The Toxicity Of Manganese On H9c2 Cardiomyocytes

ObjectiveThe current study was designed to investigate the effects of manganese exposure on oxidative stress,mitochondrial injury,apoptosis and autophagy related proteins by in vitro H9c2 cell experiments,and to explore the toxicity of manganese on H9c2 cardiomyocytes.Methods（1）H9c2 cells were exposed to 0,100,200,400μM manganese chloride（MnCl₂）for 24 hours,cell morphology was observed under the inverted phase contrast microscope,and the H9c2 cell survival rates were determined by MTT.（2）The apoptosis rates of H9c2 cells were detected by Annexin V-FITC apoptosis kit.The relative expression levels of apoptosis related proteins Bcl-2and Caspase 3 were determined by Western Blot.（3）The levels of AST and LDH were detected by Nanjing Jiangcheng kits（Jiangcheng Biochemical Inc,Nanjing,China）.（4）The levels of SOD and CAT were detected by Nanjing Jiangcheng kits.H9c2 cells were treated with 1 mM NAC in advance for 1 hour,then exposed to MnCl₂ for 24 hours,the dose of MnCl₂ were the same as mentioned above,and the cell survival rates of H9c2 cells were determined by MTT assay.（5）The levels of ROS in H9c2 cells were detected by DCFH-DA fluorescent probe and the levels of mitochondrial membrane potential were detected by JC-1 fluorescent probe.（6）The relative expression of autophagy related proteins mTOR,p-mTOR,Atg7,p62,Beclin1,Atg5,Atg3 and LC3 were determined by Western blot.In addition,H9c2 cells were treated with 50μM CQ in advance for 2 hours,and then exposed to MnCl₂（200μM）for 24 hours.The relative expression levels of proteins LC3 and p62 were detected by Western Blot,and the immunofluorescence of proteins LC3 and p62 were studied by confocal imaging.H9c2 cells were treated with 10 nM rapamycin in advance for 1 hour,and then exposed to MnCl₂（200μM）for 24 hours.The relative expression levels of proteins mTOR and p-mTOR were detected by Western Blot.Results（1）Compared with the control group,when the manganese concentration was equal to or higher than 200μM,the survival rates of the H9c2 cells were significantly decreased（P<0.05）as the concentration of manganese was increased,and caused different degrees of damage and morphological changes in H9c2 cells.When the manganese exposure time was equal to or more than 12hours,the survival rate of the H9c2 cells was significantly decreased（P<0.05）as the exposure time was increased.（2）The apoptosis rate of H9c2 cells was increased by increasing the MnCl₂concentration.The apoptosis rate in control,low dose,middle dose and high dose groups were 4.275±2.103%,5.225±2.138%,10.175±1.756%,16.625±5.847%,respectively,and there were significant differences among the groups（P<0.05）.Treatment with different concentrations of MnCl₂ did not affect the Caspase 3 protein level,but Cleaved-Caspase 3 was significantly upregulated with a dose-dependent pattern,the protein level in 400μM group was significantly higher than the other three groups（P<0.05）,while Bcl-2 was significantly decreased,the protein level in 400μM group was significantly lower than control group.（3）With an increasing exposure dose of MnCl₂,the activities of LDH in H9c2 cells increased,and the activities of LDH in control,low dose,middle dose and high dose groups were 378.162±19.243 U/L,393.297±28.602 U/L,410.378±29.219 U/L,432.432±24.526 U/L,the activities of LDH in 200μM group and 400μM group were significantly higher than in the control group（P<0.05,P<0.001,respectively）.But treatment with different concentrations of MnCl₂ did not significantly affect the AST level.（4）The survival rate of NAC+MnCl₂ treated groups were significantly higher than MnCl₂ treated groups（P<0.001）.With an increasing exposure dose of MnCl₂,the activities of SOD and CAT in H9c2 cells decreased.The activities of SOD in manganese exposed groups were significantly lower（P<0.001）.The activities of CAT in 400μM group（2.210±1.601 U/mgprot）was significantly lower than the control group（4.400±0.790 U/mgprot）（P<0.05）.（5）The ROS levels in H9c2 cells were significantly increased after MnCl₂exposure,the DCFH-DA fluorescence mean intensity in 200μM and 400μM group were significantly higher than control group（P<0.001）;while the mitochondrial membrane potential in H9c2 cells were significantly decreased,the mean intensity Red/Green ratio in 200μM and 400μM group were significantly lower than control group（P<0.001）.（6）The protein LC3 and p62 in the control group dispersed in the H9c2cytoplasm,and the LC3 spots appeared in the Mn group,studied by confocal imaging.The relative expression levels of protein mTOR and p-mTOR in 400μM group were significantly lower than the other three groups（P<0.05）.With an increasing exposure dose of MnCl₂,the relative expression level of protein Atg7 in 400μM group were significantly lower than the other three groups（P<0.05）,the relative expression level of protein Atg5 in 400μM group were significantly lower than 200μM group（P<0.05）,and the relative expression level of protein Atg3 in 400μM group were significantly lower than 100μM group（P<0.05）.The relative expression levels of protein LC3I and LC3II showed a downward trend.The relative expression level of LC3I in 400μM group was significantly lower than the other three groups（P<0.05）.The relative expression level of protein LC3II in 200μM group and 400μM group were significantly lower than those in the control group and 100μM group（P<0.05）.There was no significantly difference in the ratio of LC3II/LC3I among groups（P>0.05）.（7）Compared with control group,the fluorescence intensity of LC3 and p62 in CQ group and CQ+Mn group were higher,LC3 and p62 were obviously clustered in the cytoplasm,and those was more obvious in CQ+Mn group than in CQ group,studied by confocal imaging.Compared with control group,the expression level of protein p62 in CQ+Mn group was significantly increased（P<0.05）.The expression levels of LC3II in CQ and CQ+Mn group were significantly higher than control and Mn group,respectively（P<0.05）,and the ratio of LC3II/LC3I in CQ+Mn group was significantly higher than control and Mn group,respectively（P<0.05）.There was no significantly difference in the relative expression level of protein LC3I among groups（P>0.05）.（8）The expression levels of protein mTOR and p-mTOR in Rapa and Rapa+Mn group were significantly lower than control and Mn group,respectively（P<0.05）.There was no significantly difference between control and Mn group,or between Rapa and Rapa+Mn group（P>0.05）.Conclusions（1）MnCl₂ exposure can lead to the morphological changes of H9c2 cells,increase the cell apoptosis rates and decrease the cell survival rates.（2）MnCl₂ exposure can increase the level of LDH in H9c2 cells.（3）MnCl₂ exposure can increase the level of ROS,decrease mitochondrial membrane potential,decreased the antioxidant capacity of H9c2 cells and increase the level of oxidative stress.（4）MnCl₂ exposure can inhibit the mTOR signaling pathway and increase the relevant proteins that mediate the formation of autophagosomes,thus activating autophagy.