Henri Charrier
email: henri.charrier@pasteur-lille.fr

Marie Cuvelliez
email: marie.cuvelliez@pasteur-lille.fr

Emilie Dubois-Deruy
email: emilie.deruy@pasteur-lille.fr

Paul Mulder
email: paul.mulder@univ-rouen.fr

Vincent Richard
email: vincent.richard@univ-rouen.fr

Christophe Bauters
email: christophe.bauters@chru-lille.fr

Florence Pinet
email: florence.pinet@pasteur-lille.fr

Integrative system biology analyses identify seven microRNAs to predict heart failure

Keywords: biomarkers, miRNAs, heart failure, system biology

Heart failure (HF) has several etiologies including myocardial infarction (MI) and left ventricular remodeling (LVR), but its progression remains difficult to predict in clinical practice.

Systems biology analyses of LVR after MI predict molecular insights of this event such as modulation of microRNA (miRNA) that could be used as a signature of HF progression. To define a miRNA signature of LVR after MI, we use 2 systems biology approaches integrating either proteomic data generated from LV of post-MI rat induced by left coronary artery ligation or multi-omics data (proteins and non-coding RNAs) generated from plasma of post-MI patients from the REVE-2 study. The first approach predicts 13 miRNAs and 3 of these miRNAs were validated to be associated with LVR in vivo: miR-21-5p, miR-23a-3p and miR-222-3p. The second approach predicts 24 miRNAs among 1310 molecules and 6 of these miRNAs were selected to be associated with . We identified a signature of 7 microRNAs associated with LVR after MI that support the interest of integrative systems biology analyses to define a miRNA signature of HF progression.

Introduction

Heart failure (HF) is a major cause of mortality in occidental countries that is difficult to predict in clinical practice [START_REF] Benjamin | American Heart Association Statistics Committee and Stroke Statistics Subcommittee. Heart Disease and Stroke Statistics-2017 Update : A Report From the American Heart Association[END_REF]. HF can be the consequence of left ventricle remodeling (LVR) induced by a myocardial infarction (MI) [START_REF] Savoye | Left ventricular remodeling after anterior wall acute myocardial infarction in modern clinical practice (from the REmodelage VEntriculaire [REVE] study group)[END_REF][START_REF] Fertin | Usefulness of serial assessment of B-type natriuretic peptide; troponin I; and C-reactive protein to predict left ventricular remodeling after acute myocardial infarction (from the REVE-2 Study)[END_REF]. LVR is characterized by cardiac hypertrophy and reduction of LV wall. Although, LVR is an adaptive response early after MI, it is becoming deleterious in a long term [START_REF] St John Sutton | Left ventricular remodeling after myocardial infarction pathophysiology and therapy[END_REF]. Deciphering molecular events underlying LVR may offer new opportunities in the identification of early predictive biomarkers of LVR and HF. Omics approaches including transcriptomics, proteomics, and metabolomics have been extensively used to explore these mechanisms but the amount of complex generated data prevents from their comprehensive analysis.

Recently, systems biology opened new opportunities to understand molecular networks and identify new targets involved in HF [START_REF] Azuaje | Systems-based approaches to cardiovascular biomarker discovery[END_REF][START_REF] Mitra | Integrative approaches for finding modular structure in biological networks[END_REF]. Among those potential targets, microRNAs (miRNAs) are non-coding RNAs of 19 to 23 nucleotides that regulate gene expression by targeting messenger RNAs [START_REF] Winter | Many roads to maturity: MicroRNA biogenesis pathways and their regulation[END_REF]. MiRNAs are involved in many processes like cardiomyocyte hypertrophy, fibroblast to myofibroblast transformation and cell to cell communication [START_REF] Bang | Cardiac fibroblast-derived microRNA passenger strand-enriched exosomes mediate cardiomyocyte hypertrophy[END_REF][START_REF] Tallquist | Redefining the identity of cardiac fibroblasts[END_REF][START_REF] Dubois-Deruy | MicroRNAs regulating superoxide dismutase 2 are new circulating biomarkers of heart failure[END_REF]. The modulation of expression of a small set of miRNAs associated with LVR may define a miRNA signature to detect this process.

The present study aims to define a signature of miRNAs associated with LVR after MI to predict HF.

We analyzed two systems biology approaches that we previously developed from post MI rat model [START_REF] Dubois-Deruy | MicroRNAs regulating superoxide dismutase 2 are new circulating biomarkers of heart failure[END_REF][START_REF] Mulder | Early Versus Delayed Angiotensin-Converting Enzyme Inhibition in Experimental Chronic Heart Failure. Effects on Survival; Hemodynamics; and Cardiovascular Remodeling[END_REF][START_REF] Cieniewski-Bernard | Proteomic analysis of left ventricular remodeling in an experimental model of heart failure[END_REF][START_REF] Dubois | Decreased Serine207 phosphorylation of troponin T as a biomarker for left ventricular remodelling after myocardial infarction[END_REF] and post-MI patients from the REVE-2 study [START_REF] Pinet | Integrative network analysis reveals time-dependent molecular events underlying left ventricular remodeling in post-myocardial infarction patients[END_REF].

Results

Analysis of the protein-miRNA network derived from post-MI rats identified circulating miR-21-5p, miR-23a-3p and miR-222-3p to be associated with LVR after MI

The proteomic screening in LV of post-MI male rats [START_REF] Mulder | Early Versus Delayed Angiotensin-Converting Enzyme Inhibition in Experimental Chronic Heart Failure. Effects on Survival; Hemodynamics; and Cardiovascular Remodeling[END_REF], previously published [START_REF] Cieniewski-Bernard | Proteomic analysis of left ventricular remodeling in an experimental model of heart failure[END_REF][START_REF] Dubois | Decreased Serine207 phosphorylation of troponin T as a biomarker for left ventricular remodelling after myocardial infarction[END_REF], allowed the identification of 45 proteins modulated by LVR. Using the Qiagen's Ingenuity Pathway knowledge base, we built a protein-miRNA interaction network highlighting 13 candidate miRNAs which were prioritized to identify candidate miRNAs to detect LVR after MI (Figure 1). The 13 candidate miRNAs are predicted to interact with 8 out of the 45 proteins modulated by LVR [START_REF] Dubois-Deruy | MicroRNAs regulating superoxide dismutase 2 are new circulating biomarkers of heart failure[END_REF], testifying that they are involved in LVR after MI. Moreover, 9 out of the 13 candidate miRNAs have been described as biomarkers of HF: miR-21-3p, miR-21-5p, miR-23a-3p, miR-29b-3p, miR-122, miR-133a, miR-145-5p, miR-222-3p and miR-320a [START_REF] Dubois-Deruy | MicroRNAs regulating superoxide dismutase 2 are new circulating biomarkers of heart failure[END_REF][START_REF] Kuosmanen | MicroRNA profiling of pericardial fluid samples from patients with heart failure[END_REF][START_REF] Cortez-Dias | Circulating miR-122-5p/miR-133b Ratio Is a Specific Early Prognostic Biomarker in Acute Myocardial Infarction[END_REF][START_REF] Liu | Differential microRNA Expression and Regulation in the Rat Model of Post-Infarction Heart Failure[END_REF][START_REF] Marques | The transcardiac gradient of cardio-microRNAs in the failing heart[END_REF][START_REF] Zhang | Circulating MicroRNA-145 is Associated with Acute Myocardial Infarction and Heart Failure[END_REF][START_REF] Van Boven | Serially measured circulating microRNAs and adverse clinical outcomes in patients with acute heart failure[END_REF], confirming their potential as targets to predict HF (Figure 1A). To prioritize candidate miRNAs with high relation specificity with LVR, we evaluated in vivo the association of the 13 candidate miRNAs with LVR after MI (Figure 1B). In LV of post-MI rats, we selected 6 candidate miRNAs up-regulated by LVR at 7 days (miR-23a-3p, miR-222-3p and miR-320a) and at 2 months after MI (miR-21-3p, miR-21-5p, miR-222-3p and miR-377-5p) [START_REF] Dubois-Deruy | MicroRNAs regulating superoxide dismutase 2 are new circulating biomarkers of heart failure[END_REF] and we excluded the 7 candidate miRNAs which were not detected (miR-122 and miR-210) or not modulated by LVR (miR-29b-3p, miR-133a, miR-145-5p, miR-338-3p and miR-483-3p). In plasma of post-MI rats, we selected 3 candidate miRNAs down-regulated by LVR at 7 days and up-regulated by LVR at 2 months (miR-21-5p, miR-23a-3p and miR-222-3p) [START_REF] Dubois-Deruy | MicroRNAs regulating superoxide dismutase 2 are new circulating biomarkers of heart failure[END_REF] and we excluded the 3 candidate miRNAs which were not detected (miR-21-3p and miR-377-5p) or not measurable (miR-320a). In plasma of post-MI patients from the REVE-2 study, we show that the 3 selected candidate miRNAs: miR-21-5p, miR-23a-3p and miR-222-3p are down-regulated by LVR at baseline and are up-regulated by LVR after 3 months, especially in men, validating their potential as circulating biomarkers of adverse LVR after MI to predict HF as previously published [START_REF] Dubois-Deruy | MicroRNAs regulating superoxide dismutase 2 are new circulating biomarkers of heart failure[END_REF].

miR-21-3p ↔ ↑ X X miR-21-5p ↔ ↑ ↓ ↑ ↓ ↑ X miR-23a-3p ↑ ↔ ↓ ↑ ↓ ↑ X miR-29b-3p ↔ ↔ miR-122 X X miR-133a ↔ ↔ miR-145-5p ↔ ↔ miR-210 X X miR-222-3p ↑ ↑ ↓ ↑ ↓ ↑ X miR-320a ↑ ↔ miR-338-3p ↔ ↔ miR-377-5p ↔ ↑ X X miR-483-3p ↔ ↔

Analysis of the REVE-2 network identified miR-21-5p, miR-222-3p, miR-335-5p, miR-26b-5p, miR-375 and miR-17-5p to detect LVR after MI

The REVE-2 molecular data generated by the measurement of 24 variables in the plasma of the patients from the REVE-2 study (including miR-21-5p, miR-23a-3p and miR-222-3p) and the EdgeLeap's knowledge platform EdgeBox were used to build the REVE-2 molecular interaction network described in detail elsewhere [START_REF] Pinet | Integrative network analysis reveals time-dependent molecular events underlying left ventricular remodeling in post-myocardial infarction patients[END_REF]. The REVE-2 network contains 1310 molecules, including 24 miRNAs which were prioritized to identify candidate miRNAs to detect LVR after MI (Figure 2). Fourteen out of the 24 candidate miRNAs are described to be associated with HF: miR-21-5p, miR-222-3p, miR-423-5p, miR-26b-5p, miR-23a-3p, miR-744-5p, miR-133a-3p, miR-17-5p, miR-29c-3p, miR-145-5p, miR-29b-3p, let-7g-5p, miR-143-3p and miR-451a [START_REF] Dubois-Deruy | MicroRNAs regulating superoxide dismutase 2 are new circulating biomarkers of heart failure[END_REF][START_REF] Kuosmanen | MicroRNA profiling of pericardial fluid samples from patients with heart failure[END_REF][START_REF] Liu | Differential microRNA Expression and Regulation in the Rat Model of Post-Infarction Heart Failure[END_REF][START_REF] Marques | The transcardiac gradient of cardio-microRNAs in the failing heart[END_REF][START_REF] Jiao | Circulating microRNA signature for the diagnosis of childhood dilated cardiomyopathy[END_REF][START_REF] Wang | Reduced exosome miR-425 and miR-744 in the plasma represents the progression of fibrosis and heart failure[END_REF][START_REF] Jakob | Profiling and validation of circulating microRNAs for cardiovascular events in patients presenting with ST-segment elevation myocardial infarction[END_REF][START_REF] Fang | Circulating microRNAs as biomarkers for diffuse myocardial fibrosis in patients with hypertrophic cardiomyopathy[END_REF], confirming they are interesting targets to predict HF (Figure 2A-B). To prioritize candidate miRNAs with high relation specificity with LVR, we evaluated in silico the association of the 24 candidate miRNAs with LVR after MI through 2 criteria: active modules and betweeness centrality (Figure 2A). To avoid the selection of miRNAs associated with mechanisms not specific of LVR such as inflammation [START_REF] Pinet | Integrative network analysis reveals time-dependent molecular events underlying left ventricular remodeling in post-myocardial infarction patients[END_REF], we excluded the 15 candidate miRNAs active only at baseline. However, it could be interesting to analyze the miRNAs only active at baseline as potential biomarker of early LVR. To avoid the selection of miRNAs less significant in LVR, we excluded 13 miRNAs that were not in the top 50 molecules with the highest centrality. When combining these 2 criteria, among the 24 candidate miRNAs, only 6 remained: miR-21-5p, miR-222-3p, miR-335-5p, miR-26b-5p, miR-375 and miR-17-5p (Figure 2B). Interestingly, the 6 remaining candidate miRNAs are predicted to be active at least at 3 months after MI, when only specific mechanisms of LVR seems to be effective [START_REF] Pinet | Integrative network analysis reveals time-dependent molecular events underlying left ventricular remodeling in post-myocardial infarction patients[END_REF]. To date, only miR-21-5p and miR-222-3p, also identified by the first approach, were validated in vivo to detect LVR after MI [START_REF] Dubois-Deruy | MicroRNAs regulating superoxide dismutase 2 are new circulating biomarkers of heart failure[END_REF], testifying that the signature defined by the 6 last candidate miRNAs may be used as circulating biomarker of adverse LVR after MI to predict HF. An analysis of the 7 miRNAs targets was performed using the ClueGO [START_REF] Bindea | A Cytoscape plug-in to decipher functionally grouped gene ontology and pathway annotation networks[END_REF] and CluePedia [START_REF] Bindea | CluePedia Cytoscape plugin: Pathway insights using integrated experimental and in silico data[END_REF] 129 applications of Cytoscape (version 3.4.0). The applications used the miRecords database to identify 130 the experimentally validated targets of each miRNA which were submitted to Gene Ontology 131 enrichment (Figure 3).

REVE-2 molecular interaction network

Prediction of 1 310 molecules including [START_REF] Fang | Circulating microRNAs as biomarkers for diffuse myocardial fibrosis in patients with hypertrophic cardiomyopathy[END_REF] Thirty-one targets were predicted to interact with 5 out of 7 miRNAs (miR-21-5p, miR-222-3p, miR-23a-3p, miR-375 and miR-17-5p) (Figure 3A). The 2 remaining miRNAs (miR-335-5p and miR-26b-5p) had no validated target in miRecords. We can observe that the miRNA's targets are involved in pathways involved in LVR development such as fibroblast proliferation, regulation of reactive oxygen species metabolism and intrinsic apoptotic signaling pathways, but also embryonic heart development as embryonic heart tube development and aorta development (Figure 3B). These results testify of the involvement of the 7 miRNAs in cardiac development and LVR processes.

Discussion and Perspectives

Systems biology approaches have been shown to improve the biomarker discovery in comparison with traditional omics approaches by allowing the selection of biomarkers with a biological relevance in the pathology [START_REF] Azuaje | Systems-based approaches to cardiovascular biomarker discovery[END_REF][START_REF] Mitra | Integrative approaches for finding modular structure in biological networks[END_REF]. In this study, we described two systems biology analyses integrating both omics data to identify candidate miRNAs associated with LVR after MI and to predict HF. As interaction networks rely on the use of public databases, prediction of miRNA interactions are not always experimentally validated and may give an approximate vision of the complex mechanisms underlying LVR [START_REF] Bray | Limits of computational biology[END_REF][START_REF] Oulas | Systems Bioinformatics: increasing precision of computational diagnostics and therapeutics through network-based approaches[END_REF]. To avoid this issue, predicted miRNA interactions were either experimentally validated or predicted by at least 3 different databases in this study.

Moreover, while most systems biology approaches often integrate a single timepoint, we used different timepoints to integrate the molecular events underlying LVR progression. Finally, both approaches include a selection of candidate miRNAs associated with LVR after MI either by experimental validation in vivo or by in silico priorization before validation in post-MI patients.

The first approach, derived from post-MI rat model, predicted 13 candidate miRNAs and 3 of these candidate miRNAs were selected to be associated with LVR in vivo both in LV and in plasma of post-MI rats: miR-21-5p, miR-23a-3p and miR-222-3p. In this first approach, candidate miRNAs were selected based on the hypothesized behavior of circulating miRNAs as endocrine signal, but our results show that candidate miRNAs does not have necessarily the same profile in LV and in plasma, testifying that miRNAs secreted by LV are quantitatively and qualitatively different from their intracellular profile in LV. Moreover, although circulating miRNA profile in HF rat models may not reflect human profile [START_REF] Vegter | Rodent heart failure models do not reflect the human circulating microRNA signature in heart failure[END_REF], we validated the association of miR-21-5p, miR-23a-3p and miR-222-3p with LVR after MI both in plasma of HF rat model and HF patients, testifying that these 3 selected miRNAs define a conserved miRNA signature of LVR. The second approach predicted 24 candidate miRNAs among 1310 molecules and 6 of these miRNAs were selected to be associated with LVR in silico: miR-17-5p, miR-21-5p, miR-26b-5p, miR-222-3p, miR-335-5p and miR-375.

Although miR-21-5p and miR-222-3p were validated by the first approach, miR-335-5p, miR-26b-5p, miR-375 and miR-17-5p remain to be validated. Altogether, these two integrative systems biology analyses identified a signature of 7 microRNAs associated with LVR after MI and more especially with mechanisms underlying LVR, such as apoptosis, oxidative stress and fibroblasts proliferation.

MiR-21-5p is one of the most studied miRNAs in cardiovascular diseases. Indeed, it was shown that the failing heart was able to release miR-21-5p into the circulation [START_REF] Marques | The transcardiac gradient of cardio-microRNAs in the failing heart[END_REF]. Mir-23a-3p is less studied. However, a correlation was described between miR-23a and pulmonary function of patients with idiopathic pulmonary hypertension [START_REF] Sarrion | Role of circulating miRNAs as biomarkers in idiopathic pulmonary arterial hypertension : possible relevance of miR-23a[END_REF]. MiR-23a was also shown to regulate cardiomyocyte apoptosis by targeting SOD2 mRNA [START_REF] Long | miR-23a Regulates Cardiomyocyte Apoptosis by Targeting Manganese Superoxide Dismutase[END_REF]. MiR-222-3p is known to regulate SOD2 expression in HF patients [START_REF] Dubois-Deruy | MicroRNAs regulating superoxide dismutase 2 are new circulating biomarkers of heart failure[END_REF] and to be involved in the inhibition of myocardial fibrosis by targeting TGFβ [START_REF] Verjans | MicroRNA-221/222 Family Counteracts Myocardial Fibrosis in Pressure Overload-Induced Heart Failure[END_REF].

Recently, we showed that miR-21-5p, miR-23a-3p and miR-222-3p were decreased in the plasma of patients with high LVR at baseline and increased at one year [START_REF] Dubois-Deruy | MicroRNAs regulating superoxide dismutase 2 are new circulating biomarkers of heart failure[END_REF]. MiR-335-5p has not been yet linked with cardiovascular diseases and it has been described as a potential biomarker of osteosarcoma in children [START_REF] Allen-Rhoades | Cross-species identification of a plasma microRNA signature for detection, therapeutic monitoring; and prognosis in osteosarcoma[END_REF] and of osteoporosis [START_REF] Kocijan | Circulating microRNA signatures in patients with idiopathic and postmenopausal osteoporosis and fragility fractures[END_REF]. MiR-375 has been shown to be mainly expressed in the developing heart and to a lower extent in the adult heart [START_REF] Cao | microRNA expression profiling of the developing mouse heart[END_REF] and it is also increased in the plasma of pregnant women with fetal congenital heart defects [START_REF] Zhu | Identification of maternal serum microRNAs as novel non-invasive biomarkers for prenatal detection of fetal congenital heart defects[END_REF]. MiR-26b-5p and miR-17-5p are well known to be involved in the cardiovascular system. MiR-26b-5p has been shown to be modulated in plasma of patients presenting major cardiovascular events [START_REF] Jakob | Profiling and validation of circulating microRNAs for cardiovascular events in patients presenting with ST-segment elevation myocardial infarction[END_REF]. MiR-17-5p was shown to be increased in the plasma of patients with hypertrophic cardiomyopathy and diffuse myocardial fibrosis [START_REF] Fang | Circulating microRNAs as biomarkers for diffuse myocardial fibrosis in patients with hypertrophic cardiomyopathy[END_REF]. These results testify that miR-21-5p, miR-23a-3p, miR-222-3p, miR-320a, miR-335-5p, miR-26b-5p, miR-375 and miR-17-5p may be interesting targets to predict HF.

In conclusion, we highlighted the interest of integrative systems biology analyses to define a miRNA signature of LVR to predict HF. However, even though the building of molecular networks relies on experimental data, we showed that miRNA signature still need to be experimentally validated to be relevant in vivo.

Our study has some limitations: further experiments should be done in order to understand why there is no correlation for some miRNAs between the intracellular (LV) and extracellular (plasma) miRNA profiles. It would be important to determine the cellular origin (fibroblasts, cardiomyocytes, endothelial cells,..) of miRNAs and the characterization of their transporters from the intracellular to extracellular medium. Dubois-Deruy: 59-350253). Approval was granted by the institutional ethics review board (CEEA Nord Pas-de-Calais N°242011, January 2012). MI was induced as previously described [START_REF] Dubois-Deruy | MicroRNAs regulating superoxide dismutase 2 are new circulating biomarkers of heart failure[END_REF][START_REF] Mulder | Early Versus Delayed Angiotensin-Converting Enzyme Inhibition in Experimental Chronic Heart Failure. Effects on Survival; Hemodynamics; and Cardiovascular Remodeling[END_REF] by permanent left anterior descending coronary artery ligation in 10 weeks old Wistar male rats.

Methods

All

Hemodynamic and echocardiographic measurements were performed to detect LVR, at 7 days (n=8 in sham-and MI) and 2 months (n=8 in sham-and MI) after surgery, followed by non-infarcted area of LV sampling and plasma sampling.

The REVE-2 study

The REVE-2 study is a prospective multicenter study to analyze the association between circulating biomarkers and LVR and has been previously detailed [START_REF] Fertin | Usefulness of serial assessment of B-type natriuretic peptide; troponin I; and C-reactive protein to predict left ventricular remodeling after acute myocardial infarction (from the REVE-2 Study)[END_REF]. studies were performed at hospital discharge (baseline), 3 months and one year after MI and were used to assess LVR. LVR was defined as a >20% increase in end diastolic volume from baseline to 1 year. Serial blood samples were taken at 4 timepoint: baseline, 1 month, 3 months and 12 months.

Twenty-four molecular variables were measured in the REVE-2 plasma at 1 to 4 timepoints.

Quantification of candidate miRNAs

RNAs were extracted from non-infarcted area of LV of rats and from plasma of rats and REVE-2 patients as described elsewhere [START_REF] Dubois-Deruy | MicroRNAs regulating superoxide dismutase 2 are new circulating biomarkers of heart failure[END_REF]. Candidate miRNAs were quantified and normalized with miR-423-3p in LV and with Caenorhabditis elegans Cel-39 in plasma as decribed elsewhere [START_REF] Dubois-Deruy | MicroRNAs regulating superoxide dismutase 2 are new circulating biomarkers of heart failure[END_REF].

Functional analysis of miRNAs targets

MiRNA target prediction was performed using the ClueGO (version 2.5.2 http://apps.cytoscape.org/apps/cluego) [START_REF] Bindea | A Cytoscape plug-in to decipher functionally grouped gene ontology and pathway annotation networks[END_REF] and CluePedia (version 1.5.2 http://apps.cytoscape.org/apps/cluepedia) applications [START_REF] Bindea | CluePedia Cytoscape plugin: Pathway insights using integrated experimental and in silico data[END_REF] of Cytoscape software (version 3.4.0 http://www.cytoscape.org/). Only validated miRNAs targets from the miRecords database were selected. A Gene Ontology (GO) enrichment analysis for Biological Processes was performed for all the miRNAs targets using the ClueGO application of Cytoscape software. P-value was set at 0.05, and corrected for multiple testing using Benjamini-Hochberg adjustment.

Figure 1 .

 1 Figure 1. Identification of miR-21-5p, miR-23a-3p and miR-222-3p to detect left ventricular remodeling (LVR) after myocardial infarction (MI) and to predict heart failure (HF). (A) Design and (B) experimental selection and validation of the 13 candidate miRNAs predicted from the proteomic data obtained in LV of post-MI rats by the Ingenuity Pathway knowledge platform. Quantification of candidate miRNAs in LV and in plasma of post-MI rats at 7 days (Timepoint 0) and at 2 months and in plasma of REVE-2 patients at baseline (Timepoint 0), 3 month and 12 months after MI was published elsewhere [10]. ↑ and ↓ indicates respectively a significant increase and decrease of miRNAs (p < 0.05) detected between sham-and post-MI rats / between patients with no and high LVR, ↔ indicates no modulation, X indicates a lack of detection. MiRNAs remaining after the validation process are in bold.

  117

Figure 2 .

 2 Figure 2. Identification of miR-21-5p, miR-222-3p, miR-335-5p, miR-26ba-3p, miR-375 and miR-17-5p

126 2 . 3 .

 23 Gene ontology analysis of the 7 miRNAs targets predicted with a high relation specificity with processes 127 involved in LVR after MI 128

Figure 3 .

 3 Figure 3. Functional annotation enrichment analysis of the 7 miRNA's targets. (A) Targets of miRNAs were predicted by miRecords database. No targets have been described for miR-335-5p and miR-26b-5p. (Bb) Biological processes of miRNA's target genes were predicted by Cytoscape plugin ClueGO and Cluepedia applications (p < 0.05).

  animal experiments were performed according to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication NO1-OD-4-2-139, revised in 2011). Experimental protocols were performed under the supervision of a person authorized to perform experiments on live animals (F. Pinet: 59-350126 and E.

  miRNAs

	A	Multi-omics data	Knowledge	B
		measured in REVE-2	platform
		24 measured variables*	EdgeBox			0	1	3	12
				1	miR-21-5p*	√	√
				2	miR-222-3p*	√	√
			Association with HF 14/24 miRNAs described as	3 5 6	miR-423-5p* miR-335-5p miR-26b-5p	√ √	√ √	√ √
			biomarker of HF	9	miR-23a-3p*	√
				10	miR-124-3p	√
				18	miR-744-5p	√
		Priorization of the REVE-2
		network's molecules
		Active module	Betweenness
		Analysis	centrality analysis
		Exclusion of	Exclusion of
		miRNAs active	miRNAs with a
		only at baseline	centrality rank
		15 excluded	> 50
		miRNAs	13 excluded
			miRNAs
		Association with LVR
		6/24 miRNAs prioritized
		for their involvement in LVR

  Briefly, REVE-2 study included 226 patients with a first anterior wall Q-wave MI between February 2006 and September 2008. The research protocol was approved by the Ethics Committee of the Centre Hospitalier et Universitaire de Lille, and each patient provided written informed consent. Serial echocardiographic

miR-338-3p √ 1304 miR-424-3p √ Priorized candidate miRNAs (centrality rank) REVE-2 timepoint (months)

Acknowledgments: This work was supported by grants from "Agence Nationale de la Recherche" (ANR 15-CEA-U16), "Fondation de France" and by the "Féderation Hospital-Universitaire" FHU REMOD-VHF. We thank Jean-Paul Henry for the surgical induction of MI in rats. FP is MC substitute of COST action cardioRNA (CA1229).

Conflicts of Interest:

The authors declare no conflict of interest.


Annie Turkieh
email: ani.turkieh@pasteur-lille.fr

Henri Charrier
email: henri.charrier@pasteur-lille.fr

Emilie Dubois-Deruy
email: emilie.deruy@pasteur-lille.fr

Sina Porouchani
email: sina.porouchani@gmail.com

Marion Bouvet
email: marion.bouvet@inserm.fr

Florence Pinet
email: florence.pinet@pasteur-lille.fr

Non-coding RNAs in cardiac autophagy following myocardial infarction

Macroautophagy is an evolutionarily conserved process of the lysosome-dependent degradation of damaged proteins and organelles and plays an important role in cellular homeostasis. Macroautophagy is upregulated after myocardial infarction (MI) and seems to be detrimental during reperfusion and protective during left ventricle remodeling. Identify new regulators of cardiac autophagy may help to maintain the activity of this process and protect the heart from MI effects. Recently, it was shown that non-coding RNAs (microRNAs and long non-coding RNAs) are involved on autophagy regulation in different cell types including cardiac cells. In this review, we summarized the role of macroautophagy in the heart following MI and we focused on the non-coding RNAs and their targeted genes reported to regulate autophagy in the heart under these pathological conditions.

INTRODUCTION

Myocardial infarction (MI) is a cardiovascular event caused by obstruction of one or more arteries supplying the heart. This area of the heart is therefore no longer supplied with oxygen and nutrients leading to the death of cardiomyocytes. Coronary reperfusion is the only recognized method to reduce the size of the infarct if it is performed within hours after MI. Despite its beneficial effect, several deleterious events such as increased oxidative stress and cell death are observed during the reperfusion process. If the infarcted zone is very extensive, there is a decrease in the contractile function of the heart. In order to compensate for this loss and maintain normal blood flow, the heart will undergo structural changes such as thinning of the infarcted zone, fibrosis, cardiomyocyte hypertrophy and left ventricle (LV) dilatation [START_REF] Pfeffer | Ventricular remodeling after myocardial infarction. Experimental observations and clinical implications[END_REF] . Left ventricle remodeling (LVR) is initially a protective mechanism but in the long term can lead to heart failure (HF) [START_REF] St John Sutton | Quantitative two-dimensional echocardiographic measurements are major predictors of adverse cardiovascular events after acute myocardial infarction. The protective effects of captopril[END_REF][START_REF] De Groote | Long-term functional and clinical follow-up of patients with heart failure with recovered left ventricular ejection fraction after β-blocker therapy[END_REF][START_REF] Bauters | Longterm prognostic impact of left ventricular remodeling after a first myocardial infarction in modern clinical practice[END_REF] . Despite current therapy, acute MI and HF, remain the leading causes of death and disability worldwide. New therapeutic strategies are therefore required to protect the heart against the detrimental effects of acute ischemia/reperfusion (I/R) injury, in order to prevent cardiomyocyte death and reduce myocardial infarct size, preserve LV ventricle function, and prevent the onset of HF.

Macroautophagy is an important and non-selective proteolytic mechanism that regulates the homeostasis of long-lived proteins, macromolecules including lipids and cell organelles by surrounding them in a double-membrane vesicle, known as autophagosome in order to deliver them to lysosome for degradation [START_REF] Ohsumi | Historical landmarks of autophagy research[END_REF] . It plays an essential role for maintaining heart structure and function under baseline conditions [START_REF] Nakai | The role of autophagy in cardiomyocytes in the basal state and in response to hemodynamic stress[END_REF][START_REF] Gan | Role of FIP200 in cardiac and liver development and its regulation of TNFalpha and TSC-mTOR signaling pathways[END_REF][START_REF] Kaizuka | Atg13 Is Essential for Autophagy and Cardiac Development in Mice[END_REF] . Several studies showed that macroautophagy is upregulated in heart following MI and suggested that this process may protect heart against MI effects [START_REF] Yan | Autophagy in chronically ischemic myocardium[END_REF][START_REF] Matsui | Distinct roles of autophagy in the heart during ischemia and reperfusion: roles of AMP-activated protein kinase and Beclin 1 in mediating autophagy[END_REF][START_REF] Kanamori | The role of autophagy emerging in postinfarction cardiac remodelling[END_REF] . Recently, it was shown that non-coding RNAs (microRNAs (miRNA) and long non-coding RNAs (lncRNA)) are involved on autophagy regulation in different cell types including cardiac cells [START_REF] Gupta | Non-coding RNAs as orchestrators of autophagic processes[END_REF][START_REF] Yang | Long non-coding RNAs involved in autophagy regulation[END_REF][START_REF] Sun | MicroRNAs in Cardiac Autophagy: Small Molecules and Big Role[END_REF] . In this review, we summarized the role of macroautophagy in the heart following MI and we focused on the non-coding RNAs and their targeted genes reported to regulate autophagy in the heart under pathological conditions.

MACROAUTOPHAGY MECHANISM

Macroautophagy proceeds in several successive steps and involves different proteins as previously described [START_REF] Ohsumi | Historical landmarks of autophagy research[END_REF] . In summary, autophagy induction is mainly regulated by the ULK (unc-51-like kinase) complex which is composed of ULK1/2, ATG13 (autophagy-related gene 13), ATG101, and FIP200 (focal adhesion kinase family interacting protein with a 200 kDa mass). Activation of the PI3K complex contributes to the vesicle nucleation, the first step of autophagosome formation. This complex is composed of Beclin-1, ATG14, VPS34 (Phosphatidylinositol 3-kinase vacuolar protein sorting 34) and VPS15. Finally, two ubiquitin-like protein conjugation systems are required to the vesicle elongation, the first to form ATG12-ATG5-ATG16L1 complex and the second to form LC3II (microtubule-associated protein 1 light chain II), the lipidated form of LC3. For this latter step, ATG4 cleaves pro-LC3 to LC3I before its conjugation to phosphotidylethanolamine by ATG7, ATG3 and ATG12-ATG5-ATG16L1 complex. Several pathways were shown to regulate autophagy by activation or inactivation of one of these ATG proteins. For example, mTOR (mammalian target of rapamycin) activation inhibited autophagy by decreasing ULK1 activity [START_REF] Kim | AMPK and mTOR regulate autophagy through direct phosphorylation of Ulk1[END_REF] and ATG14/VSP34-35 complex formation [START_REF] Yuan | Regulation of PIK3C3/VPS34 complexes by MTOR in nutrient stress-induced autophagy[END_REF] . AMPK (adenosine monophosphate-activated protein kinase) positively regulated autophagy by increasing Beclin-1 phosphorylation leading to its interaction with VSP34 17 . However, Bcl-2 interacts with Beclin-1 for blocking its interaction with VSP34 [START_REF] Kang | The Beclin 1 network regulates autophagy and apoptosis[END_REF] .

1. Macroautophagy during ischemia/reperfusion

The regulation of autophagy is different during ischemia and reperfusion [START_REF] Matsui | Distinct roles of autophagy in the heart during ischemia and reperfusion: roles of AMP-activated protein kinase and Beclin 1 in mediating autophagy[END_REF] . During heart ischemia, nutrient and oxygen supplies to the cardiac cells decrease, inducing mitochondrial and cellular dysfunction that lead to cell death. To protect them, the cardiac cells induce autophagy via the AMPK/m-TOR pathway in order to degrade/eliminate damaged organelles and proteins and provide the substrates necessary for their survival. During reperfusion, there is an increase of reactive oxygen species (ROS) production inducing a strong expression of Beclin-1 which on one hand, promotes the formation of autophagosomes and on the other hand, inhibits the expression of genes involved in the fusion of autophagosomes with lysosomes [START_REF] Ma | Impaired autophagosome clearance contributes to cardiomyocyte death in ischemia/reperfusion injury[END_REF] . In addition, ROS inhibit the expression of LAMP-2, a protein involved in the fusion of autophagosomes with lysosomes. Autophagy is then induced excessively during reperfusion but is inactive. Blocking the degradation of the contents of autophagosomes promotes oxidative stress, decreases mitochondrial permeability and causes cell death. Partial inhibition of Beclin-1 expression (heterozygous mice) has been shown to protect against apoptosis induced during reperfusion while its total deletion is deleterious [START_REF] Matsui | Distinct roles of autophagy in the heart during ischemia and reperfusion: roles of AMP-activated protein kinase and Beclin 1 in mediating autophagy[END_REF] . These data showed that autophagy is a protective mechanism during ischemia but its excessive induction during reperfusion is deleterious.

Macroautophagy during LVR in post-MI

The activity of autophagy and its role in LVR post-MI have been studied in murine models with permanent ligation of the left coronary artery. Autophagy is induced in non-infarcted area of the heart during the sub-acute (1 week) and chronic (3 weeks) stages after MI [START_REF] Kanamori | The role of autophagy emerging in postinfarction cardiac remodelling[END_REF] . Inhibition of autophagy by Bafilomycin (a pharmacological agent that blocks the fusion of the autophagosome with the lysosome) promoted LVR and worsens cardiac dysfunction. In contrast, administration of Trehalose (a non-naturally reduced disaccharide) in mice after ligation, activated autophagy, reduced LVR, and improved cardiac function at 4 weeks post-MI [START_REF] Sciarretta | Trehalose-Induced Activation of Autophagy Improves Cardiac Remodeling After Myocardial Infarction[END_REF] . However, this protective effect of trehalose on the heart was not observed in mice invalidated for the Beclin-1 gene, but an increase in the activity of mTOR was observed in the non-infarcted area of the heart. It has been shown that the inhibition of mTOR activity induced autophagy leading to a decrease of LVR and an improvement in cardiac function in post-MI [START_REF] Buss | Beneficial Effects of Mammalian Target of Rapamycin Inhibition on Left Ventricular Remodeling After Myocardial Infarction[END_REF] . All these data showed a protective role of autophagy in later stages in post-MI but its activity remained insufficient to prevent LVR and cardiac dysfunction.

Macroautophagy regulation by non-coding RNAs during and following MI

About 99% of the human genome do not encode proteins, but are transcriptionally highly active and give rise to a broad spectrum of non-coding RNAs (ncRNAs) with regulatory and structural functions. Based on the size criteria of 200 nucleotides (nt), ncRNAs are divided into long (>200 nt) and short ncRNAs (<200 nt).

The ncRNAs are modulated in some cardiovascular diseases including MI [START_REF] Das | Deciphering Non-coding RNAs in Cardiovascular Health and Disease[END_REF][START_REF] Poller | Non-coding RNAs in cardiovascular diseases: diagnostic and therapeutic perspectives[END_REF] . The significant changes in their expression pattern upon MI highlighted their contribution in regulation of pathogenesis of MI. Furthermore, it was shown that ncRNAs could regulate autophagy in some cardiac disorders including MI, hypertrophy and HF [START_REF] Gupta | Non-coding RNAs as orchestrators of autophagic processes[END_REF][START_REF] Yang | Long non-coding RNAs involved in autophagy regulation[END_REF][START_REF] Sun | MicroRNAs in Cardiac Autophagy: Small Molecules and Big Role[END_REF] . In this part, we summarized the non-coding RNAs which have been reported to regulate cardiac autophagy during and following MI and highlighted their specific autophagic targets and their importance as new therapeutic targets to protect heart against I/R injury and prevent cardiac remodeling and dysfunction (Figure 1).

Macroautophagy regulation by mRNAs

MiRNAs are defined as single-stranded non-coding RNAs around 22 nucleotides and are highly conserved between species [START_REF] Das | Deciphering Non-coding RNAs in Cardiovascular Health and Disease[END_REF] . Once synthetized and matured through several steps, these miRNAs bind to the complementary 3'UTR of their target mRNA and either degrade or silence them. A near perfect match between the seed region of the miRNA (8 nucleotides at its 5' UTR end) and its target leads to complete degradation of mRNA, while a partial complementary results in the suppression of the gene expression. MiRNAs may have one or multiple mRNA targets and are involved in the regulation of numerous biological processes in the heart including autophagy.

Antiautophagic-miRNAs with protective effects

Several miRNAs were modulated during I/R and seems to have a protective effect by decreasing excessive autophagy induced-cell apoptosis by targeting one of the ATG genes. MiR-188-3p levels are reduced in cardiomyocytes treated with anoxia/reoxygenation and in MI-mice. Overexpression of miR-188-3p in MI-mice attenuated autophagy by targeting autophagy mediator Atg7 and decreased the infarcted area size [START_REF] Wang | APF lncRNA regulates autophagy and myocardial infarction by targeting miR-188-3p[END_REF] . It was shown that miR-638 suppressed the expression of Atg5 by targeting its 3'UTR region. It is down-regulated in human cardiomyocytes after hypoxia/reoxygenation (H/R) and its overexpression improve the viability of these cells. However, enforced expression of Atg5 reversed the effect of miR-638 on autophagy and cell apoptosis suggesting that miR-638 attenuated the effects of H/R treatment by regulating ATG5-mediated autophagy in human cardiomyocytes [START_REF] Zhao | Overexpression of miR-638 attenuated the effects of hypoxia/reoxygenation treatment on cell viability, cell apoptosis and autophagy by targeting ATG5 in the human cardiomyocytes[END_REF] . Also, overexpression of miR-129-5p in H9c2 cells treated by hydrogen peroxide inhibited autophagy by targeting Atg14 gene and activating PI3K/AKT/mTOR pathway resulting in decreased cell apoptosis 26 .

Other miRNAs play their protective effect by regulating one of the pathways involved on autophagy regulation. The levels of miR-223 are significantly upregulated in the heart of post-MI HFrats and in hypoxia-treated neonatal rat cardiomyocytes (NRCMs) and H9c2 cells. The increased miR-223 levels protect NRCMs and H9c2 cells from hypoxia-induced apoptosis whereas decreasing miR-223 expression had contrasting effects. This protective effect of miR-223 is explained by the decrease of its target gene expression PARP-1 (poly(ADP-ribose) polymerase 1) resulting in inhibition of excessive autophagy via the Akt/mTOR pathway [START_REF] Liu | MicroRNA-223 protects neonatal rat cardiomyocytes and H9c2 cells from hypoxia-induced apoptosis and excessive autophagy via the Akt/mTOR pathway by targeting PARP-1[END_REF] . However, miR-204 expression is decreased in the heart of rat upon I/R injury associated with increased autophagy as observed by the increased LC3II levels [START_REF] Xiao | MiR-204 regulates cardiomyocyte autophagy induced by ischemia-reperfusion through LC3-II[END_REF] . Also, it was shown that transfection of miR-204 in H9c2 cells attenuated cell apoptosis induced by H/R treatment. The protective effect of miR-204 is explained by targeting SIRT1-mediated autophagy [START_REF] Qiu | MicroRNA-204 protects H9C2 cells against hypoxia/reoxygenation-induced injury through regulating SIRT1-mediated autophagy[END_REF] . The expression of miR-34a is also decreased during I/R and overexpression of this miR decreased TNFα expression resulting in reduced autophagy and apoptosis levels on NRCMs after H/R 30 . Lower miR-29b-3p levels were found in HF patients and in hypoxia-stimulated H9c2 cells. The overexpression of miR-29b-3p inhibited autophagy and apoptosis induced in hypoxic-induced H9c2 cells through targeting SPARC and inhibiting TGFβ-1/Smad3 pathway 31 .

Antiautophagic-miRNAs with deleterious effects

Some miRNAs contribute to ischemic/reperfusion injury by inhibiting the autophagy process. The miR-497 is dramatically down-regulated in infarcted heart and in hypoxic cardiomyocytes and its overexpression in murine MI model increased the infarcted size. It was shown that miR-497 inhibited autophagy by targeting LC3B gene and induced cell apoptosis by targeting Bcl-2 gene suggesting that decreasing miR-497 levels is a protective mechanism of the heart in response to MI [START_REF] Li | Inhibition of microRNA-497 ameliorates anoxia/reoxygenation injury in cardiomyocytes by suppressing cell apoptosis and enhancing autophagy[END_REF] . The expression of miR-30e was also decreased after myocardial I/R. Its silencing in H9c2 cells increased autophagy and attenuated oxidative stress and cell apoptosis, that are reversed by treating the cells with 3methyladenine, an inhibitor of macroautophagy. These results suggest that decreasing the miR-30e levels protected the heart against I/R injury by autophagy induction [START_REF] Zheng | MicroRNA-30e protects the heart against ischemia and reperfusion injury through autophagy and the Notch1/Hes1/Akt signaling pathway[END_REF] .

Proautophagic-miRNAs with protective effects

Higashi et al. [START_REF] Higashi | MicroRNA-145 repairs infarcted myocardium by accelerating cardiomyocyte autophagy[END_REF] showed that 30 min of coronary occlusion followed by 2 days of reperfusion caused a significant decrease in the rabbit cardiac tissue expression of miR-145 in the border and infarcted areas of the myocardium compared to the remote non-infarcted area. Injection of liposomes containing miR-145 after the beginning of reperfusion reduced the infarcted area size, improved the LV function and remodeling, these beneficial effects were abolished by chloroquine treatment. Further study showed that miR-145 promoted autophagy in cardiomyocyte by directly targeting FRS2 (fibroblast growth factor receptor substrate 2) mRNA resulting in the acceleration of the transition of LC3I to LC3II, an important step of autophagosome maturation [START_REF] Higashi | MicroRNA-145 repairs infarcted myocardium by accelerating cardiomyocyte autophagy[END_REF] . The protective effect of miR-145 is also observed in H9c2 cells after H/R. In this study, the authors demonstrated that miR-145 inhibited H/R-induced apoptosis by promoting autophagy via Akt3/mTOR signaling pathway [START_REF] Yan | miRNA-145 inhibits myocardial infarction-induced apoptosis through autophagy via Akt3/mTOR signaling pathway in vitro and in vivo[END_REF] . The miR-99a was shown to be down-regulated in infarcted heart and in neonatal mice ventricle myocytes exposed to hypoxia. Lentivirus-mediated overexpression of miR-99a in infarcted heart inhibited cardiac remodeling and improved heart function at 1 and 4 weeks after its administration. It was shown that miR-99a decreased mTOR protein levels without any effect on its mRNA levels suggesting that miR-99a regulated mTOR expression at a post-transcriptional level. Consequently, the autophagy induced was associated with a decrease of cell apoptosis. This study demonstrated that overexpression of miR-99a improved post-MI cardiac function by up-regulating autophagy via targeting mTOR pathways, inhibiting apoptosis and attenuating pathological remodeling [START_REF] Li | Overexpression of microRNA-99a attenuates heart remodelling and improves cardiac performance after myocardial infarction[END_REF] . The miR-144 levels were reduced in the heart of MI mice with permanent left anterior descending artery (LAD) ligation. The miR-144 k/o mice showed a worse HF phenotype with ventricular dilatation and impaired contractility after LAD ligation. However, miR-144 administration decreased myocardial infarcted size and improved post-MI remodeling. Further study allowed authors to conclude that miR-144 increased autophagy and decreased fibrosis and apoptosis by targeting mTOR [START_REF] Li | Intravenous miR-144 reduces left ventricular remodeling after myocardial infarction[END_REF] .

Macroautophagy regulation by lncRNAs

LncRNAs are non-coding RNAs longer than 200 nucleotides that regulate both gene expression and protein translation [START_REF] Das | Deciphering Non-coding RNAs in Cardiovascular Health and Disease[END_REF] . Nuclear localized lncRNAs can regulate gene expression at both the epigenetic and transcriptional levels. Cytosol-based lncRNAs can modify protein translation by blocking, stabilizing/destabilizing, or sponging miRNAs. The lncRNAs are involved in the regulation of numerous biological processes including autophagy in cardiac and non-cardiac cells.

Antiautophagic-lncRNA with protective effects

Liu et al. [START_REF] Liu | LncRNA CAIF inhibits autophagy and attenuates myocardial infarction by blocking p53-mediated myocardin transcription[END_REF] showed that the expression of lncRNA CAIF (cardiac autophagy inhibitory factor) was significantly decreased in a mice model of I/R injury and in cardiomyocytes treated with H 2 O 2 . Conversely, overexpression of CAIF inhibited autophagy inducing cardiomyocyte cell death and cardiac dysfunction caused by I/R. In this study, the authors demonstrate that CAIF directly binds to p53 protein and blocks its interaction with the myocardin promotor. Myocardin, a smooth muscle and cardiac muscle-specific transcriptional activator, is upregulated after I/R and H 2 O 2 treatment, and is involved on autophagy regulation in cardiomyocytes by increasing Beclin 1 expression. These data suggest CAIF-P53-myocardin pathway as a novel regulator of autophagy in cardiomyocytes and as a potential therapeutic target in order to inhibit excessive autophagy and improve cardiac function after I/R 38 .

Proautophagic-lncRNA with protective effects

On the other hand, it was shown that the lncRNA H19 expression was decreased in a mice model of acute MI and that its overexpression decreased infarcted size and improved cardiac function associated with autophagy upregulation; however, the mechanisms by which autophagy is regulated by H19 is still unknown. These results suggest that H19 protects the heart from MI by increasing cardiac autophagy [START_REF] Zhou | Long non-coding RNA H19 protects acute myocardial infarction through activating autophagy in mice[END_REF] .

3.2.3. Proautophagic-lncRNAs with deleterious effects Some lncRNAs are upregulated after I/H and enhanced autophagy-target genes expression by inhibiting miRNAs expression. Yin et al. [START_REF] Yin | GATA1 activated lncRNA (Galont) promotes anoxia/reoxygenationinduced autophagy and cell death in cardiomyocytes by sponging miR-338[END_REF] showed that the lncRNA Galont (GATA1 activated lncRNA) is upregulated in neonatal mice cardiomyocytes in response to anorexia/reoxygeneation; however, miR-338 expression is downregulated. Overexpression of miR-338 directly decreased the formation of autophagic vesicules and induced cell death after anorexia/reoxygeneation treatment without any effect on control cells. The antiautophagic effect of miR-338 is explained by its direct targeting of the autophagic mediator Atg5. It was shown that Galont directly bound to miR-338 and decrease its expression. Consequently, Atg 5 expression is increased resulting in excessive cardiac autophagy and cell death [START_REF] Yin | GATA1 activated lncRNA (Galont) promotes anoxia/reoxygenationinduced autophagy and cell death in cardiomyocytes by sponging miR-338[END_REF] . Also, the lncRNA APF (Autophagy promoting factor) enhances cardiac autophagy and cell death by inhibiting miR-188-3p expression resulting in the increase of its target gene expression, Atg7 [START_REF] Wang | APF lncRNA regulates autophagy and myocardial infarction by targeting miR-188-3p[END_REF] . Furthermore, the lncRNA AK088388 is upregulated during reoxygenation in mouse cardiac myocytes associated with the decreased miR-30a expression. Overexpression of miR-30a decreased the expression of its target gene Beclin-1 resulting in inhibition of autophagy induction and decreased cell death. The co-overexpression of lncRNA AK088388 inhibited the protective effect of miR-30a. However, the mutation of the miR30-a binding site in AK088388 failed to block the effect of this miRNA on autophagy and cell survival. These results suggest that the lncRNA AK088388 regulates autophagy through miR-30a/Beclin-1 pathway to affect cardiomyocyte injury [START_REF] Wang | Long noncoding RNA AK088388 regulates autophagy through miR-30a to affect cardiomyocyte injury[END_REF] . The lncRNA HRIM (hypoxia/reoxygenation injury-related factor in myocyte) was upregulated after H/R in H9c2 cells. HRIM silencing prevented death of cells by suppressing the autophagic activity in H/R-treated cells. However, the target genes of this lncRNA and the detailed mechanism of its autophagic effect need to be elucidated [START_REF] Huang | Inhibition of LncRNA-HRIM Increases Cell Viability by Regulating Autophagy Levels During Hypoxia/Reoxygenation in Myocytes[END_REF] . Other lncRNAs were highly expressed in diabetic murine heart and contributed to I/R injury by regulating autophagy. It was shown that Neat-1 (Nuclear-enriched abundant transcript 1) and AK139328 seemed to induce autophagy by upregulating Foxo1 expression and decreasing miR-204-3p levels, respectively [START_REF] Ma | Long non-coding RNA nuclear-enriched abundant transcript 1 inhibition blunts myocardial ischemia reperfusion injury via autophagic flux arrest and apoptosis in streptozotocin-induced diabetic rats[END_REF][START_REF] Yu | Knockdown of lncRNA AK139328 alleviates myocardial ischaemia/reperfusion injury in diabetic mice via modulating miR-204-3p and inhibiting autophagy[END_REF] .

The lncRNA MALAT1 (metastasis-associated lung adenocarcinoma transcript 1) is expressed at high levels in patients with acute MI [START_REF] Vausort | Long noncoding RNAs in patients with acute myocardial infarction[END_REF] and is closely associated with the pathogenesis of myocardial I/R injury [START_REF] Zhao | Long non-coding RNA MALAT1 functions as a mediator in cardioprotective effects of fentanyl in myocardial ischemia-reperfusion injury[END_REF][START_REF] Hu | Knockdown of lncRNA MALAT1 attenuates acute myocardial infarction through miR-320-Pten axis[END_REF] . It was shown, on one hand, that MALAT1 contained binding site for miR-204 [START_REF] Li | LncRNA MALAT1 exerts oncogenic functions in lung adenocarcinoma by targeting miR-204[END_REF] and, in the other hand, that miR-204 protected the cardiomyocytes against I/R injury via inhibiting autophagic cell death [START_REF] Xiao | MiR-204 regulates cardiomyocyte autophagy induced by ischemia-reperfusion through LC3-II[END_REF] . Also, MALAT1 targeted miR-558 to enhance ULK1-mediated autophagy in isoproterenol treated-cardiomyocytes [START_REF] Guo | LncRNA MALAT1 protects cardiomyocytes from isoproterenol-induced apoptosis through sponging miR-558 to enhance ULK1-mediated protective autophagy[END_REF] . It will be important to know if lncRNA MALAT increased cardiomyocyte autophagy and myocardial injury during I/R by negatively regulating miR-204 or miR-558 expression.

CONCLUSION

Despite current therapies, acute MI and HF which often follows, remain the leading causes of death and disability worldwide. New therapeutic strategies are therefore required to protect the heart against the detrimental effects of acute ischemia/reperfusion injury. Inhibition of macroautophagy during reperfusion prevented cardiomyocyte death and reduced myocardial infarct size, however its induction during LVR preserved LV function and prevent the onset of HF. The most pharmacological agents used up to date for regulating macroautophagy are not specific and may interfere with other cellular processes, so it will be necessary to identify new therapeutic approaches to regulate autophagy. Several non-coding RNAs were shown to be modulated during I/R and involved on cardiac autophagy regulation. The tissue specific expression of some non-coding RNAs and their easy manipulation show their potential as novel targets for clinical developments to treat autophagy related-diseases. Identification of specific cardiac non-coding RNAs that regulate autophagy could be a good opportunity to protect heart from MI injury without affecting the autophagy activity in other organ.

ACKNOWLEDGMENTS

AT received a fellowship from ANR [ERA-NET Co-fund action ERA-CVD JTC2016 N° 680969) and EDD from "Fondation Lefoulon Delalande". FP is a MC substitute member of COST Action Transautophagy (CA15138) and is a MC member of COST action cardioRNA (CA1229).

Figure 1 :

 1 Figure 1: Outline summarizing the non-coding RNAs regulating cardiac autophagy, their targets and function. Green and red arrows indicate activation and inhibition, respectively

CONFLICTS OF INTEREST

The authors declare no conflict of interest


Letter by Pinet et al Regarding Article, "Comparative Analysis of Circulating Non-Coding RNAs Versus Protein Biomarkers in the Detection of Myocardial Injury"

To the Editor:

We read the article written by Schulte et al [START_REF] Schulte | Comparative Analysis of Circulating Non-Coding RNAs Versus Protein Biomarkers in the Detection of Myocardial Injury[END_REF] with great interest and appreciation. In this study, the authors performed a comparative analysis of circulating non-coding RNAs versus protein biomarkers to detect myocardial injury. Two cohorts of patients have been selected: patients undergoing transcoronary ablation of septal hypertrophy (TASH) with serial blood sampling (0-1-8-24h) and patients hospitalized for acute myocardial infarction (MI) with blood sampling (on admission, 1h and 3h). The non-coding RNAs were selected from literature or array screening for circulating RNAs. The authors treated all samples with heparinase to overcome the problem of heparin which interferes with quantification of RNAs by RT-qPCR 2 .

Their conclusion was that cardiac myosin-binding protein C was the most sensitive cardiac biomarker with miR-1 and miR-133a as promising candidates in patients with acute myocardial injury.

However, results from the study should be interpreted with the following considerations for data on the lncRNA LIPCAR presented in this article. The authors refute a predominant cardiac origin of LIPCAR, because they did not detect modulation of LIPCAR during the short-time course of blood sampling (0-1-8-24h) of TASH patients. They hypothesize that its rise in plasma may be explained by a release of mitochondria from blood cells rather than cardiac injury [START_REF] Schulte | Comparative Analysis of Circulating Non-Coding RNAs Versus Protein Biomarkers in the Detection of Myocardial Injury[END_REF] . We disagree with the authors for a number of points: First, our discovery of the potential of LIPCAR was performed in patients hospitalized for an inaugural anterior MI with serial echocardiography (admission, 3 months and 1 year post-MI) and blood sampling (5 day, 1 month, 3 months and 1 year post-MI) [START_REF] Fertin | Usefulness of serial assessment of B-type natriuretic peptide, troponin I, and C-reactive protein to predict left ventricular remodeling after acute myocardial infarction (from the REVE-2 study)[END_REF] . We observed a downregulation at early stage (5 days) but an upregulation during later stages (1, 3 months and 1 year post-MI) in patients with cardiac remodeling [START_REF] Kumarswamy | Circulating long noncoding RNA, LIPCAR, predicts survival in patients with heart failure[END_REF] . The time-course of measurements after MI is completely different to the study by Schulte et al. [START_REF] Schulte | Comparative Analysis of Circulating Non-Coding RNAs Versus Protein Biomarkers in the Detection of Myocardial Injury[END_REF] as LIPCAR seems to be regulated only at later stages.

Second, the cohorts of the investigated patients are different. In the present paper, the authors observed a modulation of miR-133a in TASH patients [START_REF] Schulte | Comparative Analysis of Circulating Non-Coding RNAs Versus Protein Biomarkers in the Detection of Myocardial Injury[END_REF] . We previously showed in patients enrolled in the REVE-2 study that circulating levels of miR-133a fail as a biomarker for left ventricular remodeling after myocardial infarction [START_REF] Bauters | Circulating miR-133a and miR-423-5p fail as biomarkers for left ventricular remodeling after myocardial infarction[END_REF] .

Third, in the myocardial tissue spike-in experiment, in which the authors spiked different amounts of human heart tissue (0 to 25 ug/100 uL plasma) into human plasma, LIPCAR was consistently detected and was among the 11 lncRNA quantified with the lowest raw Cq values (between 25 and 30) compared to the other non-coding RNAs (lncRNA, miRNAs, and circRNAs).

Fourth, LIPCAR was detectable at all-time points in samples from TASH patients, conversely to other non-coding RNAs, but not dysregulated after onset of MI.

Based on these data the authors cannot conclude about the usefulness of LIPCAR as a good or bad marker in comparison to proteins or other miRNAs in general as surprisingly broadly stated in this article. The authors cannot conclude that LIPCAR is not of predominant cardiac origin as mentioned in the abstract, only because it was not modulated in plasma taken very early after the TASH procedure.

From the data shown by the authors, LIPCAR is not a marker for detecting acute MI. Indeed, we previously have shown prognostic rather than diagnostic evidence for LIPCAR levels to identify patients developing cardiac remodeling. LIPCAR levels were independently to other risk markers associated with future cardiovascular deaths.

We think that the evidence of cardiac-tissue specific origin of LIPCAR remains to be evaluated by using cardiac and non-cardiac human cells as well as cell-type specific genetic deletion studies.

Acknowledgments

F. Pinet and T Thum conceived the study design and drafted the article. C Bär and C Bauters participated in the study design and helped to revise the article. All authors read and approved the final article.

Sources of Funding

This study was supported financially by grants from the European Union's Horizon 2020 research and innovation program under the ERA-NET Co-fund action N° 680969" and the ERC Consolidator Grant LongHeart.