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

Marie Cuvelliez

Thomas Kelder

Philippe Amouyel

Marijana Radonjic

Christophe Bauters

Integrative network analysis reveals time-dependent molecular events underlying left ventricular remodeling in post-myocardial infarction patients

Keywords: Left ventricule remodeling, echocardiography, biomarkers, system biology BBADIS-16-507-R1

Introduction

Left ventricular remodeling (LVR) after myocardial infarction (MI) is a strong predictor of heart failure and cardiovascular death [START_REF] Sutton | Quantitative two-dimensional echocardiographic measurements are major predictors of adverse cardiovascular events after acute myocardial infarction. The protective effects of captopril[END_REF] . In humans, imaging studies have confirmed a similar progressive left ventricular dilation after MI [START_REF] 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] Savoye | Left Ventricular Remodeling After Anterior Wall Acute Myocardial Infarction in Modern Clinical Practice (from the REmodelage VEntriculaire [REVE] Study Group)[END_REF] but difficulties in accessing to myocardial samples in this setting have precluded a comprehensive assessment of the time-dependent changes occurring at the tissue level.

As an alternative to directly studying human myocardial biopsies, circulating biomarkers are easy to assess and may shed light on the pathophysiologic processes involved in heart failure and LVR [START_REF] Braunwald | Biomarkers in heart failure[END_REF] . Although a reasonable number of circulating biomarkers have been associated with LVR, most studies published so far have focused on one or few markers, and in most cases at a single time-point after MI [reviewed in [START_REF] Fertin | Usefulness of circulating biomarkers for the prediction of left ventricular remodeling after myocardial infarction[END_REF] ]. Therefore, the complexity and the time-dependent nature of LVR after MI in humans have not yet been addressed adequately.

It is now recognized that complex biological processes implicated in human diseases are often the result of multiple pathways interacting through interconnected networks that can best be studied using system biology approaches [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] . Therefore, in this study we set out to combine experimental data from a serial blood sampling throughout the first year post-MI with information contained in prior knowledge databases to allow more comprehensive, network-based mapping of the dynamic of LVR process in humans.

The aim of the present study was to gain insight into molecular mechanisms that are associated with LVR at different timepoints after MI and to discover putative novel biomarker candidates for detection of LVR after a MI event. To this end, molecular data collected in the REVE-2 study, a prospective cohort of 246 patients dedicated to the study of circulating biomarkers after anterior 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] , has been integrated with known molecular interactions available in 12 public knowledge databases. The resulting network model has been analyzed to explore processes associated with LVR at different timepoints after MI and to generate hypotheses regarding putative novel biomarker candidates for timely detection of LVR after a MI event. Together, for the first time we gain mechanistic insights in post-MI LVR process in such comprehensive and dynamic way, paving the way for further testing of generated hypotheses.

Methods

The REVE-2 Study

The design of the REVE-2 study has been published in detail elsewhere [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] ; this prospective multicenter study was designed to analyze the association between circulating biomarkers and LVR.

We enrolled 246 patients with a first anterior wall Q-wave MI. Inclusion criteria were hospitalization within 24 hours after symptom onset and at least 3 akinetic left ventricular segments in the infarct zone at the predischarge echocardiography. Exclusion criteria were inadequate echographic image quality, life-limiting noncardiac disease, significant valvular disease, or prior Q-wave MI. The protocol required serial echographic studies at hospital discharge (baseline = day 3 to day 7), 3 months (3M) and 1 year (1Y) after MI to assess LVR which was defined as a change in left ventricular enddiastolic volume between baseline and 1Y >20%. Serial blood samples were taken at baseline, 1 month (1M), 3M, and 1Y after MI. The institutional Ethics Committee (Centre Hospitalier Universitaire de Lille) approved the study; written informed consent was obtained from all patients.

The characteristics of the 246 patients included in the REVE-2 study are shown in Table 1.

One-year echocardiographic follow-up was achieved in 226 (92%) patients; 87 (38.5%) patients had LVR.

Molecular data processing

The molecular REVE-2 network model was built based on the molecular data [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][START_REF] Lamblin | Circulating levels of hepatocyte growth factor and left ventricular remodelling after acute myocardial infarction (from the REVE-2 study)[END_REF][START_REF] Fertin | Circulating levels of soluble Fas ligand and left ventricular remodeling after acute myocardial infarction (from the REVE-2 study)[END_REF][START_REF] Fertin | Serum MMP-8: A Novel Indicator of Left Ventricular Remodeling and Cardiac Outcome in Patients after Acute Myocardial Infarction[END_REF][START_REF] Bauters | Circulating miR-133a and miR-423-5p fail as biomarkers for left ventricular remodeling after myocardial infarction[END_REF][START_REF] Eschalier | Extracellular Matrix Turnover Biomarkers Predict Long-Term Left Ventricular Remodeling After Myocardial Infarction: Insights From the REVE-2 Study[END_REF][START_REF] Kumarswamy | Circulating Long Noncoding RNA, LIPCAR, Predicts Survival in Patients With Heart Failure[END_REF] . This included 24 molecular variables (18 proteins, 6 non-coding RNAs (5 miRNAs and 1 long noncoding RNA)), measured at 1 to 4 time points (depending on the variable, including baseline, 1M, 3M, and 1Y).

Based on the skewed distributions, the data for all molecular variables were log2 transformed.

For variables containing zero or negative values, an offset was applied before transformation by adding the minimum and first non-zero value to the data. To test for significant changes in the measurements between the groups of patients with high LVR versus the group of patients with low LVR, an unpaired, two-sided t-test was performed for each variable at each given time point. The resulting p-values were corrected for multiple testing using the Benjamini-Hochberg FDR control procedure [START_REF] Benjamini | Controlling the False Discovery Rate : A Practical and Powerful Approach to Multiple Testing[END_REF] . Unless specified otherwise, all statistical analyses were performed in R version 3.2.0 (http://www.r-project.org/).

Building the network model

To construct the REVE-2 network model, the knowledge platform EdgeBox (EdgeLeap's proprietary knowledge platform) was used as a resource of public knowledge on molecular interactions. This result in embedding REVE-2 variables into their molecular context based on 12 public databases (Supplemental The REVE-2 network model embeds the REVE-2 molecular variables in an integrated network of known interactions ("edges") between molecules ("nodes"). To enable linking the variables measured in REVE-2 study to their corresponding molecules in the network model, the variable names were manually annotated to a structured identifiers database. Each variable was mapped to a corresponding entity in Ensembl Genes [START_REF] Hubbard | The Ensembl genome database project[END_REF] , the Human Metabolome Database [START_REF] Wishart | HMDB 3.0-The Human Metabolome Database in 2013[END_REF] , and miRBase [START_REF] Griffiths-Jones | miRBase: microRNA sequences, targets and gene nomenclature[END_REF] . In addition, the proteins were annotated to Medical Subject Headings (MeSH) [START_REF] Lipscomb | Medical subject Headings (MeSH)[END_REF] , Ensembl Phenotypes (http://www.ensembl.org/Homo_sapiens/Phenotype/All), Disease Ontology [START_REF] Schriml | Disease ontology: A backbone for disease semantic integration[END_REF] , and DrugBank [START_REF] Wishart | DrugBank: a comprehensive resource for in silico drug discovery and exploration[END_REF] . All variables were mapped to a database entity. In case multiple entities were identified for a single variable (e.g. when multiple genes encode for the measured protein), all annotations were included. These annotations were used to build the molecular network model.

Firstly, a set of seed nodes was retrieved from EdgeBox, including: 1. All molecule nodes mapping to one or more REVE-2 variables; 2. All molecule nodes that are direct neighbors of the nodes defined in step 1 and 3. All molecule nodes that are part of the shortest paths up to 3 edges between any of the nodes defined in step 1. Here "molecule nodes" are defined as nodes representing genes, miRNAs and metabolites (type "protein_coding", "processed_transcript", "miRNA", or "metabolite").

Secondly, all edges between these nodes were queried. If multiple edges between two nodes exist (e.g. multiple sources of evidence for an interaction), they were bundled into a single edge.

Furthermore, three quality criteria were applied to filter out low confidence interactions: for edges representing miRNA target interactions, edges were included only when either the target has been validated experimentally (data source is miRTarBase), or the target was predicted in at least three prediction resources (Microcosm, miRecords, and TargetScan); for edges originating from STRING, edges were included only for a STRING confidence score > 800; and for edges originating from WikiPathways, edges with type "in_group" were excluded. The resulting network forms the REVE-2 molecular network model, consisting of 1,310 nodes and 8,369 edges.

The REVE-2 molecular network model is an exploratory model which is not built towards a specific prediction, but is aimed towards uncovering potentially relevant relations ("give me all that is known about") and generating testable hypotheses based on this model.

Topology based cluster analysis

Network clustering was performed using the InfoMap algorithm as implemented in the igraph R package (version 0.7.1) (http://igraph.org/r/) for an optimal cluster structure detection in the network. As the InfoMap algorithm assigns each node in the network to a cluster, clusters with less than 5 nodes and clusters with only one or fewer edges per node within the cluster were excluded. The identified clusters were subsequently functionally annotated to biological processes. For each identified cluster an overrepresentation analysis was performed using the Fisher exact test to identify Biological Process terms from the Gene Ontology (GO) that are enriched with genes in the cluster.

Significantly enriched GO terms (p < 0.05) were added as properties to the network.

Centrality analysis

For each node in the network the centrality measure "betweenness" was calculated using the igraph R package. The betweenness of a node is the normalized count of shortest paths between all other nodes in the network passing through that node. High betweenness of a node indicates that a molecule is crucial to maintain functionality and coherence of signaling mechanisms, while a low betweenness indicates a more peripheral role for the molecule.

Assessment of tissue and/or cell type expression of molecules in the model

Expression for miRNA is based on mammalian miRNA ATLAS [START_REF] Landgraf | Resource A Mammalian microRNA Expression Atlas Based on Small RNA Library Sequencing[END_REF] , expression for proteins is based on the TISSUES database, which combines several resources (Protein Atlas, UniProt, etc.), but excluding text mining based results [START_REF] Santos | Comprehensive comparison of large-scale tissue expression datasets[END_REF] . The "tissues" column in Supplemental table 4 lists all blood, circulating cells and heart tissues that the miRNA / protein has been identified in.

Active module analysis

Active modules were extracted from the network model by including all significantly changed REVE-2 molecular variables (p<0.05) between high-and low-LVR groups for a given time point, as well as all nodes on shortest paths (up to 3 edges) connecting the significant variables, and direct neighbors of the significant variables. The cluster coverage was calculated as a percentage of molecules of each cluster that are part of active modules.

Network model visualization

All visualizations of the REVE-2 network model were performed using Cytoscape, version 3.2.1.

Results

The REVE-2 molecular network model

The REVE-2 molecular network model embeds the REVE-2 molecular variables in an integrated network of known molecular interactions and contains 1,310 nodes (1,263 protein-coding nodes, 24 miRNA nodes, 22 metabolite nodes and 1 lncRNA node), and 8,639 edges (Supplemental Table 2).

To obtain a high level overview of processes associated with REVE-2 molecular variables, the network model was clustered based on its topology and resulting clusters were functionally annotated to biological processes. Clustering algorithm identifies groups of nodes that are more highly connected to each other than to other nodes in the network, typically representing biological processes or protein complexes. In total, 33 clusters were identified in the REVE-2 molecular network model. All clusters could be significantly annotated to at least one GO term (Table 2). In total, 20 REVE-2 molecular variables are part of a cluster and 12 clusters contain at least one REVE-2 molecular variable. Table 2 lists the 33 clusters and their corresponding most significantly enriched GO term highlighting different processes important for LVR such as eukaryotic transcriptional regulation (clusters 1, 2 and 5), apoptosis (cluster 14), immune response (cluster 40), creatine and aminoacid metabolic process (cluster 41), signaling pathways (cluster 4, 21, 28 and 29) and extracellular matrix organization (clusters 3 and 9). The REVE-2 molecular network model is visualized in Figure 1. Full network cluster characterization and GO enrichment is provided in Supplemental Table 3.

Relevance of individual molecules in REVE-2 network model

To assess relevance of individual molecules in the REVE-2 network model, the betweenness centrality was calculated. High betweenness suggests that a molecule is crucial to maintain functionality and coherence of signaling mechanisms and the molecules with the highest betweenness centrality may be relevant as putative biomarker candidates. Supplemental To benchmark the relevance of REVE-2 network model in the context of known biomarkers of LVR, the collection of 52 known molecular LVR biomarkers [START_REF] Fertin | Usefulness of circulating biomarkers for the prediction of left ventricular remodeling after myocardial infarction[END_REF] has been cross-referenced to REVE-2 molecular network model. In total, of 48 biomarkers which could be annotated to either Ensembl Gene or HMDB identifier, 22 were present in the REVE-2 network model.

Time-resolved activation of network modules associated with LVR progression

The 5). These active modules represent a robust mechanistic context of changed variables and provide insight into molecular interactions and mechanisms relevant at a given time point during LVR progression. Figure 2 shows the active modules for each time point.

The most apparent observation is that the highest number of active modules is found at the baseline and 3M time points. This is reflected in both high number of clusters, as well as in total number of molecules represented in active modules at these time points (Figures 2 and3).

Specifically, while all the clusters in the REVE-2 model (n=33) are represented in active modules at baseline, there is a strong decrease of the number of clusters (n=9) in active modules at 1M. At time point 3M, again a large number of clusters (n=30) are in active modules and finally, only 3 clusters are in active modules at 1Y. Although the number of clusters highlighted at 3M (n=30) was very similar to those at baseline (n=33), a higher ratio of molecules belonging to active modules was detected at baseline (936, corresponding to 71% of the molecules of the REVE-2 network model) compared to 3M (389 molecules corresponding to 30% of the molecules of the REVE-2 network). Figure 3 details cluster coverage of the active modules at each time point.

In contrast to the general pattern of largest cluster coverage in the active module at baseline, clusters 7 and 9 involved respectively in muscle filament sliding and extracellular matrix organization, remain highly represented in active modules at 1M, with the highest cluster coverage at 3M. This pattern suggests that these two clusters and associated processes specifically represent the dynamic process of long-term pathological changes during the post-MI LVR, rather than the acute stress of the system triggered by MI (Figure 3 and Table 3). Cluster 7, which includes proteins of myofilament such as the TnT, TnC and TnI forms, myosin and tropomyosin proteins, has 33 active molecules at 1M and 3M representing the highest number of molecules, 94% compared to only 11% at baseline. Cluster 9 which includes most of the metalloproteinase (MMPs), tissue inhibitors of metalloproteinases (TIMPs) and laminin proteins has a ratio of nearly 60% of active molecules (n=28) at 3M compared to baseline (n=17) and 1M (n=7).

Discussion

Due to the difficulties in accessing human myocardial samples after MI, little is known about long-term processes associated with LVR in humans. Studies so far focused on a handful of measurable variables, typically assessed only at the baseline [START_REF] Fertin | Usefulness of circulating biomarkers for the prediction of left ventricular remodeling after myocardial infarction[END_REF] . Our objective was therefore to build a more comprehensive view on processes associated with LVR up to one year after MI, by enriching our dataset obtained from serial blood samples during the LVR progression (baseline, 1M, 3M, 1Y) with prior knowledge on molecular interactions. Network analysis allowed us to identify robust mechanisms associated with the measured variables, as this approach considers not only list of isolated molecules, but also accounts for their mechanistic context. Importantly, availability of time-resolved dataset allowed us to determine relevance of LVR-associated mechanisms over time after an MI event.

Interestingly, muscle filament sliding and extracellular matrix disassembly have been identified to be specifically (unlike other identified mechanisms) more activated at the 3M timepoint compared to the baseline, suggesting their relevance for the long-term pathological changes during the post-MI LVR.

To the best of our knowledge, here we for the first time can discriminate between mechanisms associated with acute stress response triggered by MI and the mechanisms associated with dynamic process of long-term pathological changes during the post-MI LVR.

Another important result of our study is the prioritization of putative LVR-relevant molecules, including those that have not yet been measured or previously associated with LVR. The undertaken knowledge-based network modelling approach therefore stimulates novel discovery and hypothesis forming, which is particularly relevant for identification of putative novel biomarker candidates for timely detection of LVR after a MI event. In line with this motivation, we provide a shortlist of putative novel biomarker candidates prioritized based on their high betweeness centrality and expression in accessible tissues, which may be considered for a follow up biomarker studies.

Circulating biomarkers may provide information on changes occurring in the myocardium after MI, specifically in humans due to the difficulties in accessing to human myocardial samples after MI.

MMPs, TIMPs, and cytokines are known to be implicated in the myocardium during the remodeling process [START_REF] Spinale | Myocardial matrix remodeling and the matrix metalloproteinases: influence on cardiac form and function[END_REF][START_REF] Nian | Inflammatory cytokines and postmyocardial infarction remodeling[END_REF] , and are also modulated in a similar time-dependent fashion in the circulation [START_REF] Fertin | Serum MMP-8: A Novel Indicator of Left Ventricular Remodeling and Cardiac Outcome in Patients after Acute Myocardial Infarction[END_REF][START_REF] Webb | Specific temporal profile of matrix metalloproteinase release occurs in patients after myocardial infarction: relation to left ventricular remodeling[END_REF][START_REF] Orn | The chemokine network in relation to infarct size and left ventricular remodeling following acute myocardial infarction[END_REF] . In a biomarkers and LVR were cited [START_REF] Fertin | Usefulness of circulating biomarkers for the prediction of left ventricular remodeling after myocardial infarction[END_REF] . Therefore -although this approach can certainly not be viewed as equivalent to direct studies on myocardial tissue, the assessment of circulating biomarkers may provide useful information on LVR and heart failure.

The REVE-2 network model

Systems and network biology approaches are being applied with the aim to capture the molecular complexity of the disease by data integration, ultimately achieving goals of systems medicine [START_REF] Chan | The Emerging Paradigm of Network Medicine in the Study of Human Disease[END_REF] . Integrative approaches have been classified into four broad categories: identification of active modules through integration of networks and molecule profiles, identification of conserved modules across multiple species, identification of differential network modules through integration of different interaction types, and identification of composite functional modules through integration of different interaction types [START_REF] Mitra | integrative approaches for finding modular structure in biological networks[END_REF] . Recently, a MI-specific protein-protein-interaction (MIPIN) network has been built using 38 seed proteins from published human studies [START_REF] Nguyen | Integrative computational and experimental approaches to establish a post-myocardial infarction knowledge map[END_REF] . Based on statistical analysis of individual molecules, the MIPIN network was constituted of 613 proteins that associated 4443 edges interactions. In the present study, we used the molecular variables measured in the REVE-2 study, their direct neighbors and molecules on the shortest paths between these molecules as the seed nodes for building a network model. The interactions between molecules were derived from 12 public resources, including functional links such as physical interaction, pathway interactions, transcriptional regulation and enzyme activation. The resulting network model constitutes 1,310 nodes, including not only 1,263 proteins but also 24 miRNAs, 22 metabolites and 1 lncRNA, connected through 8639 edges. A network topology of clustering the REVE-2 network model identified 33 clusters annotated to Gene Ontology processes. Therefore, the REVE-2 network model provides robust insight in LVRassociated physiopathological mechanisms and facilitates identification of novel potentially relevant molecules to be studied further, which is as mentioned above, an important resource for discovery of novel putative biomarkers. For instance, betweenness centrality of the nodes indicates relevance of individual molecules within the model, where high betweenness suggests that a molecule is crucial to maintain functionality and coherence of signaling mechanisms. Interestingly, we identified as highly central molecules 1 miRNA, miR-335-5p and 2 proteins, the histone acetylase p300 (EP300) and the transcriptional repressor CCCTC-binding factor (CTCF). The latter proteins have not yet been associated with LVR but they have been described indirectly to have a potential role in the heart.

SIRT-1, which is acetylated by EP300 has been described to be implicated in dystrophic cardiomyopathy [START_REF] Kuno | The effects of resveratrol and SIRT1 activation on dystrophic cardiomyopathy[END_REF] and a CTCF binding site has been described to be a novel heart enhancer [START_REF] Schultz | Enhancers compete with a long noncoding RNA for regulation of the Kcnq1 domain[END_REF] .

Importantly, most of the molecules with high betweenness centrality are expressed in blood, circulating cells or heart tissues, further qualifying them for the follow up biomarker studies. For example, three miRNAs (hsa-miR-26b-5p, hsa-miR-124-3p, hsa-miR-744-5p) would be interesting candidates for further testing based on their high expression in circulating cells and novelty.

Temporal changes associated with LVR

Strength of the present study is the availability of serial blood samples during the LVR progression (baseline, 1M, 3M, 1Y). Imaging studies in humans have shown that LV dilation begins early after MI and may continue up to 1 year after the initial insult [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] Gaudron | Progressive left ventricular dysfunction and remodeling after myocardial infarction. Potential mechanisms and early predictors[END_REF] . In animal models, the cellular and molecular changes that occur within the myocardium can last weeks or months after MI [START_REF] Spinale | Myocardial matrix remodeling and the matrix metalloproteinases: influence on cardiac form and function[END_REF][START_REF] Nian | Inflammatory cytokines and postmyocardial infarction remodeling[END_REF] After the initial changes at baseline, at 1M the changes in processes underlying LVR are less prominent, and are reactivated at 3M (although including a smaller subset of molecules in these processes). Interestingly, as outlined above, large number of molecules from the clusters associated with muscle filament sliding and extracellular matrix disassembly, represented in active modules at 3 months confirms a specific role for these processes in the later stage of LVR. The statistical significance of changes between high-and low-LVR group in REVE-2 molecules associated with these clusters (troponin and MMPs) is in line with this hypothesis, with increase in changes from baseline up to 3M (supplemental Table 5). Finally, the low number of active molecules and clusters detected at 1Y indicates a shut-down of all processes associated to LVR. In summary, our results suggest that many molecular events and pathways are activated during LVR in a time-dependent manner and allow better understanding of which mechanisms are linked to acute stress of the system upon MI, and which are associated with long-term pathological changes associated with LVR.

Conclusion

In conclusion, our results improve our knowledge about the LVR process after MI by identifying the temporal activation profiles of REVE-2 molecules along with their associated mechanistic context. This provides more robust insight in LVR processes compared to purely statistical methods based on limited set of variables, facilitates mechanistic hypotheses and prioritizes molecules to be further studied as putative biomarker for timely detection of LVR after MI. The REVE-2 network model is a promising tool to capture the molecular complexity of the disease and can be used as a resource for discovery of LVR-relevant interactions in the context of development of therapeutic interventions aiming at reducing adverse LVR (e.g., which target? at which time point after MI?). Finally, another implication of interest would be to explore whether the same type of proteins (nodes) segregate into close neighborhood of proteins associated for other diseases, providing clues for investigation of comorbidities [START_REF] Menche | Disease networks. Uncovering disease-disease relationships through the incomplete interactome[END_REF] .

BBADIS-16-507-R1

  REVE-2 network model is based on prior knowledge of molecular interactions and as such provides insight into molecular mechanisms associated with variables measured in REVE-2 study, and identifies key molecules that may be relevant in the context of LVR. To assess which of these mechanisms change under LVR conditions in REVE-2 study, and which mechanisms are relevant at which time point after MI, an active module analysis was performed (see methods). The active modules were extracted from the REVE-2 network model for each time point (baseline, 1M, 3M, 1Y), enabling insight into time-resolved molecular changes associated with LVR progression. Each active module highlights the part of the network model associated with REVE-2 molecules which are differentially expressed between high-and low-LVR groups at each time point (supplemental Table

  . The baseline sample (3-7 days) likely combines information on initial myocardial necrosis and on the early steps of LVR. Later samples (1M to 1Y) are at a significant distance from initial MI and are probably more indicative of the late remodeling process. Our data summarized in Figures 2 and 3 show the active modules of the REVE-2 network model associated with each time point, enabling insight into time-dependent molecular changes associated with LVR progression. Examination of temporal molecular patterns reveals a clear dynamic signature of biological processes. Most of the LVR-relevant processes are activated at the baseline and include molecules which have been previously reviewed 3 as potential heart failure biomarkers such as markers of inflammation and oxidative stress, extracellular matrix remodeling, and myocyte injury.

  Figure 1

Table 1 )

 1 : ENCODE (

http://encodenets.gersteinlab.org),

	EnsemblGenes	(http://www.ensembl.org),	HMDB	(http://www.hmdb.ca),	Microcosm
	(http://ebi.ac.uk/enright-srv/microcosm),	miRBase	(http://mirbase.org),	miRecords
	(http://c1.accurascience.com/miRecords), miRTarBase (http://mirtarbase.mbc.nctu.edu.tw), Reactome
	(http://www.reactome.org), STRING (http://string-db.org), TargetScan (http://www.targetscan.org),
	TFe (http://www.cisreg.ca/cgi-bin/tfe/home.pI) and WikiPathways (http://www.wikipathways.org).

Table 4

 4 (hsa-miR-335-5p, EP300, CTCF). Interestingly, the 20 nodes with the highest betweenness centrality are linked to 8 clusters (1, 2, 3, 5, 10, 12, 13 and 16), with cluster 1 represented by 8 nodes (Histone acetyltransferase p300 (EP300), Myc proto-oncogene protein (MYC), Fos (FOS) and CCAAT/enhancer-binding protein beta (CEBPB)). To further determine if the molecules with high betweenness centrality could qualify as putative biomarker candidates we assessed whether these miRNAs and proteins are expressed in blood, circulating cells or heart tissues.Of the top 20 molecules, 17 can be mapped to one or more of these tissues, further narrowing down the list of putative novel biomarker candidates. For example, three miRNAs (hsa-miR-26b-5p, hsa-

	lists the nodes with

REVE-2 variables (e.g. miR-21-5p, miR-222-3p, miR-423-5p,) as well as novel highly central molecules that were not measured yet but could be good candidates for further investigation as putative novel biomarkers miR-124-3p, hsa-miR-744-5p) would be interesting candidates for further testing based on their high betweenness centrality, expression in circulating cells and novelty (i.e. not yet measured among REVE-2 variables). The expression of molecules with the highest betweenness centrality in the tissues of interest is provided in the Supplemental Table

4

.

Table 1 . Characteristics of the patients included in the REVE-2 study 1

 1

	Age, y (mean±SD)

[START_REF] Sutton | Quantitative two-dimensional echocardiographic measurements are major predictors of adverse cardiovascular events after acute myocardial infarction. The protective effects of captopril[END_REF]

Of the 226 patients with echocardiographic follow-up; 2 Defined as a >20% change in left ventricular end-diastolic volume between baseline and 1 year.

Table 2 . List of the identified clusters of the REVE-2 network model. Cluster number Nodes 1 Edges 2 REVE-2 variables 3 Most significant GO category 4

 2 Nodes represent molecules such as proteins, non-coding RNAs and metabolites; 2 Edges represent known interactions between molecules (nodes); 3 REVE-2 variables represent molecules measured in plasma of REVE-2 patients; 4 Full network cluster characterization and GO (Gene Ontology) is provided in supplemental Table 3. Number corresponds to the cluster listed in Table 2; 2 REVE-2 variables measured in plasma of REVE-2 patients; 3 Number of nodes (molecules) present within the cluster; Genes in bold indicates the molecules present in active modules at each time point; Accession number in UniProtKB (http://www.uniprot.org/) is provided for proteins under bracket.

	1	191	1013	ST2	sequence-specific DNA binding
	2	203	302	Mir-21-5p	intrinsic component of membrane
	3	57	978	ICTP, P1NP,	endoplasmic reticulum lumen
				P3NP
	4	65	403	HGF	transmembrane receptor protein tyrosine kinase signaling pathway
	5	98	123	miR-222-3p	RNA binding
	6	37	495	TIMP1	platelet alpha granule lumen
	7	35	392	troponin	muscle filament sliding
	8	30	271		nuclear-transcribed mRNA catabolic process
	9	47	154	MMP1, MMP2,	extracellular matrix disassembly
				MMP3, MMP8,
				MMP9, TIMP2,
				TIMP4
	10	38	75		ATP binding
	12	30	75		mitotic cell cycle phase transition
	13	32	33		O-glycan processing
	14	22	111	FasL	activation of cysteine-type endopeptidase activity involved in apoptotic
					process
	15	17	67		G2/M transition of mitotic cell cycle
	16	23	33		G-protein coupled receptor signaling pathway
	18	14	61		RNA splicing, via transesterification reactions
	19	16	29		positive regulation of intrinsic apoptotic signaling pathway
	20	22	23		phosphatase activity
	21	14	32		toll-like receptor 2 signaling pathway
	22	15	17		steroid hormone receptor activity
	24	11	19		regulation of cell differentiation
	25	10	18		cell surface
	26	11	16		cellular component disassembly involved in execution phase of apoptosis
	28	9	14		negative regulation of transforming growth factor beta receptor signaling
					pathway
	29	8	15		small GTPase mediated signal transduction
	30	11	12	BNP	protein targeting to mitochondrion
	32	9	12		DNA repair
	33	9	10		nuclear pore
	35	7	11		Mitochondrial inner membrane
	37	7	8		DNA-directed RNA polymerase II, holoenzyme
	39	6	11		posttranscriptional gene silencing
	40	7	12	CRP	complement activation
	41	6	11	Creatine kinase creatine metabolic process

1 1

Funding Sources

This work was supported by grants from the E.U. FP7 HOMAGE (305507), from CHRU de Lille (Bonus H2013) and from ANR-15-CEA-016/DGOS 15-013.

Disclosure

FP, MC, PA and CB disclose no conflicts of interest.

TK and MR are founders and owners of EdgeLeap B.V.

Author Contributions

FP, CB conceived and designed the experiments; FP, MC, CB acquired the data; FP, CB, TK, MR designed and executed analysis workflows and interpreted the data; FP, MC, TK, MR, CB drafted the manuscript; FP, TK, PA, MR, CB revised the manuscript for important intellectual content; FP, PA, CB obtained funding; FP, CB supervised the study.


PhD Annie Turkieh

MD, PhD Marie Fertin

PhD Marion Bouvet

PhD Paul Mulder

MSC Hervé Drobecq

MD, PhD Gilles Lemesle

MD, PhD Nicolas Lamblin

MD, PhD Pascal De Groote

MSC a , Sina Porouchani

MSC a , Maggy Chwastyniak

MSC a , Olivia Beseme

MD, PhD Philippe Amouyel

MD Frédéric Mouquet

MD, PhD Jean-Luc Balligand

PhD Vincent Richard

MD Christophe Bauters

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

Expression and implication of clusterin in left ventricular remodeling after myocardial infarction

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

INTRODUCTION

Chronic heart failure (HF) remains a major public health problem [START_REF] Benjamin | Heart Disease and Stroke Statistics-2017 Update: A Report From the American Heart Association[END_REF] with a high rate of mortality in spite of recent therapeutic improvements 2 . The most common cause of HF is coronary artery disease and particularly myocardial infarction (MI) 3 .

Left ventricular remodeling (LVR) following myocardial infarction is a dynamic and complex process that occurs in response to myocardial damage and is associated with an increased risk of HF and cardiovascular death 4,5 . Post-MI LVR can be divided in two phases. The early phase (occurring few hours to several weeks after MI onset) involves the expansion of the infarct zone, including dilatation and thinning of the infarct zone 6 . The late phase (months, years) involves globally the LV and is associated with time-dependent dilatation, distortion of ventricular shape, mural hypertrophy, as well as structural tissue changes 7 . Although LVR may be considered as a protective mechanism to maintain systolic flow and cardiac "pump" function, this phenomenon leads to alterations of global LV function and ultimately aggravates HF. In two independent prospective echocardiographic studies (REmodelage VEntriculaire, REVE and REVE-2), we observed a 30-40% LVR rate after a first acute anterior wall MI despite high acute reperfusion rates and widespread prescription of secondary prevention medications 8,9 . However, LVR remains difficult to predict in clinical practice and its severity cannot be fully predicted based on its known determinants such as those related to infarct size, anterior infarct location or LV ejection fraction 10 . Circulating biological markers called "biomarkers" may facilitate the diagnosis and the prognosis of LVR and HF [START_REF] Dubois-Deruy | Interplay between troponin T phosphorylation and O-N-acetylglucosaminylation in ischaemic heart failure[END_REF] . The only biomarker clinically used in MI patients is B-type natriuretic peptide (BNP) whose concentration helps to predict the risk of HF 12 , but data on its ability to predict LVR are discordant 9,13,14,15 .

Patients

The studies were approved by the Ethics Committee of the "Centre Hospitalier et Universitaire de Lille" and comply with the Declaration of Helsinski. All patients gave written informed consent.

REVE-2 study

The design of the REVE-2 study has been published in detail elsewhere 9 . We enrolled 246 patients with a first anterior wall Q-wave MI between February 2006 and September 2008. The protocol required serial echographic studies at baseline (day 3 to day 7) and 3 and 12 months after MI. Left ventricular remodeling (LVR) was defined as percent change in EDV from baseline to 1 year. Serial blood samples were taken at discharge (day 3 to day 7), and 1, 3, and 12 months after MI.

INCA study

All patients referred for evaluation of systolic HF (LVEF <45%) in our institution between November 1998 and May 2010 were included in a prospective cohort on prognostic indicators [START_REF] De Groote | B-type natriuretic peptide and peak exercise oxygen consumption provide independent information for risk stratification in patients with stable congestive heart failure[END_REF][START_REF] De Groote | Impact of diabetes mellitus on long-term survival in patients with congestive heart failure[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 beta-blocker therapy[END_REF] . From this population, we selected 198 patients included between November 1998 and December 2005, as previously described 24 . Blood sample was obtained at inclusion.

Human heart biopsies

CIRCHF/2017/004838-R3 6 Tissues from failing and non-failing human hearts were obtained respectively from Lille University Hospital (France) and from Catholic University of Leuven (Belgium).

Animal models

All 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). MI was induced in 10-week-old male Wistar rats (Janvier, Le Genest St isle, France) by ligation of the left anterior descending coronary artery [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] Pfeffer | Survival after an experimental myocardial infarction: beneficial effects of long-term therapy with captopril[END_REF] . Shamoperated and MI rats were then further separated in two subgroups that were sacrificed at 7 days (n=8/group) or 2 months (n= 9 to 11/ group) post-MI. Haemodynamic and echocardiographic measurements were taken 2 months after surgery, followed by heart excision, as previously described [START_REF] Dubois | Decreased serine207 phosphorylation of troponin T as a biomarker for left ventricular remodelling after myocardial infarction[END_REF][START_REF] Mulder | Long-term heart rate reduction induced by the selective I(f) current inhibitor ivabradine improves left ventricular function and intrinsic myocardial structure in congestive heart failure[END_REF] .

Cellular models

H9C2 cells and primary cultures of neonate rat cardiomyocytes (NCM) were prepared and cultured as previously described [START_REF] Dubois-Deruy | Interplay between troponin T phosphorylation and O-N-acetylglucosaminylation in ischaemic heart failure[END_REF] .

Proteomic analysis of plasma samples from REVE-2 patients

Plasma samples underwent no more than two freeze/thaw cycles and were treated combinatorial peptide ligand library (CPLL) method to allow for the enrichment in medium-and low-abundance proteins 30 . 2D-DIGE electrophoresis was performed and analyzed as previously described [START_REF] Acosta-Martin | Impact of incomplete DNase I treatment on human macrophage proteome analysis[END_REF] . Differentially regulated spots were excised manually from a preparative gel performed with 500 µg of proteins and in-gel digested with trypsin [START_REF] Shevchenko | Linking genome and proteome by mass spectrometry: large-scale identification of yeast proteins from two dimensional gels[END_REF] .

Western blot analysis

CIRCHF/2017/004838-R3

7

The primary antibodies and dilution used for western blot analysis are detailed in the Supplemental Table 1).

Quantification by multiplex immunoassay

B-type natriuretic peptide (BNP) was measured in plasma samples using a Multi Analyte Profiling Kit for simultaneous quantitative detection of rat cardiovascular biomarkers (Rat CVD1 Panel 1, RCVD1-89K Millipore) according to the manufacturer's instructions as previously described [START_REF] Dubois-Deruy | Circulating plasma serine208-phosphorylated troponin T levels are indicator of cardiac dysfunction[END_REF] .

Quantification of clusterin in plasma from REVE-2 and INCA studies

Clusterin was quantified in plasma of REVE-2 patients by western blot. Clusterin was quantified in plasma of INCA patients by LC-MRM MS with SIS peptides and by ELISA (R&D systems, Minneapolis, MN, USA). The endogenous protein concentrations in the patient samples are determined through linear regression [START_REF] Percy | Advances in multiplexed MRM-based protein biomarker quantitation toward clinical utility[END_REF][START_REF] Mohammed | Qualis-SIS: automated standard curve generation and quality assessment for multiplexed targeted quantitative proteomic experiments with labeled standards[END_REF] .

Statistical analysis

Data summarized as medians with interquartile range (IQR) were analyzed with GraphPad Prism version 6.01 (GraphPad Software, San Diego, CA).

RESULTS

Clusterin is increased in patients with high LVR following MI

We performed differential proteomic analysis by comparing 10 pools of 5 patients from the REVE-2 study 9 , the pools (1 to 5) and (37-41) corresponding respectively to the lowest (-17.0 % ± 6.2) and the highest (+ 37.2 % ± 13) LVR (Figure 1A). Because inflammatory response and cytokines are particularly active in early phase after MI, we selected, for each patient pool, the plasma samples collected at 3 months post-MI for the differential 2D-DIGE based proteomic analysis. We found 7 spots differentially expressed (P<0.05) between low and high LVR pooled patients. The MS analyses identified 3 spots as being clusterin (Figure 1B). Detailed 2D-spots corresponding to clusterin are presented for each pool of plasma analyzed (5 for no LVR and 5 for high LVR groups) (Supplemental Figure 1, Supplemental Table 3). We observed that the spots 842, 853 and 873 identified as being clusterin (CLU) gathered as a cluster in this gel within a range of Mw (37-45 kDa) and of pI (4.7-5.0).

To confirm these results, we analyzed CLU plasma levels by 1D-western blotting from individual plasma samples of 45 REVE-2 patients at 3 months post-MI. Patients were divided by tertile of ΔEDV with mean ΔEDV of -7.4% (-13.9 to 1%) for T1, 11.0% (5.3 to 14%) for T2 and 22.6% (18.7 to 29.5%) for T3. We observed in crude plasma one band with a molecular mass of 36 kDa corresponding to CLU (s-CLU) that is significantly increased in plasma of patients with LVR (P<0.01) (Figure 1C). We also identified clusterin as being significantly increased in plasma from patients with LVR at 1 year post-MI (Supplemental Figure 2).

CLU is increased in the LV of rats after MI and correlated positively with LVR

CIRCHF/2017/004838-R3 9

To determine if increased plasma CLU levels has a cardiac origin, we analyzed CLU expression in the LV of rats at 7 days and 2 months post-MI. In this model, cardiac remodeling was characterized by LV dilatation as shown by significantly increased LV end-diastolic (LVEDD) and end-systolic (LVESD) diameters in the 2 months post-MI rats (Supplemental Table 4). Cardiac hypertrophy was also increased in post-MI: LV weight/Body weight (LVW)/BW) and heart weight (HW)/BW ratios were significantly increased in 2 months post-MI rats (Supplemental Table 4). Also, mRNA levels of ANP and BNP genes were significantly increased in the LV of rats at 7 days and 2 months post-MI (Supplemental Figure 3A) but only BNP plasma levels were significantly increased at 7 days post-MI (Supplemental Figure 3B).

We first analyzed CLU mRNA and its intracellular protein levels in the LV of rats after MI (Figure 2A). Quantitative polymerase chain reaction (qPCR) analysis showed a significant increase of CLU mRNA levels in the LV of rat at 7 days (median [IQR]: 1. The mature form (m-CLU) was the predominant intraventricular form suggesting that CLU maturation is active in the heart independently of MI (Supplemental Figure 3C). We also observed that p-CLU was only significantly increased at 7 days post-MI (0.05 [0.03-0.06] vs 0.03 Next, we verified if increased CLU expression in the LV was associated with cardiac remodeling. CLU mRNA level was positively correlated with ANP and BNP expression at 7 days (r=0.76, P=0.002 / r=0.85, P <0.001, respectively) and 2 months post-MI (r=0.78, P <0.001/ r=0.67, P=0.004, respectively) (Figure 2B). Cardiac p-CLU and m-CLU levels were positively correlated with plasma BNP levels (r=0.69, P=0.004/ r=0.59, P=0.018, respectively) at 7 days post-MI (Figure 2C, left panel). The cardiac m-CLU levels were positively correlated with LVEDD (r=0.59, P=0.004), LVW/BW ratio (r=0.48, P=0.034) and HW/BW ratio (r=0.52, P=0.02; data not shown) at 2 months post-MI (Figure 2C, right panel). These data show that CLU expression is increased in LV after MI and is correlated with cardiac remodeling. However, CLU expression is time-dependent after MI, suggesting different mechanisms of CLU regulation and different roles of this protein in cardiac remodeling.

Clusterin expression and secretion are increased in hypertrophied cardiomyocytes

Chronic activation of the adrenergic system with associated up-regulation of the reninangiotensin-aldosterone (RAA) system plays a major role in cardiac remodeling after MI. To determine the implication of these mechanisms on CLU secretion, we treated H9c2 cells for 48h by Angiotensin II (AII, 2 µmol/L), phenylephrine (PE, 20 µmol/L) and isoproterenol (ISO, 10 µmol/L), agonist of alpha-and beta-adrenergic receptors, respectively. Western blots analysis showed increased s-CLU level in the culture media of H9c2 treated cells compared to untreated cells with the strongest effect with ISO treatment (1.7 [1.1-1.8] vs 1, P =0.03). The increased s-CIRCHF/2017/004838-R3 11 CLU level was confirmed at 72h after ISO treatment (2.7 [2-5.3] vs 1, P =0.02) (Supplemental Figure 4A).

To determine the implication of hypertrophy on CLU regulation, we treated primary cultures of neonatal rat cardiomyocytes (NCM) by ISO (10 µmol/L) for 72h, and characterized cell hypertrophy by increased cell surface area (Figure 3A) and significant increased expression of ANP and BNP genes (Figure 3B). As shown in Figure 3C 3D). These data confirm that CLU expression and secretion are increased by hypertrophied cardiomyocytes and suggest that increased CLU in plasma after MI has at least in part a cardiac origin.

CLU silencing in cardiomyocytes inhibits cell hypertrophy induced by isoproterenol

Since CLU expression and secretion were increased in hypertrophied cardiomyocytes, we studied its role on hypertrophy by silencing it in order to understand its involvement in cardiac remodeling post-MI. We verified that ISO treatment increased significantly phospho Akt (Ser473)/total Akt (pAkt/Akt) ratio, phospho ERK1/2 (Thr202/Tyr204)/total ERK1/2 (pERK1-2/ERK1-2) ratio as previously described [START_REF] Ueyama | Requirement of activation of the extracellular signal-regulated CIRCHF/2017/004838-R3 26 kinase cascade in myocardial cell hypertrophy[END_REF][START_REF] Yano | A novel signaling pathway for beta-adrenergic receptor-mediated activation of phosphoinositide 3-kinase in H9c2 cardiomyocytes[END_REF][START_REF] Chowdhury | Prohibitin confers cytoprotection against ISO-induced hypertrophy in H9c2 cells via attenuation of oxidative stress and modulation of Akt/Gsk-3β signaling[END_REF] with no significant difference on pGSK-3β (Ser9)/ total GSK-3β (pGSK/GSK) ratio (Supplemental figure 4B). CLU silencing in hypertrophied H9c2 cells (Figure 4A) decreased cell size (Figure 4B) and was associated with significant decreased pERK1/ERK1 (0.79 [0.71-0.9] vs 1.01 [0.93-1.07], P<0.001) and pERK2/ERK2 (0.87 CIRCHF/2017/004838-R3 12 [0.77-0.98] vs 1.01 [0.93-1.07], P<0.001) ratios. However, CLU silencing increased significantly pAkt/Akt ratio (1.31 [1.2-1.5] vs 0.97 [0.9-1.1], P =0.002) with no significant difference on pGSK/GSK ratio (Figure 4C). On another hand, CLU silencing in hypertrophied NCM by using as with the siClu-pool (Figure 4F). These results suggest that CLU silencing inhibits isoproterenol induced hypertrophy by inhibition of ERK1/2 pathway.

CLU is a predictive marker of cardiac mortality in heart failure patients

Since CLU protein levels were higher in the LV of HF rat model than in control rats, we sought to evaluate whether this was also the case in myocardial tissue of HF patients who underwent heart transplantation. We observed an increased level of p-CLU (1.34 [1.17-1.93] vs 0.99 [0.87-1.13], P=0.009) and m-CLU (5.34 [3.83-7.9] vs 2.68 [2.27-3.28], P=0.009) in LV of HF patients compared to controls (Figure 5A). Using immunohistochemistry, we also observed an increased CLU staining in failing hearts compared to control hearts with no difference on CLU cellular localization (Figure 5B).

To evaluate whether the secreted form of CLU (s-CLU) may be a predictor of adverse effects in HF patients before heart transplantation, we quantified, by two methods (quantitative mass spectrometry and ELISA), s-CLU in plasma of chronic systolic HF patients (99 alive and 99 dead CIRCHF/2017/004838-R3 13 patients) from the INCA study recently described 24 . The baseline characteristics of the patients included were matched on age, sex, HF etiology, diabetes. There was no significant difference in treatment at inclusion in the study. The patients who died from a cardiovascular cause during the 3-year follow-up showed significantly higher NYHA class, BNP level, creatinine level, and lower peak VO 2 as compared to patients alive at 3 years. Plasma CLU was increased significantly (73.6

DISCUSSION

LVR post-MI is associated with an increased risk of HF and death [START_REF] Bauters | Long-term prognostic impact of left ventricular remodeling after a first myocardial infarction in modern clinical practice[END_REF] . It remains a frequent event despite the modern therapeutic approach and difficult to predict in clinical practice. For this, we designed the REVE-2 study which included 246 patients after a first anterior MI with serial echocardiography examinations and serial samplings in the first year after MI 9 . Our aim was to identify new biomarkers of LVR post-MI and understand their involvement in this evolutive process.

The development of proteomic analysis techniques makes possible to use new strategies without a priori for the identification of proteins involved in cardiovascular pathologies [START_REF] Lindsey | Transformative Impact of Proteomics on Cardiovascular Health and Disease: A Scientific Statement From the American Heart Association[END_REF] . To identify new biomarkers of LVR post-MI, we performed a differential proteomic analysis, by 2D-DIGE [START_REF] Dupont | Application of saturation dye 2D-DIGE proteomics to characterize proteins modulated by oxidized low density lipoprotein treatment of human macrophages[END_REF] , on enriched plasma prepared as previously described 30 , obtained from REVE-2 patients with high LVR and patients with no LVR evaluated 1 year after an anterior inaugural MI. We identified several polypeptidic spots corresponding to CLU. The observed molecular mass differences for CLU isoforms in our proteomic study can be explained by the potential Nglycosylation of CLU, consisting of associated complex sugars contributing to 20-30% of its molecular weight [START_REF] Burkey | Intracellular processing of apolipoprotein J precursor to the mature heterodimer[END_REF] . We validated our results by western blot analysis showing that s-CLU isoform with a molecular mass of ~36 kDa is positively associated with high LVR post-MI.

CLU is constitutively expressed in most mammalian tissues and is induced in many organs where tissue injury or remodeling occurs. CLU levels were shown to be increased in plasma of patients after 24h of acute MI 17 and recently described in the exosomes isolated from pericardial fluids collected from MI patients [START_REF] Kuzyk | Development of MRM-based assays for the absolute quantitation of plasma proteins[END_REF] . Our proteomic data is the first study showing that CLU is increased in late stage of MI and is associated with LVR. To verify if increased CLU levels in CIRCHF/2017/004838-R3 15 plasma (s-CLU) of LVR patients has in part a cardiac origin, we first used an experimental MI rat model [START_REF] Mulder | Early versus delayed angiotensin-converting enzyme inhibition in experimental chronic heart failure. Effects on survival, hemodynamics, and cardiovascular remodeling[END_REF] in order to analyze the temporal CLU expression in the LV following MI. s-CLU is obtained by proteolytic cleavage of its precursor (p-CLU, 60 kDa) to generate alpha and beta chains of 40 kDa that are glycosylated and assembled to give the mature form, m-CLU that will be then secreted (s-CLU). Our results show that CLU mRNA and its intracellular levels are increased in non-infarcted LV area of MI rat with the mature form as being predominant, suggesting that LV cells are able to express and secrete CLU into the bloodstream. Our results also show that CLU is increased as early as 7 days after MI and correlated with LVR parameters. This finding suggests an implication of CLU in LVR.

To verify if increased CLU expression in the LV is associated with increased CLU secretion, we induced hypertrophy in NCM, a mechanism observed in the non-infarcted area after MI. The 72h-isoproterenol treatment increased CLU expression as shown by its mRNA and intracellular levels. Our results are in agreement with a previous study showing increased CLU levels in the LV of mouse after chronic β-adrenergic stimulation [START_REF] Dostanic | Chronic beta-adrenoreceptor stimulation CIRCHF/2017/004838-R3 27 in vivo decreased Bcl-2 and increased Bax expression but did not activate apoptotic pathways in mouse heart[END_REF] . Interestingly, we also observed increased CLU in the culture media of hypertrophied NCM. All these data show that CLU expression and secretion are increased in hypertrophic cardiomyocytes and suggests that the increased CLU plasma levels in REVE 2 patients with high LVR after MI has, in part, a cardiac origin.

The physiological role of CLU is complex depending on CLU isoforms, localization and cellular types [START_REF] Rohne | The CLU-files: disentanglement of a mystery[END_REF] . Several data suggest an anti-apoptotic role of CLU in early phase after MI. In ischemia/reperfusion rat model, the administration of CLU for 3 days reduced both infarct size and death of rats [START_REF] Van Dijk | Intravenous clusterin administration reduces myocardial infarct size in rats[END_REF] . In addition, infusion of CLU after acute MI reduced the cardiomyocyte apoptosis and increased the induction of epithelial mesenchymal transition, angiogenesis and CIRCHF/2017/004838-R3 16 preservation of myocardial function [START_REF] Kuzyk | Development of MRM-based assays for the absolute quantitation of plasma proteins[END_REF] . In vitro, the addition of CLU in culture media inhibited H9c2 apoptosis induced by ischemic/reperfusion [START_REF] Krijnen | Clusterin: a protective mediator for ischemic cardiomyocytes?[END_REF] . However, in MI rats, CLU was also detected in peri-infarct zone [START_REF] Silkensen | Temporal induction of clusterin in the peri-infarct zone after experimental myocardial infarction in the rat[END_REF] . These data in addition to our results lead us to study the role of CLU on hypertrophy. In our model, CLU silencing in cardiomyocytes decreased cells size, ANP and BNP expression associated with decreased ERK1/2 activity suggesting a pro-hypertrophic role of CLU in LVR via ERK1/2 pathway. This is supported by previous studies showing, in one hand, a decrease of ERK1/2 pathways after CLU silencing in cancer cells [START_REF] Tang | Knockdown of clusterin sensitizes pancreatic cancer cells to gemcitabine chemotherapy by ERK1/2 inactivation[END_REF] and in another hand, a protective effect of some molecules against hypertrophy via inhibition of ERK1/2 activity [START_REF] Song | Cardiac ankyrin repeat protein attenuates cardiac hypertrophy by inhibition of ERK1/2 and TGF-β signaling pathways[END_REF] .

Finally, we validate that CLU expression is also increased in the LV of HF patients that underwent heart transplantation. We evaluated whether the secreted form of CLU may be a predictor of adverse effects in HF patients before heart transplantation, and we showed significantly increased CLU levels in HF patients who died from a cardiovascular cause during the 3-year follow-up compared to survivors, and it is not depending on the HF etiology.

However, a recent prognostic study showed a decreased of CLU levels in the plasma of HF patients who died compared to survivors 20 . The discrepancy results observed between the two studies can be explained by a different design of the study. In our case, we performed a casecontrol study and they included consecutive older HF patients hospitalized, with only 1/3 of the patients with altered ejection fraction (<30% vs 100% in our study), a higher female/male ratio and more HF patients with NYHA class III and IV vs no class IV patients in our study. A recent study confirmed our data with increased levels of CLU found in circulating extracellular vesicles derived from dilated cardiomyopathy patients compared to controls [START_REF] Roura | 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] . As in our study, the patients were relatively young (62.3±11.5 years), mostly men (90%) with reduced ejection fraction (36.1 ± 11.7) and a ratio NYHA II/III (80/20). Our results suggest that CLU is a new predictive marker of early cardiac mortality.

In conclusion, our data show that CLU is increased in non-infarcted LV at late stage of MI in the rat model. CLU expression is time-dependent in post-MI suggesting different mechanisms of CLU regulation and different roles of this protein in cardiac remodeling. Our data suggest that CLU is involved in the compensatory hypertrophy but its role in HF is not yet elucidated. Since the role of CLU is complex depending on CLU isoforms, localization and cellular types, it will be important to study the mechanisms involved in CLU expression, maturation, secretion and degradation in cardiomyocytes in order to better understand its regulation and its role in different phase after MI. In addition, our study is the first showing that circulating levels of CLU are associated with LVR post-MI and is a new predictive marker of cardiac mortality in HF patients with reduced EF. To consider CLU as a clinically useful biomarker, it should be validated in other cohorts of patients. Our results were obtained in selected patients: 1) with first anterior MI and substantial residual akinesia at predischarge echocardiography; they may not apply to all post-MI patients and; 2) relatively young HF patients with reduced EF. Finally, a gold standard and accurate measurement of CLU usable at hospital should be developed.

WHAT IS NEW?

This is the first study analyzing the relationship of plasma clusterin levels with left ventricular remodeling following myocardial infarction, discovered by proteomic analysis.

CIRCHF/2017/004838-R3 18 Our findings suggest that clusterin is involved in compensatory hypertrophy and played a role in early and late phase following myocardial infarction.

Modulation of plasma clusterin levels is in part of cardiac origin following hypertrophy of cardiomyocytes.

We show a pro-hypertrophic role of clusterin via ERK1/2 pathway in cardiomyocyte under beta-adrenergic stress.

WHAT ARE THE CLINICAL IMPLICATIONS?

Despite modern therapeutic approaches, left ventricular remodeling following myocardial infarction remains relatively frequent and difficult to predict in clinical practice.

Clusterin plasma levels are associated with left ventricular remodeling development following myocardial infarction.

Our findings suggest that clusterin is involved in compensatory hypertrophy and played a role in early and late phase following myocardial infarction.

Clusterin plasma levels is new predictive marker of early cardiac mortality in heart failure patients with reduced ejection fraction.

A significant increase of intracellular clusterin was found in failing human hearts. 4] and then digested by using pancreatin and collagenase type II as previously described [START_REF] Dubois-Deruy | Interplay between troponin T phosphorylation and O-N-acetylglucosaminylation in ischaemic heart failure[END_REF] . NCMs were purified by centrifugation of the cells at 2800 rpm for 30 min on Percoll gradients (58.5% and 40.5% in the balanced salt solution).

SOURCE OF FUNDINGS

NCMs were seeded at a density 6.5 × 10 5 cells/well in 6-well plates coated with 10% of collagen (Sigma-Aldrich) and cultured in a medium containing 4 parts of Dulbecco's modified Eagle's medium (DMEM, D1152, Sigma-Aldrcih) and 1 part of Medium199 (M199, M2520, Sigma), 10% FBS and 1% P/S at 37°C under 5% CO 2 atmosphere.

Hypertrophic treatment

After 24h of seeding, cells were serum deprived for 24 h and then treated with isoproterenol (10 µmol/L, Tocris Bioscience, 1747), Angiotensin II (2 µmol/L, A9525, Sigma-Aldrich) or phenylephrine (20 µmol/L, 2838, Tocris Bioscience) for 48 h or 72h in the presence of 1% FBS.

Transfection studies

To inhibit the expression of Clusterin, cells were transfected by 25 nmol/L of siRNA non target (D-001810-10-20, Dharmacon) as a control or ON-TARGETplus Rat Clu siRNA (Smart pool, or the first individual siRNA, Dharmacon) using Darmaphect according to the manufacturer's instructions. For treatment of transfected cells with isoproterenol, the transfection was occurred in serum and P/S free medium 24h before the treatment.

Immunostaining studies

Immunohistochemistry

CIRCHF/2017/004838-R2

Frozen human heart tissues were included in Richard-Allan Scientific Neg-50 TM (ThermoFisher Scientific). Tissue Sections (5 µm thickness) were fixed with acetone/methanol (V:V) for 20 min at -20°C. The endogenous peroxidase activity was blocked in 0.3% hydrogen peroxide (H 2 O 2 ) in methanol for 30 min at room temperature. After washing with TN (Tris 2.42 g/L and NaCl 29.22 g/L in water), the sections were saturated with TNO (TN with ovalbumin at 5 g/L) for 30 min at room temperature and then incubated with polyclonal rabbit clusterin antibody (ab42673, Abcam) diluted at 1/50 in TNO overnight at 4°C. After washing with TNO, Sections were incubated with anti-rabbit biotinylated secondary antibody (BA-1000, Vector laboratories) diluted at 1/200 in TNO for 1h at room temperature, washed and then incubated in Vectastain Elite ABC Kit (vector laboratories) for 30 min. Finally the sections were developed in ABC staining kit (sigma) for 5 min, counter-stained with hematoxylin to identify nuclei and mounted in glycerol. Staining was visualized with the x20 and x40 objectives of color Axioplan 2 microscope followed by Zen image acquisition.

Immunofluorescence

For measuring cell surface area, the cells were plated at a density of 2 x 10 5 cells on coverslip (24 mm x 24 mm) for H9c2 cells and 8x10 8 cells on coverslip coated with collagen for NCM in a 6-wells culture plate. After treatment, the cells were incubated with the complete medium containing Hoechst (33342, Invitrogen) for 30 min at 37°C to color nuclei, then with the Cell Mask plasma membrane stain (C10046, Life Technology) at a final concentration of 1X in the medium for 10 min at 37°C. The cells were then fixed with 3.75 % paraformaldehyde (43368, Thermofisher) for 10 min at room temperature and then wash three times for 5 min with PBS 1X.

The coverslips were mounted on slides with glycerol 90%. Staining was visualized with the x40 objectives of LSM710 confocal microscope followed by Zen image acquisition.

aligned with the image of comigrating standard). All gels were compared with the reference gel to normalize spots'values. An average of 429 spots per gel was detected. Each spot was normalized by the ratio between intensities of Cy3-REVE-2 sample and Cy5-standard. We then compared the normalized spots (Cy3-REVE-2/Cy5-standard). Fold values as well as p-values of all spots were calculated by SamesSpots software using one way ANOVA analysis. Differential expression of a protein present in the gels was considered significant when the p value was below 0.05 (one-way ANOVA analysis).

Differentially regulated spots were excised manually from a preparative gel performed with 500 µg of proteins and in-gel digested with trypsin 16

RNA extraction and quantitative real time-polymerase chain reaction (qRT-PCR)

RNA was extracted from LV with TRI Reagent (Ambion, Austin, TX, USA) and from NCM with QIAGEN RNeasy Mini Kit (Qiagen), as described by the manufacturers's instructions.

CIRCHF/2017/004838-R2

Then, 500 ng of LV RNA and 250 ng of NCM RNA were reverse-transcribed using the miScript II RT kit (Qiagen). qRT-PCR was performed with the miScript SYBR Green PCR kit (Qiagen) on a Mx3005P Q-PCR system (Agilent Technologies), according to the manufacturer's instructions. The sequences of the different primers (Eurogentec) used were: rat ANP (sense:

CCGGTACCGAAGATAACAGC and antisense: CTCCAGGAGGGTATTCACCA), rat BNP (sense:CTGGGAAGTCCTAGCCAGTCTCCA and antisense:

GCGACTGACTGCGCCGATCCGGTC), rat Clu (sense: GCTCCATAGCCCAGCTTTAC and antisense: ACTTCTCACACTGGCCCTTC), rat GAPDH (sense:

GGCATTGCTCTCATTGACAA and antisense: TGTGAGGGAGATGCTCAGTG), and rat HPRT (sense: ATGGGAGGCCATCACATTGT and antisense:

ATGTAATCCAGCAGGTCAGCAA-. ∆∆CT method was used for data analysis.

Western blot analysis

Antibodies

The primary antibodies used for western blot analysis were: Clusterin (sc-6419) and Proteins from culture medium (2 mL) were precipitated with 8 mL acetone at -20°C overnight and then centrifuged at 2700 g, 4°C, for 15 min. The pellet was then washed 3 times with 3 mL of cold ethanol and a centrifugation at 2700 g, 4°C, for 10 min is performed after each wash. The protein pellets were then dried and resuspended in 300 µL of RIPA buffer and the samples were stored at -80°C.

Protein concentrations were determined with a Bradford-based method protein assay (Biorad, 500-0006).

Western blot

Proteins (20 µg of NCM, 40 µg of culture media protein, 50 µg of LV protein and 1µl of plasma) were separated on 10% SDS-PAGE and transferred on 0.22 µm nitrocellulose membranes (Trans-Blot® Turbo TM Transfert Pack, Bio-rad). Equal total proteins loads were confirmed by Ponceau red [0.1% Ponceau red, 5% acetic acid (v/v) (Sigma-Aldrich)] staining of the membranes. After blocking with 5% non-fat dry milk in TBS-Tween 0.1% buffer for 1 h at room temperature, the membranes were incubated with primary antibodies at 4°C with gentle shaking overnight as described in supplemental table 1. The membranes were then washed three between 3.4-3.7%.by ELISA (R&D systems, Minneapolis, MN, USA). The sensitivity was 0.189 ng/mL and the variability between plates estimated between 3.4-3.7%.

LC-MRM MS with SIS peptides

The peptides had been previously validated for its use in LC-MRM experiments following the CPTAC guidelines for assay development (https://assays.cancer.gov). The tryptic peptide was selected to serve as molecular surrogate for clusterin according to a series of peptide selection rules (e.g., sequence unique, devoid of oxidizable residues; see [START_REF] Kuzyk | Development of MRM-based assays for the absolute quantitation of plasma proteins[END_REF] for detailed criteria) and previous detectability in pooled plasma samples. To help compensate for matrix-induced suppression or variability in LC-MS performance, 13 C/ 15 N-labeled peptide analogues were used as internal standards. The peptides (light and SIS) were synthesized via Fmoc chemistry, purified through RP-HPLC with subsequent assessment by MALDI-TOF-MS, and characterized via amino acid analysis, (AAA), and capillary zone electrophoresis (CZE) with an average CZEderived purity of 92.4%.

The plasma proteolytic digests were prepared in an automated manner on a Tecan Freedom EVO 150 on a 96-well microplate (Axygen). In the sample preparation, 10 µL of raw plasma was subjected to 9 M urea, 20 mM dithiothreitol, and 0.5 M iodoacetamide (all in Tris buffer at pH 8.0). Denaturation and reduction occurred simultaneously at 37°C for 30 min; with alkylation occurring thereafter in the dark at room temperature for 30 min. Proteolysis was initiated by the addition of TPCK-treated trypsin (35 µL at 1 mg/mL; Worthington) at a 20:1 substrate: enzyme ratio. After overnight incubation at 37°C, proteolysis was quenched with 1% FA. The SIS peptide mixture was then spiked into the digested samples, the standard curve samples, and the QC samples and concentrated by solid phase extraction (SPE) (Oasis HLB, 2 mg sorbent; Waters).

After SPE, the concentrated eluate was frozen, lyophilized to dryness, and rehydrated in 0.1% FA (final concentration: 0.5 µg/µL digest) for LC-MRM/MS.

The standard curve was prepared using a mix of light peptides that was spiked into a human plasma tryptic digest in which the peptides were dimethylated (to shift their masses) from a high concentration of 1000X the Lower limit of quantitation (LLOQ) over 8 dilutions to the lowest point of the curve which was the LLOQ for the assay. The QC samples were prepared from the same light peptide mix and diluted in dimethylated human plasma digest at 4X, 50X, and 500X

the LLOQ for each peptide.

Twenty microliters injections of the plasma tryptic digests were separated with a Zorbax Eclipse Plus RP-UHPLC column (2.1 x 150 mm, 1.8 µm particle diameter; Agilent) that was contained within a 1290 Infinity system (Agilent). Peptide separations were achieved at 0.4 mL/min over a 60 min run, via a multi-step LC gradient (2-80% mobile phase B; mobile phase compositions: A was 0.1% FA in H 2 O while B was 0.1% FA in ACN). The column was maintained at 40˚C. A post-gradient equilibration of 4 min was used after each sample analysis. multiplier was set to -20 kV for improved ion detection efficiency and signal-to-noise ratios.

Specific LC-MS acquisition parameters were employed for optimal peptide ionization/fragmentation and scheduled MRM (see supplemental

  7 [1.3-2.7] vs 1 [0.8-1.2], P =0.001) and 2 months (1.8 [1.6-2.1] vs 1.2 [1.1-1.4], P =0.010) post-MI (Figure 2A, left panel). Western blot analysis detected 2 bands with a molecular mass of 60 and 40 kDa corresponding respectively to the precursor (p-CLU) and the mature (m-CLU) forms of clusterin.

[ 0 .

 0 02-0.03], P=0.037) (Figure 2A, middle panel). However, m-CLU was significantly increased at 7 days (0.65 [0.54-0.81] vs 0.29 [0.28-0.4], P=0.001) and 2 months post-MI (0.49 [0.37-0.58] vs 0.37 [0.31-0.41], P=0.035) compared to the sham-rats (Figure 2A, right panel). However, a significant decrease of p-CLU (0.025 [0.02-0.034] vs 0.05 [0.027-0.061], P=0.033) and m-CLU CIRCHF/2017/004838-R3 10 (0.5 [0.38-0.59] vs 0.65 [0.54-0.81], P=0.032) levels was observed in the LV of MI rats at 2 months compared to 7 days post-MI (Figure 2A, middle and right panels).

  the siRNA-Clu-1 (the first siRNA of pool used in panels A-C) decreased significantly CLU expression (0.24 [0.19-0.34] vs 0.8 [0.66-1], P<0.001) and CLU secretion (0.04 [0.04-0.07] vs 1.02 [0.79-1.21], P<0.001) (Figure 4D) and decreased significantly ANP (0.63 [0.41-0.73] vs 0.97 [0.84-1.07], P<0.001) and BNP (0.35 [0.31-0.55] vs 0.7 [0.48-0.87], P<0.001) expression (Figure 4E) associated with significantly decreased pERK1/ERK1 (0.74 [0.59-0.85] vs 0.95 [0.78-1.19], P=0.004) and pERK2/ERK2 (0.56 [0.42-0.72] vs 0.98 [0.79-1.21], P<0.001) ratios

[

  58.3-82.4] x 10 3 vs 30.3 [28.5-73] x 10 3 , P<0.001 (MS), 150 [103-185] vs 134 [99-164], P=0.03 (ELISA) in patients with early cardiac mortality compared to survivors (Figure 5C), independently of HF etiology (not shown).

Figure 1 :

 1 FIGURE LEGENDSFigure 1: Discovery of clusterin as biomarker of high LVR by differential 2D-DIGE

Figure 2 :

 2 Figure 2: Clusterin expression is increased in the left ventricle after MI and correlated

Figure 3 :

 3 Figure 3: Clusterin expression and secretion are increased in hypertrophied

Figure 4 :

 4 Figure 4: Clusterin silencing inhibited cardiomyocytes hypertrophy induced by

Figure 5 :

 5 Figure 5: Clusterin protein levels in human myocardial and plasma samples from HF

  . Identifications were performed by MALDI-TOF with a Voyager DE STR mass spectrometer (PerSeptive Biosystems, Framingham, MA, USA) equipped with a 337.1 nm nitrogen laser and a delayed extraction facility (125 msec). All spectra were acquired in a positive ion reflector mode under 20 kV voltage, 61% grid. The mass spectra were then calibrated before protein identification by peptide mass fingerprinting, conducted by running the MASCOT web searcher (http://www.matrixscience.com, Matrix Science, UK) against the NCBInr 20080718 (6833826 sequences; 2363426297 residues) with the following parameters: Fixed modifications: CyDye-Cy3 (C); Variable modifications: Oxidation (M); Peptide Mass Tolerance: ± 50 ppm; Peptide Charge State: 1+; Max Missed Cleavages: 1 as previously described15 . Some spots were identified with nano-LC-MS/MS (on an Ultraflex TM TOF/TOF instrument).

The

  LC system was interfaced to a triple quadrupole mass spectrometer (Agilent 6490) via a standard-flow ESI source, operated in the positive ion mode. The general MRM acquisition parameters employed were as follows: 3.5 kV capillary voltage, 300 V nozzle voltage, 11 L/min sheath gas flow at a temperature of 250°C, 15 L/min drying gas flow at a temperature of 150°C, 30 psi nebulizer gas pressure, 380 V fragmentor voltage, 5 V cell accelerator potential, and unit mass resolution in the first and third quadrupole mass analysers. The high energy dynode (HED)

  Comparisons were made by Kruskal-Wallis test for REVE-2 and INCA, Wilcoxon-Mann-Whitney test and Wilcoxon-signed rank test for animal and in vitro experiments, as appropriate. Correlations were carried out by Spearman correlation test. Results were considered statistically significant if the p<0.05.

	CIRCHF/2017/004838-R3
	8

Table 2

 2 ) for the dynamic MRM

	CIRCHF/2017/004838-R2

Table 1 :

 1 List and dilution of antibodies used for western blot analysis

	Primary antibodies	Reference	Incubation buffer Sample :Dilution	Secondary	Dilution
					antibodies
	Clusterin	sc-6419, Santa Cruz	TBST-0.1% + 5%	H9c2 : 1/500	Anti-goat antibody	1/10000
			Milk	NCM Media : 1/2000	(sc-2020, Santa	1/10000
				NCM Lysate : 1/500	Cruz)	1/10000
				Rat LV : 1/1000		1/5000
				Human cardiac biopsies :		1/40000
				1/20000
	Clusterin	RD182034110,		Human plasma : 1/2000	Anti-mouse antibody	1/5000
		Biovendor			(NA931, GE
					Healthcare)
	pAkt (Ser473)	9271, Cell Signalling TBST-0.1% + 5%	1/500	Anti-rabbit antibody	1/5000
	Akt	9272, Cell Signalling	BSA	1/2000	(NA934V, GE	1/5000

Table 2 .

 2 Specific acquisition parameters for the target peptides for clusterin (P10909) measured by bottom-up LC-MRM/MS.

				NAT		SIS
	Compound	Compound					RT	CE
			Ion	Q1	Q3	Q1	Q3
	Name	Group					(min)	(V)
				m/z	m/z	m/z	m/z
	Clusterin	ELDESLQVAER	y3.heavy	644.83 602.33 649.83 385.21	21	12.65
	Clusterin	ELDESLQVAER	Y5.heavy	644.83 1046.52 649.83 612.33	21	12.65
	Clusterin	ELDESLQVAER	Y9.heavy	644.83 375.21 649.83 1056.52	25	12.65
	Clusterin EPQDTYHYLPFSLPHR y12+++.heavy 500.75 549.28 503.25 514.27	9	21.76
	Clusterin EPQDTYHYLPFSLPHR y13+++.heavy 500.75 409.23 503.25 552.61	9	21.76
	Clusterin EPQDTYHYLPFSLPHR	y3.heavy	500.75 510.93 503.25 419.24	17	21.76
	Clusterin	TLLSNLEEAK	b2.heavy	559.31 790.39 563.32 215.14	17	19.65
	Clusterin	TLLSNLEEAK	y7.heavy	559.31 903.48 563.32 798.41	17	19.65
	Clusterin	TLLSNLEEAK	y8.heavy	559.31 215.14 563.32 911.49	17	19.65
	The fragmentation and cell acceleration voltages were 380 and 5 V, respectively, for all
	transitions.

Table 3 :

 3 Identification by MALDI-MS of the 2D spots selected in Figure1.

	Spot	Accession	Protein	Mr (Da)		pI		MASCOT	Number of	Sequence	Peptides
	number*	number	name	Theo	Exp	Theo	Exp	score	matched	coverage	Mr (Exp)
									peptides/total	(%)
									peptides
	842			40000		4.75	65	6 / 24	12	1887.8214
	853										1903.8205
	873			59184							1392.6909
		P10909	Clusterin			5.89
											1074.5735
											949.4149
											1872.9244

*Number corresponds to the spots visualized in Figure

1B

Proteomic signature of circulating vesicles in dilated cardiomyopathy. Lab Investig. 2018;

EXPANDED MATERIALS AND METHODS

Patients

The studies were approved by the Ethics Committee of the "Centre Hospitalier et Universitaire de Lille" and comply with the Declaration of Helsinski. All patients gave written informed consent.

REVE-2 study

The design of the REVE-2 study has been published in detail elsewhere [START_REF] Benjamin | Heart Disease and Stroke Statistics-2017 Update: A Report From the American Heart Association[END_REF] . We enrolled 246 patients with a first anterior wall Q-wave MI between February 2006 and September 2008.

Inclusion criteria were hospitalization within 24 hours after symptom onset and at least 3 akinetic LV segments in the infarct zone at the predischarge echocardiography. The protocol required serial echographic studies at baseline (day 3 to day 7) and 3 and 12 months after MI. Serial blood samples were taken at baseline (day 3 to day 7), and 1, 3, and 12 months after MI.

A standard echographic imaging protocol was used based on apical 4-and 2-chamber views.

All echocardiograms were analyzed at the Lille Core Echo Laboratory (Lille, France), as previously described [START_REF] Benjamin | Heart Disease and Stroke Statistics-2017 Update: A Report From the American Heart Association[END_REF] . LV end-diastolic volume (EDV), LV end-systolic volume (ESV), and LV ejection fraction (EF) were calculated with a modified Simpson's rule. Left ventricular remodeling (LVR) was defined as percent change in EDV from baseline to 1 year. Blood samples were collected in EDTA-containing tubes. Plasma was obtained by centrifugation at 800g for 10 min. Samples were aliquot, and stored at -80 °C until further analysis.

INCA study

All patients referred for evaluation of systolic HF (LVEF <45%) in our institution between November 1998 and May 2010 were included in a prospective cohort on prognostic indicators 2,3,4,5 . All patients were ambulatory and clinically stable for at least 2 months, and received CIRCHF/2017/004838-R2 optimal medical therapy with maximal tolerated doses of angiotensin-converting enzyme inhibitors and betablockers. At inclusion, patients underwent a prognostic evaluation including: BNP level assessment, echocardiography, and cardiopulmonary exercise testing as previously described 2,4 . In addition, patients underwent a coronary angiogram to help define the etiology of left ventricular (LV) systolic dysfunction as either ischemic or nonischemic. A follow-up was performed at 3 years to assess clinical outcome. All events were adjudicated by two investigators and by a third one in case of disagreement. Cardiovascular death included cardiovascular-related death, urgent transplantations defined as United Network for Organ Sharing (UNOS) status 1), and urgent assist device implantation.

From this population, we selected 198 patients included between November 1998 and December 2005. Ninety nine patients who died from cardiovascular cause within 3 years after the initial prognostic evaluation (cases) were individually matched for age, sex, and HF etiology with 99 patients who were still alive at 3 years (controls) as previously described 6 .

At inclusion, peripheral blood was collected in tubes containing EDTA and plasma samples were stored at -80°C.

Human heart biopsies

Tissues from failing and non-failing human hearts were obtained respectively from Lille University Hospital (France) and from Catholic University of Leuven (Belgium). Explanted heart tissues were obtained from patients undergoing heart transplantation for end-stage ischemic heart failure and from patients died of non-cardiac causes. Samples were quick-frozen and stored at -80°C. All materials from patients and controls were recovered as surgical waste with informed consent of the donors and with approval of the local ethical boards and according to the Declaration of Helsinki.

Animal models

All 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). Animals were used and experimental protocols performed under the supervision of a person authorized to perform experiments on live animals (F. Pinet: 59-350126). Approval was granted by the institutional ethics review board (CEEA Nord Pas-de-Calais N°242011, January 2012).

Before surgery, rats were anaesthetized (sodium methohexital, 50 mg/kg intraperitoneally (IP)), while analgesia was administered before (xylazine 5 mg/kg IP) and 1 hr after surgery (xylazine 50 mg/kg subcutaneously) as described 7 . Anesthesia and sedation were controlled by monitoring heart rate. MI was induced in 10-week-old male Wistar rats (Janvier, Le Genest St isle, France) by ligation of the left anterior descending coronary artery 7,8 . Sham-operated and MI rats were then further separated in two subgroups that were sacrificed at 7 days (n=8/group) or 2 months (n= 9 to 11/ group) post-MI. Haemodynamic and echocardiographic measurements were taken 2 months after surgery, followed by heart excision, as previously described 9,10 .

Cellular models

H9C2 cells

Rat cardiac myoblasts (H9c2 cells, CRL-14146, ATCC) were cultured in Dulbecco's Modified

Eagle's Medium (DMEM Glutamax, 31966, Gibco, Life Technologies) with addition of 10% (v/v) fetal bovine serum (FBS, 30-2020, ATCC) and 1% penicillin and streptomycin (P/S, 10,000 U/mL) (15140-122, Invitrogen™, Life Technologies). Cells were cultured at 37°C under 5% CO 2 atmosphere.

Primary cultures of neonate rat cardiomyocytes (NCM)

Proteomic analysis of plasma samples from REVE-2 patients

Constitution of pooled plasma samples

The follow-up of REVE-2 study was completed for 205/246 patients (serial echography and blood samples). The 205 patients were ranked by the increasing percentage of LVR, and then grouped together by 5, defining 41 pools. The pools with the lowest (1 to 5) and with the highest (37-41) percent of LVR were constituted by mixing an equal volume of plasma from the 5 patients in the same group (Figure 1A).

Processing of pooled plasma

Prior to the proteomic study, plasma samples underwent no more than two freeze/thaw cycles.

One mL of each plasma sample was treated with the ProteoMiner™ kit (Bio-Rad Laboratories, Hercules, CA, USA) as previously described 12,13 . This combinatorial peptide ligand library (CPLL) method has been shown to be reproducible and to allow for the enrichment in mediumand low-abundance proteins 14 . Samples were eluted by adding 100 µL elution reagent, repeated three times and the pooled samples were stored at -20°C, before the protein concentration was determined for 2D analysis.

2D-DIGE electrophoresis

Two gels (technical replicates) of each sample were run with a total of 10 µg of mixed labelled protein per gel. These protein mixtures contained 5 µg of protein from each REVE 2 sample group (pool of 4 patients and pool of 4 healthy donors) randomly labelled with 400 pmol saturation CyDye DIGE fluors (Cy3 or Cy5) and 50 µg of the two pools mix (25 µg from patients' pool and 25 µg from healthy donors' pool) labeled with 400 pmol Cy2. Five micrograms of each sample were reduced with 2 mM Tris (2-carboxyethyl)phosphine (TCEP) for 1 h at 37 °C. Cyanine 3 (Cy3) and cyanine 5 (Cy5) sulfhydryl-reactive dyes (0.8 nmol/µg protein) were added respectively to REVE2 and standard samples and incubated for 30 min at 37 °C in the dark. The reaction was stopped with the addition of an equal volume of sample buffer containing 7 mol/L urea, 2 mol/L thiourea, 4% CHAPS, 130 mmol/L dithiothreitol (DTT) and, 2% pharmalytes (GE Healthcare).The Cy3-labelled REVE samples were then mixed with Cy5-labelled standard samples and left for 15 min on ice in the dark. Immobilized pH gradient (IPG) strip (240 mm, pH 4-7 linear gradient) was rehydrated with 450 µl of labelled mixed samples in rehydration buffer containing 1% Pharmalytes, 1300 mmol/L DTT, 7 mol/L urea, 2 mol/L thiourea and 4% CHAPS on a Protean IEF cell system (Biorad) for 24 h without applying any current. After rehydration, IEF was performed at 300 volts (V) for 3 h, and then at a gradient to 1000 V for 6 h, at a gradient to 8000 V for 3 h and finally at 8000 V for 3 h with the temperature maintained at 20 °C. After IEF, the IPG strips were incubated in equilibration buffer containing 6 mol/L urea, 0.1 mmol/L Tris-HCl pH 8, 2% w/v sodium dodecyl sulfate (SDS) and 30% v/v glycerol for 10 min at room temperature. The equilibrated IPG strips were transferred for the second dimension (SDSpolyacrylamide gel electrophoresis (PAGE)) onto 12% PAGE gels and seal with low-melting agarose. Electrophoresis was carried out at 20 °C using an Ettan Dalt 6 system (GE Healthcare) at a constant voltage of 70 V overnight followed by 300 V in the dark.

Following electrophoresis, gels were scanned with an Ettan DIGE Imager (GE Healthcare) at excitation/emission wavelengths of 532/580 nm for Cy3 and 633/670 nm for Cy5 to yield images with a pixel size of 100 µm. Gel images were processed with Progenesis SameSpots (v 3.0) from Nonlinear Dynamics Ltd. (Newcastle, UK) as previously described 15 . First, the images were classified by pairs which corresponded to comigration of one standard and one REVE2 sample in the same gel. After choosing the image of reference, the whole 2D-images were automatically aligned in 2 steps (all the standards' images were aligned then each REVE2 sample's image is times for 10 min with TBS-Tween 0.1% buffer and incubated with the corresponding horseradish peroxidase-labelled secondary antibody for 1h in the blocking solution (see details in the Supplemental table 1). After three washes with TBS-Tween 0.1% buffer, the membranes were incubated with enhanced chemiluminescence (ECL™) western blotting detection reagents (GE Healthcare) according to the manufacturer's instructions.

The Chemidoc® camera (Biorad) was then used for imaging the membranes and densitometric measurements of the bands were analysed with the Image Lab software (Bio-Rad).

Quantification by multiplex immunoassay

B-type natriuretic peptide (BNP) was measured in plasma samples using a Multi Analyte Profiling Kit for simultaneous quantitative detection of rat cardiovascular biomarkers (Rat CVD1 Panel 1, RCVD1-89K Millipore) according to the manufacturer's instructions as previously described 17 .

Quantification of clusterin in plasma from REVE-2 and INCA studies

Western blot

Western blot detection of clusterin in plasma of REVE-2 patients was performed with the mouse primary antibody (Hs-3, Biovendor) (1/2000). Briefly, 1 µL of crude plasma proteins was resolved on 12% SDS-PAGE (NuPAGE 10% Bis-Tris gel, Life Technologies), transferred as described above before incubation with horseradish peroxidase-labelled secondary antibody (1/5000) for 1 h in blocking solution.

ELISA

Clusterin in plasma of INCA patients was quantified by ELISA (R&D systems, Minneapolis, MN, USA). The sensitivity was 0.189 ng/mL and the variability between plates estimated method. Note that the peptide optimizations were empirically optimized previously by direct infusion of the purified SIS peptides. In the quantitative analysis, the targets (with 3 transitions/peptide form) were monitored over 500 ms cycles and 1 min detection windows.

The MRM data was visualized and examined with MassHunter Quantitative Analysis software (version B.07.00; Agilent). This involved peak inspection to ensure accurate selection, integration, and uniformity (in terms of peak shape and retention time) of the SIS and NAT peptide forms. Thereafter, the processed response (i.e., peak area) data was inputted into our inhouse developed software tool -Qualis-SIS -for quantitative analysis. After defining a small number of criteria (i.e., 1/x 2 regression weighting, <20% deviation in a given level's precision and accuracy) for each concentration level of the standard curve, the tool automatically generates and extracts assay-related information from each standard curve. The endogenous protein concentrations in the patient samples are determined through linear regression 19,20 .

Statistical analysis

Data summarized as medians with interquartile range (IQR) were analyzed with GraphPad Prism version 6.01 (GraphPad Software, San Diego, CA).