycyclic aromatic hydrocarbons like tobacco procarcinogens. Inactive AHR is present in the cytoplasm and forms a complex with HSP90. In addition to toxic compounds, it has been reported that AHR is activated by kynurenine (Kyn) and Kyn-derived metabolites generated from Tryptophan (Trp) metabolism in the Kynurenine pathway (KP), the diet and the gut microbiota-derived metabolites (Opitz et al., 2011;[START_REF] Taleb | Tryptophan Dietary Impacts Gut Barrier and Metabolic Diseases[END_REF]. Binding of endogenous or exogenous ligands mediates its activation explained by its translocation from the cytoplasm to the nucleus where it dissociates from the chaperone complex, then dimerizes with the AHR nuclear translocator (ARNT) also known as HIF-1β, and combines with the xenobiotic-responsive element (XRE) in the target gene promoter region followed by transactivation of the downstream signaling pathways related to cellular toxicology [START_REF] Huang | AhR expression and polymorphisms are associated with risk of coronary arterial disease in Chinese population[END_REF]. Some examples of AHR target genes include cytochrome P450 1A1 (cyp1a1), cytochrome P450 1B1 (cyp1b1), and the gene coding for NOX [START_REF] Zhang | The role of endogenous aryl hydrocarbon receptor signaling in cardiovascular physiology[END_REF].

AHR plays an important role in cardiovascular physiology including heart development and function, vascular development, and BP regulation. For instance, studies have shown that both mice harboring total ahr deletion and those with EC-specific ahr deletion were hypotensive (Agbor et al., 2011;[START_REF] Zhang | An activated renin-angiotensin system maintains normal blood pressure in aryl hydrocarbon receptor heterozygous mice but not in null mice[END_REF]. Wang and colleagues demonstrated that mice harboring ahr specific deletion in CMs were protected against heart dysfunction due to NKX2.5 haploinsufficiency, where Nkx2.5 gene is specific for CMs and is implicated in the regulation of cardiac myogenesis and heart morphogenesis (Wang et al., 2017a). Moreover, AHR was found to be implicated in CM apoptosis and cardiotoxicity induced by chemotherapeutic drug and toxic compound [START_REF] Ansari | The role of aryl hydrocarbon receptor signaling pathway in cardiotoxicity of acute lead intoxication in vivo and in vitro rat model[END_REF]. Notably, lead (Pb 2+ ), a naturally toxicant heavy metal, induced cardiotoxicity and HF through AHR/CYP1A1-mediated mechanism. This was explained by an increased level of cardiac lactate dehydrogenase which is a biomarker for cardiotoxicity, in addition to an increased interstitial fibrosis, and dilated intermyofibrillar areas in the rat heart [START_REF] Ansari | The role of aryl hydrocarbon receptor signaling pathway in cardiotoxicity of acute lead intoxication in vivo and in vitro rat model[END_REF]. Another study demonstrated that CMs exposure to TCDD, resulting in AHR/CYP1A1 overexpression, led to an attenuated mitochondrial activity and an increased mitochondrial oxidative stress in these cells [START_REF] Zhou | Mitochondrial activity and oxidative stress functions are influenced by the activation of AhR-induced CYP1A1 overexpression in cardiomyocytes[END_REF].

AHR is also implicated in the development of CVDs such as atherosclerosis and MI. In atherosclerosis, three mechanisms have been proposed to be involved in AHR mediated effects on inflammation and disease progression including monocyte chemotaxis through the VCAM-1 via the AHR/nuclear factor-κB (NF-κB) signaling pathway, macrophages absorption of ox-LDL and foam cell formation, in addition to VSMCs proliferation via the activation of the NF-κB signaling pathway and ROS production which also participates to ECs damage and reduction of vascular endothelium integrity [START_REF] Zhu | Aryl hydrocarbon receptor pathway: Role, regulation and intervention in atherosclerosis therapy (Review)[END_REF]. Moreover, a study in Chinese population comprising 939 patients with CAD and 868 control subjects revealed that AHR expression and polymorphisms are associated with risk of CAD [START_REF] Huang | AhR expression and polymorphisms are associated with risk of coronary arterial disease in Chinese population[END_REF].

Interestingly, Xue and coworkers found that AHR inhibition by baicalin, an ingredient found in a Chinese herb used for its antithrombotic, antihyperlipidemic, anti-inflammatory, antiarrhythmic and antihypertensive properties, attenuated myocardial injury and inflammatory response in a mouse model of myocardial ischemia [START_REF] Xue | Baicalin inhibits inflammation and attenuates myocardial ischaemic injury by aryl hydrocarbon receptor[END_REF]. Remarkably, ahr ablation ameliorated ischemia-induced angiogenesis in a mouse model of femoral artery occlusion due to an increased activity of the HIF-1α-ARNT heterodimer and enhanced expression of its target gene, VEGF [START_REF] Ichihara | A role for the aryl hydrocarbon receptor in regulation of ischemiainduced angiogenesis[END_REF].

Therefore, these findings let us consider the AHR as a potential target in the pathology of MI.

Interestingly, they open new pharmacological perspectives in limiting adverse ventricular remodeling following MI by inhibiting this receptor.

II. Indoleamine 2, 3-dioxygenase 1 (IDO)

Indoleamine 2, 3-dioxygenase 1 (IDO, EC1.13.11.42) was first discovered in rabbit small intestine by Hayaishi andcolleagues in 1967 (Higuchi andHayaishi, 1967;[START_REF] Yamamoto | Tryptophan pyrrolase of rabbit intestine. D-and Ltryptophan-cleaving enzyme or enzymes[END_REF]. IDO [UniProt ID P14902 (human), P28776 (mouse), Q9ERD9 (rat), P47125

(yeast)] is a non-secreted intracellular enzyme widely distributed throughout the organism. It is active under inflammatory states and mainly induced by IFN-γ [START_REF] Ball | Characterization of an indoleamine 2,3-dioxygenase-like protein found in humans and mice[END_REF]. IDO catalyzes the first and rate limiting step of Trp metabolism via KP, involving oxidation of L-Trp to N-formylkynurenine (NFK). Activation of KP participates to different physiological and pathological effects. In pregnancy, for example, it suppresses the immune response. In chronic inflammatory diseases including cancer, it suppresses effector T cells and stabilizes Treg cells, thus suppressing antitumor immunity to promote tumorigenesis [START_REF] Lemos | Immune control by amino acid catabolism during tumorigenesis and therapy[END_REF]. KP also mediates host response to microbial infection as well as non-immune biological effects like in neuropathology, control of vascular tone and BP [START_REF] Yeung | Role of indoleamine 2,3-dioxygenase in health and disease[END_REF]. Most recently, evidence of significant activation of the KP had been found in the recent COVID-19 pandemic, where derived-metabolites Kynurenate, Kyn, and 8-methocykynurenate were elevated in patients suffering from this disease [START_REF] Shen | Proteomic and Metabolomic Characterization of COVID-19 Patient Sera[END_REF].

In this chapter, I will explain Trp metabolism via KP, then describe IDO starting from the molecular mechanisms controlling its expression, passing by its enzymatic activity and catalytic mechanism, and ending by discussing its role in physiological and pathophysiological settings.

A. Tryptophan metabolism

Amino acids (AAs) are organic compounds containing amine (-NH2) and carboxyl (-COOH) functional groups, along with a side chain (R group) specific to each AA. Currently, ~500 AAs are known. Among them, only 20 standard AAs appear in the genetic code. They are naturally incorporated into polypeptides and are called proteinogenic. Hence, they serve as basic components for protein synthesis and constitute an important energy source in addition to glucose and fatty acids [START_REF] Nitz | Amino Acids and Their Metabolism in Atherosclerosis[END_REF]. Nine of them are essential because they cannot be synthesized endogenously, and are therefore supplied in the diet. TAT1 encoded by the SLC16A10 gene found in the basolateral epithelial membrane. Then it passes into the bloodstream where its transport requires a plasma albumin binding due to the fact that it is highly lipophilic. It is then delivered to tissues including muscles and liver before it is taken by cells where it serves for protein synthesis. Of note, its passage into the central nervous system (CNS) requires a transporter formed by a protein belonging to the superfamily of L-type AA transporter 1 (LAT1), and an accessory protein, the cell surface antigen CD98

(heavy chain 4F2), which is highly expressed in the barrier capillary endothelium [START_REF] Palego | Tryptophan Biochemistry: Structural, Nutritional, Metabolic, and Medical Aspects in Humans[END_REF].

Trp exists in two forms, L-Trp and D-Trp. It is the only AA that contains the structure of an indole which is a bicyclic compound consisting of a benzene ring fused to a N-containing pyrrole group [START_REF] Roager | Microbial tryptophan catabolites in health and disease[END_REF]. Trp is implicated in protein synthesis. However, its participation accounts for less than 1% of its dietary degradation (Badawy, 2017b). In addition to protein synthesis, dietary Trp is metabolized by 3 known pathways (Figure 8). The quantitatively most important is the KP which accounts for ~95% of overall Trp degradation [START_REF] Taleb | Tryptophan Dietary Impacts Gut Barrier and Metabolic Diseases[END_REF]. This major route of L-Trp catabolism leads ultimately to the formation of oxidized nicotinamide adenine dinucleotide (NAD + ), selectively in liver. Of note, niacins are alternative sources of precursor to form NAD. The first and rate limiting step of KP is O2-dependent and involves oxidation of L-Trp to NFK. In liver cells, it is constitutively catalyzed by tryptophan 2, 3-dioxygenase (TDO, also known as tryptophan oxygenase and L-tryptophan pyrrolase; EC 1.13.11.11). In other cell types, Trp can be metabolized by the inducible IDO (EC 1.13.11.17). Another less important pathway is the serotonin pathway, which accounts for ~1-2%. It consists on a Trp hydroxylation, by Trp Hydroxylase 1, resulting in its conversion into the neurotransmitter, serotonin or 5-hydroxytryptamin (5-HT) and melatonin, mainly in the gastrointestinal tract [START_REF] Taleb | Tryptophan Dietary Impacts Gut Barrier and Metabolic Diseases[END_REF]. In the human CNS, 5-HT regulates mood-anxiety, cognition, nociception, impulsivity, aggressiveness, libido, feeding behavior, and body temperature. Among its numerous roles, 5-HT is also known to modulate the gut function, the immune and inflammatory responses, the differentiation process of blood stem cells, and the hemodynamic function. Remarkably, alterations affecting 5-HT transmission could result in many disorders such as mood-affective disorders, autism, anorexia or bulimia nervosa [START_REF] Palego | Tryptophan Biochemistry: Structural, Nutritional, Metabolic, and Medical Aspects in Humans[END_REF]. The third pathway is the indole pathway which accounts for ~5% of overall Trp degradation. Trp is converted by gut microbiota into Tryptamine via the action of decarboxylase and indole metabolites via the action of tryptophanase. Gut microbiota constitutes an important barrier and the first defense line against harmful pathogens in the intestine where digestion and absorption of nutrients occur. Of note, Trp metabolism is a key modulator of host-gut microbiota cross-talk and is of major importance in health and diseases [START_REF] Borghi | Tryptophan as a Central Hub for Host/Microbial Symbiosis[END_REF]. Notably, in homeostatic condition, the host converts Trp into Kyn, and the microbiota converts Trp into indole metabolites. Indoles are key effectors in gut homeostasis. It has been reported that they participate to mucosal defense against pathogens by activating AHR, resulting in IL-22 production and antimicrobial peptide induction. They also promote epithelial barrier function by stimulating mucin production and enhancing tight junction proteins. Moreover, indoles are involved in hyperglycemia where they induce the production of incretin in enteroendocrine L cells [START_REF] Chimerel | Bacterial metabolite indole modulates incretin secretion from intestinal enteroendocrine L cells[END_REF][START_REF] Zelante | Tryptophan catabolites from microbiota engage aryl hydrocarbon receptor and balance mucosal reactivity via interleukin-22[END_REF].

Incretin is a glucagon-like peptide 1, which stimulates insulin secretion, appetite suppression, and inhibits gastrointestinal motility and secretion. In obesity, Trp metabolism dysregulation, explained by an increased IDO activity in the host, may cause microbiota dysbiosis due to a decreased indole production, which has as consequence, an enhanced intestinal permeability and inflammation (Laurans et al., 2018).

B. Tryptophan metabolism in kynurenine pathway

KP is the major route for Trp metabolism. It is important for detoxification of excess Trp. This function is exclusively insured by TDO mainly expressed in the liver where it constitutively oxidizes Trp, under normal physiologic conditions. Liver cells contain a complete set of enzymes necessary for nicotinamide (NAM or vitamin B3) and oxidized NAD + synthesis and complete oxidation of Trp to carbon dioxide (CO2) and water (Badawy, 2017b). In this case, KP constitutes 90% of total Trp metabolism [START_REF] Wang | Tryptophan-kynurenine pathway is dysregulated in inflammation, and immune activation[END_REF]. KP is also widely distributed extrahepatically but its contribution to Trp degradation is normally minimal (5%-10%). In this case, Trp is catalyzed by an alternative inducible IDO, under certain pathophysiological conditions, especially inflammatory conditions, which consequently increases the formation of Kyn metabolites.

In the KP, either TDO in the liver or IDO extrahepatically converts L-Trp, by opening pyrrole moiety, to NFK which is then hydrolyzed to Kyn by NFK formidase (Figure 9). Kyn is then converted to anthranilic acid (AA) by kynureninase or kynurenic acid (Kna) by kynurenine aminotransferase (KAT) and most importantly to 3-hydroxykynurenine (3-OHKyn) by Kynurenine monooxygenase (KMO). 3-OHKyn is further metabolized into 3hydroxyanthranilic acid (3-HAA) by Kynureninase. Then quinolinic acid (Quina) is formed, and the NAD + is produced as an end product of the KP (Badawy, 2017a(Badawy, , 2017b)).

NAD plays an essential role in oxidoreduction reactions in energy metabolism, where oxidized NAD + is reduced to NADH. This latter is produced by krebs cycle and constitutes the major electron donor to the electron transport chain in the mitochondria. In addition, NAD is transformed to nicotinamide adenine dinucleotide phosphate (NADP) by the action of NAD kinase-mediated phosphorylation or the mitochondrial nicotinamide nucleotide transhydrogenase. NADPH is the reduced form of NADP. It is the essential coenzyme of the enzymatic pathways implicated in ROS detoxification. Moreover, NAD is a substrate for signaling enzymes including poly-ADP-ribose-polymerases (PARPs) implicated in DNA damage repair, sirtuins, and CD38 ectoenzymes implicated in Ca 2+ signaling [START_REF] Mericskay | Nicotinamide adenine dinucleotide homeostasis and signalling in heart disease: Pathophysiological implications and therapeutic potential[END_REF]. In mammals, 4 pathways lead to NAD synthesis: de novo from Trp in the liver, the Preiss-Handler pathway from nicotinic acid (NA), the salvage pathway from NAM, and the nicotinamide riboside (NR) kinase pathway from NR. It has been shown that NAD metabolism is tissue-specific. The liver synthesizes NAD from Trp and orally delivered NR, then excretes it in the form of NAM in the circulation where it is taken by the other tissues to synthesize their need of NAD with a capacity for the muscle to produce NAD from NR after intravenous administration of this latter. Moreover, researchers found that NAD production and consumption rates (fluxes) were high in small intestine and spleen and low in skeletal muscle [START_REF] Liu | Quantitative Analysis of NAD Synthesis-Breakdown Fluxes[END_REF]. Given the fundamental biological function of NAD, alterations of its homeostasis has been implicated in physiological conditions like aging, as well as pathological conditions. It has been reported that NAD depletion leads to mitochondrial dysfunction and nuclear damage [START_REF] Fang | Nuclear DNA damage signalling to mitochondria in ageing[END_REF]. Moreover, NR kinase 2 deficiency in aged mice resulted in a maladaptive metabolic response in cardiac and skeletal muscle following an endurance exercise training [START_REF] Deloux | Aged Nicotinamide Riboside Kinase 2 Deficient Mice Present an Altered Response to Endurance Exercise Training[END_REF]. Another study showed that NR protects against excitotoxicity-induced axonal degeneration and that this protection implies intracellular NAD + homeostasis [START_REF] Vaur | Nicotinamide riboside, a form of vitamin B3, protects against excitotoxicity-induced axonal degeneration[END_REF]. In addition, Diguet and colleagues found a 30% decrease in NAD + levels in two mouse models of HF: the dilated cardiomyopathy and cardiac hypertrophy.

Remarkably, a significant increase in the NR kinase 2, implicated in NR phosphorylation, was observed in a mouse model of dilated cardiomyopathy, in addition to human failing hearts.

Interestingly, NR supplementation in food preserves cardiac function, by stabilizing myocardial NAD + levels [START_REF] Diguet | Nicotinamide Riboside Preserves Cardiac Function in a Mouse Model of Dilated Cardiomyopathy[END_REF]. Furthermore, it has been demonstrated that AF mediated-DNA damage and atrial CM loss activates PARP1 leading to NAD + depletion and further DNA damage and CM dysfunction [START_REF] Zhang | DNA damage-induced PARP1 activation confers cardiomyocyte dysfunction through NAD+ depletion in experimental atrial fibrillation[END_REF].

C. IDO Biochemistry i. Molecular mechanisms a. Ido-1 gene

Ido-1 gene also known as Indo is located in a pericentromeric region on chromosome 8. More specifically, on chromosome 8p12-p11 in humans and 8 A2 in mice. This gene measures about 15 kilobase pairs (kb) with ten exons and nine introns [START_REF] Burkin | Localization of Human Indoleamine 2,3-Dioxygenase (IDO) Gene to the Pericentromeric Region of Human Chromosome 8[END_REF]. Ido-1 gene encodes a monomeric protein of ~45 kDa constituted of 403 AAs [START_REF] Yeung | Role of indoleamine 2,3-dioxygenase in health and disease[END_REF]. In humans, some genetic variants of Ido-1 like single nucleotide polymorphisms (SNPs) and base pair deletions lead to modifications in IDO protein expression and activity. In human patients, these genetic variants are associated with conditions like autoimmune systemic sclerosis and aspergillosis [START_REF] Napolioni | Genetic Polymorphisms Affecting IDO1 or IDO2 Activity Differently Associate With Aspergillosis in Humans[END_REF] or correlated with reduced risk of recurrent vulvovaginal candidiasis [START_REF] Yeung | Role of indoleamine 2,3-dioxygenase in health and disease[END_REF].

b. Ido-1 gene transcription 1. Interferon-γ -dependent transcription

Ido-1 gene expression requires a stimulus like IFN-γ. This latter is the most potent for Ido-1 induction in vitro [START_REF] Kadoya | Gene structure of human indoleamine 2,3-dioxygenase[END_REF] and in vivo [START_REF] Jeong | Curcumin suppresses the induction of indoleamine 2,3-dioxygenase by blocking the Janus-activated kinase-protein kinase Cdelta-STAT1 signaling pathway in interferon-gamma-stimulated murine dendritic cells[END_REF]. IFN-γ activates the Janus kinase/signal transducer and activator of transcription (JAK/STAT) and protein kinase C-delta (PKCδ) signaling pathways. Together with IRF1, they are required for an optimal IFNγ -induced Ido-1 expression. Interaction of IRF1 and STAT1, respectively with two interferonstimulated response elements (ISREs) and three IFN-γ -activated sites (GASs) present in Ido-1 promoter region, initiates Ido-1 gene transcription [START_REF] Yeung | Role of indoleamine 2,3-dioxygenase in health and disease[END_REF].

TNFα is another inducer of Ido-1 gene transcription. While it cannot exert its role alone, TNFα participates synergically with IFN-γ to maximize the signal. It binds to its receptor and stimulates NF-κB pathway. This is accompanied with an increase in IRF1 transcription and an activation of STAT1 followed by an up-regulation of IFN-γ receptor expression and a potentiation of IFN-γ signaling. Moreover, stimuli like type I IFNs (IFNα and IFNβ), IFNλ, IL-1, IL-6, TLR 3, 4, 7, 8 and 9 ligands, thymosin α1, estrogen, prostaglandin E2 (PGE2), muramyl tripeptide, α-galactosylceramide, glucocorticoids, haemoglobin and certain infectious pathogens are other inducers of Ido-1 gene expression. In this case, induction can be dependent or independent from IFN-γ [START_REF] Yeung | Role of indoleamine 2,3-dioxygenase in health and disease[END_REF].

Cyclo-oxygenase 2 (COX2)-derived PGE2 is also a possible mechanism to induce Ido-1 expression. It has been reported that COX inhibitors prevent Ido-1 induction in mouse lung slices treated with IFN-γ [START_REF] Sayama | Inhibition of interferon-mediated induction of indoleamine 2,3-dioxygenase in mouse lung by inhibitors of prostaglandin biosynthesis[END_REF]. Moreover, it has been shown that COX2derived PGE2 is responsible for IDO increase in tumor cells as in breast cancer [START_REF] Basu | Cyclooxygenase-2 inhibitor enhances the efficacy of a breast cancer vaccine: role of IDO[END_REF]. Ido-1 gene transcription can also be induced by ligation of CD80/86 with cytotoxic Tlymphocyte-associated protein 4 (CTLA-4) as shown in DCs, in an IFN-γ-dependent or independent mechanism. Another way is via interaction of the glucocorticoid-induced TNF receptor-related protein ligand (GITRL) with its receptor GITR expressed on Treg cells (de [START_REF] De Araújo | The IDO-AhR Axis Controls Th17/Treg Immunity in a Pulmonary Model of Fungal Infection[END_REF][START_REF] Li | Tolerogenic Phenotype of IFN-γ-Induced IDO+ Dendritic Cells Is Maintained via an Autocrine IDO-Kynurenine/AhR-IDO Loop[END_REF][START_REF] Yeung | Role of indoleamine 2,3-dioxygenase in health and disease[END_REF].

Interferon-γ-independent transcription

Ido-1 expression can also be achieved by IFN-γ-independent mechanisms. In addition to its role in inducing Ido-1 transcription in combination with IFN-γ, NF-κB pathway can act independently from IFN-γ when stimulated with lipopolysaccharide (LPS) and TGF-β [START_REF] Fujigaki | The signal transducer and activator of transcription 1alpha and interferon regulatory factor 1 are not essential for the induction of indoleamine 2,3-dioxygenase by lipopolysaccharide: involvement of p38 mitogen-activated protein kinase and nuclear factor-kappaB pathways, and synergistic effect of several proinflammatory cytokines[END_REF], CpG oligodeoxynucleotides (CpG-ODNs) [START_REF] Volpi | A GpC-rich oligonucleotide acts on plasmacytoid dendritic cells to promote immune suppression[END_REF], viral infection [START_REF] Liu | Epstein-Barr virus infection induces indoleamine 2,3-dioxygenase expression in human monocyte-derived macrophages through p38/mitogen-activated protein kinase and NF-κB pathways: impairment in T cell functions[END_REF][START_REF] Yeung | Flavivirus infection induces indoleamine 2,3-dioxygenase in human monocyte-derived macrophages via tumor necrosis factor and NF-κB[END_REF] and CD80/86 ligation [START_REF] Koorella | Novel regulation of CD80/CD86-induced phosphatidylinositol 3-kinase signaling by NOTCH1 protein in interleukin-6 and indoleamine 2,3-dioxygenase production by dendritic cells[END_REF].

It is important to note that following induction, IDO enzymatic activity is maintained thanks to IDO-Kyn/AhR-IDO feed-forward loop [START_REF] Li | Tolerogenic Phenotype of IFN-γ-Induced IDO+ Dendritic Cells Is Maintained via an Autocrine IDO-Kynurenine/AhR-IDO Loop[END_REF].

Ido-1 gene repression

While stimuli, like the ones mentioned above, induce Ido-1 gene expression, some others down-regulate it. Some examples of negative regulators are IL-4, IL-6, TGF-β as well as the transcription repressor B lymphocyte-induced maturation protein-1 (BLIMP1) activated in response to L-Trp deficiency [START_REF] Barnes | Amino acid deprivation links BLIMP-1 to the immunomodulatory enzyme indoleamine 2,3-dioxygenase[END_REF]. It is important to note first that regulator stimuli of Ido-1 gene expression may exert opposing effects depending on the existing conditions. Munn and colleagues found that IFN-γ was responsible for constitutive Ido-1 expression inhibition in CD40L-matured CD123+ human DCs, but treatment with IL-10 reversed this down-regulation [START_REF] Munn | Potential regulatory function of human dendritic cells expressing indoleamine 2,3-dioxygenase[END_REF]. However, it has been shown that LPSinduced Ido-1 gene expression was inhibited by IL-10 in murine BM-derived DCs [START_REF] Jung | Blockade of indoleamine 2,3-dioxygenase protects mice against lipopolysaccharide-induced endotoxin shock[END_REF]. In addition, Ido-1 gene expression is cell-specific. For instance, TGFβ has been found

to inhibit IFN-γ -induced Ido-1 expression in cultured FBs [START_REF] Yuan | Modulation of cellular tryptophan metabolism in human fibroblasts by transforming growth factor-beta: selective inhibition of indoleamine 2,3-dioxygenase and tryptophanyl-tRNA synthetase gene expression[END_REF] and to induce its expression in plasmacytoid dendritic cells (pDCs) [START_REF] Pallotta | Indoleamine 2,3-dioxygenase is a signaling protein in long-term tolerance by dendritic cells[END_REF].

c. IDO protein structure

In 2006, Sugimoto and colleagues were the first to identify the crystal structure of the human IDO protein [START_REF] Sugimoto | Crystal structure of human indoleamine 2,3-dioxygenase: catalytic mechanism of O2 incorporation by a heme-containing dioxygenase[END_REF]. IDO is a monomeric enzyme that belongs to the mammalian heme dioxygenase enzymes. It contains two α-helical domains: a large domain and a small one connected by a long loop [START_REF] Rafice | Oxidation of Ltryptophan in biology: a comparison between tryptophan 2,3-dioxygenase and indoleamine 2,3-dioxygenase[END_REF]) (Figure 10). Within its large domain, IDO enzyme contains a cavity for the active-site heme covered by the small domain. Ser 263 are essential for positioning the substrate, as L-Trp, within the distal active-site heme pocket [START_REF] Yeung | Role of indoleamine 2,3-dioxygenase in health and disease[END_REF].

Figure 10 Crystal structure of human IDO. IDO monomeric protein with two α-helical domains: the large one (red, orange, yellow and green) and the small one (blue and violet). This scheme is from Yeung and colleagues' manuscript [START_REF] Yeung | Role of indoleamine 2,3-dioxygenase in health and disease[END_REF].

Catalytic mechanism

The reaction involves L-Trp substrate oxidation to NFK by IDO enzyme. When produced, IDO enzyme exists under its inactive form, a heme-free apoenzyme. When the heme group binds to the apoenzyme, this latter becomes an inactive holoenzyme with an inactive ferric (Fe III ) state.

Its activity requires both dioxygen (O2) and substrate binding notably to L-Trp. IDO becomes an active holoenzyme with an active ferrous (Fe II ) state, able to produce NFK from L-Trp (Figure 11). Reaction is initiated when the ternary complex Fe III -superoxo-Trp is formed. It This scheme is from Yeung and colleagues' manuscript [START_REF] Yeung | Role of indoleamine 2,3-dioxygenase in health and disease[END_REF].

Post-translational IDO regulation

IDO control at transcriptional and post-translational levels is substantial. Indeed, an unbalanced IDO production could be detrimental to the host. For instance, an excessive IDO activity leads to a high depletion of the essential AA, L-Trp, and an overproduction of KP metabolites which could have cytotoxic effects [START_REF] Yeung | Role of indoleamine 2,3-dioxygenase in health and disease[END_REF]. It is possible that IDO protein is expressed in the cell where it stays inactive or becomes active under a specific condition. Here are some evidences supporting the post-translational control of IDO. For example, inactive IDO protein has been reported to be expressed in the lung of mice with chronic granulomatous disease (CGD) suffering from fungal infection (Romani et al., 2008). Moreover, IDO enzyme in human DC remains inactive until it is activated upon stimulation with IFNγ [START_REF] Munn | Potential regulatory function of human dendritic cells expressing indoleamine 2,3-dioxygenase[END_REF].

IDO activity regulation is exerted through different modes such as heme supply, IDO enzyme cofactors, redox control of IDO, NO, and hydrogen peroxide (H2O2). For instance, heme-free apoenzyme proportion compared with heme-containing holoenzyme is an important modulator of IDO activity. The heme being a principal factor in the regulation of IDO activity, its biosynthesis and degradation rates are determinant. Heme synthesis is performed by deltaaminolevulinic acid synthase (δ-ALAS) which has the role of converting succinyl-CoA to δaminolevulinic acid, the heam precursor. Heme degradation by heme oxygenase 1 (HO-1) results in carbon monoxide (CO), iron and biliverdin production. Moreover, IDO enzyme activation involves reduction of its active-site heme from inactive ferric (Fe III ) state to active ferrous (Fe II ) state. To be achieved, reduction needs a reductant intervention. Notably, O2 is a major intracellular reducing cofactor for IDO. For instance, in the 18 O2-labelling studies aiming to measure oxygen consumption by L-Trp during its oxidation by rabbit IDO enzyme, Hayaishi and colleagues found that O2 • ‾ was incorporated into L-Trp [START_REF] Hayaishi | Indoleamine 2,3-dioxygenase: incorporation of 18O2--and 18O2 into the reaction products[END_REF]. Another important intracellular reductant is cytochrome b5 reductase, which is a protein expressed in the outer membrane of the endoplasmic reticulum. Other reductants are NADPH cytochrome P450 reductase, flavine mononucleotide and tetrahydrobiopterin. In addition, IDO is a heme enzyme rich in redox-active cysteine and tyrosine residues which are subjects for redox signaling by reactive nitrogen species (RNS) and ROS, namely NO, peroxynitrite and H2O2

produced by the cell during inflammation. For instance, H2O2, by oxidizing protein cysteine residues, controls the enzyme activity. In turn, NO binds to heme active sites or reacts with cysteine residues to form protein-S-nitrosothiols via S-nitrosylation. It is known as an IDO inhibitor. For instance, it was found to inhibit IDO activity in mononuclear phagocytes [START_REF] Thomas | Nitric oxide inhibits indoleamine 2,3dioxygenase activity in interferon-gamma primed mononuclear phagocytes[END_REF]. Different mechanisms have been supposed to support NO-mediated inhibition, notably its reversible binding to the active-site heme, proteasomal degradation of IDO, and suppression of Ido-1 gene transcription.

D. IDO inhibitors

The necessity for developing IDO inhibitors was emerged from the fact that anticancer effects of certain targeted drugs as imatinib can be due to IDO blockade [START_REF] Prendergast | Discovery of IDO1 Inhibitors: From Bench to Bedside[END_REF].

However, the usefulness of IDO inhibition in cancer therapy remains uncertain. Many side and off-target effects should be better understood to explain the results of ongoing or discontinued clinical trials [START_REF] Günther | Limitations and Off-Target Effects of Tryptophan-Related IDO Inhibitors in Cancer Treatment[END_REF] .

1-Methyltryptophan (1 MT

) is an IDO inhibitor that exists in two stereoisomers which are levo-1-Methyltryptophan (L-1MT) and dextro-1-methyltryptophan (D-1MT). Of note, L-1MT but not D-1MT inhibits IDO activity as shown in human DCs [START_REF] Lob | Levo-but not dextro-1-methyl tryptophan abrogates the IDO activity of human dendritic cells[END_REF]. In turn, D-1MT preferentially inhibits indoleamine 2, 3-dioxygenase 2 (IDO2) (Metz et al., 2007). In addition, the racemic compound D/L-1MT is also an IDO inhibitor with a Ki for IDO of 34 µmol/L.

According to Prendergast and colleagues, this latter is the most used in the preclinical investigations [START_REF] Prendergast | Discovery of IDO1 Inhibitors: From Bench to Bedside[END_REF]. It has been shown that treatment with L-1MT and melanoma, where it ameliorated vaccine responses explained by a significant reduction of tumor size. Actually, navoximod is in phase I clinical trials tested as a monotherapy or in combination with another drug [START_REF] Nayak-Kapoor | Phase Ia study of the indoleamine 2,3-dioxygenase 1 (IDO1) inhibitor navoximod (GDC-0919) in patients with recurrent advanced solid tumors[END_REF].

iii. Epacadostat (INCB024360)

It is a high selective Trp-competitive IDO inhibitor with a half maximal inhibitory concentration (IC50) of 12 nmol/L. Epacadostat is developed from hydroxyamidine chemotype which is supposed to exert inhibition by directly binding to the iron of the heme in the IDOactive site [START_REF] Prendergast | Discovery of IDO1 Inhibitors: From Bench to Bedside[END_REF]. Many studies demonstrated epacadostat efficacy in ameliorating immunity. It has been reported that more effector T cells, natural killer (NK) cells and CD86 high DCs, and less Treg cells were observed in a co-culture of human allogeneic lymphocytes with DCs or tumor cells treated with epacadostat [START_REF] Liu | Selective inhibition of IDO1 effectively regulates mediators of antitumor immunity[END_REF]. Actually, epacadostat is in phase III trial in melanoma where the results of its combination with an antiprogrammed cell death-1 (anti-PD-1) are really encouraging [START_REF] Prendergast | Discovery of IDO1 Inhibitors: From Bench to Bedside[END_REF].

However, another trial showed that administration of epacadostat and pembrolizumab, a PD-1 inhibitor, to patients with unresectable stage III or IV melanoma did not improve survival in these patients compared to control ones receiving placebo and pembrolizumab [START_REF] Long | Epacadostat plus pembrolizumab versus placebo plus pembrolizumab in patients with unresectable or metastatic melanoma (ECHO-301/KEYNOTE-252): a phase 3, randomised, double-blind study[END_REF].

iv. BMS-986205 and other IDO inhibitors

It is an IDO irreversible inhibitor. Actually, it entered a phase I/II study in melanoma patients.

It is also being tested in other types of cancer [START_REF] Prendergast | Discovery of IDO1 Inhibitors: From Bench to Bedside[END_REF].

PF-06840003 is a Trp noncompetitive, non-heme binding IDO inhibitor. Its capacity to increase human half-life and penetrate to the central nervous system makes of it a promising candidate able to fight cancer especially brain metastases. Moreover, PF-06840003 efficacy was explained by amplifying antitumor effects of anti-PD-1/PD-L1 blockade. Of note, this compound was first tested in 2016 in patients suffering from malignant gliomas [START_REF] Prendergast | Discovery of IDO1 Inhibitors: From Bench to Bedside[END_REF].

Some other IDO inhibitors including IOM2983, RG-70099, and benzylsulfonyl hydrazide series are still in preclinical development [START_REF] Prendergast | Discovery of IDO1 Inhibitors: From Bench to Bedside[END_REF].

E. IDO Tissue expression

IDO is widely expressed in the organism. It is expressed intracellularly in a constitutive manner, mostly to prevent bacterial or viral infection and to suppress T-cell-mediated immune responses against self-antigens, allogeneic antigens, or fetal antigens. Notably, Kamimura and colleagues found that IDO is localized in fetal-derived syncytiotrophoblasts in the human placenta [START_REF] Kamimura | Localization and developmental change of indoleamine 2,3-dioxygenase activity in the human placenta[END_REF]. Moreover, in their work published in 1998, Munn and colleagues detected IDO transcripts in placenta of pregnant female mice [START_REF] Munn | Prevention of allogeneic fetal rejection by tryptophan catabolism[END_REF].

Other organs also express IDO, such as epididymis, prostate, esophagus, intestine, colon, cecum, kidney, spleen, thymus, heart, lung, brain and skin [START_REF] Dai | Indoleamine 2,3-dioxygenase tissue distribution and cellular localization in mice: implications for its biological functions[END_REF][START_REF] Yoshida | Induction of indoleamine 2,3dioxygenase in mouse lung during virus infection[END_REF].

During inflammation, IDO is induced most importantly in DCs [START_REF] Lob | Levo-but not dextro-1-methyl tryptophan abrogates the IDO activity of human dendritic cells[END_REF] and monocytes/macrophages [START_REF] Carlin | Interferon-induced indoleamine 2,3-dioxygenase activity in human mononuclear phagocytes[END_REF][START_REF] Munn | Inhibition of T cell proliferation by macrophage tryptophan catabolism[END_REF] in response to specific proinflammatory stimuli mainly TNF-α, IL-6, and IFN-γ (Chon et al., 1996;O'Connor et al., 2009). Moreover, IDO was found to be importantly induced in SMCs (Cuffy et al., 2007;[START_REF] Sakash | Cytokines Induce Indoleamine 2,3-Dioxygenase Expression in Human Atheroma-Associated Cells: Implications for Persistent Chlamydophila pneumoniae Infection[END_REF], ECs (Wang et al., 2014), FBs [START_REF] Cui | Tumor-Associated Fibroblasts and Microvessels Contribute to the Expression of Immunosuppressive Factor Indoleamine 2, 3-Dioxygenase in Human Esophageal Cancers[END_REF], epithelial cells [START_REF] Mailankot | Induction of indoleamine 2,3-dioxygenase by interferon-gamma in human lens epithelial cells: apoptosis through the formation of 3hydroxykynurenine[END_REF], neurons, astrocytes, microglia (Cuartero et al., 2014;[START_REF] Guillemin | Expression of indoleamine 2,3-dioxygenase and production of quinolinic acid by human microglia, astrocytes, and neurons[END_REF], and many tumor cell lines like ovarian tumor cells [START_REF] Qian | Efficacy of levo-1methyl tryptophan and dextro-1-methyl tryptophan in reversing indoleamine-2,3dioxygenase-mediated arrest of T-cell proliferation in human epithelial ovarian cancer[END_REF].

F. Mechanisms of action of IDO

IDO-mediated degradation of Trp through KP was originally proposed as an innate immune defense mechanism to protect the host against bacterial or viral infection and to regulate adaptive immune responses. Later, it was shown that IDO exerts its effects not only through

Trp depletion but also via the generation of bioactive Trp catabolites in the KP.

i. Defense against pathogens

First studies hypothesized that IDO plays a role in inhibiting eukaryotic intracellular pathogens proliferation. For instance, in his study published in 1984, Pfefferkorn found that IFN-γinduced Trp degradation by human FBs was responsible for blocking the intracellular parasite Toxoplasma gondii growth in these cells [START_REF] Pfefferkorn | Interferon gamma blocks the growth of Toxoplasma gondii in human fibroblasts by inducing the host cells to degrade tryptophan[END_REF]. As shown, Trp radioactivity decrease was accompanied with Trp conversion in to other radioactive metabolites, mostly Kyn and NFK with higher radioactive levels in the medium and in the cells, respectively. After 9 years, Habara-Ohkubo proved a role of mouse IDO expression in mediating antitoxoplasma state [START_REF] Habara-Ohkubo | Establishment of an antitoxoplasma state by stable expression of mouse indoleamine 2,3-dioxygenase[END_REF]. Notably, ZnSO4 IDO induction in mouse rectal cancer (CMT-93) cells transfected with a mouse IDO expression plasmid containing a heavy metalresponsive metallothionin promoter resulted in a complete Trp depletion from the medium accompanied with a blockade of Toxoplasma gondii growth within these cells. This was then confirmed with the work of Gupta and colleagues who found that IDO enzyme, expressed within human FBs was responsible for antiparasitic and antiproliferative effects [START_REF] Gupta | Antiparasitic and antiproliferative effects of indoleamine 2,3-dioxygenase enzyme expression in human fibroblasts[END_REF]. Remarkably, Chlamydia psittaci and Toxoplasma gondii growth was inhibited in cadmium (Cd 2+ ) or zinc (Zn 2+ )-treated human FBs expressing IDO independently of IFN-γ stimulation after transfection with an expression vector containing cloned cDNA for human IDO with metallothionein promoter in comparison with IDO negative human FBs. Growth inhibition was accompanied with Trp degradation and was reversed by its excess addition to the medium. Similarly, [ 3 H] thymidine incorporation was also inhibited and resulted in human FBs cytotoxicity upon IDO induction.

ii.

Immune regulation

IDO is recognized as a central mediator of immune tolerance. In 1998, Munn, Mellor and colleagues found that Trp catabolism prevent allogeneic fetal rejection [START_REF] Munn | Prevention of allogeneic fetal rejection by tryptophan catabolism[END_REF].

First, IDO transcripts were detected in placenta of pregnant female mice. Remarkably, treatment of these mice with IDO pharmacological inhibitor, 1-MT, resulted in inflammation and progressive deterioration followed by rejection after 9.5 days post coitus of only allogeneic embryos but not syngeneic ones. Notably, maternal lymphocytes were responsible for this rejection. In fact, 1-MT-treated female RAG-1 -/-pregnant mice, with impaired lymphocytes development, delivered healthy litters of normal size. However, they lost their embryos after reconstitution with a population of H-2K b -specific T cells from BM3 T receptor (TCR) transgenic mice. Moreover, 1-MT-treated females mated with syngeneic males, carrying a recombinant H-2K b transgene expressed under the control of promoter elements from the human HLA-G gene in trophoblasts in placenta, lost their embryos. Hence, this work proved that IDO expression and Trp catabolism during murine pregnancy are required to prevent rejection of the allogeneic fetus by maternal T cells. Later in 1999, Munn and colleagues found that macrophage Trp catabolism inhibited T cells proliferation [START_REF] Munn | Inhibition of T cell proliferation by macrophage tryptophan catabolism[END_REF]. Moreover, it has been shown that IDO expression in cancers, like ovarian cancer, could be associated with immune tolerance via suppression of T-cell proliferation. Remarkably, CD4 + Th1, CD8 + T cells, and NK cells, from tumor-derived and peripheral blood cells of ovarian cancer patients, presented a reduced proliferation when they are co-cultured with IDO-transfected human embryonic kidney cell line 293 or IDO + ovarian cancer cells like IDO + DCs [START_REF] Qian | Efficacy of levo-1methyl tryptophan and dextro-1-methyl tryptophan in reversing indoleamine-2,3dioxygenase-mediated arrest of T-cell proliferation in human epithelial ovarian cancer[END_REF].

Interestingly, IDO mediates suppression of effector T-cell function and favors the differentiation of Treg cells (Mellor and Munn, 2004;[START_REF] Munn | IDO in the Tumor Microenvironment: Inflammation, Counter-Regulation, and Tolerance[END_REF].

iii.

AHR-mediated effects

Interestingly, a study by Sinclair and colleagues showed that Kyn crosses the T-cell membrane by the system L transporter SLC7A5 [START_REF] Sinclair | Single cell analysis of kynurenine and System L amino acid transport in T cells[END_REF]. It can then activate the AHR by binding to its hydrophobic ligand-binding pocket. Besides Kyn, other Trp-derived metabolites can act as AHR activators. Recently, Seok and colleagues identified two trace-active derivatives of Kyn named trace-extended aromatic condensation products (TEACOPs) that act as high affinity AHR ligands [START_REF] Seok | Trace derivatives of kynurenine potently activate the aryl hydrocarbon receptor (AHR)[END_REF]. In their study, Opitz and colleagues identified a role for TDO-derived Kyn in suppressing antitumor immune responses and promoting tumorcell survival and motility through the AHR in an autocrine/paracrine manner (Opitz et al., 2011). Moreover, it has been shown that IDO controls Th17/Treg immunity via AHR in pulmonary paracoccidioidomycosis which is a fungal infection [START_REF] De Araújo | The IDO-AhR Axis Controls Th17/Treg Immunity in a Pulmonary Model of Fungal Infection[END_REF]. In their study, researchers found that infected-Ido1 -/-mice had an increased influx of activated Th17 cells to the lungs, and a reduction in Treg cells. This was also accompanied with a reduced expression of AHR.

In addition to the regulation of immune response, IDO was found to contribute to arterial vessel relaxation and regulation of BP in septic shock (Wang et al., 2010). Moreover, Mohib and coworkers identified a role of IDO expression in promoting renal I/R injury (Mohib et al., 2008). Furthermore, it has been reported that Kyn-derived metabolites promote inflammation, oxidative stress, and apoptosis (Metghalchi et al., 2018;Wang et al., 2014[START_REF] Wang | Tryptophan-kynurenine pathway is dysregulated in inflammation, and immune activation[END_REF].

G. Other Enzymes implicated in Tryptophan metabolism

TDO and IDO are common in the fact that they are the first rate-limiting heme-containing cytosolic enzymes controlling the KP more precisely Trp availability. But at the same time, other characteristics make them different in many respects. Moreover, IDO2 is an isoform of IDO that participates to Trp degradation.

TDO was initially discovered in mammalian liver in 1936 (Kotake, Y. andMasayana, I. (1936) Z. Physiol. Chem. 243,[237][238][239][240][241][242][243][244] and first characterized in rat liver in 1955 [START_REF] Knox | The hormonal control of tryptophan peroxidase in the rat[END_REF]. TDO is mainly a liver-specific enzyme. It has also been identified in the skin [START_REF] Ishiguro | Skin L-tryptophan-2,3dioxygenase and rat hair growth[END_REF]. It also exists in the brain (Cuartero et al., 2014). It has been characterized in many species including humans, rabbits, mice, insects such as Drosophila melanogaster as well as bacteria like Escherichia coli and yeast [START_REF] Rafice | Oxidation of Ltryptophan in biology: a comparison between tryptophan 2,3-dioxygenase and indoleamine 2,3-dioxygenase[END_REF]. TDO and IDO have a very low sequence identity which is estimated to around 10% [START_REF] Rafice | Oxidation of Ltryptophan in biology: a comparison between tryptophan 2,3-dioxygenase and indoleamine 2,3-dioxygenase[END_REF]. TDO is a tetrameric enzyme with four subunits. It is of ~190 kDa in eukaryotes and ~120 kDa in prokaryotes. Inactive form of TDO (apoenzyme) has a half-life of 2 hours. Its activity is induced by dietary Trp and steroid hormones [START_REF] Ball | Characterization of an indoleamine 2,3-dioxygenase-like protein found in humans and mice[END_REF]. TDO uses O2 as a co-substrate and heme as a cofactor. It oxidizes L-Trp to ultimately synthetize NAD + and NADP + . Trp analogues like 5-fluorotryptophan and 6-fluorotryptophan can serve as substrates too. Unlike IDO, TDO has a lower affinity but higher capacity for Trp (Badawy, 2017b).

Ido-2 gene was first discovered in human then in mouse by Metz and colleagues (Metz et al., 2007). It is located on chromosome 8p12 downstream of the Ido-1 gene. Human and mouse IDO2 proteins are 420 and 405 amino acids, respectively, and are more conserved than IDO proteins. Like IDO, IDO2 requires heme and substrate binding, however these two enzymes are only 43% identical. Compared to IDO, IDO2 has a lower affinity to L-Trp. IDO2 showed higher Km than IDO in different species. For instance, mouse IDO has a Km of 29µM whereas IDO2 has a Km of 12mM (Reyes [START_REF] Ocampo | Kynurenines with Neuroactive and Redox Properties: Relevance to Aging and Brain Diseases[END_REF]. While IDO is inhibited by L-1MT, IDO2 is inhibited by D-1MT. According to Metz and colleagues, human IDO2 is expressed in liver, small intestine, spleen, placenta, thymus, lung, brain, kidney and colon. However, mouse

Ido-2 transcripts were only detected in liver and kidney (Metz et al., 2007)

H. IDO role in pathologies

i.

Stroke

Stroke is the second leading cause of death after IHD. It affects millions of people every year.

It is due to ischemia which results from cerebral arterial occlusion or hemorrhage in the brain resulting in motor, sensory and cognitive neurological symptoms [START_REF] Colpo | Systematic Review on the Involvement of the Kynurenine Pathway in Stroke: Pre-clinical and Clinical Evidence[END_REF].

Inflammation, excitotoxicity, oxidative stress and apoptosis participate to brain damage [START_REF] Brouns | The role of tryptophan catabolism along the kynurenine pathway in acute ischemic stroke[END_REF]. Under inflammatory conditions, toxic KP-derived metabolites are produced. For instance, 3-OHKyn induces neuronal apoptosis, 3-OHKyn and 3-HAA participate to oxidative stress and QA activates N-methyl-D-aspartic acid (NMDA)-receptor [START_REF] Brouns | The role of tryptophan catabolism along the kynurenine pathway in acute ischemic stroke[END_REF][START_REF] Colpo | Systematic Review on the Involvement of the Kynurenine Pathway in Stroke: Pre-clinical and Clinical Evidence[END_REF]. Both pre-clinical studies in experimental models and clinical studies in human patients demonstrated KP involvement in stroke as well as its contribution to clinical outcome. They also considered KP metabolites as potential therapeutic targets for the development of neuroprotective strategies to prevent brain damage after stroke.

In an observational study, part of the Middelheim's Interdisciplinary Stroke Study, Brouns and coworkers found that IDO activity correlates with stroke severity and long-term stroke outcome in patients with ischemic stroke or transient ischemic attack [START_REF] Brouns | The role of tryptophan catabolism along the kynurenine pathway in acute ischemic stroke[END_REF]. Of note, from admission to day 7 following stroke, a decrease in Kyn/Trp ratio and the neurotoxic 3-HAA levels as well as an increase in the neuroprotective KA levels were observed. Results show a positive correlation between plasma Kyn/Trp ratio with the infarct volume at the admission.

Moreover, patients with poor long-term outcome had a higher plasma Kyn/Trp ratio compared to the ones with favorable outcome. This ratio was also more elevated in patients who died compared to the stroke survivors. Interestingly, associations between plasma Kyn/Trp ratio and inflammatory markers like plasma C-reactive protein levels, erythrocyte sedimentation rate and neutrophil/lymphocyte count were also found in patients suffering from stroke.

An experimental study, conducted by Cuartero and colleagues, described a role of Kyn/AHR pathway in mediating brain damage after stroke (Cuartero et al., 2014). An increase in AHR expression and activity were observed in mouse brain following ischemic stroke induced by permanent middle cerebral artery occlusion. Remarkably, AHR was found to be involved in acute ischemic damage. Its pharmacological inhibition with its antagonists 6,2',4'-trimethoxyflavone (TMF) or CH223191 (CH) or genetic loss-of-function reduced infarct size.

This deleterious effect was mediated by Kyn which was directly increased in the brain after middle coronary artery occlusion (MCAO). Notably, Kyn administration after MCAO, resulting in its high levels in the brain and plasma, was associated with a worse infarct volume in treated compared to vehicle-treated animals. This deleterious effect of Kyn in cerebral ischemia was found to be mediated by AHR because it was abolished after TMF administration or with ahr deletion. Comparably, in vitro study showed increased AHR expression and activation in rat cortical neurons exposed to oxygen-glucose deprivation (OGD). Researchers found that AHR inhibits cAMP response element-binding protein (CREB) induced prosurvival signaling after ischemic stroke. Moreover, Kyn supplementation was responsible for induction of AHR expression and activation as well as a decrease in CRE-mediated transcription.

Treatment with TMF reversed neurons damaged and lactate dehydrogenase release induced by Kyn after OGD (Cuartero et al., 2014).

ii.

Cardiovascular diseases a. Abdominal Aortic Aneurysm

AAA is an age-associated disease with considerable consequences on mortality. For instance, 9% of adults >65 years old suffer from AAA responsible for ~ 15000 deaths per year, in the United States. AAA is a permanent localized dilation of the abdominal aorta characterized by elastin degradation and loss, VSMC apoptosis, compensatory collagen deposition as well as chronic aortic wall inflammation (Metghalchi et al., 2018;Wang et al., 2017b). Development and progression of this disease is principally mediated by AngII. In 2014, Wang and colleagues found that IFN-γ mediated AngII effect powerfully induced IDO through the production of ROS and increased its derived-metabolites in the aortas of mice (Wang et al., 2014). In this elegant study, authors described a role of 3-OHKyn in mediating endothelial apoptosis and dysfunction through the activation of Nox. Markedly, as a result of AngII infusion, IFN-γ was increased and IDO expression in aortas was induced accompanied with plasma Kyn and 3-OHKyn upregulation. Moreover, AngII was found to be responsible for enhancing ROS production and promoting EC apoptosis and endothelial dysfunction in aortas of only WT mice but not mice harboring Nox subunits deficiency (p47 phox , gp91 phox ), or Ido-1 deletion.

Interestingly, these effects were reversed after IFN-γ neutralization and this was associated with inhibition of AngII-induced Kyn-metabolites generation. Of note, 3-OHKyn supplementation resulted in increased superoxide anions generation by Nox in ECs leading to aggravated apoptosis and endothelial dysfunction. These findings highlighted an important role of the KP, through its derived-metabolites, in mediating EC apoptosis, and endothelium dysfunction through NADPH-derived superoxide upregulation (Wang et al., 2014).

It was only 3 years later that Wang and colleagues identified, for the first time, a direct role of IDO in AAA (Wang et al., 2017b). AngII-infusion was responsible for increased maximal abdominal aortic diameter and total aortic weight in addition to increased disruption and degradation of elastic laminas as well as collagen deposition in hypercholesterolemic apolipoprotein E deficient mice (Apoe -/-), but not in Apoe -/-Ido-1 -/-mice. Moreover, IFN-γ expression was increased in the aortas of both groups resulting in up-regulation of IDO expression in Apoe -/-mice. In addition, AngII-infused Apoe -/-Ido-1 -/-mice had lower MMP2 messenger ribonucleic acid (mRNA) level, protein expression and activity in their suprarenal aortas in comparison with those of Apoe -/-counterparts which had increased levels of Kynureninase and 3-HAA in their aortas. In turn, 3-HAA administration increased MMP2 expression and activity in mouse aortas. Interestingly, Kynureninase knockdown via siRNA transfection reduced MMP2 mRNA level, protein expression and activity in these mice and attenuates AngII-induced AAA formation. Wang and coworkers found that IDO-deficiency protection against AAA formation was mediated by non-BM derived cells, thus suggesting a possible role of KP in vascular cells like VSMCs. In vitro results show that IFN-γ treatment induced IDO in Human Aortic Smooth Muscle Cells (HASMCs), increased 3-HAA and Kynureninase, accompanied with increased MMP2 expression via the transcription factor NF-κB. Researchers found elevated MMP2 protein level associated with IDO and Kynureninase up-regulation as well as a stronger 3-HAA staining in human AAA sections compared with adjacent non-aneurysmal aortic sections (Wang et al., 2017b).

In line with these findings, my team demonstrated a protective effect of IDO deficiency against AngII-induced AAA in Low-density lipoprotein receptor deficient mice (Ldlr -/ -) fed a high cholesterol (HC) diet (Metghalchi et al., 2018). Plasma Kyn was found to be increased in AngII-infused Ldlr -/ -mice without any change in Trp concentrations following 1 week of treatment. Moreover, IDO deficiency reduced apoptotic area as revealed by lower terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) positive staining accompanied with an increased α-actin staining within the media of Ldlr -/ -Ido1 -/-mice, thereby suggesting a preservation of VSMCs in IDO-deficient mice. Inflammation was not affected since no differences were observed in macrophage and T lymphocyte infiltration. Of note, IDO was not implicated in AngII-independent elastase-induced-aneurysm phenotype. As shown, this model was not able to induce Kyn production in the aorta of C57Bl/6 mice and IDO deficiency did not impact aneurysm progression. Finally, these findings demonstrated that the deleterious role of IDO in AAA formation is explained by AngII-induced IDO expression which then affects VSMC survival through KP.

b. Atherosclerosis

Atherosclerosis is a major pathology contributing to CVDs, notably CAD and cerebrovascular disease (Polyzos et al., 2015). As explained in the first chapter, it is due to an increased blood lipid levels, namely hypercholesterolemia as well as a retention and an accumulation of oxLDL in the subendothelial space of large-and medium-sized arteries leading to a persistent inflammatory response. Hence, this chronic inflammatory disease is driven by various cytokines notably IL-6 and IFN-γ. It involves innate and adaptive ICs as well as vascular cells like ECs which participate to its progression by amplifying inflammation and disrupting vascular homeostasis. As IDO is induced by inflammatory mediators, Trp catabolism may play an important role in atherosclerosis [START_REF] Nitz | Amino Acids and Their Metabolism in Atherosclerosis[END_REF]Polyzos et al., 2015). According to Eussen and colleagues, human studies reflected a negative association between plasma Kyn/Trp ratio and HDL cholesterol, and a positive association with LDL cholesterol and triglycerides. It has been also reported that plasma Kyn/Trp ratio was positively associated to carotid intima-media thickness in individuals with advanced atherosclerosis (Eussen et al., 2015). Recently, IDO was shown to play a critical role in atherogenesis in different mouse models.

In their study published in 2012, Zhang and coworkers evaluated the role of 3-HAA in modulating atherosclerosis (Zhang et al., 2012). They found that the administration of supraphysiological levels of 3-HAA reduced atherosclerosis as explained by a reduction in atherosclerotic lesion area in the aorta of HC diet-fed Ldlr -/-treated mice compared to PBS treated mice. 3-HAA was also found to modulate adaptive immune responses and reduce systemic inflammation. In addition, 3-HAA had a potential lipid-lowering effector marked by a reduction in total plasma cholesterol and triglycerides in treated Ldlr -/-mice. This was accompanied with an increased mRNA level of peroxisome proliferator-activated receptor beta/delta (PPAR β/δ) known to be involved in lipoprotein metabolism. These results let researchers to consider 3-HAA as a novel anti-atherosclerotic agent having beneficial effects on lipid metabolism and vascular inflammation (Zhang et al., 2012). Of note, these results

suggest that the pharmacological treatment with supra-physiological levels of 3-HAA inhibits atherosclerosis development rather than revealing its endogenous role in atherosclerosis.

It was only in 2015 that Polyzos and colleagues examined, for the first time, the role of IDO and Trp metabolism in atherosclerosis development (Polyzos et al., 2015). They found that IDO inhibition with 1-methyl-DL-tryptophan (D/L 1-MT) increased atherosclerosis in Apoe -/ mice explained by 58 and 54% bigger atherosclerotic lesion in the aortic arch and root, respectively. It also enhanced vascular inflammation accompanied with an up-regulation of VCAM-1, macrophage chemotactic protein CCL2, and an increased CD68 + macrophage infiltration into the intima of the vessel wall (Polyzos et al., 2015). However, this may result from nonspecific effects of D/L 1-MT administration since no significant differences were observed in plasma Kyn/Trp ratio, indicating non efficient inhibition of IDO activity in D/L 1-MT treated mice.

In this context, my team showed another aspect of the role of IDO-dependent Trp metabolism in atherosclerosis (Metghalchi et al., 2015). They found an increased Kyn/Trp ratio in the plasma, accompanied with increased concentrations of Kna in Ldlr -/-mice fed with HC diet compared to their controls receiving chow diet. Interestingly, IDO deficiency reduced the development of atherosclerosis in the aorta of Ldlr -/-Ido-1 -/-mice compared to Ldlr -/-Ido1 +/+ littermates. Similar results were obtained in Ldlr -/-mice with Ido-1 deletion in BM-derived cells compared with their controls reconstituted with Ido1 +/+ both on HC diet. It has been shown that IDO activity was required for the regulation of IL-10 production. Of note, Ldlr -/-Ido1 -/-mice supplemented with Kna showed decreased IL-10 expression in their spleens associated with a marked increase in plaque size. These effects have been explained by an activation of cyclic adenosine monophosphate (cAMP)-dependent pathway and inhibition of Erk1/2 phosphorylation. In vitro study indicated that IDO deficiency in cultured BM-derived macrophages (BMDM) was associated with enhanced IL-10 production which was abolished by Erk1/2 inhibition. Of interest, inhibition of IL-10 broke immune homeostasis of IDOdeficient mice resulting in a severe spontaneous colitis and increased susceptibility to atherosclerosis. Notably, Ldlr -/-Ido1 -/-Il-10 -/-mice revealed a more important lesion size indicating a susceptibility of these animals to atherosclerosis. Importantly, high plasma Kna levels were predictive of a higher risk of death or recurrent MI in patients with CAD (Metghalchi et al., 2015).

c. Ischemic heart disease

A study showed that KP was early activated after cardiac arrest in rats, pigs, and humans [START_REF] Ristagno | Early kynurenine pathway activation following cardiac arrest in rats, pigs, and humans[END_REF]. Notably, Trp was decreased accompanied with an increase in 3-HAA and Kna levels in plasma during 3-5 days in the post-resuscitation period. Changes in these metabolites reflected adverse outcome following cardiac arrest as MI in rats, cerebral injuries in pigs and systemic inflammation and mortality in humans. However, there was any study before 2011 that related it to clinical outcomes in case of IHD.

In the large Bergen Coronary Angiography Cohort including 3314 patients with stable angina pectoris, Pedersen and colleagues identified associations of systemic markers of IFN-γ, neopterin and plasma Kyn/Trp ratio, with increased risk of major coronary events and mortality in patients with a mean age of 63.7 years, suffering from CAD [START_REF] Eva Ringdal | Systemic Markers of Interferon-γ-Mediated Immune Activation and Long-Term Prognosis in Patients With Stable Coronary Artery Disease[END_REF]. In their study published in 2013, Pedersen and coworkers found that urinary inflammation marker, Kyn/Trp ratio, is a strong predictor of major coronary events, acute MI and mortality in patients with CAD from the same cohort [START_REF] Pedersen | Urinary excretion of kynurenine and tryptophan, cardiovascular events, and mortality after elective coronary angiography[END_REF]. Then in 2015, Pedersen and collaborators proved associations of elevated plasma kyn metabolites notably, Kna, 3-OHKyn, AA, and 3-HAA, with increased risk of acute MI in 4122 patients with a mean age of 62 years having a stable angina pectoris (Pedersen et al., 2015). Another proof was provided by the Hordaland Health Study including 2819 participants aged 71-74 years without a known history of cardiac heart disease. Notably, plasma Kyn and 3-OHKyn levels were associated with an increased risk of acute coronary events (Eussen et al., 2015). Later in 2019, the first Mendelian randomization study discussing about the association of IDO with IHD was published. Unlike the previous studies, a recent proteome genome-wide association study (GWAS) in 3301 healthy participants of European descent with a mean age of 43.7 years, revealed an inversed association linking IDO to IHD [START_REF] Li | Indoleamine 2,3dioxygenase and ischemic heart disease: a Mendelian Randomization study[END_REF]. Recently in 2020, the published PREGICA cohort consisting of 292 patients showed that plasma Kyn/Trp ratio was predictive of LV remodeling after MI [START_REF] Saadi | Plasma Indoleamine 2,3-dioxygenase is predictive of left ventricular remodeling after myocardial infarction[END_REF].

Together these studies, let us consider IDO as a potential therapeutic target in IHD worthy of further investigation. Of note, the direct role of IDO in IHD more specifically acute MI remains unexplored, and no randomized controlled trials of IDO or IDO inhibitors to prevent or treat IHD have been conducted. Our hypothesis is that Kyn/Trp ratio, which is a surrogate marker of IDO activity, is induced after acute MI and is instrumental in the regulation of cardiac remodeling and function.

Results

IDO is an enzyme that catalyzes the degradation of the essential amino acid L-Trp to NFK leading to the generation of Kyn and its derived metabolites. We found an increased Kyn/Trp ratio in the heart and plasma after MI. Moreover, Ido-1 -/-mice, subjected to LAD coronary artery ligation in order to induce MI, displayed attenuated cardiac dysfunction represented by less decreased LVEF and adverse remodeling assessed by smaller infarct size, less interstitial fibrosis, and an increased capillaries number/CM, as well as a preserved CM contractility compared to WT mice. Interestingly, the reduction in infarct size was explained by a less apoptosis in the LV peri-infarcted area in Ido-1 -/-mice. Using a pharmacological inhibitor of IDO, L-1MT, we succeeded to show similar beneficial effects as observed in mice with total IDO deletion. Our results did not show any significant differences in the infiltration of inflammatory cells such as monocytes or lymphocytes in the infarcted LV area of Ido-1 -/-mice compared to their controls. Moreover, our findings show that only IDO deletion in non-BMderived cells was responsible for the preservation of cardiac function and LV remodeling. We wanted to know which cell type was implicated in IDO-mediated deleterious effects, following MI. We found that IDO specific deletion in CM, as confirmed by decreased Ido-1 mRNA expression, did not affect neither cardiac function nor LV remodeling, after MI. Interestingly, we showed an IDO expression in the infarcted LV vessel wall of WT mice but not in Ido-1 -/- mice. As IDO is known to be expressed in the vascular cells such as VSMC and EC, we were interested in studying the implication of IDO specific deletion in these cell types. The results revealed no effects of IDO specific deletion in VSMC on cardiac function or LV remodeling, after MI. However, IDO specific deletion in EC (EC IDOKO) resulted in attenuated cardiac dysfunction and adverse remodeling as well as a less apoptosis in the LV peri-infarcted zone and a preserved CM contractility. Importantly, we found that Ido-1 mRNA expression and Kyn/Trp ratio were much higher in EC compared to CM. Consistently, Kyn concentrations were more elevated in the supernatants of cultured Non-CM compared to that of CM isolated from control mice, after MI. We further wanted to explore which metabolite was implicated in IDO-mediated deleterious effects in MI. Our results indicated that Kyn was the only metabolite that importantly increased in the heart and plasma of WT mice after MI compared to shamoperated mice. Remarkably, we found that Kyn supplementation in Ido-1 -/-mice abrogated the protective effects of IDO deletion. Kyn is known to be an endogenous ligand of the AHR. We postulated that Kyn pro-apoptotic effects are mediated through this receptor. Our results

showed that the administration of the AHR antagonist, CH-223191, to Ido-1 -/-mice supplemented with Kyn decreased Kyn-mediated apoptosis in the LV of peri-infarcted area.

We also demonstrated that Kyn supplementation enhanced CM oxidative stress and apoptosis which were abolished in the presence of AHR antagonist. We then speculated that IDO expressed in EC leads to Kyn production, which can affect CM viability in a paracrine manner.

Notably, Kyn concentrations were markedly decreased in the cultured Non-CM supernatants from EC IDOKO mice indicating that ECs are the main cells producing Kyn. Interestingly, CMs incubated in the presence of supernatants of Non-CM isolated from EC IDOKO mice after MI exhibited less apoptosis compared to CMs incubated with supernatants of Non-CM isolated from control mice, following MI. We also found that Ido-1 -/-CM isolated after MI and supplemented with Kyn had a reduced relaxation compared to their controls.

In conclusion, we demonstrated that following MI, the induction of Kyn/Trp ratio within the EC alters cardiac function and LV remodeling through paracrine effects of Kyn on CM.

D i s c l a i m e r : T h e m a n u s c r i p t a n d i t s c o n t e n t s a r e c o n f i d e n t i a l , i n t e n d e d f o r j o u r n a l r e v i e w p u r p o s e s o n l y , a n d n o t t o b e f u r h e r d i s c l o s e d .

Disclaim er: The m anuscript and it s cont ent s are confident ial, int ended for j ournal review purposes only, and not t o be furt her disclosed.

URL: ht t ps: / / circ-subm it .aha-j ournals.org

D i s c l a i m e r : T h e m a n u s c r i p t a n d i t s c o n t e n t s a r e c o n f i d e n t i a l , i n t e n d e d f o r j o u r n a l r e v i e w p u r p o s e s o n l y , a n d n o t t o b e f u r t h e r d i s c l o s e d .

Olivier

D i s c l a i m e r : T h e m a n u s c r i p t a n d i t s c o n t e n t s a r e c o n f i d e n t i a l , i n t e n d e d f o r j o u r n a l r e v i e w p u r p o s e s o n l y , a n d n o t t o b e f u r t h e r d i s c l o s e d .

3

Clinical Perspective

What Is New?

 Indoleamine 2, 3-dioxygenase 1 (IDO), an enzyme involved in Tryptophan catabolism, precipitates adverse cardiac remodeling after acute myocardial infarction.

 The specific deletion of IDO in endothelial cells enhances cardiomyocyte survival and contractility leading to cardiac function improvement.

 IDO dependent effects are mediated by endothelial cell production of kynurenine.

What Are the Clinical Implications?

 Our study provides novel insights into the complex cross-talk between the cardiac endothelial cells and cardiomyocytes during cardiac repair after myocardial infarction and suggests a deleterious role for endothelial IDO through kynurenine production in cardiac repair.

 Therapeutic strategy targeting cardiac IDO could constitute an innovative approach to curb cardiac dysfunction following acute myocardial infarction.

D i s c l a i m e r : T h e m a n u s c r i p t a n d i t s c o n t e n t s a r e c o n f i d e n t i a l , i n t e n d e d f o r j o u r n a l r e v i e w p u r p o s e s o n l y , a n d n o t t o b e f u r t h e r d i s c l o s e d .

Introduction

Myocardial infarction (MI), by promoting adverse remodeling of the left ventricle (LV), is a leading cause of morbidity and mortality in humans worldwide. Following MI, intricate interactions among different cellular entities such as cardiomyocytes, inflammatory and vascular cells are instrumental in the regulation of tissue remodeling and govern the prevalence of heart failure, arrhythmias, or sudden death 1 . Therefore, it is of great importance to unravel the mechanisms underlying adverse cardiac remodeling and subsequently to explore new therapeutic approaches in patients with MI.

Indoleamine 2-3 dioxygenase 1 (IDO) is an enzyme that catalyzes the degradation of the essential amino acid L-tryptophan (Trp) to N-formylkynurenine leading to the generation of several active metabolites including kynurenine (Kyn), , anthranilic acid (AA), 3hydroxyanthranilic acid (3-HAA), kynurenic acid (Kna) and quinolinic acid. IDO-related pathways have been involved in various diseases ranging from chronic granulomatous disease 2 to neurodegenerative diseases 3 , and more recently in cardiometabolic diseases including metabolic syndrome 4 , aneurysm 5, 6 and atherosclerosis 7-11 . In these mouse models, either protective 4-6, 11 or deleterious 7-10 effects of IDO-generated metabolites were reported. Yet, in human studies, IDO activity was constantly found to be associated with worse cardiovascular outcome in patients with coronary artery disease 12-14 and atherosclerosis progression in patients with end-stage renal disease 15 .

During inflammation, IDO is up-regulated mostly in dendritic cells and macrophages by pro-inflammatory stimuli such as tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) 16 , and interferon-gamma (IFNγ) 17 . IDO exerts its biological effects through the generation of downstream metabolites that suppress effector T-cell function and favor the differentiation of regulatory T cells (Tregs) 18 . However, the biological effects of IDO extend beyond its role in the regulation of the immune response.

Interestingly, IDO activity has been shown to contribute to arterial vessel relaxation and regulation of blood pressure in septic shock 19 , and to promote renal ischemia-reperfusion injury 20 . Recent studies also demonstrate that Kyn metabolites may increase inflammation 21 , oxidative stress, as well as apoptosis of smooth muscle and endothelial cells 6, 22 .

Given the potential link between IDO-dependent pathways and MI, we hypothesized that IDO activity is induced after acute MI and is instrumental in the regulation of cardiac remodeling and function.

Here, we show that, deficiency or pharmacological inhibition of IDO protects against MI-induced cardiac dysfunction. Moreover, conditional loss of function of IDO in endothelial cells (EC), but not in smooth muscle cells (SMC), or in cardiomyocytes (CM) or in bone marrow-derived inflammatory cells, reduces infarct size and limits cardiac injury after MI. Furthermore, we demonstrate that Kyn is responsible for the deleterious effects observed in IDO-deficient mice. Collectively, our results reveal the biological importance of Kyn produced by EC in the context of ischemic cardiac injury. These

D i s c l a i m e r : T h e m a n u s c r i p t a n d i t s c o n t e n t s a r e c o n f i d e n t i a l , i n t e n d e d f o r j o u r n a l r e v i e w p u r p o s e s o n l y , a n d n o t t o b e f u r t h e r d i s c l o s e d .

5 findings could have implications for the development of novel therapeutic strategies aimed at inhibiting IDO activity for the treatment of patients with ischemic cardiac diseases.

D i s c l a i m e r : T h e m a n u s c r i p t a n d i t s c o n t e n t s a r e c o n f i d e n t i a l , i n t e n d e d f o r j o u r n a l r e v i e w p u r p o s e s o n l y , a n d n o t t o b e f u r t h e r d i s c l o s e d .

6

Methods

Animals

Ido1 -/-mice were from the Jackson Laboratory and are raised in our facility. Cardiomyocyte deficiency was achieved by crossbreeding transgenic B6129-Tg (Myh6-cre/Esr1)1Jmk/J, in which the cardiomyocyte-specific α-myosin heavy chain (Myh6) promoter drives expression of a 4-OHtamoxifen inducible Cre recombinase fusion protein with Ido-1 flox/flox mice (Ido-1 flox/flox αMHC-Cre).

Myocardial Infarction

MI was induced by left coronary ligation as previously described 24 . Mice were anesthetized using ketamine (100 mg/kg body weight) and xylazine (10 mg/kg body weight) via intraperitoneal injection,

D i s c l a i m e r : T h e m a n u s c r i p t a n d i t s c o n t e n t s a r e c o n f i d e n t i a l , i n t e n d e d f o r j o u r n a l r e v i e w p u r p o s e s o n l y , a n d n o t t o b e f u r t h e r d i s c l o s e d .

intubated, and ventilated using a small-animal respirator. The chest wall was shaved and a thoracotomy was performed in the fourth left intercostal space. The pericardial sac was removed, and the left anterior descending artery was permanently ligated with 7-0 monofilament suture at the site of its emergence close to the left atrium. The thoracotomy was closed with 6-0 monofilament sutures.

The exact same procedure was performed for the sham-operated mice except that the ligation was not tied. The endotracheal tube was removed once spontaneous respiration resumed, and mice were placed on a warm pad at 37°C until awakened. All MI experiments were performed in a blinded manner with regard to the genotype of mice.

Echocardiographic Measurements

Transthoracic echocardiography was performed using a VEVO 2100 (Visual sonics) with a cardiac probe (MS-440D). Mice were anesthetized with isoflurane (1.5-1% in air), shaved with the use of

Cell isolation and culture

Cardiomyocytes (CM) isolation protocol was performed as previously described 25 . Briefly, heart was For the study of cardiomyocyte contractility, cells were isolated as previously described 26 . In some experiments, Kyn (100 μM) was added in the medium for 90 min to test its effect on CM contractility. Cell shortening and calcium concentration were studied using CM stimulation at 0.5 Hz (20 V, 1 ms) at room temperature. Sarcomere length (SL) and fluorescence (405 and 480 nm) were simultaneously recorded using Ion Wizard/IonOptix 6.6 software (IonOptix system, Hilton, USA)

connected to an inverted fluorescence microscope.

Smooth muscle cells (SMC) were prepared from mouse thoracic aorta as previously described 27 .

Briefly, thoracic aorta was dissected and incubated with enzyme solution containing collagenase type II (1mg/ml, Worthington Biochemical) and elastase (0.744 units/ml, Worthington Biochemical), at 37°C for 15 minutes. SMC were grown for 7 days at 37°C in DMEM/F12 20%FBS then stimulated with LPS (1 μg/ml) and IFN-γ (100 U/ml) for 24 hours. Cells were then lysed for RNA extraction.

Flow Cytometry and cell sorting

Hearts were collected and the left ventricle was isolated, minced with fine scissors, and gently passed through the Bel-Art Scienceware 12-Well Tissue Disaggregator (Fisher Scientific). Cells were collected, filtered through 40-μm nylon mesh, and washed with PBS. Red blood cells were removed

D i s c l a i m e r : T h e m a n u s c r i p t a n d i t s c o n t e n t s a r e c o n f i d e n t i a l , i n t e n d e d f o r j o u r n a l r e v i e w p u r p o s e s o n l y , a n d n o t t o b e f u r t h e r d i s c l o s e d .

9

by incubation in hemolysis buffer (Sigma Life Science). Cells were stained for flow cytometry at 4°C.

Total cardiac cells were gated on Alexa Fluor 700 anti-mouse CD45 Biolegend) Quina on heart and Kyn and Trp on cell homogenates were performed as previously described 28 . IDO activity was assessed as the kynurenine/tryptophan ratio. Heart and cell lysis were achieved by mechanical disruption in an ice-cold modified Tyrode buffer (3013m Osm/l, pH 7.40) using a Teflonglass homogenizer (5 min, 2200 rpm at 4°C), followed by an -SH-activated toxin treatment as previously described 29 . Briefly, 150 hemolytic units of streptolysin o or alveolysin (equivalent to 375 ng or 6 pmoles, of protein) were added (10 µl) to tissue homogenates at 4°C and the suspensions warmed to 37°C for 10 minutes. This amount of toxin binds cell membranes at 4°C and elicits complete cell lysis at 37°C. Separate experiments showed that the addition of equivalent amounts of toxin to cells already disrupted by homogenization produced no change in the metabolites measured here.

Statistical analysis

Groups of animals were pre-defined according to genotype and active treatment versus vehicle, and were matched for age and sex. Differences between values were examined using Student t-test or Mann Whitney test. We used one-way ANOVA repeated measures for assessment of differences between more than 2 groups.

D i s c l a i m e r : T h e m a n u s c r i p t a n d i t s c o n t e n t s a r e c o n f i d e n t i a l , i n t e n d e d f o r j o u r n a l r e v i e w p u r p o s e s o n l y , a n d n o t t o b e f u r t h e r d i s c l o s e d .

Results

Up-regulation of IDO activity after MI alters cardiac function and remodeling

To investigate the time-dependent regulation of IDO activity, we assessed the Kyn/Trp ratio in Wild-Type (WT) mice challenged with either sham surgery or MI for 1, 3, 5, 7 or 14 days. As shown in Figure 1A, IDO activity is markedly induced in the heart as early as day 1 after MI compared to sham-operated WT mice. IDO activity is also increased in plasma (Figure 1B) at day 1 after MI, and peaks at day 7 after the onset of ischemia. We next evaluated the role of IDO on cardiac function in WT and IDO-deficient (Ido-1 -/-) mice either sham-operated or subjected to MI. Remarkably, we found that loss of function of IDO attenuated cardiac dysfunction after MI, as revealed by a significant improvement of left ventricle (LV) ejection fraction and a decrease in LV systolic and diastolic endocardial volumes (Figure 1C). We next assessed whether pharmacological inhibition of IDO activity by 1-Methyl-L-tryptophan (L-1MT) could recapitulate the beneficial impact of IDO deficiency. IDO activity inhibition by L-1MT, as revealed by a decrease in plasma Kyn/Trp ratio (Figure 1D), improved cardiac function in comparison with control mice subjected to MI (Figure 1E).

Attenuation of cardiac dysfunction in absence of IDO activity was associated with a decrease in adverse LV remodeling. We observed a smaller infarct size as well as less interstitial fibrosis in Ido-1 -/- and L-1MT-treated animals in comparison with WT and untreated mice, respectively, 14 days after MI (Figure 2A-B). Moreover, IDO deficiency, but not L-1MT treatment, improved capillary numbers as compared with WT mice (Figure 2C). This discrepancy could be accounted by the marked decrease in Kyn/Trp ratio in IDO deficient mice compared to L-1MT treated animals.

Interestingly, cardiac lactate levels (supplementary Fig. 1) as well as apoptotic cell number, as assessed by TUNEL staining, were also decreased in Ido-1 -/-and L-1MT-treated mice compared to WT animals (Figure 2D).

Ido-1 deficiency could decrease infarct size and attenuate cardiac dysfunction through modulation of inflammation. Accordingly, since IDO is known to be expressed in inflammatory cells such as macrophages 18 , we first examined whether the number of infiltrating inflammatory cells in the infarcted heart was modulated by IDO deficiency. Using flow cytometry, we evaluated the number of Ly-6C high and Ly-6C low monocytes, neutrophils and T lymphocytes within the myocardium at day 0, 3 or 7 after coronary ligation. As shown in supplementary Fig. 2A, no significant differences in the number of cardiac T CD4+ cells, monocytes and neutrophils were observed between WT and Ido-1 -/- mice. Similarly, cardiac Tregs number was comparable in WT and Ido-1 -/-mice (supplementary Fig. 2B). Next, we assessed the role of bone marrow (BM)-derived inflammatory cells expressing IDO. For this purpose, we generated WT and Ido-1 -/-chimeras lethally irradiated and transplanted with BMderived cells isolated from WT or Ido-1 -/-mice. After induction of MI, no differences in cardiac function, infarct size and capillary density were observed between WT mice transplanted with BM from either WT or Ido-1 -/-cells (Figure 3A-B). In contrast, cardiac function was improved in Ido-1 -/-

D i s c l a i m e r : T h e m a n u s c r i p t a n d i t s c o n t e n t s a r e c o n f i d e n t i a l , i n t e n d e d f o r j o u r n a l r e v i e w p u r p o s e s o n l y , a n d n o t t o b e f u r t h e r d i s c l o s e d .

12 mice transplanted with BM isolated from either WT or Ido-1 -/-cells, compared to WT chimeras (Figure 3A). Consistently, infarct size and capillary number were also improved (Figure 3B).

Altogether, these results suggest that inflammatory cells expressing IDO do not participate to the regulation of cardiac function and support a crucial role for IDO expressed in non BM-derived cells.

We then aimed to determine whether IDO was expressed in cardiomyocytes (CM) and affected cardiac homeostasis. Loss of function of IDO improved ex vivo contractility of cardiomyocytes isolated after acute MI, as assessed by high contraction and relaxation velocities (Supplementary Fig. 3A). We therefore hypothesized that IDO expressed in CM was a major regulator of cardiac remodeling and function. Cardiomyocyte deficiency was achieved by crossbreeding α-MHC-MerCre animals in which the cardiomyocyte-specific α-myosin heavy chain (Myh6) promoter drives expression of a 4-OHtamoxifen-inducible Cre recombinase fusion protein with Ido-1 flox/flox mice (Ido-1 flox/flox αMHC-Cre or CM IDO KO). After tamoxifen treatment, IDO expression was markedly decreased in CM (Figure 3C). However, as shown in Figure 3D and E, cardiac function and remodeling were unaffected in CM IDO KO when compared to their wild-type littermates (CM IDO), suggesting that CM are not the major source of IDO in heart and that non-CM IDO-expressing cells govern IDO-related effects on the cardiac tissue. We then postulated that vascular cells such as SMC and EC, which are known to express IDO 19, 30 , are involved in IDO-related effects on cardiac tissue. Consistently, IDO expression was observed in the vessels of infarcted heart (Supplementary Fig. 3B). To test the role of IDO expressed in SMC, we developed mice with a specific deletion of IDO in this cell type (Ido-1 flox/flox SMC22-Cre or SMC IDO KO) and challenged them with MI. Although a marked reduction in IDO expression was observed in SMC, cardiac function and healing were unchanged in SMC IDO KO mice when compared to their wild-type littermates (Supplementary Fig. 3C), indicating that IDO expressed in SMC was not involved in the regulation cardiac function in our experimental conditions.

IDO expressed by EC alters cardiac function and remodeling

We next speculated that IDO expressed in EC could participate to the cardiac phenotype.

Interestingly, IDO activity (Kyn/Trp) was higher in isolated cardiac EC compared to CM or leukocytes, after MI (Figure 4A). Consistently, Ido-1 mRNA was 13-fold higher in isolated cardiac EC compared to CM (Figure 4B), unravelling the importance of IDO expressed in EC. We then analyzed the role of EC expressing IDO using mice with specific conditional deletion of IDO in EC (Ido-1 flox/flox VECad-CreERT2 + or EC IDO KO). We found that Ido-1 mRNA expression was dampened in EC isolated from heart of EC IDO KO animals (Figure 4B). Moreover, Kyn/Trp ratio was significantly reduced in LV heart extracts of EC IDO KO compared to control animals, after MI (Figure 4C). Interestingly, cardiac function of EC IDO KO mice subjected to MI was markedly improved compared to their wild-type controls (EC IDO) (Figure 4D). This was associated with a reduction in infarct size, interstitial fibrosis, and apoptosis (Figure 4E-G). Altogether, these results

D i s c l a i m e r : T h e m a n u s c r i p t a n d i t s c o n t e n t s a r e c o n f i d e n t i a l , i n t e n d e d f o r j o u r n a l r e v i e w p u r p o s e s o n l y , a n d n o t t o b e f u r t h e r d i s c l o s e d .

13 indicated that cardiac function recovery was dependent on IDO expression in EC but not in SMC, inflammatory cells or CM.

Kyn alters cardiac function and remodeling

Then, we analyzed the molecular and cellular mechanisms associated with IDO detrimental effects on cardiac tissue. We measured the IDO activity-related metabolites in the cardiac tissue (Supplementary Fig. 4A). Kyn levels were significantly increased in the heart and plasma as early as day 1 after MI (Supplementary Fig. 4B), compared to sham-operated WT mice. Concentrations of Kyn-derived metabolites including anthranilic acid, 3-HAA, Kna and quinolinic acid, were 100 to 1000-fold lower when compared to that of Kyn. Furthermore, their cardiac levels did not significantly change after MI or were even decreased as for 3-OHKyn (Supplementary Fig. 4C), suggesting a minor role for those metabolites in acute MI.

We thus hypothesized that IDO exerted its deleterious role on cardiac outcome through Kyn production. Ido-1 -/-mice were supplemented with Kyn and subjected to MI. As shown in (Figure 5A

D i s c l a i m e r : T h e m a n u s c r i p t a n d i t s c o n t e n t s a r e c o n f i d e n t i a l , i n t e n d e d f o r j o u r n a l r e v i e w p u r p o s e s o n l y , a n d n o t t o b e f u r t h e r d i s c l o s e d .

14 CM cells isolated from EC IDO KO mice (Figure 6B). Interestingly, the specific deletion of IDO in EC improved ex vivo contractility of CM cells isolated from EC IDOKO mice 14 days post MI (Figure 6C). Moreover, as shown in supplementary Figure 6, in vitro Kyn supplementation reduced relaxation velocity of IDO KO CM cells. These results suggest that the increase in Kyn production within EC exerted deleterious effects on CM, which may contribute to worse cardiac outcome after MI.

D i s c l a i m e r : T h e m a n u s c r i p t a n d i t s c o n t e n t s a r e c o n f i d e n t i a l , i n t e n d e d f o r j o u r n a l r e v i e w p u r p o s e s o n l y , a n d n o t t o b e f u r t h e r d i s c l o s e d .

Discussion

In the present study, we show for the first time that IDO deficiency attenuates cardiac dysfunction after MI and that Kyn, one the major IDO-related metabolites, conveys the deleterious effects of IDO on cardiac remodeling. Previous studies have already underlined the damaging impact of IDOdependent pathways on cardiovascular system. IDO has been shown to promote the development of atherosclerosis 11 , through inhibition of a major immune-regulatory and athero-protective cytokine, interleukin (IL)-10 33 . The induction of IDO by dietary microbial oxazoles also reduced IL-10 production in intestinal epithelial cells 21 . On the same note, clinical studies showed that circulating Kyn, a major IDO-related metabolites, was associated with cardiovascular risk factors 34, 35 and with worse cardiovascular outcome 12-14 .

IDO is induced in the context of inflammatory diseases 18 . MI is associated with an intense inflammatory reaction through the production of cytokines, such as TNF-α, and IL-6, that are known to foster IDO expression and activity 16, 36 . TNF-α, and IL-6 upregulation in the acute post-infarction period is under the control of hypoxia and ROS. The increased cardiac IDO activity, within the first day after MI, may therefore depend on the induction of such ischemia-associated inflammatory cytokines. IDO is known as an immunosuppressive factor through breakdown of tryptophan and generation of some metabolites, which induced inactivation or apoptosis of effector T cells 18 .

However, in our study, we did not find any significant differences in the infiltration of T cells or Tregs within the infarcted hearts of IDO KO mice as compared to controls, suggesting that such mechanisms were not involved in the deleterious IDO-related effects on cardiac remodeling. In line with these results, the lack of IDO in BM-derived inflammatory cells did not affect post-MI cardiac outcome.

Vascular cells such as SMC and EC have been shown to express IDO 19, 30 . We thus postulated that IDO localized in the vascular compartment could impact cardiac remodeling and function. 6, 19 .

D i s c l a i m e r : T h e m a n u s c r i p t a n d i t s c o n t e n t s a r e c o n f i d e n t i a l , i n t e n d e d f o r j o u r n a l r e v i e w p u r p o s e s o n l y , a n d n o t t o b e f u r t h e r d i s c l o s e d .

Kyn has been identified as an endogenous ligand for AHR 32 , and Kyn/AHR interaction exerted deleterious role during cerebral ischemia 38 . On the same note, EC-specific AHR knockout mice exhibited hypotension and blunted response to angiotensin II 39 . Moreover, AHR may have deleterious effects in myocardial ischemia 40 . In our experimental conditions, we showed that the apoptotic effects on CM, as well as ROS induction by Kyn, was dependent on AHR since its inactivation prevented the Kyn-mediated effects. The in vivo pro-apoptotic effects of Kyn were also blunted after administration of the AHR antagonist. Altogether, our data underscore the paracrine effects of ECproducing Kyn on CM apoptosis.

In conclusion, our findings show that IDO inhibitor, as well as the total and the specific endothelial deletion of IDO, protect against the deleterious cardiac effects of MI-induced ischemia. In contrast, Kyn supplementation, a major IDO-related metabolite, abolishes the protective effects of IDO deficiency in this setting. Inhibition of IDO activity might represent a novel potential therapeutic strategy to mitigate ischemic cardiac injury.

D i s c l a i m e r : T h e m a n u s c r i p t a n d i t s c o n t e n t s a r e c o n f i d e n t i a l , i n t e n d e d f o r j o u r n a l r e v i e w p u r p o s e s o n l y , a n d n o t t o b e f u r t h e r d i s c l o s e d .

D i s c l a i m e r : T h e m a n u s c r i p t a n d i t s c o n t e n t s a r e c o n f i d e n t i a l , i n t e n d e d f o r j o u r n a l r e v i e w p u r p o s e s o n l y , a n d n o t t o b e f u r t h e r d i s c l o s e d .

D i s c l a i m e r : T h e m a n u s c r i p t a n d i t s c o n t e n t s a r e c o n f i d e n t i a l , i n t e n d e d f o r j o u r n a l r e v i e w p u r p o s e s o n l y , a n d n o t t o b e f u r t h e r d i s c l o s e d .

D i s c l a i m e r : T h e m a n u s c r i p t a n d i t s c o n t e n t s a r e c o n f i d e n t i a l , i n t e n d e d f o r j o u r n a l r e v i e w p u r p o s e s o n l y , a n d n o t t o b e f u r t h e r d i s c l o s e d .

D i s c l a i m e r : T h e m a n u s c r i p t a n d i t s c o n t e n t s a r e c o n f i d e n t i a l , i n t e n d e d f o r j o u r n a l r e v i e w p u r p o s e s o n l y , a n d n o t t o b e f u r t h e r d i s c l o s e d .

21

D i s c l a i m e r : T h e m a n u s c r i p t a n d i t s c o n t e n t s a r e c o n f i d e n t i a l , i n t e n d e d f o r j o u r n a l r e v i e w p u r p o s e s o n l y , a n d n o t t o b e f u r t h e r d i s c l o s e d .

D i s c l a i m e r : T h e m a n u s c r i p t a n d i t s c o n t e n t s a r e c o n f i d e n t i a l , i n t e n d e d f o r j o u r n a l r e v i e w p u r p o s e s o n l y , a n d n o t t o b e f u r t h e r d i s c l o s e d .

D i s c l a i m e r : T h e m a n u s c r i p t a n d i t s c o n t e n t s a r e c o n f i d e n t i a l , i n t e n d e d f o r j o u r n a l r e v i e w p u r p o s e s o n l y , a n d n o t t o b e f u r t h e r d i s c l o s e d .

D i s c l a i m e r : T h e m a n u s c r i p t a n d i t s c o n t e n t s a r e c o n f i d e n t i a l , i n t e n d e d f o r j o u r n a l r e v i e w p u r p o s e s o n l y , a n d n o t t o b e f u r t h e r d i s c l o s e d .

D i s c l a i m e r : T h e m a n u s c r i p t a n d i t s c o n t e n t s a r e c o n f i d e n t i a l , i n t e n d e d f o r j o u r n a l r e v i e w p u r p o s e s o n l y , a n d n o t t o b e f u r t h e r d i s c l o s e d .

D i s c l a i m e r : T h e m a n u s c r i p t a n d i t s c o n t e n t s a r e c o n f i d e n t i a l , i n t e n d e d f o r j o u r n a l r e v i e w p u r p o s e s o n l y , a n d n o t t o b e f u r t h e r d i s c l o s e d .

Discussion

This work shows that IDO deficiency prevents cardiac function deterioration and adverse LV remodeling, following MI. We demonstrated that Kyn, an IDO activity-resulting metabolite, mediates its deleterious effects, and this was attributed to an important production of Kyn by EC.

Previous cohorts conducted in patients with or without a history of CHD have considered urinary or plasma Kyn/Trp ratio as an important predictor of major acute coronary events (Eussen et al., 2015;[START_REF] Pedersen | Urinary excretion of kynurenine and tryptophan, cardiovascular events, and mortality after elective coronary angiography[END_REF]Pedersen et al., , 2015;;[START_REF] Eva Ringdal | Systemic Markers of Interferon-γ-Mediated Immune Activation and Long-Term Prognosis in Patients With Stable Coronary Artery Disease[END_REF], as well as a marker of adverse LV remodeling, following MI [START_REF] Saadi | Plasma Indoleamine 2,3-dioxygenase is predictive of left ventricular remodeling after myocardial infarction[END_REF]. Only, a proteome genome-wide association study revealed an inversed association linking plasma IDO to the risk of developing IHD [START_REF] Li | Indoleamine 2,3dioxygenase and ischemic heart disease: a Mendelian Randomization study[END_REF]. This result is not consistent with the previous observational studies. Surprisingly, the authors analyzed plasma IDO protein instead of the metabolites Kyn/Trp that reflect its activity. Commonly, in the plasma, we measured Trp metabolites rather than IDO itself, which is expressed in cells. Moreover, other experimental studies have also underscored the critical role of IDO in the progression of cardiovascular diseases. For instance, IDO has been shown to be implicated in the development of atherosclerosis through the inhibition of IL-10, known to be atheroprotective (Mallat et al., 1999;Metghalchi et al., 2015), as well as in AngII-induced AAA by promoting VSMC apoptosis (Metghalchi et al., 2018;Wang et al., 2017b). On the other hand, other studies showed a protective role of IDO or an IDO activity-resulting metabolite, 3-HAA, in atherosclerosis (Cole et al., 2015;Polyzos et al., 2015;Zhang et al., 2012). The discrepancy in the results could be attributed to the different mouse models used and the efficiency of IDO inhibitor to effectively inhibit IDO activity in vivo.

IDO is mainly induced in DCs and macrophages by pro-inflammatory stimuli, mainly TNF-α, IL-6, and IFN-γ (Chon et al., 1996;[START_REF] Jiang | Indoleamine-2,3-Dioxygenase Mediates Emotional Deficits by the Kynurenine/Tryptophan Pathway in the Ethanol Addiction/Withdrawal Mouse Model[END_REF]O'Connor et al., 2009). It has also been

shown that IDO expression is enhanced in DCs by hypoxia [START_REF] Song | Hypoxia enhances indoleamine 2,3-dioxygenase production in dendritic cells[END_REF]. Of note, MI is accompanied with conditions of hypoxia, oxidative stress and inflammation associated with the release of pro-inflammatory cytokines like TNF-α, IL-6 and IL-1β [START_REF] Shimizu | Physiological and pathological cardiac hypertrophy[END_REF], thus suggesting that these conditions may be responsible for cardiac IDO activity induction observed at day 1 post-MI. Of note, it has been shown that HIF-1 β encoded by the ARNT (Aryl Hydrocarbon Receptor Nuclear Translocator) gene interacts with HIF-1α to form a heterodimer [START_REF] Ichihara | A role for the aryl hydrocarbon receptor in regulation of ischemiainduced angiogenesis[END_REF], suggesting a cross-talk between HIF-1α and AHR pathways. Interestingly, activated AHR was also shown to induce IDO expression in DCs [START_REF] Vogel | Aryl hydrocarbon receptor signaling mediates expression of indoleamine 2,3-dioxygenase[END_REF]. Our results showed an increase in cardiac expression of an AHR target gene, cyp1a1, early after MI, suggesting an induction of AHR activity that may stimulate IDO expression within the heart.

Trp is an essential amino-acid found in the diet. After food digestion, it is absorbed by the gut where it crosses enterocytes thanks to two distinct carrier molecules: a Na + -dependent transmembrane protein-SLC6A19 found in the apical membrane, and SLC16A10 in the basolateral epithelial membrane. When IDO is induced, it catabolizes Trp into Kyn and Kynderived metabolites via the KP which accounts for 95% of Trp catabolism [START_REF] Taleb | Tryptophan Dietary Impacts Gut Barrier and Metabolic Diseases[END_REF]. This major route of L-Trp catabolism leads ultimately to the formation of NAD + , selectively in liver which then excretes it in the form of NAM in the circulation where it is taken by other tissues to synthesize their need of NAD [START_REF] Liu | Quantitative Analysis of NAD Synthesis-Breakdown Fluxes[END_REF]. In the heart, we found that Quina, which is the NAD + precursor in the KP, was weakly produced after MI and no difference has been found between mice with MI and sham-operated mice. This excludes the possibility of NAD + production in the heart due to IDO activation following MI. The second pathway is the 5-HT pathway, which accounts for ~1-2% of Trp catabolism. A recent study deciphered an important role of platelet-derived 5-HT in aggravating myocardial I/R injury via enhancing neutrophil degranulation and that its long-term reuptake inhibition protected patients with depression from cardiovascular events [START_REF] Mauler | Platelet Serotonin Aggravates Myocardial Ischemia/Reperfusion Injury via Neutrophil Degranulation[END_REF]. We analyzed 5-HT concentrations after MI in the LV of mice harboring total or specific IDO deletion in CM, EC, VSMC and their controls. The results showed a homogenous increase in 5-HT concentrations at day 1 post-MI in all IDO deficient mouse models, indicating a shift of Trp catabolism toward 5-HT in absence of IDO.

However, despite the increase in 5-HT in all these mouse models only IDOKO and EC IDOKO but not IDO deletion in CM neither VSMC showed protective roles after MI, suggesting that IDO effects in MI are independent of serotonin pathway. The third pathway is the indole pathway which accounts for ~5% of overall Trp degradation. Indole metabolites are known to activate AHR [START_REF] Seok | Trace derivatives of kynurenine potently activate the aryl hydrocarbon receptor (AHR)[END_REF], however, they are unlikely to be involved in the observed effects in the heart given that they are produced by bacteria in the gut.

IDO biologic function relies on Kyn generation and Trp consumption. It is known that IDO through catabolizing Trp induces the activation of stress-response pathways such as general control nondepressible 2 (GCN2) kinase [START_REF] Munn | Indoleamine 2,3 dioxygenase and metabolic control of immune responses[END_REF]. In T cells, this leads to cell-cycle arrest and functional anergy, blocks T helper (TH) 17 differentiation and promotes de novo Tregs differentiation. In our project, we did not find any significant differences in the infiltration of T cells or Tregs within the infarcted LV of Ido-1 -/-mice when compared to their controls, suggesting that GCN2 pathway is unlikely involved in the phenotype. This is also consistent with the fact that chimeric mice displaying IDO deletion in BM-derived cells did not show any effects on post-MI outcome.

Principal cells constituting the heart are CMs, ECs, VSMCs and FBs [START_REF] Wagner | Cellular cross-talks in the diseased and aging heart[END_REF]. We wondered which cell type could be implicated in IDO-mediated effects following MI.

In order to answer this question, we generated different mouse strains with cell specific IDO deletion. Notably, we found an increased Kyn/Trp ratio in CM isolated from mice with MI compared to sham-operated mice. However, IDO specific deficiency in CM did not show any effects on cardiac function and LV remodeling, following MI. It is known that vascular cells such as VSMC (Cuffy et al., 2007;[START_REF] Sakash | Cytokines Induce Indoleamine 2,3-Dioxygenase Expression in Human Atheroma-Associated Cells: Implications for Persistent Chlamydophila pneumoniae Infection[END_REF] and EC (Wang et al., 2014) express IDO. We found that IDO specific deletion in EC, but not in VSMC, attenuated cardiac dysfunction and adverse LV remodeling, following MI, indicating the importance of IDO expressed in EC. Other important effectors participating to the post-MI reparative response, mainly fibrosis, are FBs which are also known to express IDO [START_REF] Cui | Tumor-Associated Fibroblasts and Microvessels Contribute to the Expression of Immunosuppressive Factor Indoleamine 2, 3-Dioxygenase in Human Esophageal Cancers[END_REF]. Although FBs are probably implicated in IDO activity-mediated effects following MI, the generation of a mouse strain displaying a specific IDO deletion in these cells is not feasible. Notably, FBs do not have a specific marker because many of the proteins highly expressed in these cells are also expressed in other cell types [START_REF] Wagner | Cellular cross-talks in the diseased and aging heart[END_REF] Our results showed that total IDO deletion or its specific deletion in EC attenuated adverse LV remodeling. Ischemic cell death during myocardial infarction leads to a reparative response encompassing multiple molecular and cellular pathways in which the damaged tissue is replaced with a fibrotic scar. The smaller infarct size was associated with a less apoptosis in the LV peri-infarcted area which could explain, in turn, the observed decreased fibrosis. It has been shown that ECs promote CMs viability by enhancing their organization thanks to capillary-like networks formed by ECs, thereby resulting in a synchronized CMs contraction (Narmoneva [START_REF] Daria | Endothelial Cells Promote Cardiac Myocyte Survival and Spatial Reorganization[END_REF]. Particularly, NO secreted by EC has been found to have an anti-apoptotic effect on CM by suppressing caspase activity via S-nitrosylation [START_REF] Maejima | Nitric oxide inhibits myocardial apoptosis by preventing caspase-3 activity via S-nitrosylation[END_REF]. Interestingly, our results showed that IDO specific deletion in EC protected against CM apoptosis. We also found a significant more capillaries/CM in Ido-1 -/-mice and a trend to have more angiogenesis in EC IDOKO mice. This could be explained by the reduced number of apoptotic CMs resulting in more VEGF production. Of note, CMs are important sources of VEGF in the heart and their loss may significantly alter myocardial paracrine signaling and negatively influence post-MI ventricular remodeling [START_REF] Giordano | A cardiac myocyte vascular endothelial growth factor paracrine pathway is required to maintain cardiac function[END_REF]. Indeed, it has been reported that CMs secrete VEGF-A which mediates EC activation and proliferation by binding to its receptor VEGFR2 in these cells [START_REF] Talman | Cardiomyocyte-Endothelial Cell Interactions in Cardiac Remodeling and Regeneration[END_REF]. We found a higher VEGF-A concentrations at day 3 post-MI in the LV of EC IDOKO mice compared to their controls. This could explain the tendency for an increased capillaries/CM in these mice.

Furthermore, Ido-1 -/-and EC IDOKO mice had a preserved CM contractility that may participate to the observed attenuated cardiac dysfunction after MI, which can be also explained by the reduced CM apoptosis in the LV peri-infarcted zone. Altogether, these results suggest that IDO activity within the EC exerts its harmful effects on CM apoptosis and contractility. This is supported by previous studies showing a role of IDO activity in promoting cell apoptosis and decreasing contractility (Metghalchi et al., 2018;Wang et al., 2014;[START_REF] Watts | Indoleamine 2,3diooxygenase in periaortic fat: mechanisms of inhibition of contraction[END_REF].These effects seemed to be mediated by Kyn, the only IDO activity-resulting metabolite presenting a significant increase in the heart and plasma after MI. In vivo, Kyn supplementation reversed the protective effects of IDO deficiency on cardiac function and LV remodeling associated with an increased apoptosis in the LV peri-infarcted zone in treated mice. In vitro, Kyn increased CM apoptosis which could be the consequence of an increase in oxidative stress production in these cells.

Communication between cardiac cells is of great importance to perform the essential function of the heart. However, loss of cells occurring in some conditions such as acute MI is largely irreversible depending on cell type and dysregulation of cell communications constitute a big health burden [START_REF] Bergmann | Dynamics of Cell Generation and Turnover in the Human Heart[END_REF]. Particularly, CMs and ECs are the most abundant cardiac cells [START_REF] Bergmann | Dynamics of Cell Generation and Turnover in the Human Heart[END_REF][START_REF] Perbellini | Heterocellularity and Cellular Cross-Talk in the Cardiovascular System[END_REF]Pinto et al., 2016;[START_REF] Wagner | Cellular cross-talks in the diseased and aging heart[END_REF] and communication between them, by both autocrine and paracrine mechanisms, is of major importance for cardiac repair post-MI [START_REF] Perbellini | Heterocellularity and Cellular Cross-Talk in the Cardiovascular System[END_REF]Pinto et al., 2016;[START_REF] Talman | Cardiomyocyte-Endothelial Cell Interactions in Cardiac Remodeling and Regeneration[END_REF]. We highlighted Kyn as a new mediator orchestrating the communication between EC and CM. Our in vitro study showed that non-CMs isolated from the infarcted LV of mice harboring a specific IDO deletion in EC preserved against CMs apoptosis. We demonstrated that Ido-1 mRNA expression or Kyn/Trp ratio was higher in the EC than the CM. Also, Kyn production was more elevated in the supernatant of cultured ECs than CMs, suggesting that EC is the major reservoir of Kyn in the heart following MI.

  At PARCC, special thanks to Dr. Jean-Sébastien SILVESTRE and team 6 for the fruitful collaboration. Thank you so much José VILAR for your availability and generosity in providing explanations, Mathilde LEMITRE for the help, Ivana ZLATANOVA and Christina PINTO for teaching me the technique of left anterior descending coronary artery ligation. I am also grateful for Ingrid GOMEZ and the lovely Yanyi SUN for the help. Thank you Angélique LEVOYE, Vincent DUVAL, Paul ALAYRAC, and Stéphane CAMUS. Big thanks to Pr. Jean-Sébastien HULOT for the confidence and freedom that he gave me every time I needed to do experiments in his laboratory. I would thank Marion BOUVET who taught me how to isolate cardiomyocytes. Thank you Clément DELACROIX for sharing your bench, Nihar MASURKAR for your kindness, and all team 7 members for your welcome. I such as "Destination labo" scientific communication operation and the launch of "Apprentis Chercheurs" project organized by INSERM and "Arbre des connaissances" foundation. I would also thank you and Dr. Xavier LOYER for accepting to be members of my thesis monitoring committee and evaluate my work. Thank you both for your kindness and the numerous advices. A special thank you for Nicolas PEREZ, the head of the animal facility, and the staff for the great care brought to my mice. Many thanks to Françoise RUELLE, Aziz TAOURI, Corina SULDAC, Guillaume GUEGUIN, Laura BOGUET, Gwendoline LUQUÉ and REX. A thought for Elizabeth HUC and Emma PAURON who already left the PARCC. Stéphanie LAMOUREUX, how can I forget the "commande magasin bonjour" with the beautiful and unique smile? I am so grateful for Isabelle GALY-FAUROUX, for her kindness, generosity in providing explanations and help during the confocal microscopy training. Corinne LESAFFRE, for her kindness and availability. Dominique CHARUE, Corinne TANCHOT, Estelle ROBIDEL, Irmine FERREIRA LOISEL, for their kindness and the long discussions. A big thank you to all the administration staff: Cyrille MAHIEUX, Bruno PILLARD, Muriel DELACROIX, Véronique OBVERWEISS, Annette DERUEDA, Laurie-Louis JOSEPH and Martine JOLIBIS. A special thought for Catherine TRITSCHER, Annie GALTIER and Martine AUTRAN who already left the PARCC.

Figure 1 32 Figure 2 35 Figure 3 38 Figure 4 39 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11

 132235338439567891011 Figure 1 Hospitalization for acute myocardial infarction in the era of COVID-19………….32 Figure 2 Classification of acute myocardial infarction……………… ……….........……….35 Figure 3 Diagnosis of acute myocardial infarction.................................................................38 Figure 4 Temporal changes and LV remodeling after acute myocardial infarction.……..….39 Figure 5 LAD coronary artery surgery…………………………………………….…...……48 Figure 6 Excitation-contraction coupling in the CM..........................................................….51 Figure 7 Tissue reparative response following ischemia………………………..…………...63 Figure 8 Tryptophan catabolism.………………………………….………………...……….72 Figure 9 Kynurenine pathway…………………….…………………………………............74 Figure 10 Crystal structure of human IDO.………………….………………..…………......77 Figure 11 Oxidation of L-Trp to NFK catalyzed by human IDO.…………………………...78

  exercise on cardiovascular health. Interestingly, it can have antiatherogenic effects in the vasculature, reduce the risk of arrhythmias, induce cardioprotection against ischemiareperfusion (I/R) injury, promote an anti-inflammatory milieu through the release of musclederived myokines, and ameliorate age-related loss of muscle mass and strength. Notably, regular exercise exerts its numerous beneficial effects through different protective mechanisms. For instance, it ameliorates vascular endothelial function through enhanced vasodilatation, protects against endothelial injury and atherosclerosis, and increases coronary artery size thus reducing the risk of flow-limiting stenosis. Moreover, exercise training displays various favorable effects in the myocardium. It enhances revascularization and development of coronary collateral blood vessels leading to a better perfusion which in turn limits MI lesion size. In addition, it limits vascular and cardiac stiffness, thus protecting against heart failure (HF), and ameliorates contractility. It also improves cardiac parasympathetic regulation and protects against ventricular fibrillation (VF). Furthermore, exercise facilitates resistance to oxidative stress, as well as mitochondrial adaptations explained by a resistance to injury via an improved tolerance to apoptotic stimuli or better capacity for scavenging reactive oxygen species (ROS). Finally, exercise ameliorates insulin resistance and promotes a healthy gut microbiota[START_REF] Fiuza-Luces | Exercise benefits in cardiovascular disease: beyond attenuation of traditional risk factors[END_REF].

Figure 1

 1 Figure 1 Incidence of hospitalization for acute MI before and during the COVID-19 pandemicin 2020 and during the same period in 2019, relative to the incidence of hospitalization for COVID-19. This scheme is fromSolomon et al., manuscript (Solomon et al., 2020)

Figure 2

 2 Figure 2 Classification of acute myocardial infarction

Figure 3

 3 Figure 3 Pathologic and clinical ST-Segment Elevation Acute Myocardial Infarction (STEMI) and Non-Segment Elevation Acute Myocardial Infarction (NSTEMI).

Figure 4

 4 Figure 4 Temporal changes and LV remodeling following acute MI.

Figure 5

 5 Figure 5 LAD coronary artery surgery. A) Intubated and ventilated mouse during the surgery. B) The pattern of LCA in the mouse and position of the ligation. C) An infarcted heart at day one post-MI. D) An infarcted heart at day 14 post-MI. LAD: left anterior descending coronary artery, LA: left atrium, LV: Left ventricle, RA: right atrium, RV: right ventricle.

Figure 6

 6 Figure 6 Excitation-contraction coupling in the CM. Depolarization due to the opening of voltage-gated Na + -channels (1). Ca 2+ entry via activated L-type voltage-gated Ca 2+ channels

  Macrophages and DCs are found in the normal cardiac valves. Macrophages are also present in a high concentration in the atrioventricular node where they interact with CMs through Cx43-containing gap junctions[START_REF] Hulsmans | Macrophages Facilitate Electrical Conduction in the Heart[END_REF]. Moreover, monocytes, neutrophils, B and T cells circulate in the blood within the coronary artery and macrophages, mast cells as well as DCs interact with the CMs neighboring the coronary vessel. This specific localization of leukocytes suggests a specific intercellular communication between these ICs and the other resident cells in the heart like CMs, FBs and ECs by secreting cytokines, chemokines and growth factors and expressing receptors recognizing these products. For instance, communication between CMs and neutrophils through IL-6 results in neutrophils activation via the expression of intercellular adhesion molecule 1 (ICAM1). In addition, TGF-β, VEGF

  as FB-to-myofibroblast and endothelial-to-mesenchymal transition (EndoMT). This latter is explained by EC acquisition of cellular motility and contractility associated to the loss of endothelial-specific genes expression such as vascular-endothelial cadherin and CD31, and expression of SM22α, S100A4, fibronectin, N-cadherin, type I and III collagen, which participate to fibrosis through TGF-β production. Macrophages also foster angiogenesis through interaction with ECs and secretion of growth factors such as VEGF-A and VEGF-C.In turn, ECs promote expansion and differentiation of hematopoietic progenitor cells towards an M2-macrophage phenotype. Moreover, TNFα secretion by mast-cells and IL-17 secretion by T cells result in ECs and cardiac FBs activation, respectively. Moreover, T cell-derived IL-10 reduces the ratio of pro-inflammatory M1 to M2 macrophages and activates FBs.[START_REF] Swirski | Cardioimmunology: the immune system in cardiac homeostasis and disease[END_REF][START_REF] Wagner | Cellular cross-talks in the diseased and aging heart[END_REF].FBs respond to pro-inflammatory factors by proliferating, differentiating into myofibroblasts and producing matrix proteins. In turn, they participate to myeloid cells recruitment thanks to FB-derived granulocyte-macrophage colony stimulating-factor (GM-CSF). Moreover, since FBs regulate ECM by producing proteases, they indirectly influence CMs which are physically linked to the ECM via integrin molecules.

Figure 7

 7 Figure 7 Tissue reparative response following ischemia. Phase 1: Thrombus formation and damaged cells start to die. Phase 2: Inflammatory phase featuring immune cells recruitment for the removal of dead cells. Phase 3: Revascularization consisting in new vessel formation and/orremodeling of preexisting vasculature and formation of scar tissue in order to replace necrotic cells. This scheme is fromSilvestre et al., manuscript (Silvestre et al., 2013).

  from the membrane and involved in intercellular communication[START_REF] Sánchez-Alonso | Extracellular Vesicle-Mediated Immune Regulation of Tissue Remodeling and Angiogenesis After Myocardial Infarction[END_REF]. As explained above revascularization is also possible thanks to EPCs mainly derived from the BM such as BM-derived mononuclear cells, mesenchymal/multipotent stroma cells and smooth muscle progenitor cells. These cells are classified into early and late EPCs regarding the culture duration. Early EPCs or circulating angiogenic cells are obtained after 4-7 days of culture, and have a weak proliferative potential. They consist of a mixture of T cells and CD14 + monocytes. Late EPCs or endothelial colony forming cells are obtained after 1-3 weeks of culture. Unlike early EPCs, late EPCs have a strong proliferative potential with a real potential for vascular reconstruction(Silvestre et al., 2013).

  Cardiac lymphatic vessels consist of a monolayer of lymphatic ECs. They are organized into capillaries, precollectors and collectors or collecting vessels which contain a muscular layer of autonomously contracting lymphatic smooth muscle cells (SMCs). Capillaries are responsible for absorbing interstitial fluid and solutes from the extracellular space. Precollectors, localized between absorbing initial capillaries and large collectors, transport lymph along the cardiac surface before they merge with collectors at the base of the heart. Collectors or collecting vessels run outside the heart and have the role to transport lymph via the lymph nodes towards ducts that empty into the superior vena cava at the junctions between the left and the right subclavian and internal jugular veins (Jankowska-Steifer et al., 2020).

Figure 8

 8 Figure 8 Tryptophan catabolism.

Figure 9

 9 Figure 9 Kynurenine pathway.

  consists on radical addition of the ferric iron-bound superoxide to the C2=C3 (C: carbon) bond of L-Trp. This results in ferryl-oxo and indole 2, 3 epoxide (L-Trp-epoxide) species formation.The following step leads to the ring opening after proton transfer from the NH3 + group of L-Trp to the epoxide oxygen. Then, nucleophilic attack of the Fe IV = O oxygen to C2 of L-Trp occurs. Finally, C2-C3 bond is cleaved and proton is transferred back from the C3 oxygen to the NH3 + group of L-Trp.

Figure 11

 11 Figure 11 Oxidation of L-Trp to NFK catalyzed by human IDO. Reaction mechanism of L-Trp oxidation to NFK by human IDO1 involving ferryl-oxo and Trp-epoxy species formation.

  Tit le: Endot helial cell indoleam ine 2, 3-dioxygenase 1 alt ers cardiac funct ion following m yocardial infarct ion t hrough kynurenine Manuscript num ber: CI RCULATI ONAHA/ 2020/ 050301D-A Aut hor( s) : Soraya Taleb, I nserm U970 Nadaj oe Melhem , I nserm U970 Mouna Chaj adine, I nserm Gom ez I ngrid, I nserm U698 Kiave-Yune Howangyin, I NSERM UMR-S 970 Marion Bouvet , I nserm Cam ille Knosp, PARCC Yanyi Sun, I nserm Marie Rouanet , I nserm U970 Ludivine Laurans, I NSERM U970

  ien Hulot , I cahn School of Medicine at Mount Sinai Jacques Callebert , Lariboisiere Hospit al Jean-Marie Launay, I NSERM UMR-S 942 Jerem y Fauconnier, I nserm U1046 Jean-Sébast ien Silvest re, I NSERM UMR-

  Smooth muscle cell deficiency was achieved by crossbreeding transgenic B6.Cg-Tg(Tagln-cre)1Her/J, in which the mouse smooth muscle protein 22-alpha promoter drives expression of a Cre recombinase with Ido-1 flox/flox (Ido-1 flox/flox SM22-Cre). Endothelial cell deficiency was obtained by crossbreeding transgenic Tg (Cdh5-cre/ERT2 + ), in which the vascular endothelial cell-specific cadherin promoter drives expression of a tamoxifen inducible Cre recombinase with Ido-1 flox/flox mice (Ido-1 flox/flox VECad-CreERT2 + ). In both cardiomyocyte and endothelial cell deficiency, the Cre-mediated excision of floxed Ido-1 alleles was induced by treatment with Tamoxifen containing diet (Harlan) for 15 days.Ido1 -/-, Ido-1 flox/flox αMHC-Cre (CM IDOKO), Ido-1 flox/flox SM22-Cre (SMC IDOKO) and Ido-1 flox/floxVECad-CreERT2 (EC IDOKO) mice and their respective littermate controls (Ido1 +/+ , CM IDO, SMC IDO and EC IDO) were on a C57Bl/6J background. All experiments were performed in 10-to 12week-old male animals, because of their higher susceptibility to MI compared to females23 , and were carried out using age and littermate-matched groups without randomization. In some experiments, WT mice were treated with the IDO inhibitor, 1-Methyl-L-tryptophan (L-1MT, 2mg/mL in drinking water), during 4 weeks before ligature and then 14 days after MI until sacrifice. Other Ido1 -/-mice were supplemented with Kynurenine (Kyn, 2mg/mL in drinking water), and some of these mice were injected with Aryl Hydrocarbon Receptor (AHR) antagonist CH-223191 (100µg/mouse) or its vehicle, 3 times during 1 week before MI. Some WT and Ido1 -/-mice were also subjected to medullar aplasia by 9.5 gray lethal total body irradiation and were repopulated 24 hours later with an intravenous injection of 1×10⁷ total bone marrow (BM) cells isolated from femurs and tibias of age-and sex-matched WT and Ido1 -/-mice. Mice were then challenged with acute myocardial infarction (MI) 4 weeks after BM reconstitution. Experiments were conducted according to the French and European community guidelines for experimental animal use and to the institutional guidelines approved by the local ethics committee of the French authorities, the 'Comité d'Ethique en Experimentation Animale' (CEEA) under the following number 01373.03. All the animals were maintained under identical standard conditions including housing, regular care, and normal chow in the same animal facility throughout the duration of the experiments.

  depilatory cream, and placed on a dedicated heating plate in the supine position. During the procedure, heart rate (HR) and temperature(35-37°C) were monitored. Two-dimensional parasternal long-axis views at the level of the largest Left Ventricle (LV) diameter were obtained for guided B-mode (mice subjected to myocardial infarction) and M-mode (sham-operated mice) measurements at the end of the diastole and systole. Endocardium contours were drawn from end-systolic and end-diastolic long-axis views; left ventricular end-diastolic and-systolic volumes were measured and percentage ejection fraction (EF) was then calculated (EF=[(Vd-Vs)/Vd] ×100). The echocardiographic measurements were assessed in an external imaging platform by external experts, blinded to the genotype or the treatment of mice.ImmunohistochemistryFourteen days after MI, hearts were perfused with PBS. Left ventricles were mounted in Cryomatrix (Thermo Scientific) and dropped into frozen isopentane. Sections (7 μm) were cut. For evaluation of apoptosis, heart sections and cardiomyocytes were stained with terminal deoxynucleotidyl transferase dUTP nick-end labeling technology kit (Roche Diagnostics, Meylan, France) according to the manufacturer's instructions. Immunohistochemistry was performed using antibodies for IDO (clone 4B7, Merck), CD31 (clone MEC 7.46, Abcam) and cleaved caspase 3 (clone 9661, Cell Signaling).Masson trichrome and Red Sirius stainings were completed to analyze infarct size and collagen content, respectively. Infarct size was expressed as the ratio of endocardial left ventricular scar length to the total endocardial left ventricular length. Collagen content and the number of capillaries were quantified in the border zone of the infarct scar on a Axioimager Carl Zeiss microscope. Collagen content was measured as the ratio of the total area stained by Red Sirius to the total area of the tissue section on the Image J software. Capillaries were stained with TRITC-conjugated Griffonia simplicifolia lectin (Sigma-Aldrich, Evry, France) and cardiomyocyte membranes with FITCconjugated wheat germ agglutinin lectin (Sigma-Aldrich). Results were expressed as the ratio of cardiomyocytes. All quantifications were performed in a blinded manner without knowledge of mice genotype and treatment.

  removed and perfused with buffer (130 mM NaCl, 5 mM KCl, 0.5 mM NaH₂PO₄, 10 mM HEPES, 10 mM glucose, 10 mM 2, 3-Butanedione Monoxime (BDM), 10 mM Taurine and 1.5 mM EDTA), then perfusion buffer (130 mM NaCl, 5 mM KCl, 0.5 mM NaH₂PO₄, 10 mM HEPES, 10 mM glucose, 10 mM BDM, 10 mM Taurine and 1 mM MgCl₂) after enzyme solution containing collagenase II (245 U/mg, Worthington Biochemical), collagenase IV (235 U/mg, Worthington Biochemical) and protease XIV (0.05 mg/mL, Sigma) for 80 ml/hour. CM cells were pelleted and the supernatant containing non-CM fraction was centrifuged at 200 x g for 10 minutes. CM in the pellet were suspended in calcium buffer containing CaCl₂ (0.125 µM). Gradual calcium increments were done by adding 5 µl, 5 µl, 10 µl, 30 µl and 50 µl of CaCl₂ (100 mM) with 4 minutes delay between each increase. For some conditions, CM were lysed for RNA extraction. For other conditions, CM (~10 5 cells) were incubated in 24-well plate previously pre-coated with Laminin (50 µg/ml, Gibco TM ), in the presence or not of an AHR antagonist, CH-223191 (10µM), (Sigma-Aldrich) and/or in the presence ofKyn (10µM, Sigma) or vehicles. In other experiments, CM cells were incubated in the presence of either non-CM cells (CM/non-CM ratio= 1) or non-CM supernatants after 24 H of culture. Reactive Oxygen Species (ROS) were visualized in red using CellRox Deep Red Reagent Kit (Life technologies).

  , and the following antibodies were used: Brilliant Violet (BV) 605 anti-mouse/human CD11b (M1/70, Biolegend), phycoerythrin (PE)-conjugated anti-Hu/Mo CD45R (B220) (RA3-6B2, eBioscience), PE-Cy7 Hamster Anti-Mouse CD3e (145-2C11, BD Pharmingen, BD Biosciences), BV 650 Rat Anti-Mouse CD4 (RM 4-4, BD Opti Build, BD Biosciences), PerCP-Cy5,5 Rat Anti-Mouse CD8a (53-6.7, BD Pharmingen, BD Biosceinces), allophycocyanin-conjugated anti-F4/80 (MCA497, AbD Serotec, Bio-Rad), fluorescein isothiocyanate (FITC) Rat Anti-Mouse Ly-6C (AL-21, BD Pharmingen, BD Biosciences), BUV 395 Rat Anti-Mouse Ly6G (1A8, BD Horizon, BD Biosciences). The total number of cells was then normalized to heart weight. Events were acquired on an LSR II flow cytometer (BD Biosciences) and results were analyzed on FlowJo software. In other experiments, non-CM cells were stained with Rat Anti-Mouse CD16/CD32 (2.4G2, BD Pharmingen TM ) antibody in order to block Fc receptors. Then cells were stained with phycoerythrin (PE)-conjugated Rat anti-CD31 (MEC 13.3, BD Biosciences) and fluorescein isothiocyanate (FITC) Rat anti-CD45 (30-F11, BD Biosciences). Endothelial cells (CD45-CD31+) and leukocytes (CD45+CD31-) in non-CM fraction were isolated by using FACSAria II (BD Biosciences).Quantitative Real time PCRCells were lysed in detergent buffer RLT (1:100 β-Mercaptoethanol) and then subjected to RNA extraction and reverse transcription (Qiagen). Quantitative real-time PCR was performed on an ABI PRISM 7700 (Applied Biosystems) in triplicates. Cycle threshold for Gapdh (primers: Gapdh-R, 5′-CGTCCCGTAGACAAAATGGTGAA-3′; Gapdh-L, 5′-GCCGTGAGTGGAGTCATACTGGAACA-3′) was used to normalize gene expression. Primers for Ido-1 are: Ido-1-R, 5′-. PCR conditions were 10 min at 95°C; 35 cycles of 95°C for 15 s, 60°C for 20 s, and 72°C for 20 s; and a final extension at 72°C for 20s.HPLC QuantificationsSome plasma, heart, supernatant and cell samples were subjected to HPLC quantifications, as previously described4 . The plasma samples and standards containing equal volume of 3-nitro-Ltyrosine (1M) were deproteinized by 25 l of 2 M tricholoroacetic acid. The proteins were pelleted by centrifugation at 10,000 rpm for 6 minutes. The supernatants were injected in autosampler (Waters) for detection of tryptophan (Trp) by its autofluorescence at an excitation wavelength of 286 nm and emission wavelength of 366 nm. Kynurenine (Kyn) was detected by UV absorption at 360 nm. The standards were prepared with 7% BSA containing 100 M Trp and 10 M Kyn and the height of the peak was referenced to the height of the internal standard, 3-nitro-L-tyrosine. The samples were eluted in a mobile phase of 15 mM acetic acid-sodium acetate buffer (pH 4.0) containing 27 ml/L acetonitrile rate of 0.9 ml/min on a purosphere STAR RP-18endcapped (3 m) column (Merck). The measurements of Tryptophan (Trp), Kynurenine (Kyn), Anthranilic Acid (AA), Kynurenic Acid (Kna), 3-Hydroxykynurenine (3-OHKyn), 3-Hydroxyanthranilic Acid (3HAA) and (Quinolinic Acid)

  Fig.5E) or supernatants of cultured non-CM cells (Figure6B) isolated from infarcted heart as compared to sham-operated heart. CM apoptosis was reduced in the presence of supernatant of non-

Figure 1 .

 1 Figure 1. IDO deficiency or inhibition limits cardiac dysfunction after MI A-B, Time-dependent monitoring of cardiac (A) and plasma (B) IDO activity (Kyn/Trp) in wild-type (WT) mice challenged with MI compared to sham-operated mice at day 1, 3, 5, 7 or 14 after surgery (n=3-5 mice per time point). C, Quantitative evaluation of left ventricle (LV) ejection fraction (left), LV systolic (middle) and diastolic (right) volumes in WT and Ido-1 -/-mice either sham-operated (n=5 per group) or challenged with MI (n=10 per group). D, Circulating IDO activity (Kyn/Trp) measured before MI, in 1-Methyl-L-Tryptophan (L-1MT)-treated WT mice (n=5) compared to non-treated WT mice (n=8). E, Quantitative evaluation of LV ejection fraction (left), LV systolic (middle) and diastolic (right) volumes in L-1MT-treated WT mice compared to non-treated WT mice challenged with MI (n=14 per group). Cardiac function was evaluated at day 14 after MI. Individual data are presented as aligned dot plots, with the mean and s.e.m. *P<0.05, **P<0.01, ***P<0.001.

Figure 2 .

 2 Figure 2. IDO deficiency or inhibition improves cardiac healing after MI A, Quantification and representative photomicrographs of infarct size in wild-type (WT) (n=29), Ido-1 -/-(n=11) and 1-Methyl-L-Tryptophan (L-1MT)-treated (n=13) mice challenged with myocardial infarction (MI). B-C, Quantitative evaluation of interstitial fibrosis (B) and capillary density (C) in WT and Ido-1 -/-mice either sham-operated (n=10 for WT and n=5 for IDOKO mice) or challenged with MI as well as in L-1MT-treated WT mice after MI. D, TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling) quantification and representative images (right) in the peri-infarcted area of the LV of Ido-1 -/-mice and L-1MT-treated WT mice compared to their control littermates at day 2 post-MI. Arrows point to apoptotic nuclei. Representative images are presented. Cardiac remodeling was evaluated at day 14 after MI. Individual data are presented as aligned dot plots, with the mean and s.e.m. *P<0.05, **P<0.01, ***P<0.001.

Figure 3 .

 3 Figure 3. IDO specific deficiency in bone morrow-derived cells or cardiomyocytes does not control cardiac function and remodeling after MI A-B, Quantitative analysis of left ventricle (LV) ejection fraction, LV systolic and diastolic volumes (A), infarct size and capillary density (B) in wild-type (WT) and Ido-1 -/-lethally irradiated and transplanted with bone marrow (BM)-derived cells isolated from WT or Ido-1 -/-mice and then challenged with myocardial infarction (MI) (n=6-14 per group). C, Ido-1 mRNA in cardiomyocytes isolated from the left ventricle (LV) of IDO deficient mice (CM IDOKO) (n=4) compared to their control littermates (CM IDO) (n=4). D-E, Quantitative evaluation of left ventricle (LV) ejection fraction, LV systolic and diastolic volumes (D), infarct size, interstitial fibrosis and capillary density (E) in cardiomyocyte IDO deficient mice (CM IDOKO) (n=12) compared to their control littermates (CM IDO) (n=13), challenged with MI. Representative images are presented. Cardiac

Figure 4

 4 Figure 4. IDO specific deficiency in endothelial cell limits cardiac dysfunction and deleterious remodeling after MI A, IDO activity (Kyn/Trp) in fluorescence-activated cell-sorted cardiac endothelial cells (EC) (CD31+CD45-), leukocytes (CD31-CD45+) and cardiomyocytes (CM) isolated from the left ventricle (LV) of WT mice (n=9), 1 day after myocardial infarction (MI) (n=3/per group). Each point represents Kyn/Trp ratio of pooled cells isolated from 3 WT mice challenged with MI. B, Ido-1 mRNA expression in pooled CM isolated from the left ventricle (LV) of WT mice (n=9) and in fluorescence-activated cell-sorted cardiac EC isolated from non-CM fraction recovered from the LV extracts of Ido-1 -/-mice (EC IDOKO) (n=3) and control littermates (n=7). C, IDO activity (Kyn/Trp) in LV extracts of EC IDOKO mice (n=4) compared to their control littermates (EC IDO) (n=5), at day 1 after MI. D-F, Quantitative evaluation of left ventricle (LV) ejection fraction, LV systolic and diastolic volumes (D), infarct size (E) and interstitial fibrosis (F) of EC IDOKO mice compared to their control littermates (EC IDO) (n=10 per group), challenged with MI. G, Representative images and quantifications of TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling) staining in the peri-infarcted area of the LV of EC IDOKO mice and control littermates (EC IDO) at day 2 post MI. Arrows point to apoptotic nuclei. Cardiac function and remodeling were evaluated at day 14 after MI. Representative images are presented. Individual data are presented as aligned dot plots, with the mean and s.e.m. *P<0.05, **P<0.01, ***P<0.001.

Figure 5 .

 5 Figure 5. Kynurenine metabolite alters cardiac function and remodeling after MI A-D, Kynurenine (Kyn) levels at day 3 after myocardial infarction (MI), left ventricle (LV) ejection fraction, LV systolic and diastolic volumes (A), capillary density (B), infarct size (C) and interstitial fibrosis (D) in Ido-1 -/-mice supplemented with Kyn (Ido-1 -/-+ Kyn) (n= 9) compared to Ido-1 -/-mice (n=7), challenged with MI. E, TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling) staining and quantification in the peri-infarcted area of the LV of Ido-1 -/-+ Kyn mice compared to Ido-1 -/-at day 2 post MI. F, Representative images and quantification of reactive oxidative species (ROS) staining in cultured cardiomyocytes (CM) treated with either Kyn (10µM) or vehicle (DMSO, 1/3000), (n=4 wells per condition, the result shown is representative of 4 independent experiments). The pictures represent merged images of CM in green and ROS staining in red. ROS staining was normalized on CM area. G, TUNEL staining and quantification in the peri-infarcted area of the LV of Ido-1 -/-+ Kyn mice compared to Ido-1 -/-, pre-treated with either aryl hydrocarbon receptor (AHR) antagonist (CH-223191) or its vehicle control (vehicle CH-223191), at day 2 post MI. Arrows point to apoptotic nuclei. Representative

Figure 6 .

 6 Figure 6. IDO specific deficiency in endothelial cell limits cardiomyocyte apoptosis and improves cardiomyocyte contractility. A, Kynurenine (Kyn) levels in supernatants after 24-hours of culture of cardiomyocytes (CM) or non-cardiomyocytes (non-CM) isolated from the left ventricle (LV) of endothelial cell (EC) IDO deficient mice (EC IDOKO) and their control-matched animals, at day 1 after MI or either after sham condition (n= 3 per group). B, Representative images and quantification of TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling) staining in cultured CM incubated with supernatants from cultured non-CM isolated from LV non-infarcted area of either EC IDOKO or their littermate controls challenged with MI or sham-operated mice (n=4 wells per condition, the result shown is representative of 2 independent experiments). Arrows point to apoptotic nuclei. Representative images are presented. C, Contraction and relaxation velocities of isolated CM from EC IDOKO mice (n=3) compared to their control littermates (n=3), after 14 days post-MI. Each point represents a value of CM contractility. Individual data are presented as aligned dot plots, with the mean and s.e.m. **P<0.01, ***P<0.001.

Résumé

  L'infarctus du myocarde (IM) survient principalement suite à la rupture d'une plaque d'athérosclérose aboutissant à une occlusion de l'artère coronaire par un thrombus. Les cardiomyocytes (CM) meurent par manque d'oxygène et de nutriments. Une réponse réparatrice consistant en une inflammation suivie d'une fibrose se déclenche permettant d'induire un remodelage cardiaque. L'indoléamine 2,3 dioxygénase 1 (IDO), induite par des stimuli pro-inflammatoires comme l'interféron-gamma (IFN)-γ, catalyse la dégradation du L-Tryptophane (Trp) en kynurénine (Kyn) et d'autres métabolites impliqués dans différentes pathologies telles que l'athérosclérose. Plusieurs études ont montré que l'activité plasmatique d'IDO, définie par le ratio Kyn/Trp, est associée à un mauvais pronostic cardiovasculaire chez les patients souffrant de maladie coronaire. Notre hypothèse est qu'IDO est induite après IM et que son activité locale participe au remodelage et la fonction cardiaque. Notre modèle de souris consiste en une ligature de l'artère coronaire gauche afin d'induire l'IM. Nos résultats montrent que l'activité d'IDO augmente dans le coeur et le plasma de souris sauvages (WT) dès le premier jour suivant l'IM. L'invalidation génétique d'IDO ainsi que l'inhibition pharmacologique de son activité préservent la fonction cardiaque et réduisent le remodelage cardiaque délétère suite à l'IM. Afin d'étudier le rôle d'IDO dans les cellules inflammatoires, des souris chimères WT ou Ido-1 -/-irradiées et transplantées par des cellules de moelle osseuse de souris WT ou Ido-1

-/-ont été générées. Nos résultats montrent que l'expression d'IDO dans les cellules non-dérivées de la moelle osseuse a des effets délétères, suite à l'IM. Nous avons trouvé que les CM expriment IDO et que son activité augmente après IM. De plus, les CM isolés de souris Ido-1 -/-avaient des vitesses de contraction et de relaxation plus accélérées que celles des souris Ido-1 +/+ . Afin d'étudier le rôle d'IDO dans les CM, nous avons généré des souris déficientes en IDO dans les CM. Cependant, cette délétion n'avait pas d'effet ni sur la fonction cardiaque ni sur le remodelage cardiaque après IM. Afin d'étudier le rôle des cellules vasculaires exprimant IDO dans les effets observés, des souris Ido-1 flox/flox SM22-Cre et Ido-1 flox/flox VECad-CreERT2 + déficientes en IDO respectivement dans les cellules musculaires lisses (CML) et les cellules endothéliales (CE) ont été générées. Nous n'avons observé aucun effet significatif dans le modèle de souris invalidées pour IDO dans les CML.

  It occurs in conditions of hyperglycemia defined as raised blood sugar. It is caused by either an insufficient insulin production by the pancreas or an incapacity of the body to effectively use the secreted insulin. Based on the latest statistics published by the WHO, diabetes caused 1.6 million deaths in 2016. It increases the risk of death due to CVDs, mainly IHD or stroke.

	Of note, increase in overweight, obesity, unhealthy diet and physical inactivity is a major cause
	for increased prevalence of diabetes. In France, for example, prevalence of diabetes among
	adults aged 20-79 years was estimated to 4.8%, in 2017. The highest one was in Egypt (17.3%)
	and the lowest one in Ireland (3.3%). Based on ESC: Cardiovascular Disease Statistics 2019,
	the prevalence of diabetes in people aged 20-79 across ESC member countries was greater in
	middle income countries (7.7%) compared to high income countries (5.6%) (Timmis et al.,
	2020).

  The active-site heme consists of two pockets: one proximal and another distal. Identified residues in the active site, particularly histidine His 303 , His 346 and aspartic acid Asp 274 , are important for IDO heme binding. His 346 and Asp 274 are linked via a hydrogen-bonding network within the proximal heme pocket. Moreover, other residues, notably Phe 226 , Phe 227 , Arginine Arg 231 and

  . Moreover, IDO2 was found to be expressed in DCs. Like IDO, it has a cytoplasmic expression. Based on what has been presented in the Atlas of Genetics and Cytogenetics in Oncology and Haematology in 2009, SNPs were found in human genome and were responsible for IDO2 enzymatic activity suppression, by either making a critical contact with the indole ring of Trp or by generating a premature stop codon. Individuals with one of these SNPs, display an altered immune function.

We used two-way ANOVA to compare values of sham and MI conditions

. Values are expressed as means ± s.e.m. Values were considered significant at P<0.05. All statistical tests were performed using GraphPad PRISM software version 8.3.1.

, CM isolated from EC IDO KO mice displayed better contractility highlighting the preeminent role of the crosstalk between EC and CM in the regulation of cardiac function.

  Mice with endothelial-, but not SMC-specific IDO deletion, subjected to coronary ligation exhibited less interstitial fibrosis, and cardiomyocyte apoptosis, leading to greater preservation of LV systolic function as compared to control mice, thereby revealing the importance of IDO expression in cardiac EC. Total and EC-specific deletion of IDO were associated with a decrease in apoptosis in ischemic heart after MI. This was substantiated by in vitro studies showing that IDO deletion in EC protected against CM apoptosis, and that Kyn was responsible for these pro-apoptotic effects, most likely through ROS production. In vivo, Kyn reversed the protective effects of IDO deficiency, suggesting that the increase in Kyn via upregulation of IDO in EC was involved in the detrimental effects on cardiac remodeling after MI. Moreover

16

  Cardiac cells communicate with each other through direct cell interactions and paracrine pathways, to shape signaling mechanisms in the injured cardiac tissue. Particularly, CM and EC are two of the most abundant cardiac cell types and they also play central roles in cardiac remodeling. Recent evidence underlined the importance of EC, not only as the most abundant non-myocyte cell type in the heart, but also as key players in post-infarction remodeling 37 . In our study, despite the fact that IDO expression and activity were detected in CM, IDO expression in these cells did not affect cardiac outcome after MI. Interestingly, IDO expression or activity, as assessed by Kyn/Trp ratio, in cardiac EC was much higher than that in CM, suggesting that IDO in EC, but not in CM, is a major reservoir of Kyn in the cardiac tissue. Nevertheless, EC and CM communicate with each other and this could account for the effect of EC-IDO derived metabolites on CM apoptosis and contractility, although the mechanisms of this crosstalk are not completely elucidated. The importance of EC-derived IDO was further highlighted by the fact that the non-CM fraction obtained from mice with the specific IDO deletion in EC after MI markedly decreased Kyn levels and protected CM against apoptosis. Hence, EC-derived IDO exerted pro-apoptotic effects on CM. Our results are consistent with previous observations showing that Kyn or Kyn-derived metabolites have detrimental effects on apoptosis

Acknowledgments

Research is a long trail of work, effort and perseverance. This path is full of doubts. It is the failure that leads to success. But not only this! Because after the endurance, there is the discovery: a sunshine of hope. The researcher is this candle that illuminates the darkness of ignorance. He is the ambassador of knowledge! Will be me one day?

Maybe yes because YOU are helping me dream about it. YOU are letting me grow. YOU are pushing me higher and higher. YOU are the reason for my past, my present and my future.

Acknowledgments ……………………………………………………………...6 Table of contents ……………………………………………………………...12

Acknowledgments and sources of funding

This work was supported by Inserm and French National Research Agency (ANR to S.T.). N-J.M. was the recipient of fellowships from the Medical Research Foundation (FRM FDT201904008169) and Lebanese Association for Scientific Research (LASeR) for thesis and GRRC (Groupe de Reflexion sur la Recherche Cardiovasculaire) travel grant. M.C. was the recipient of fellowship from ANR (IDOAMI). We are grateful to the imaging platform for small animals of Paris Descartes University (Carmen Marchiol, Franck Lager and Gilles Renault) for echocardiographic analysis and Camille Brunaud for performing cell sorting experiments.

Disclosures: None.

National institute for health and medical research I/P: Intraperitoneal I/R: Ischemia/reperfusion

D/L-1MT but not with D-1MT reversed IDO expressing cells-induced suppression of T cells proliferation of ovarian cancer patients, and that L-1MT and D/L-1MT are more efficient than D-1MT in inhibiting IDO enzyme activity in these cancer cells [START_REF] Qian | Efficacy of levo-1methyl tryptophan and dextro-1-methyl tryptophan in reversing indoleamine-2,3dioxygenase-mediated arrest of T-cell proliferation in human epithelial ovarian cancer[END_REF]. Unlike L-1MT, D-1MT was not able to restore Trp levels in the supernatant of IDO + expressing cells but Kyn levels were identical in both. Hence, the degree to which Trp depletion and Kyn production are abrogated by 1-MT is important to overcome IDO-induced blockade of T-cell proliferation [START_REF] Qian | Efficacy of levo-1methyl tryptophan and dextro-1-methyl tryptophan in reversing indoleamine-2,3dioxygenase-mediated arrest of T-cell proliferation in human epithelial ovarian cancer[END_REF]. Moreover, D/L-1MT power exists in ameliorating the efficacy of chemotherapeutic agents in limiting recalcitrant tumors. It has been reported that D/L-1MT effects are dependent on T cell-mediated immunity because CD4+ and CD8+ T cells depletion abolished the effects [START_REF] Muller | Inhibition of indoleamine 2,3-dioxygenase, an immunoregulatory target of the cancer suppression gene Bin1, potentiates cancer chemotherapy[END_REF]. This observation led to consider it useful in cancer therapy.

Indoximod, navoximod, epacadostat, and BMS-986205 are pharmacological IDO inhibitors actually in clinical investigation. They all trigger antitumor immunity and act in most of the time synergistically with chemotherapies [START_REF] Prendergast | Discovery of IDO1 Inhibitors: From Bench to Bedside[END_REF].

i. Indoximod (D-1MT; NLG-8189)

While D-1MT does not bind nor inhibit IDO, it is considered as an indirect inhibitor of IDO pathway [START_REF] Prendergast | Indoleamine 2,3-dioxygenase pathways of pathogenic inflammation and immune escape in cancer[END_REF]. It contributes to the majority of the antitumor effects of 1MT [START_REF] Hou | Inhibition of indoleamine 2,3-dioxygenase in dendritic cells by stereoisomers of 1-methyl-tryptophan correlates with antitumor responses[END_REF] by relieving suppression and autophagy in T cells resulting from Trp deprivation [START_REF] Metz | IDO inhibits a tryptophan sufficiency signal that stimulates mTOR: A novel IDO effector pathway targeted by D-1-methyl-tryptophan[END_REF]. Given its efficacy in combination with chemotherapy, indoximod is now a drug candidate in phase II clinical trials where results from its combination with other drugs in melanoma, metastatic prostate and breast cancers were promising [START_REF] Prendergast | Discovery of IDO1 Inhibitors: From Bench to Bedside[END_REF].

ii.

Navoximod (NLG-919)

It is developed from 4-phenylimidazole and imidazoisoindole series. Navoximod is a noncompetitive inhibitor of IDO enzyme activity with a half maximal effective concentration (EC50) of 75 nmol/L. It also inhibits TDO but its selectivity against this enzyme is only about 10-to 20-fold [START_REF] Prendergast | Discovery of IDO1 Inhibitors: From Bench to Bedside[END_REF]. Inhibition is accomplished by binding to the heme We wondered how EC could exert its effects on CM through Kyn. It is known that Kyn in most cell types diffuses passively through the cell membrane. It has been shown that, in T cells, Kyn crosses the cell membrane by the system L transporter SLC7A5 [START_REF] Sinclair | Single cell analysis of kynurenine and System L amino acid transport in T cells[END_REF]. It can then activate the AHR by binding to its hydrophobic ligand-binding pocket (Opitz et al., 2011).

Of note, it has been reported that IDO enzymatic activity is maintained thanks to IDO-Kyn/AhR-IDO feed-forward loop [START_REF] Li | Tolerogenic Phenotype of IFN-γ-Induced IDO+ Dendritic Cells Is Maintained via an Autocrine IDO-Kynurenine/AhR-IDO Loop[END_REF]. Besides Kyn, other Trp-derived metabolites such as the Trp photo-metabolite 6-fromylindolo [3, 2-b] carbazole (FICZ), as well as indoles can also act as AHR activators [START_REF] Seok | Trace derivatives of kynurenine potently activate the aryl hydrocarbon receptor (AHR)[END_REF]. Recently, Seok and colleagues identified two trace-active derivatives of Kyn named trace-extended aromatic condensation products (TEACOPs) which act as high affinity potent AHR ligands [START_REF] Seok | Trace derivatives of kynurenine potently activate the aryl hydrocarbon receptor (AHR)[END_REF]. It is important to note that in this present project, we are not able to know whether it is Kyn or its trace-active derivatives which bind to the AHR to mediate the observed effects. Importantly, a study in Chinese population revealed that AHR expression and polymorphisms are associated with risk of CAD [START_REF] Huang | AhR expression and polymorphisms are associated with risk of coronary arterial disease in Chinese population[END_REF]. AHR has been proposed to participate to myocardial I/R injury, and myocardial ischemia where its deletion attenuated the injury and the inflammatory response [START_REF] Xue | Baicalin inhibits inflammation and attenuates myocardial ischaemic injury by aryl hydrocarbon receptor[END_REF]. Moreover, Kyn was found to exert deleterious role during stroke through interaction with AHR (Cuartero et al., 2014). In our project, we demonstrated that Kyn-induced ROS production and CM apoptosis were mediated by AHR since its inhibition prevented the Kyn-mediated effects. This was also confirmed after AHR antagonist in vivo administration in Ido-1 mice treated with Kyn. This result sheds light on the importance of the AHR through Kyn activation as a potential target in the pathology of MI. Notably, its inhibition could importantly limit adverse LV remodeling following MI, thus preventing poorer outcomes like heart failure and mortality.

Pharmacological inhibition of IDO activity with a specific IDO inhibitor, L-1MT, mimics the same beneficial effects on cardiac function and LV remodeling observed in IDO KO mice as well as EC IDOKO. Actually, IDO inhibitors are in clinical investigations used as anti-cancer monotherapy or in combination with other anti-cancer drugs. Although some clinical trials were encouraging, some others failed to show beneficial effects as for Epacadostat inhibitor actually in phase III clinical trial. Hence, the usefulness of IDO inhibition in cancer therapy remains uncertain, and many side and off-target effects should be better understood. More hope should be given for further clinical investigation in IHD as no randomized controlled trials of IDO inhibitors to prevent or treat this disease have been conducted.