Loss Of GATA4 At The C-terminus Promotes Cardiomyocyte Apoptosis By Enhancing The Expression Of AKT/FOXO

Aim GATA4 is an important transcription factor which takes part in cardiac development.Our group previously found a special type of GATA4 mutation(p.S335X)in the myocardium in subjects with ventricular septal defect(VSD),which could produce termination codon in advance,leading to deletion of GATA4 C-terminal.To further understand the effects of p.S335X on cardiomyocyte and the mechanisms,we used rat cardiomyocyte H9C2 cells cultured in vitro,transfected with wild-type GATA4(pcDNA6-GATA4-WT,WT)and mutant GATA4 p.S335X(pcDNA6-GATA4-MU,MU)vectors,validated transfection efficacy,studied the effects of GATA4 p.S335X on apoptosis and the involvement of classical apoptosis pathway AKT/FOXO/Bcl-2.On the basis,we further screened the regulatory effects of GATA4 p.S335X on the apoptosis-related down-stream molecules and pathways other than classical Bcl-2 pathway.Method We used rat cardiomyocyte H9C2 cells cultured in vitro,transfected with WT,MU and EV vectors,validated transfection efficacy by RT-PCR and Western Blot which was performed to detected the expression of GATA4.TUNEL was carried out to investigate the effects of GATA4 p.S335X on apoptosis.RT-PCR and Western Blot were observed respectively to determine the mRNA,protein and its phosphorylated products of the classical apoptosis pathway-AKT/FOXO/Bcl-2-related factors.PCR Array was used to screen the regulatory effects of GATA4 p.S335X on the apoptosis-related down-stream molecules and pathways other than classical Bcl-2 pathway.Results Real-Time PCR and Western blot indicated that the mRNA and protein expression of GATA4 in transfected with WT and MU H9C2 cells was significantly increased,and protein molecular weight in WT was more than MU(Group WT:50KDa,Group MU:40KDa).Without TNF,clearly apoptosis can be observed in H9C2 cells transfected MU.In TNF-introduced group,the apoptosis of MU and EV group was significantly higher than the WT group,indicating GATA4 WT inhibite effects on apoptosis.Western Blot revealed the protein expression of GSK3β and p-GSK,FOX03、BAX in MU are higher than in WT,and AKT,p-AKT and Bcl-2 in MU are lower in MU.While the RNA expression of these genes by Real-Time PCR showed no difference.PCR Array showed that,compared with group WT,in group MU,the RNA expression of p53,p53bp2,Bcl-10,Casp3,Gadd45α,Jun,Mapk1,Mapk8,Pdgfra,Pdk2,Pdpkl and Tscl were up-regulated,Casp7,Ltbr,Mapk8ip1,Tnfsf10,Pdk1 and Pik3rl were decreased.Conclusion GATA4 p.S335X did not exert inhibitory effects on apoptosis as wild-type GATA4 did,and this might be related to classical AKT/FOXO/Bcl-2 pathway.Additionally,GATA4 p.S335X with deletion of C-terminal might also regulate non-classical apoptosis pathways,including p53/Tnfsfl 0,MAPK/Gadd45α,JNK/c-jun,and PDK1/PKC/Bcl10/caspase7.