| IntroductionAcute myocardial infarction(AMI) is a kind of acut myocardial ischemic disease based on atherosclerosis and has high morbidity and mortality rate in the global scope, seriously harmful to human health. It may lead to vascular occlusion causing by thrombosis or plaque rupture. A lot of researches showed that cell hypoxia condition could activate the oxidative stress reaction and the produce of reactive oxygen species(ROS) could be used as signal molecules mediated apoptosis. At the same time, the accumulation of ROS can cause DNA chain rupture, DNA mutation, and other forms of DNA damage. In order to maintain the integrity and stability of the genome, the organism would initiate a series of DNA damage response(DDR).DDR is a complex reaction process and classical DDR pathways include the ATR/Chk1 and ATM/ Chk2.The ATR and ATM are members of PIKKs family, which can cause a series of phosphorylation cascade by activating downstream cell cycle checkpoint Chk1 and Chk2, mediating cell cycle arrest and DNA repair. When the repair fails, it can furtherly activate P53 and other death signaling pathways, ultimately lead to cell apoptosis.It has been reported that after myocardial infarction, the free DNA in blood circulation would rise significantly. So it can be used as a sensitive detection index of myocardial cell injury, playing an important role on monitoring and assessment of the disease.In the plaques of atherosclerosis patients, ATM level rose obviously, and it was related to the severity of disease. In the myocardial infarction tissues, whether the hypoxia of myocardial cells would initiate the DDR, and what role it piayed was not fully clear at present. ObjectiveWe established myocardial infarction model in rats and detected the participation of DDR in the myocardial ischemia anoxic injury. We also established thehypoxia models ofrat myocardial cell line H9C2 and neonatal rat ventricle myocardial cells(NRVMs), detecting the existence of DDR in myocardial hypoxia injury. Then we explored whether inhibition of DNA damage response on cardiac hypoxic damage would have obvious improvement on cardiac hypoxic damage. Methods(1) Male Wistar rats of six to eight weeks were randomly divided into control group and myocardial infarction group. Anterior descending branch of left coronary artery was ligated to set up AMI model in rats. HE staining, TUNEL was used to verify the correctness of the hypoxic model. Immunohistochemical was used to detect the expression of p-H2 A.X, the marker protein of DNA damage.(2) Hypoxia workstation was used to establish hypoxia model of H9C2 cell, and we verified whether the hypoxic model was successful by methods of MTT, LDH, FCM and WB. Immunofluorescence was used to detect the expression of p-H2 A.X and WB was used to the detection of DDR correlative protein, including p-H2 A.X, p-Chk1 and p-Chk2 at different time point.(3) Aftera lack of oxygen for 6h, WB was used to text the expression of p-H2 A.X by using Chk1 inhibitor UCN–01 on different concentrations. MTT, LDH were used to detect cellular damage and flow cytometry was used to detect the apoptosis rate.(4)With ATM inhibitor KU-55933 in different concentrations, immunofluorescence was used to detect the changes of p-H2 A.X after 6h anoxic. Wealso detected whether the expression of DNA damage response protein p-ATM, p-H2 A.X and p-Chk2 were changed by WB. LDH, FCM, TUNEL and Caspase3/7 activity were used on the detection of cell injury and apoptosis.(5) H9C2 was transfected with Chk2 si RNA and WB was used to detect the interference effect. FCM, LDH and Caspase3/7 activitywere used to detect the change of anoxic damage after hypoxia for 6h.(6) NRVMs wereseparated by percoll density gradient centrifugation method. MTT, LDH, Hoechst and WB were used to verify whether the hypoxic model was successful.Then blocking the ATM/Chk2 pathway byATM inhibitor KU-55933 and we texted the expression of p-H2 A.X by WB, cellular damageby LDH and TUNEL. Results(1) HE results showed that compared with the control group, in myocardial infarction 6h group cardiac muscle fiber were swollen,arranged in disorder and even broke. The nuclei stainied by TUNEL were dyed tan, and immunohistochemical results showed that the expression of p-H2 A.X increased significantly in infarction group.(2) With the hypoxia time prolonged, cellular proliferative capacity was gradually reduced determined by MTT. LDH and apoptosis rate were increased, the same with the expression of p-H2 A.X in H9C2. WB resules showed that the expression of DNA damage response protein p-H2 A.X,p-Chk1 and p-Chk2 were increased with the extended hypoxia time. Immunofluorescence method for p-H2 A.X got the same result.(3) H9C2 was treated in different concentrations of Chk1 inhibitor UCN-01 and anoxic for 6h, the results showed that there were no significant changes on the expression of p-H2 A.X, LDH and apoptosis rate after dosing.(4) Treating H9C2 on different concentrations of ATM inhibitor KU-55933 and oxygen deficit for 6 h, it was found that in 30μM group, p-ATM, p-H2 A. X and p-Chk2 phosphorylation levels were significantly lower, and it was consistent with immunofluorescence. The results of FCM, TUNEL, LDH, and Caspase3/7 activity showed that the cell apoptosis rate was significantly reduced, the same with LDH value,Caspase3/7 activity and positive staining cells of TUNEL.(5) H9C2 weretransfected with Chk2 siRNA. According to the WB results, both 60 nM and 100 nM could significantly inhibit the expression of Chk2, and the inhibition effect of 100 nMwas more apparent. FCM, LDH and Caspase3/7activity results showed, compared with the hypoxia 6h group, after transfection with 100 nm Chk2 siRNA, cell apoptosis rate was significantly reduced, in accordance with LDH value and Caspase3/7 activity.(6) Compared with control group, cell viability was signifieantly decreased and LDH was markedly increased in cells hypoxia treatment. Moreover, the effects were positively correlated with the increasing exposure time of hypoxia. Hoechst 33342 staining showed that NRVMs presented typical morphological changes of apoptosis after hypoxia 6h and apparently over time. WB showed that with hypoxia time prolonged, the expression of Hif-1α increased gradually. Immunityfluorescence, TUNEL showed that the number of p-H2 A.X and TUNEL positive staining cells significantly reduced, and LDH value significantly decreased after adding KU-55933 30μM.Conclusions1.DDR participates in the myocardial cell injury induced by hypoxia.2. Blocking ATM/Chk2 pathway can significantly reduce the cell damage induced by hypoxia. However, there is no obvious effect of blocking the ATR/Chk1 pathway. |
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