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Modifications post-traductionnelles des protéines contractiles cardiaques : nouveaux biomarqueurs du remodelage ventriculaire post-infarctus

Abstract : Despite significant improvements in management of myocardial infarction (MI), left ventricular remodelling (LVR) remains a major complication and a strong predictor of both heart failure (HF) and death after MI. Although several variables, such as MI size, have been identified as risk factors, LVR remains difficult to predict in clinical practice. Better prediction could allow an individualized approach with more intense therapy and follow-up for such high-risk patients. The aim of my work is to identify molecular determinants of LVR to have a better understanding of physiopathological mechanisms of LVR. For that purpose, we studied post-translational modifications of contractile proteins in particular, phosphorylation and O-N-acetylglucosaminylation (O-GlcNAc). Then, we studied particularly troponin T (TnT) for which we could highlight a decrease of phosphorylation of serine 208 in LV and plasma of MI-rats. These results suggest that the level of circulating phosphorylated troponin T could be new biomarker of LVR and may help to predict the development of heart failure after MI. For this study, we worked in collaboration with INSERM unit U644 at Rouen using an experimental model of HF. MI was induced in rat by left coronary ligation and, the control rats undergoing the surgery without ligation. Initially, we performed differential phosphoproteomic study of LV in the late phase of the LVR (2 months post-MI). For this purpose, LV proteins were extracted and separated by two-dimensional electrophoresis. Gels were first stained by Pro-Q®Diamond (specific of phosphorylated proteins) and then by Sypro®Ruby (specific of total proteins). By bioinformatic analysis, we showed that 69 polypeptidic spots were modulated for their phosphorylation levels. We analyzed these spots by mass spectrometry and identified 30 proteins corresponding to 53 spots with modulationof phosphorylation. Among these proteins, we have chosen to study 6 contractile proteins: TnT, alpha-tropomyosin 1 (Tm-α1), desmin, αB-crystallin and myosin light chains 1 and 2 (MLC). For each described proteins, we have validated the modulation of phosphorylation and determined the aminoacid involved in the phosphorylation modulation using immunoprecipitation techniques with specific antibodies against the proteins and phospho-Tyrosine, -Threonine and –Serine antibodies confirming the screening performed by 2D-electrophoresis for the detection of phosphoproteins. We observed a significant decrease of phosphorylation on serine for Tm-α1, TnT and MLC-2 and on tyrosine residues for αB-crystallin as well as a significant increase in phosphorylation on tyrosine for MLC-1 and on serine residues for desmin, thus confirming the results obtained in two-dimensional electrophoresis. In order to complete analysis of the post-translational modifications, we studied the modifications of O-GlcNAc for each one of these proteins. We thus observed a significant decrease in O-GlcNAcylation of Tm-α1, αB-crystallin and desmin as well as an increase in O-GlcNAcylation of the MLC-3 and TnT. In addition, we have correlated these modulations of phosphorylation and O-GlcNAcylation levels with modulations of the activity of enzymes implied in these modulations. Indeed, by bioinformatic analysis of the TnT sequence and literature review, we highlighted that the protein kinase C and the protein phosphatase 2A could be implied in these modulations. We observed a decrease of protein kinase C epsilon isoform expression in the LV of MI- rats without modulation of protein phosphatase 2A activity. In addition, we showed an increase in the activity of O-GlcNAc transferase and a decreaseof O-GlcNAcase activity in LV of MI rats. [...]
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Emilie Dubois. Modifications post-traductionnelles des protéines contractiles cardiaques : nouveaux biomarqueurs du remodelage ventriculaire post-infarctus. Médecine humaine et pathologie. Université du Droit et de la Santé - Lille II, 2010. Français. ⟨NNT : 2010LIL2S023⟩. ⟨tel-00580405⟩



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