Identification of the Receptor for ORAIP That Mediates Cardiac Response to Oxidative Stresses and Development of Treatment

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1.Production of recombinant ORAIP, a bioactive protein that mediates cardiac response to oxidative stresses :We subcloned a DNA sequence consisting of ORAIP cDNA+FLAG+(6X His) into an expression vector, then transfected the cDNA clone into an appropriate cell line. We purified recombinant ORAIP from the cell lysates with Ni column and an affinity column for FLAG protein.2.Identification of the ORAIP receptor :We collected the plasma membrane fraction from cultured cardiac myocyte lysates and solubilized it, then immunoprecipitated ORAIP receptor with recombinant ORAIP. Hypoxia/reoxygenation significantly increased the plasma membrane content of ORAIP receptor within 60 min of reoxygenation. Myocardial ischemia/reperfusion also significantly increased the expression of ORAIP receptor as well as ORAIP on the plasma membrane of cardiac myocytes within 30 min of reperftision.3.Identification of the cytoprotective factor that mediates cardiac response to oxidative stresses :We identified cyclophilin A (CyPA) as one of bioactive proteins released from cardiac myocytes in response to oxidative stresses. CyPA activated MAPK family members and Akt as well as increased Bcl-2 in cardiac myocytes. CyPA also increased Bcl-2 and Bcl-XL in cardiac fibroblasts. Taken together, CyPA seems to play a role in cardiac remodeling after ischemia/reperfusion by facilitating the proliferation of cardiac fibroblasts.