Résumé :
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Acute Myocardial Infarction causes cardiomyocytes cell death in the ischemic ventricle, followed by a wound healing response, which includes migration of inflammatory cells into the affected myocardium, extracellular matrix degradation, fibroblasts proliferation and angiogenesis. Cell surface-associated proteolysis plays an important role in the extracellular matrix remodeling as a wound healing response. We are studying extracellular proteases/receptor partners which, through regulated interactions, might contribute to the regeneration of the ischemic heart. Indeed, it is well documented that components of the plasminogen activation system are required for the repairing process of infarcted myocardium. Furthermore, the glycolytic enzyme alpha-enolase can act as a plasminogen receptor when located at the cell surface. Alpha-enolase/ plasminogen interaction enhances plasminogen activation allowing surface-associated proteolytic activity. We have analyzed alpha-enolase expression and function as a plasminogen receptor in H9C2 cells (an embryonic rat heart-derived cell line) and cultured neonatal ventricular cardiomyocytes. Cells were subjected to metabolic inhibition as an in vitro model for ischemia/reperfusion. To do this, cells were submitted to glucose and serum deprivation and sodium cyanide treatment, followed by reperfusion by control medium (normal glucose and serum). First, we have demonstrated by western blotting(WB) on whole cardiomyocytes cell extracts that alpha-enolase expression was up-regulated during metabolic inhibition and was back to basal levels after reperfusion of the cells in normal medium. Furthermore, cell-surface expression of alpha-enolase was detected on both H9C2 and primary cardiomyocytes by three different methods: flow cytometry, immunofluorescence(IF) using anti-alpha-enolase antibody, and WB analysis of cell surface membranes isolated from H9C2. In addition, we have confirmed that Fluorescein isothiocyanateplasminogen( FITC-Plg) was able to bind to the cell-surface of cardiomyocytes by flow cytometry and IF. Finally, we also have shown that metabolic inhibition increases both the binding of FITC-Plg at the cell surface of cardiomyocytes (analyzed by flow cytometry) and alpha-enolase cell-surface expression, (flow cytometry, IF and WB on membrane extracts). In summary, our results have shown that alpha-enolase expression and plasminogen recruitment on the cell surface were both up-regulated by simulated ischemia in cardiomyocytes.
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