The Function And Mechanism Of Septin4 Regulated PARP1 Cleavage By Cleaved Caspase3 In Promoting Oxidative Stress-induced Cardiomyocyte Apoptosis

Objective:Cardiovascular disease（CVD）is currently one of the most important causes of death in the world,and the incidence is still increasing year by year,with a younger trend.Current studies have found that oxidative stress is related to the occurrence and development of various cardiac abnormal states and diseases.Oxidative stress is an abnormal state caused by the imbalance of oxidative capacity and antioxidant capacity of the human body,which can cause a variety of abnormal reactions and diseases of the cardiovascular system.Oxidative stress is mainly caused by two types of active substances in the body:reactive oxygen species（ROS）and reactive nitrogen species（RNS）.Under physiological conditions,these active substances are involved in maintaining normal physiological activities.But when external stimuli upset the balance,secondary oxidative stress damage can have devastating effects on cells.Apoptosis is a kind of programmed cell death,which plays an important role in various physiological processes.However,uncontrolled apoptosis can cause severe damage to the body.The inhibition of apoptosis of immune cells can lead to autoimmune diseases in the body.Excessive apoptosis of nerve cells is the basic feature of various neurodegenerative diseases.Acute myocardial infarction（AMI）and subsequent reperfusion induce apoptosis of cardiomyocytes.Therefore,maintaining the balance of apoptosis contributes to the prevention and treatment of diseases.Apoptosis can be divided into caspase-dependent and caspase-independent pathways.In the Caspase-dependent pathway,activation of Caspase3 generates cleaved Caspase3,which cleaves poly（ADP-ribose）polymerase 1（PARP1）and promotes the production of cleaved PARP1.In a caspase-independent apoptotic pathway known as"Parthanatos",severe stimulation can overactivate PARP1,promoting the synthesis of poly（ADP-ribose）（PAR）,which can promote apoptosis induction factor（AIF）enters the nucleus,causing massive DNA breaks and chromatin condensation.And cleaved PARP1 was recently found to enhance the effect of PAR on AIF.Septin4 is an apoptosis-related protein that can inhibit the occurrence and development of diseases such as liver fibrosis and tumors,and interacts with PARP1.Moreover,Septin4,cleaved Caspase3 and PARP1 are unified in phase and space,which suggests that the three may influence each other and jointly participate in and regulate apoptosis.This study explored the role and related mechanisms of Septin4 in oxidative stress-induced cardiomyocyte injury.Methods:1.To investigate whether H₂O₂ stimulation can induce ischemia-reperfusion injury in H9C2 cardiomyocytes and its effect on Septin4 expression.We first divided H9C2 cardiomyocytes into 4 groups and stimulated the cells with 0μmol/L,50μmol/L,100μmol/L and 150μmol/L of H₂O₂ for 90 minutes,and then detected cell viability,apoptosis rate and the expression level of Septin4,cleaved Caspase3,cleaved PARP1.We then divided H9C2 cardiomyocytes into 4 groups,stimulated cells with 150μmol/L H₂O₂ for 0 min,30 min,60 min,and 90 min respectively,and then detected the expression levels of Septin4,cleaved Caspase3 and cleaved PARP1.2.To investigate the effect of Septin4 overexpression on H₂O₂-induced H9C2cardiomyocyte injury.We divided H9C2 cardiomyocytes into 4 groups:Flag Vector group,Flag Vector+H₂O₂（150μmol/L,90 min）group,Flag Septin4 group,and Flag Septin4+H₂O₂（150μmol/L,90 min）group,and then detected the cell apoptosis rate and the expression levels of Septin4,cleaved PARP1,cleaved Caspase3 and the level of PARylation.We then divided H9C2 cardiomyocytes into 8 groups:Flag Vector group,Flag Septin4 group,Flag Vector+H₂O₂（150μmol/L,30 min）group,Flag Septin4+H₂O₂（150μmol/L,30 min）group,Flag Vector+H₂O₂（150μmol/L,60 min）group,Flag Septin4+H₂O₂（150μmol/L,60 min）group,Flag Vector+H₂O₂（150μmol/L,90 min）group,Flag Septin4+H₂O₂（150μmol/L,90 min）group,and then detected the cell viability.3.To investigate the effect of Septin4 overexpression on H₂O₂-induced injury of primary neonatal rat cardiomyocytes.We first tested whether Flag Septin4 was successfully transfected into cells.Next,we divided the cells into 4 groups:Flag Vector group,Flag Vector+H₂O₂（150μmol/L,90 min）group,Flag Septin4 group,Flag Septin4+H₂O₂（150μmol/L,90 min）group,and then detected the level of apoptosis and the expression levels of Septin4,cleaved PARP1 and cleaved Caspase3.4.To investigate the effect of knockdown of Septin4 on H₂O₂-induced H9C2cardiomyocyte injury.We divided H9C2 cardiomyocytes into 4 groups:sh con group,sh con+H₂O₂（150μmol/L,90 min）group,sh Septin4 group,and sh Septin4+H₂O₂（150μmol/L,90 min）group,and then detected the cell apoptosis rate and the expression levels of Septin4,cleaved PARP1,cleaved Caspase3 and the level of PARylation.We divided H9C2 cardiomyocytes into 8 groups:sh con group,sh Septin4 group,sh con+H₂O₂（150μmol/L,30 min）group,sh Septin4+H₂O₂（150μmol/L,30 min）group,sh con+H₂O₂（150μmol/L,60 min）group,sh Septin4+H₂O₂（150μmol/L,60 min）group,sh con+H₂O₂（150μmol/L,90 min）group,sh Septin4+H₂O₂（150μmol/L,90 min）group,and then detected the cell viability.5.To investigate the binding of Septin4 to cleaved Caspase3 and PARP1 under normal conditions and H₂O₂ stimulation conditions.We divided the proteins into four major groups according to the added antibodies,and each major group included two groups:Septin4antibody group and IgG group;cleaved Caspase3 antibody group and IgG group;Septin4antibody group and IgG group;PARP1 antibody group and IgG group.Then we detected the expression levels of cleaved Caspase3,Septin4,PARP1,and Septin4 respectively.We then divided the proteins into four groups according to the added antibodies and stimulation conditions and each group included three groups:Septin4 antibody group,Septin4 antibody+H₂O₂（150μmol/L,90 min）group and IgG group;cleaved Caspase3antibody group,cleaved Caspase3 antibody+H₂O₂（150μmol/L,90 min）group and IgG group;Septin4 antibody group,Septin4 antibody+H₂O₂（150μmol/L,90 min）group and IgG group;PARP1 antibody group,PARP1 antibody+H₂O₂（150μmol/L,90 min）group and IgG group.Then we detected the expression levels of cleaved Caspase3,Septin4,PARP1,and Septin4 respectively.6.To explore the role of Septin4 in the cleavage of PARP1 by cleaved Caspase3.We divided the proteins into four major groups according to the added antibodies and whether Septin4 was overexpressed,each of which includes three groups:cleaved Caspase3antibody group,cleaved Caspase3 antibody+Flag Septin4 group and IgG group;PARP1antibody group,PARP1 antibody group+Flag Septin4 group and IgG group;cleaved Caspase3 antibody group,cleaved Caspase3 antibody+sh Septin4 group and IgG group;PARP1 antibody group,PARP1 antibody+sh Septin4 group and IgG group.Then we detected the expression levels of PARP1,cleaved Caspase3,PARP1,and cleaved Caspase3respectively.We then divided H9C2 cardiomyocytes into four groups,and the cells were transfected with 0μg,2μg,4μg and 6μg of Flag Septin4 respectively.We detected the expression levels of cleaved PARP1,Caspase3 and cleaved Caspase3.Next,we divided the H9C2 cardiomyocytes with Septin4 stably knocked down into four groups according to the knockdown sequence:sh con group,sh Septin4 target sequence 1 group,sh Septin4target sequence 2 group,sh Septin4 target sequence 3 group,and then detected the expression levels of cleaved PARP1,Caspase3 and cleaved Caspase3.Finally,we divided H9C2 cardiomyocytes into four groups,and the cells were transfected with si con,si Septin4 target sequence 1,si Septin4 target sequence 2 and si Septin4 target sequence 3respectively.We then detected the expression levels of cleaved PARP1,Caspase3 and cleaved Caspase3.7.To investigate the effect of Septin4 on the accumulation of ROS in H9C2cardiomyocytes.We divided H9C2 cardiomyocytes into 4 groups:Flag Vector group,Flag Vector+H₂O₂（150μmol/L,90 min）group,Flag Septin4 group,Flag Septin4+H₂O₂（150μmol/L,90 min）group,and then detected the accumulation of ROS and the expression levels of 3-Nitrotyrosine,8-oxoguanine DNA glycosylase（OGG1）and superoxide dismutase 1（SOD1）.We then divided H9C2 cardiomyocytes into 4 groups:sh con group,sh con+H₂O₂（150μmol/L,90 min）group,sh Septin4 group,sh Septin4+H₂O₂（150μmol/L,90 min）group,and then detected the accumulation of ROS and the expression levels of 3-Nitrotyrosine,OGG1 and SOD1.Results:1.With the increase of H₂O₂ stimulation intensity,the activity of H9C2cardiomyocytes gradually decreased,the apoptosis rate of H9C2 cardiomyocyte gradually increased,and the expression of Septin4,cleaved Caspase3 and cleaved PARP1 gradually increased in H9C2 cardiomyocytes.2.Compared with the cells in the non-overexpression group,after being stimulated by the same degree of H₂O₂,H9C2 cardiomyocytes overexpressing Septin4 had lower activity,higher apoptosis rate,higher expression levels of cleaved PARP1 and cleaved Caspase3,and higher degree of PARylation.3.Compared with the cells in the non-overexpression group,after being stimulated by the same degree of H₂O₂,the primary neonatal rat cardiomyocytes overexpressing Septin4 had more serious apoptosis,and higher expression levels of cleaved PARP1 and cleaved Caspase3.4.Compared with the cells in the control group,after being stimulated by the same degree of H₂O₂,H9C2 cardiomyocytes in which Septin4 was stably knocked down had higher activity,lower apoptosis rate,lower expression levels of cleaved PARP1 and cleaved Caspase3,and lower degree of PARylation.5.Septin4 in H9C2 cardiomyocytes can bind to cleaved Caspase3 and PARP1.This binding was enhanced after cells were stimulated with H₂O₂.6.Overexpression of Septin4 can promote the binding of cleaved Caspase3 and PARP1.Knockdown of Septin4 can inhibited the binding of cleaved Caspase3 to PARP1.With the increase of Septin4 content in cells,the expression of cleaved PARP1 increased,and the expression of Caspase3 and cleaved Caspase3 did not change significantly.With the decrease of Septin4 content in cells,the expression of cleaved PARP1 decreased,and the expression of Caspase3 and cleaved Caspase3 did not change significantly.7.Compared with the cells in the non-overexpression group,after being stimulated by the same degree of H₂O₂,H9C2 cardiomyocytes overexpressing Septin4 had more ROS,higher expression levels of 3-Nitrotyrosine and OGG1,and lower expression level of SOD1.Compared with the cells in the control group,after being stimulated by the same degree of H₂O₂,H9C2 cardiomyocytes in which Septin4 was stably knocked down had less ROS,lower expression levels of 3-Nitrotyrosine and OGG1,and higher expression level of SOD1.Conclusions:1.Septin4 participates in and promotes H₂O₂-induced cardiomyocyte apoptosis.2.Septin4 promotes the ability of cleaved Caspase3 to cleave PARP1 by forming the cleaved Caspase3-Septin4-PARP1 regulatory axis,thereby promoting cardiomyocyte apoptosis.