The Role And Mechanism Of Mitochondrial Dysfunction In Cardiomyocyte Apoptosis Induced By Excessive Copper

Background:Copper is one of the trace elements necessary for the normal operation of organisms.When there is too much copper in the body,it will produce heavy metal toxicity to the heart.This study aims to investigate the effect of excessive copper on mitochondrial damage and apoptosis of cardiomyocytes.Purpose:The toxicological effects of excessive copper on myocardium and The Mechanism of Mitochondrial energy metabolism disorder and Mitochondrial-mediated apoptosis were observed.Methods:Twelve 4-week-old male Kunming mice were used for the in vivo experiment.The model was made with 1.08g/kg copper chloride-containing feed and 0.1%copper chloride-containing water.The modeling duration was 8 weeks and 14 weeks,each group 6 mice in each group,and 12 normal Kunming mice were taken as the 8-week control group and the 14-week control group,with 6 in each group.After successful modeling,the heart tissue,serum and urine were collected,and the copper content in the heart tissue,serum and urine was measured.Observe the echocardiogram,morphological changes and changes of HW/BW in each group of mice.HE and Masson staining were used to observe the pathological changes of the mouse heart,and the ultrastructure changes of cardiac mitochondria were observed by transmission electron microscope.ELISA was used to determine the serum NT-pro-BNP and c Tn I of each group of mice.Western blot was used to detect Cyt-c、Bcl-2、Bax and Cleaved-Caspase-3 apoptosis-related protein expression in myocardial tissue,mitochondria and cytoplasm.In the in vitro experiment,human cardiomyocytes（AC16）were used.In order to explore the concentration and time of Cu Cl₂to treat cardiomyocytes,CCK-8 was used to detect cell viability to determine the time and concentration of copper treatment.Atomic absorption method detects the copper content in cardiomyocytes and mitochondria.Fluorescence microscope was used to observe the changes of mitochondrial membrane potential,and the kit was used to detect mitochondrial respiratory chain complex I-IV activity expression,ATP Horizontal and SDH activity expression.Flow cytometry and fluorescence microscopy to detect ROS levels.ELISA was used to determine the NT-pro-BNP and c-Tn I of each group of cells.Flow cytometric detection of apoptosis and Western blotting were used to detect the expression of Cyt-c,Bcl-2,Bax and Cleaved-Caspase-3 apoptosis-related proteins in AC16 cells,mitochondria and cytoplasm.Results:In in vivo experiments,compared with the control group,the heart copper concentration of the model group increased,and c-Tn I and NT-pro-BNP increased.Analysis of echocardiographic results showed that the model group severely affected the cardiac function parameters of the mice.The heart weight index results showed that8 weeks after the model was created,the heart was injured,and the injury in the14-week model group was more obvious.Histopathological and ultrastructural observations showed that CVF of mouse myocardium and the cross-sectional area of mouse myocardial cells in the model group were significantly higher than those in the control group.Transmission electron microscopy observed that the mitochondrial bilayer membrane of the membrane type group was not Intact,the mitochondria swelled obviously,and the mitochondrial crest was blurred or even disappeared.The results of in vitro experiments showed that compared with the control group,the copper content in the myocardial mitochondria of the model group was significantly increased,the cell viability was weakened,and c-Tn I and NT-pro-BNP were increased,similar to the in vivo results.It was observed through fluorescence microscope and transmission electron microscope that the mitochondrial membrane potential of the model group was significantly reduced,and the mitochondria were damaged.The test results of the kit analyzed the activity of COX I,II,IV and ATP.The results of flow cytometry showed that the cells of the model group had different degrees of apoptosis.Fluorescence microscopy and flow cytometry results show that the model group has a large increase in ROS.NAC+Cu Cl₂group can significantly inhibit the increase in ROS.WB results shows that compared with the control group,Cyt-c in the mitochondria of the model group is down-regulated,Cyt-c in the cytoplasm is up-regulated,Bcl-2 is down-regulated,and Bax is up-regulated.Compared with the model group,the NAC+Cu Cl₂group can inhibit Cyt-c in the mitochondria.The release of Cyt-c also inhibits the expression of Bax and Cleaved-Caspase-3.Conclusions:The results of this study indicate that excessive copper can cause heart damage.The mechanism may be related to copper damage to the mitochondrial respiratory chain,mitochondrial energy metabolism disorder,Cyt-c leakage,activation of caspase-dependent apoptotic pathways and further heart damage.