The Mechanism Of Induce Myocardial Cells Injury By Combined Effect Of Homocysteine And Copper

Objective:Numerous studies show that hyperhomocysteinemia and high serum Cu are two independent risk factors for cardiovascular disease.When these two factors occur together,the damage on the cardiovascular system can be greatly enhanced.There are several Hcy-Cu complexes that are formed depending on the molar ratio of Hcy : Cu.We sought to explore the effects of varying concentrations of Hcy,Cu,and Hcy : Cu mixed at various ratios on cardiovascular damage,and further explore to the pattern of the above factors-induced rat neonatal cardiomyocytes and the protective effects of the adding antioxidants.Methods:Isolated primary porcine hepatocytes were divided into several experimental groups.MTT experimental group is divided into Hcy50,100,200μM,Cu50,100,200μM,Hcy / Cu ratio of 4 / 1,2 / 1,1 / 1 group treated 72 h,DCF-DA,Mitosox-Red and rhodamine 123 dye component Hcy100μM + Cu50μM processing2,4,8,16,24 h,Hcy50,100,200μM,Cu50,100,200μM,and Hcy100μM,Hcy100μM +Cu25μM,Hcy100μM + Cu50μM,Hcy100μM + Cu100μM group treated 24 h,NOX were divided into a control group,Hcy100μM + Cu50μM set of processing 24 h,the rate of oxygen consumption were divided into control group and treatment group Hcy100μM + Cu50μM 8h,qRT-PCR experiments into Hcy100μM +Cu50μM2,4,8,12,24 h group,western blot were divided into a control group,H200μM + Cu100μM 8h,12 h,24h,48 h,H200μM + Cu50μM 12 h,24h,48 h,72h,H100μM + Cu50μM 24 h,H100μM + Cu50μM + 3MA4 m M 24 h,H100μM +Cu50μM + NH4Cl20 m M 24 h peer group.Results:1.we observed a significant change in rat neonatal cardiomyocytes morphology over time.some cardiomyocytes began to beat after 24 h of culture.72 hours later,most of the cells synchronized pulsation and frequency in 60-100 beats per minute,after treatment of Hcy and Cu cell morphology and structure change.The cells began to circle,and the vacuoles appeared in the cell.We acquired images by using a inverted microscope.2.MTT assay results showed that Hcy or Cu treatment alone resulted in a very minor effect on cell injury after 72 h of treatment.However,the combination treatment greatly induced myocardial cell death,and with increasing in the molar ratio of Hcy : Cu,damage effect is more obvious.3.DCF-DA staining experiments: whether cardiomyocytes were treated by Hcy or Cu or Hcy plus Cu,no obvious changes were observed in the levels of mitochondrial ROS in these groups.4.Mitosox-Red staining experiment: the change of mitochondrial ROS is not marked after Hcy or Cu treatment alone.However,mitochondrial ROS was significantly increased after Hcy plus Cu treatment,and increased with increasing molar ratio of Hcy : Cu within a defined time period.5.HET dyeing experiments: the ultra oxygen anion in the cytoplasm is increased after Hcy and Cu work together.6.Rhodamine 123 staining experiments: the cell survival rate was decreased with increasing Hcy concentrations,processing time,and the molar ratio of Hcy : Cu,and it was significantly greater decrements after Hcy plus Cu treatment than after Hcy.7.NADPH oxidase(NOX)activity: the activity of NOX was not increased after Hcy plus Cu treatment.8.mitochondrial oxygen consumption rate: mitochondrial respiratory reserve capacity was decreased after Hcy plus Cu treatment.9.the TUNEL,DAPI stain experiments: staining with TUNEL or DAPI was negative,and the merged figure is also negative.10.The results of qRT-PCR: p53 and SCO2 mRNA expression was decreased rapidly after Hcy plus Cu treatment.11.The expression of LC3: the expression of LC3 II in cardiomyocytes which were treated with Hcy plus Cu increased gradually with time and increasing concentration of Hcy plus Cu.LC3 II expression continued to increase after adding3-MA or ammonium chloride.Conclusion:The Hcy or Cu treatment alone resulted in a very minor effect on cell injury.The combination treatment had decreased survival in rat neonatal cardiomyocytes.The pattern of cell death was autophagy rather than apoptosis.The main reason for cell death was that the combined effect of Hcy and Cu impaired the mitochondrial respiratory chain complex,and then the enhanced production of reactive oxygen species.The decrease of cell survival rate was substantially attenuated when cell were treated with antioxidants which scavenges some free radicals.