LIPCAR levels in plasma-derived extracellular vesicles is associated with left ventricle remodeling post-myocardial infarction.

BACKGROUND: Long Intergenic noncoding RNA predicting CARdiac remodeling (LIPCAR) is a long noncoding RNA identified in plasma of patients after myocardial infarction (MI) to be associated with left ventricle remodeling (LVR). LIPCAR was also shown to be a predictor of early death in heart failure (HF) patients. However, no information regarding the expression of LIPCAR and its function in heart as well as the mechanisms involved in its transport to the circulation is known. The aims of this study are (1) to characterize the transporter of LIPCAR from heart to circulation; (2) to determine whether LIPCAR levels in plasma isolated-extracellular vesicles (EVs) reflect the alteration of its expression in total plasma and could be used as biomarkers of LVR post-MI. METHODS: Since expression of LIPCAR is restricted to human species and the limitation of availability of cardiac biopsy samples, serum-free conditioned culture media from HeLa cells were first used to characterize the extracellular transporter of LIPCAR before validation in EVs isolated from human cardiac biopsies (non-failing and ischemic HF patients) and plasma samples (patients who develop or not LVR post-MI). Differential centrifugation at 20,000g and 100,000g were performed to isolate the large (lEVs) and small EVs (sEVs), respectively. Western blot and nanoparticle tracking (NTA) analysis were used to characterize the isolated EVs. qRT-PCR analysis was used to quantify LIPCAR in all samples. RESULTS: We showed that LIPCAR is present in both lEVs and sEVs isolated from all samples. The levels of LIPCAR are higher in lEVs compared to sEVs isolated from HeLa conditioned culture media and cardiac biopsies. No difference of LIPCAR expression was observed in tissue or EVs isolated from cardiac biopsies obtained from ischemic HF patients compared to non-failing patients. Interestingly, LIPCAR levels were increased in lEVs and sEVs isolated from MI patients who develop LVR compared to patients who did not develop LVR. CONCLUSION: Our data showed that large EVs are the main extracellular vesicle transporter of LIPCAR from heart into the circulation independently of the status, non-failing or HF, in patients. The levels of LIPCAR in EVs isolated from plasma could be used as biomarkers of LVR in post-MI patients.

Lim Domain Binding 3 (Ldb3) Identified as a Potential Marker of Cardiac Extracellular Vesicles.

Extracellular vesicles (EVs) are considered as transporters of biomarkers for the diagnosis of cardiac diseases, playing an important role in cell-to-cell communication during physiological and pathological processes. However, specific markers for the isolation and analysis of cardiac EVs are missing, imposing limitation on understanding their function in heart tissue. For this, we performed multiple proteomic approaches to compare EVs isolated from neonate rat cardiomyocytes and cardiac fibroblasts by ultracentrifugation, as well as EVs isolated from minced cardiac tissue and plasma by EVtrap. We identified Ldb3, a cytoskeletal protein which is essential in maintaining Z-disc structural integrity, as enriched in cardiac EVs. This result was validated using different EV isolation techniques showing Ldb3 in both large and small EVs. In parallel, we showed that Ldb3 is almost exclusively detected in the neonate rat heart when compared to other tissues, and specifically in cardiomyocytes compared to cardiac fibroblasts. Furthermore, Ldb3 levels, specifically higher molecular weight isoforms, were decreased in the left ventricle of ischemic heart failure patients compared to control groups, but not in the corresponding EVs. Our results suggest that Ldb3 could be a potential cardiomyocytes derived-EV marker and could be useful to identify cardiac EVs in physiological and pathological conditions.

Identification of additional proteins in differential proteomics using protein interaction networks.

In this study, we developed a novel computational approach based on protein-protein interaction networks to identify a list of proteins that might have remained undetected in differential proteomic profiling experiments. We tested our computational approach on two sets of human smooth muscle cell protein extracts that were affected differently by DNase I treatment. Differential proteomic analysis by saturation DIGE resulted in the identification of 41 human proteins. The application of our approach to these 41 input proteins consisted of four steps: (i) Compilation of a human protein-protein interaction network from public databases; (ii) calculation of interaction scores based on functional similarity; (iii) determination of a set of candidate proteins that are needed to efficiently and confidently connect the 41 input proteins; and (iv) ranking of the resulting 25 candidate proteins. Two of the three highest-ranked proteins, beta-arrestin 1, and beta-arrestin 2, were experimentally tested, revealing that their abundance levels in human smooth muscle cell samples were indeed affected by DNase I treatment. These proteins had not been detected during the experimental proteomic analysis. Our study suggests that our computational approach may represent a simple, universal, and cost-effective means to identify additional proteins that remain elusive for current 2D gel-based proteomic profiling techniques.

Circulating plasma serine208-phosphorylated troponin T levels are indicator of cardiac dysfunction.

Heart failure (HF) following myocardial infarction (MI) is characterized by progressive alterations of left ventricular (LV) structure and function, named LV remodelling. Although several risk factors such as infarct size have been identified, HF remains difficult to predict in clinical practice. Recently, using phosphoproteomic technology, we found that serine(208)-phosphorylated troponin T (P-Ser(208)-TnT) decreases in LV of HF rats. Our aim was to determine the performance of P-Ser(208)-TnT as plasma biomarker of HF compared to conventional cardiac biomarkers such as B-type natriuretic peptide (BNP), cardiac troponin I (cTnI), C-reactive protein (CRP) or tissue inhibitor of metalloproteinase I (TIMP-1) measured by x-MAP technology, as well as its capacity to reflect a pharmacological improvement of HF. We observed a significant increase of BNP, TnT and cTnI levels and a significant decrease of P-Ser(208)-TnT and TIMP-1 in the plasma of 2-month-MI rats compared with control rats with no modulation of CRP level. Circulating levels of P-Ser(208)-TnT were shown to be associated with most of the echocardiographic and haemodynamic parameters of cardiac function. We verified that the decrease of P-Ser(208)-TnT was not because of an excess of phosphatase activity in plasma of HF rats. Two-month-MI rats treated with the heart rate reducing agent ivabradine had improved LV function and increased plasma levels of P-Ser(208)-TnT. Thus, circulating phosphorylated troponin T is a highly sensitive biological indicator of cardiac dysfunction and has the potentiality of a new biomarker of HF post-MI, and of a surrogate marker for the efficacy of a successful treatment of HF.

CRISPR/Cas9-mediated inactivation of the phosphatase activity of soluble epoxide hydrolase prevents obesity and cardiac ischemic injury.

INTRODUCTION: Although the physiological role of the C-terminal hydrolase domain of the soluble epoxide hydrolase (sEH-H) is well investigated, the function of its N-terminal phosphatase activity (sEH-P) remains unknown. OBJECTIVES: This study aimed to assess in vivo the physiological role of sEH-P. METHODS: CRISPR/Cas9 was used to generate a novel knock-in (KI) rat line lacking the sEH-P activity. RESULTS: The sEH-P KI rats has a decreased metabolism of lysophosphatidic acids to monoacyglycerols. KI rats grew almost normally but with less weight and fat mass gain while insulin sensitivity was increased compared to wild-type rats. This lean phenotype was more marked in males than in female KI rats and mainly due to decreased food consumption and enhanced energy expenditure. In fact, sEH-P KI rats had an increased lipolysis allowing to supply fatty acids as fuel to potentiate brown adipose thermogenesis under resting condition and upon cold exposure. The potentiation of thermogenesis was abolished when blocking PPARγ, a nuclear receptor activated by intracellular lysophosphatidic acids, but also when inhibiting simultaneously sEH-H, showing a functional interaction between the two domains. Furthermore, sEH-P KI rats fed a high-fat diet did not gain as much weight as the wild-type rats, did not have increased fat mass and did not develop insulin resistance or hepatic steatosis. In addition, sEH-P KI rats exhibited enhanced basal cardiac mitochondrial activity associated with an enhanced left ventricular contractility and were protected against cardiac ischemia-reperfusion injury. CONCLUSION: Our study reveals that sEH-P is a key player in energy and fat metabolism and contributes together with sEH-H to the regulation of cardiometabolic homeostasis. The development of pharmacological inhibitors of sEH-P appears of crucial importance to evaluate the interest of this promising therapeutic strategy in the management of obesity and cardiac ischemic complications.

Mitochondrial-Targeted Therapies Require Mitophagy to Prevent Oxidative Stress Induced by SOD2 Inactivation in Hypertrophied Cardiomyocytes.

Heart failure, mostly associated with cardiac hypertrophy, is a major cause of illness and death. Oxidative stress causes accumulation of reactive oxygen species (ROS), leading to mitochondrial dysfunction, suggesting that mitochondria-targeted therapies could be effective in this context. The purpose of this work was to determine whether mitochondria-targeted therapies could improve cardiac hypertrophy induced by mitochondrial ROS. We used neonatal (NCMs) and adult (ACMs) rat cardiomyocytes hypertrophied by isoproterenol (Iso) to induce mitochondrial ROS. A decreased interaction between sirtuin 3 and superoxide dismutase 2 (SOD2) induced SOD2 acetylation on lysine 68 and inactivation, leading to mitochondrial oxidative stress and dysfunction and hypertrophy after 24 h of Iso treatment. To counteract these mechanisms, we evaluated the impact of the mitochondria-targeted antioxidant mitoquinone (MitoQ). MitoQ decreased mitochondrial ROS and hypertrophy in Iso-treated NCMs and ACMs but altered mitochondrial structure and function by decreasing mitochondrial respiration and mitophagy. The same decrease in mitophagy was found in human cardiomyocytes but not in fibroblasts, suggesting a cardiomyocyte-specific deleterious effect of MitoQ. Our data showed the importance of mitochondrial oxidative stress in the development of cardiomyocyte hypertrophy. We observed that targeting mitochondria by MitoQ in cardiomyocytes impaired the metabolism through defective mitophagy, leading to accumulation of deficient mitochondria.

Desmin aggrephagy in rat and human ischemic heart failure through PKCζ and GSK3ß as upstream signaling pathways.

Post-translational modifications of cardiac proteins could participate to left contractile dysfunction resulting in heart failure. Using a rat model of ischemic heart failure, we showed an accumulation of phosphorylated desmin leading to toxic aggregates in cardiomyocytes, but the cellular mechanisms are unknown. The same rat model was used to decipher the kinases involved in desmin phosphorylation and the proteolytic systems present in rat and human failing hearts. We used primary cultures of neonate rat cardiomyocytes for testing specific inhibitors of kinases and for characterizing the autophagic processes able to clear desmin aggregates. We found a significant increase of active PKCζ, no modulation of ubitiquitin-proteasome system, a defect in macroautophagy, and an activation of chaperone-mediated autophagy in heart failure rats. We validated in vitro that PKCζ inhibition induced a significant decrease of GSK3ß and of soluble desmin. In vitro activation of ubiquitination of proteins and of chaperone-mediated autophagy is able to decrease soluble and insoluble forms of desmin in cardiomyocytes. These data demonstrate a novel signaling pathway implicating activation of PKCζ in desmin phosphorylation associated with a defect of proteolytic systems in ischemic heart failure, leading to desmin aggrephagy. Our in vitro data demonstrated that ubiquitination of proteins and chaperone-mediated autophagy are required for eliminating desmin aggregates with the contribution of its chaperone protein, α-crystallin Β-chain. Modulation of the kinases involved under pathological conditions may help preserving desmin intermediate filaments structure and thus protect the structural integrity of contractile apparatus of cardiomyocytes by limiting desmin aggregates formation.

Profile of macrophages in human abdominal aortic aneurysms: a transcriptomic, proteomic, and antibody protein array study.

Abdominal aortic aneurysms (AAA) are defined by an increased aortic diameter and characterized by impairment of the extracellular matrix, macrophages infiltration and decreased density of smooth muscle cells. Our aim is to identify the key molecules involved in the pathogenesis of AAAs. This study investigated transcriptomic and proteomic profiles of macrophages from AAA patients (>50 mm aortic diameter) (n = 24) and peripheral arterial occlusion (PAO) patients without AAA detected (n = 18), who both needed a surgery. An antibody protein microarray, generated by printing antibodies onto membranes against proteins selected from the transcriptomic and proteomic analysis, was performed to validate the proteins differentially expressed specifically in macrophages and plasma from the same patients. We found a restricted number of proteins differentially expressed between AAA and PAO patients: TIMP-3, ADAMTS5, and ADAMTS8 that differ significantly in plasma of AAA patients compared to PAO patients, as found in the macrophages. In contrast to plasma MMP-9, soluble glycoprotein V (sGPV) and plasmin-antiplasmin complex levels, plasma TIMP-3 levels were not correlated to AAA size but interestingly correlated to sGPV, a platelet activation marker. Combining transcriptomic and proteomic is a valid approach to identify diseases causing proteins and potential biomarkers.

Combinatorial peptide ligand library plasma treatment: Advantages for accessing low-abundance proteins.

Depletion of major blood proteins is one of the most promising approaches to accessing low-abundance biomarkers. This study compared the use of combinatorial peptide ligand library (CPLL) and albumin and immunoglobulins (IgGs) depletion technology for accessing these low-abundance proteins in plasma using 2-DE in an acidic restricted pH range (4-7). Compared with native plasma, both techniques enlarge the visibility of other proteins than albumin and IgG, but there were marked differences in their composition. An increase of the number of spots was detected compared with native plasma (157 spots) with 427 and 557 spots, respectively, detected with albumin and IgG depletion, and CPLL treatment. We selected 70 spots to be identified by MALDI-TOF related to their absence in the 2-D gels from native or albumin and IgG-depleted plasma. The 42 spots identified corresponded to 24 different proteins, with more than half of the proteins which did not belong to the major plasma proteins. CPLL treatment allowed the accessibility to proteolytic fragments obtained from major plasma proteins. We found a large superiority of the CPLL approach over the albumin and IgG depletion process. These findings show the utility of depleting major blood proteins to be able to access low-abundance proteins and the potential of CPLL to select and identify candidate biomarkers in clinical studies.

Apolipoprotein Proteomic Profiling for the Prediction of Cardiovascular Death in Patients with Heart Failure.

PURPOSE: Risk stratification in chronic systolic heart failure (HF) is critical to identify the patients who may benefit from advanced therapies. It is aimed at identifying new biomarkers to improve prognosis evaluation and help to better understand HF physiopathology. EXPERIMENTAL DESIGN: Prognostic evaluation is performed in 198 patients with chronic systolic HF: 99 patients who died from cardiovascular cause within three years are individually matched for age, sex, and HF etiology (ischemic vs not) with 99 patients who are alive after three years of HF evaluation. A proteomic profiling of 15 apolipoproteins (Apo) is performed: Apo-A1, -A2, -A4, -B100, -C1, -C2, -C3, -C4, -D, -E, -F, -H, -J, -L1, and -M using LC-MRM-MS. RESULTS: In univariate analysis, the levels of Apo-B100 and -L1 are significantly lower and the levels of Apo-C1, -J, and -M are significantly higher in patients who died from cardiovascular cause as compared with patients alive. In the final statistical model, Apo-C1, Apo-J, and Apo-M improve individually the prediction of cardiovascular death. Ingenuity pathway analysis indicates these three Apo in a network associated with lipid metabolism, atherosclerosis signaling, and retinoid X receptor activation. CONCLUSIONS: Proteomic profiling of apolipoproteins using LC-MRM-MS might be useful in clinical practice for risk stratification of HF patients.

Increased clusterin levels after myocardial infarction is due to a defect in protein degradation systems activity.

Clusterin (CLU) is induced in many organs after tissue injury or remodeling. Recently, we show that CLU levels are increased in plasma and left ventricle (LV) after MI, however, the mechanisms involved are not yet elucidated. On the other hand, it has been shown that the activity of the protein degradation systems (PDS) is affected after MI with a decrease in ubiquitin proteasome system (UPS) and an increase in macroautophagy. The aim of this study was to decipher if the increased CLU levels after MI are in part due to the alteration of PDS activity. Rat neonate cardiomyocytes (NCM) were treated with different modulators of UPS and macroautophagy in order to decipher their role in CLU expression, secretion, and degradation. We observed that inhibition of UPS activity in NCM increased CLU mRNA levels, its intracellular protein levels (p-CLU and m-CLU) and its secreted form (s-CLU). Macroautophagy was also induced after MG132 treatment but is not active. The inhibition of macroautophagy induction in MG132-treated NCM increased CLU mRNA and m-CLU levels, but not s-CLU compared to NCM only treated by MG132. We also demonstrate that CLU can be degraded in NCM through proteasome and lysosome by a macroautophagy independent pathway. In another hand, CLU silencing in NCM has no effect either on macroautophagy or apoptosis induced by MG132. However, the overexpression of CLU secreted isoform in H9c2 cells, but not in NCM decreased apoptosis after MG132 treatment. Finally, we observed that increased CLU levels in hypertrophied NCM and in failing human hearts are associated with proteasome inhibition and macroautophagy alteration. All these data suggest that increased CLU expression and secretion after MI is, in part, due to a defect of UPS and macroautophagy activities in the heart and may have a protective effect by decreasing apoptosis induced by proteasome inhibition.

Circulating proteomic signature of early death in heart failure patients with reduced ejection fraction.

Heart failure (HF) remains a main cause of mortality worldwide. Risk stratification of patients with systolic chronic HF is critical to identify those who may benefit from advanced HF therapies. The aim of this study is to identify plasmatic proteins that could predict the early death (within 3 years) of HF patients with reduced ejection fraction hospitalized in CHRU de Lille. The subproteome targeted by an aptamer-based technology, the Slow Off-rate Modified Aptamer (SOMA) scan assay of 1310 proteins, was profiled in blood samples from 168 HF patients, and 203 proteins were significantly modulated between patients who died of cardiovascular death and patients who were alive after 3 years of HF evaluation (Wilcoxon test, FDR 5%). A molecular network was built using these 203 proteins, and the resulting network contained 2281 molecules assigned to 34 clusters annotated to biological pathways by Gene Ontology. This network model highlighted extracellular matrix organization as the main mechanism involved in early death in HF patients. In parallel, an adaptive Least Absolute Shrinkage and Selection Operator (LASSO) was performed on these 203 proteins, and six proteins were selected as candidates to predict early death in HF patients: complement C3, cathepsin S and F107B were decreased and MAPK5, MMP1 and MMP7 increased in patients who died of cardiovascular causes compared with patients living 3 years after HF evaluation. This proteomic signature of 6 circulating plasma proteins allows the identification of systolic HF patients with a risk of early death.

Predicting left ventricular remodeling after a first myocardial infarction by plasma proteome analysis.

Recent improvements in therapeutic strategies did not prevent left ventricular remodeling (LVR), which remains a common event (30%) after acute myocardial infarction (AMI). We report the use of a systematic approach, based on comparative proteomics, to select circulating biomarkers that may be associated with LVR. We selected 93 patients enrolled in a prospective study. These patients with anterior wall Q-wave AMI underwent echocardiographic follow-up at hospitalization, 3 months and 1 year after AMI. They were divided into three groups (no, low, or high remodeling). Plasma samples of these patients (day 5 of hospitalization) were processed and stored at -80 degrees C within 2 h and analyzed using SELDI-TOF protein chip technology. This systematic approach allowed to select candidate proteins modulated by LVR: post-translational variants of alpha1-chain of haptoglobin (Hpalpha1) corresponding to m/z 9493, 9565, and 9623, which were more elevated in remodeling patients. The peak 9493 m/z was shown having a receiving-operating characteristic (ROC) value of 0.71 between non- and remodeling patients. SELDI-TOF approach may lead to the identification of circulating proteins associated with LVR. Whether these candidate proteins will help to identify patients who are at high risk of heart failure after AMI will have to be tested in future studies.

Glycoproteomics and glycomics investigation of membrane N-glycosylproteins from human colon carcinoma cells.

Aberrant glycosylation of proteins is known to profoundly affect cellular adhesion or motility of tumoral cells. In this study, we used HT-29 human colon epithelial cancer cells as a cellular model of cancer progression, as they can either proliferate or differentiate into enterocyte phenotype. A glycoproteomic approach based on Con A lectin-affinity chromatography, SDS-PAGE and MS analysis, allowed the identification of membrane N-glycoproteins from Triton X-100-solubilized proteins from membrane preparation. Among them, 65% were membrane proteins, and 45% were known to be N-glycosylated, such as alpha chains integrin and dipeptidyl isomerase IV. By lectin-blot analysis, significant changes of alpha-2,3- and alpha-2,6-sialylation of membrane glycoproteins were observed between proliferating and differentiated HT-29 cells. From these results, nano-LC-MS/MS analysis of the tryptic digests of the corresponding bands was performed and led to the identification of several transmembrane glycoproteins, like members of the solute carrier family and adhesion proteins. Finally, we compared N-glycans profiles and monosaccharide composition of proliferating and enterocyte-like HT-29 cells using MALDI-MS and GC-MS analyses of permethylated derivatives. This glycomic approach allowed to underscore significant changes in N-glycans structure, in particular the expression of atypical N-acetylglucosamine (GlcNAc)-ended N-glycans in enterocyte-like HT-29 cells.

Interplay Between Phosphorylation and O-GlcNAcylation of Sarcomeric Proteins in Ischemic Heart Failure.

Post-translational modifications (PTMs) of sarcomeric proteins could participate to left ventricular (LV) remodeling and contractile dysfunction leading in advanced heart failure (HF) with altered ejection fraction. Using an experimental rat model of HF (ligation of left coronary artery) and phosphoproteomic analysis, we identified an increase of desmin phosphorylation and a decrease of desmin O-N-acetylglucosaminylation (O-GlcNAcylation). We aim to characterize interplay between phosphorylation and O-GlcNAcylation for desmin in primary cultures of cardiomyocyte by specific O-GlcNAcase (OGA) inhibition with thiamet G and silencing O-GlcNAc transferase (OGT) and, in perfused heart perfused with thiamet G in sham- and HF-rats. In each model, we found an efficiency of O-GlcNAcylation modulation characterized by the levels of O-GlcNAcylated proteins and OGT expression (for silencing experiments in cells). In perfused heart, we found an improvement of cardiac function under OGA inhibition. But none of the treatments either in in vitro or ex vivo cardiac models, induced a modulation of desmin, phosphorylated and O-GlcNAcylated desmin expression, despite the presence of O-GlcNAc moities in cardiac desmin. Our data suggests no interplay between phosphorylation and O-GlcNAcylation of desmin in HF post-myocardial infarction. The future requires finding the targets in heart involved in cardiac improvement under thiamet G treatment.

Publisher Correction: MicroRNAs regulating superoxide dismutase 2 are new circulating biomarkers of heart failure.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Expression and Implication of Clusterin in Left Ventricular Remodeling After Myocardial Infarction.

BACKGROUND: Left ventricular remodeling (LVR) after myocardial infarction is associated with an increased risk of heart failure and death. In spite of a modern therapeutic approach, LVR remains relatively frequent and difficult to predict in clinical practice. Our aim was to identify new biomarkers of LVR and understand their involvement in its development. METHODS AND RESULTS: Proteomic analysis of plasma from the REVE-2 study (Remodelage Ventriculaire)-a study dedicated to the analysis of LVR which included 246 patients after a first anterior myocardial infarction-identified increased plasma levels of CLU (clusterin) in patients with high LVR. We used a rat model of myocardial infarction to analyze CLU expression in the LV and found a significant increase that was correlated with LVR parameters. We found increased CLU expression and secretion in primary cultures of rat neonate cardiomyocytes hypertrophied by isoproterenol. Silencing of CLU in hypertrophied neonate cardiomyocytes induced a significant decrease in cell size, ANP (atrial natriuretic peptide), and BNP (B-type natriuretic peptide) expression, associated with a decreased ERK (extracellular signal-regulated kinase) 1/2 activity, suggesting a prohypertrophic role of CLU. We then confirmed a significant increase of both intracellular p-CLU (precursor form of CLU) and m-CLU (mature form of CLU) in failing human hearts. Finally, the circulating levels of CLU (secreted form) were increased in patients with chronic heart failure who died from cardiovascular cause during a 3-year follow-up (n=99) compared with survivors (n=99). CONCLUSIONS: Our results show for the first time that plasma CLU levels are associated with LVR post-myocardial infarction, have in part a cardiac origin, and are a predictor of early death in heart failure patients.

MicroRNAs regulating superoxide dismutase 2 are new circulating biomarkers of heart failure.

Although several risk factors such as infarct size have been identified, the progression of heart failure (HF) remains difficult to predict in clinical practice. Using an experimental rat model of post-myocardial infarction (MI), we previously identified 45 proteins differentially modulated during HF by proteomic analysis. This study sought to identify microRNAs (miRNAs) able to regulate these proteins and to test their relevance as biomarkers for HF. In silico bioinformatical analysis selected 13 miRNAs related to the 45 proteins previously identified. These miRNAs were analyzed in the rat and in cohorts of patients phenotyped for left ventricular remodeling (LVR). We identified that 3 miRNAs, miR-21-5p, miR-23a-3p and miR-222-3p, and their target Mn superoxide dismutase (SOD2) were significantly increased in LV and plasma of HF-rats. We found by luciferase activity a direct interaction of miR-222-3p with 3'UTR of SOD2. Transfection of human cardiomyocytes with miR-222-3p mimic or inhibitor induced respectively a decrease and an increase of SOD2 expression. Circulating levels of the 3 miRNAs and their target SOD2 were associated with high LVR post-MI in REVE-2 patients. We demonstrated for the first time the potential of microRNAs regulating SOD2 as new circulating biomarkers of HF.

Increased level of phosphorylated desmin and its degradation products in heart failure.

Although several risk factors such as infarct size have been identified, the progression/severity of heart failure (HF) remains difficult to predict in clinical practice. Using an experimental rat model of ischemic HF and phosphoproteomic technology, we found an increased level of phosphorylated desmin in the left ventricle (LV) of HF-rats. The purpose of the present work is to assess whether desmin is a circulating or only a tissue biomarker of HF. We used several antibodies in order to detect desmin, its proteolytic fragments and its phosphorylated form in LV and plasma by western blot, phosphate affinity electrophoresis, mass spectrometry and immunofluorescence. Plasma was treated with combinatorial peptide ligand library or depleted for albumin and immunoglobulins to increase the sensitivity of detection. We found a 2-fold increased serine-desmin phosphorylation in the LV of HF-rats, mainly in the insoluble fraction, suggesting the formation of desmin aggregates. Desmin cleavage products were also detected in the LV of HF rats, indicating that the increased phosphorylation of desmin results in more susceptibility to proteolytic activity, likely mediated by calpain activity. The native desmin and its degradation products were undetectable in the plasma of rat, mouse or human. These data suggest the potential of serine-phosphorylated form of desmin and its degradation products, but not of desmin itself, as tissue but not circulating biomarkers of HF.

Multimarker proteomic profiling for the prediction of cardiovascular mortality in patients with chronic heart failure.

Risk stratification of patients with systolic chronic heart failure (HF) is critical to better identify those who may benefit from invasive therapeutic strategies such as cardiac transplantation. Proteomics has been used to provide prognostic information in various diseases. Our aim was to investigate the potential value of plasma proteomic profiling for risk stratification in HF. A proteomic profiling using surface enhanced laser desorption ionization - time of flight - mass spectrometry was performed in a case/control discovery population of 198 patients with systolic HF (left ventricular ejection fraction <45%): 99 patients who died from cardiovascular cause within 3 years and 99 patients alive at 3 years. Proteomic scores predicting cardiovascular death were developed using 3 regression methods: support vector machine, sparse partial least square discriminant analysis, and lasso logistic regression. Forty two ion m/z peaks were differentially intense between cases and controls in the discovery population and were used to develop proteomic scores. In the validation population, score levels were higher in patients who subsequently died within 3 years. Similar areas under the curves (0.66 - 0.68) were observed for the 3 methods. After adjustment on confounders, proteomic scores remained significantly associated with cardiovascular mortality. Use of the proteomic scores allowed a significant improvement in discrimination of HF patients as determined by integrated discrimination improvement and net reclassification improvement indexes. In conclusion, proteomic analysis of plasma proteins may help to improve risk prediction in HF patients.