The Role Of Oxidized CaMKⅡ On Apoptosis Induced By High Uric Acid In H9C2 Cardiomyocytes

AimsIn this study,we test the effect of high uric acid(UA)on cardiomyocytes apoptosis in vitro,and investigate the role and mechanism of oxidative stress and oxidized Ca MKⅡ on apoptosis induced by high uric acid in H9C2 cardiomyocytes.Methods1.H9C2 cardiomyocytes were co-cultured with 0,5,10,15,20mg/dl UA for 36 h,or were cultured in UA of 20mg/dl for 12 h,24h,36 h,48h.Then the protein expression of ox-Ca MKⅡ and Cleaved Caspase-3 were detected by Western Blot.Which were used to determine the most suitable concentration and exposure period.2.H9C2 cardiomyocytes were co-cultured with 0,5,10,15,20mg/dl UA for 12 h,24h,36 h,48h.The cell viability of H9C2 cardiomyocytes was detected by CCK-8 assay.3.H9C2 cardiomyocytes were cultured in UA of 20mg/dl for 36 h,and TUNEL staining was used to determine the apoptosis rate of H9C2 cardiomyocytes.4.H9C2 cardiomyocytes were cultured in UA of 20mg/dl for 36 h,with or without pretreating with NAC for 30 min.Then H9C2 cardiomyocytes were stained with DCFH-DA for30 min,and the untreated cells were control group.The fluorescence intensity of DCF in cells were determined by fluorescence microscope and flow cytometry.5.H9C2 cardiomyocytes were cultured in UA of 20mg/dl for 36 h,with or without pretreating with NAC or KN93 for 30 min.Then the protein expression of ox-Ca MKⅡ and Cleaved Caspase-3 were detected by Western Blot.And flow cytometry was used to determine the apoptosis rate of H9C2 cardiomyocytes.Results1.The CCK-8 assay confirmed that with the increase of uric acid concentration and the prolongation of treatment time,the cell viability was reduced gradually.2.TUNEL staining results showed that high uric acid could promote the apoptosis of H9C2 cardiomyocytes.3.After the cells were stained with DCFH-DA for 30 min,the fluorescence microscope and flow cytometry results showed that high uric acid could increase production of reactive oxygen species in H9C2 cardiomyocytes,which was blocked by the NAC.4.The results of Western Blot confirmed that high uric acid could increase the protein expression of ox-Ca MKⅡ and Cleaved Caspase-3,and these effects were blocked by NAC or KN93.5.Flow cytometry results showed that high uric acid could promote the apoptosis of H9C2 cardiomyocytes,and which could be inhibited by NAC or KN93.ConclusionHigh uric acid induces apoptosis of cardiomyocytes through ox-Ca MKⅡ pathway via oxidative stress.