Protective Effect And Mechanism Of CaN/NFAT3-mediated Mitochondrial Aldehyde Dehydrogenase 2 On High Glucose-induced Myocardial Injury

Objective:To investigate whether CaN/NFAT3 signaling pathway mediates the protective effect of mitochondrial aldehyde dehydrogenase 2 on myocardial injury in diabetic rats and its possible molecular mechanism.Methods:The heart of SD rats within 3 days was taken out under aseptic conditions,The apex was cut and digested with trypsin and collagenase 2 to form a single cell.After differential adherence,5-Brdu was added to the culture solution.The ventricular myocytes are treated when the growth is in a fused state.I mmunofluorescence was used to detect the?-SA protein in cultured neonatal rat cardiomyocytes to identify the purity of primary cultured ventricular myocytes.The experiment involved the following groups:5.5mmol/L glucose control group?M?,30mmol/L high glucose group?MH?,30mmol/L high glucose with acetaldehy de dehydrogenase 2 agonist?Alda-1?group?MHA?,30mmol/L high glucose with acetaldehyde dehydrogenase 2 inhibitor?Daidzin?group?MHD?,30mmol/L high glucos e with acetaldehyde dehydrogenase 2 agonist?Alda-1?and inhibitor?Daidzin?group?MHAD?,30mmol/L high glucose with acetaldehyde dehydrogenase 2 agonist?Ald a-1? and NFAT3 inhibition Agent?11R-VIVIT?group?MHAV?;CCK8 kit measures the optimal concentration of NFAT3 inhibitor;fluorescence probe detects intrace llular Ca²⁺concentration;ELISA measures intracellular CaN content;immunofluor escence detects intracellular CaNA? content;The expression of protein of ALDH2,CaN and NFAT3 were detected by Western blot;CCK8 kit was used to detect the activity of every group;hydroxyproline reagent kit was used to detect the content of hydroxyproline in cell culture supernatant;DHE probe was used to detect the oxidative stress level in every group.Result:1.Identification of cardiomyocytes:High-purity cardiomyocytes were obtained by differential adherence with 5-Brdu,and immunofluorescence staining was performed on?-striated muscle actin specifically expressed in cardiomyocytes and skeletal muscle cells.Myocardial?-SA antigen was positive for immunofluorescence,and it was green,located in the cytoplasm,and cells were stained blue by DAPI.2.The optimal concentration of NFAT3 inhibitor:According to the experimental results,the cell activity increased with the increase of NFAT3 inhibitor11R-VIVIT concentration.When the drug concentration reached to 2uM,the cell activity was the highest?P<0.01?.And increasing the concentration of the drug again,the cell activity is instead reduced.3.Ca²⁺concentration in cardiomyocytes:Compared with M group,the concentration of Ca²⁺in MH group was increased?P<0.01?;compared with MH group,the concentration of Ca²⁺in MHA group was decreased?P<0.05?,and the concentration of Ca²⁺in MHD group was increased.?P<0.01?,while the Ca²⁺concentration in the MHAD group was lower than that in the MHD group.4.CaN content in cardiomyocytes:Compared with group M,the intracellular CaN content was significantly increased?P<0.01?in MH group.Compared with MH group,the intracellular CaN content was decreased?P<0.01?in MHA group,the intracellular CaN content increased?P<0.05?in MHD group.Compared with the MHD group,the intracellular CaN was significantly decreased in the MHAD group?P<0.01?.5.Cardiomyocyte intracellular CaNA?content:The content of CaNA?in cardiomyocytes was the lowest in the normal group M.Compared with the M group,the content of CaNA?in the cardiomyocytes was significantly increased in the MH group,MHD group and MHAD group?P<0.01?.Compared with the MH group,The content of CaNA?in the MHA group was significantly decreased?P<0.01?.Compared with the MHA group,the content of CaNA?in the MHAD group was increased again?P<0.01?.Compared with the MH group,the content of CaNA?in the cardiomyocytes was higher than that in the MH MHD group.6.The expression of ALDH2,CaN,NFAT3 protein in cardiomyocytes:Compared with M group,the expression ALDH2 protein was decreased in MH group?P<0.05?,CaN protein was increased?P<0.05?,and NFAT3 protein was increased?P<0.01?.Compared with MH group,the expression of CaN protein and NFAT3 protein was decreased?P<0.05?,and the expression of ALDH2 protein was increased?P<0.05?in MHA group.Compared with MHA group,the expression of ALDH2 protein in MHAV group had no significant change?p>0.05?,the expression of NFAT3 protein was decreased?P<0.05?.7.Cardiomyocyte activity:The activity of cardiomyocytes was significantly lower in MH,MHAD and MHD groups than in M group?P<0.01?.Compared with MH group,the activity of MHA group was increased?P<0.05?.Myocardium viability was also significantly increased?P<0.05?in MHV group.Compared with MHAD group,the myocardial activity of MHD group was significantly decreased?P<0.05?.8.The content of hydroxyproline in the culture supernatant of cardiomyocytes:Compared with the M group,the content of hydroxyproline in the culture supernatant of MH group was significantly higher;compared with the MH group,the content of hydroxyproline in the culture supernatant was significantly decreased in MHA,while the content of hydroxyproline in the MHD group wassignificantly increased.Compared with the MHA group,the content of hydroxyproline in MHAD was significantly increased?P<0.01?.9.Oxidative stress levels in cardiomyocytes:DHE fluorescence density was significantly increased in MH group compared with normal group;fluorescence density was significantly reduced in MHA and MHV groups compared with MH group,and further increased in MHD group.compared with MHD group,the fluorescence density of MHAD group was significantly reduced?P<0.01?.Conclusion:1.Excited mitochondria ALDH2 can increase myocardial cell viability,reduce oxidative stress and Hydroxyproline levels in cardiomyocytes;2.After inhibition of CaN/NFAT3 signaling pathway,the expression of mitochondrial ALDH2 had no significant effect,myocardial cell viability increased,and oxidative stress and Hydroxyproline levels in cardiomyocytes were weakened.3.Mitochondrial ALDH2 plays a protective role in high glucose-induced neonatal rat cardiomyocytes,and its mechanism may be related to the negative regulation of ALDH2 on Ca²⁺-CaN-NFAT3 signaling pathway.