Role Of Src Tyrosine Kinase In Zinc Induced Cardiomyocyte Protection

Objective To explore the function and mechanism of Src tyrosine kinase in zinc ion(Zn²⁺)-induced cardiomyocyte protection.Methods H9c2 cells were routinely cultured.Endoplasmic reticulum stress（ERS）in H9c2cells was induced by using ERS inducers tunicamycin（TM）and 2-deoxy-D-glucose（2-DG）.And the cells were pre-treated with exogenous zinc chloride（Zn Cl2）,Src tyrosine kinase inhibitor PP2,or Src si RNA.Mitochondrial membrane potential（△Ψm）was measured by using Tetramethylrhodamine Methyl Ester（TMRE）and JC-1 fluorescence probes to observe mitochondrial permeability transition pore（m PTP）opening by laser confocal microscope.The concentration of cytosol and mitochondrial Zn²⁺was analyzed with Newport Green DCF fluorescent dye and Rodzin-3 fluorescent dye respectively.Cell viability was detected by CCK8.The expressions of ERS chaperone glucose regulated protein 78（GRP 78）,GRP 94,Cleaved-Caspase 3,mitochondrial related endoplasmic reticulum membranes（MAMs）protein mitochondrial fusion protein 1/2（Mfn1/2）,and phosphorylated Src tyrosine kinase were detected by western bloting.The expression and location of GRP 94 was detected by immunofluorescence staining.The binding of Src and Mitofusin 1/2 were detected by immunoprecipitation.Results 1 Exogenous zinc inhibited the decrease of cytosolic and mitochondrial Zn²⁺which caused by ERS.Confocal results showed that 2-DG significantly reduced the green fluorescence intensity of Newport Green DCF and the red fluorescence intensity of Rodzin-3.Exogenous Zn Cl2 significantly inhibited the effect of 2-DG on the cytosolic and mitochondrial Zn²⁺concentration.2 Zinc ions inhibited 2-DG-induced ERS and cell apoptosis through Src tyrosine kinase.Western blotting and immunofluorescence staining results showed that 2-DG significantly increased the expressions of ERS molecule Marker GRP 78,GRP 94 and apoptotic protein Cleaved-Caspase-3 compared with the control group.Exogenous Zn Cl2 significantly inhibited the protein level elevation caused by 2-DG and PP2markedly reversed the effect of Zn Cl2.The effect of Zn Cl2 was also blocked by Src si RNA.3 CCK8 results showed that 2-DG significantly reduced cell viability.Zn Cl2 pretreatment significantly inhibited the change caused by 2-DG and PP2 significantly inhibited the effect of Zn Cl2.4 Zinc ions prevented the 2-DG-induced m PTP opening through Src tyrosine kinase.Confocal results showed that 2-DG significantly reduced the red fluorescence intensity and increased the green fluorescence of JC-1.Exogenous Zn Cl2 significantly inhibited the change of JC-1,while PP2 reversed the effect of Zn Cl2.5 TM treatment caused ERS and m PTP opening.Confocal and Western blotting results showed that treatment of H9c2 cells with different concentrations of TM（50,100,200,300,500 ng/ml）decreased the red fluorescence intensity of TMRE and increased the expressions of GRP 78 and GRP 94with the peak effect at 200 ng/ml.Thus,200 ng/ml TM was used in the subsequent experiments.6 Zinc ions inhibited TM-induced ERS through Src tyrosine kinase.TM treatment significantly increased the expressions of GRP 78 and GRP 94,Zn Cl2 significantly inhibited the change caused by TM and PP2 significantly reversed the effect of exogenous Zn Cl2.7 Zinc ions prevented the m PTP opening through Src tyrosine kinase.Confocal results showed that 2-DG and TM significantly reduced the red fluorescence intensity of TMRE,the m PTP opening,Zn Cl2 significantly inhibited the red fluorescence intensity of TMRE caused by 2-DG and TM.PP2 and Src si RNA reversed the effect of Zn Cl2.8 Zinc ions activated Src tyrosine kinase to exert myocardial protection.Compared with the control group,2-DG significantly reduced the phosphorylation of Src tyrosine kinase in the cytosol and mitochondria,exogenous Zn Cl2 significantly inhibited the change caused by 2-DG and PP2 also inhibited the effect of Zn Cl2.9 Zinc ion induced the binding of Src tyrosine kinase and Mfn1/2 to inhibit ERS.Western blotting results showed that 2-DG significantly reduced the expressions of Mfn1 and Mfn2 compared with the control group,exogenous Zn Cl2significantly inhibited the decrease caused by 2-DG and PP2 significantly inhibited the effect of Zn Cl2.The immunoprecipitation results showed that Src formed a complex with Mfn1and Mfn2 in H9c2 cell.Conclusion Zinc ions activated Src tyrosine kinase to protect cardiomyocytes from ERS and m PTP opening partially through Mfn1/2.Figure 15;Table 1;Reference 152...