Regulation Of Myocardial Remodeling Induced By Hypoxia Reoxygenation In H9C2 Cells By AKAP5

Objective: After si RNA transfection of H9c2 cardiomyocytes down regulated AKAP5,the changes of hypertrophy related proteins and surface area of H9c2 cardiomyocytes were observed after hypoxia reoxygenation,and the intracellular interaction between AKAP5 PKA and CAN was detected by immunofluorescence and immunoprecipitation techniques.In order to explore the mechanism of AKAP5 on hypoxia reoxygenation remodeling of H9c2 cardiomyocytes.Methods: si RNA was transfected into H9c2 cells with Lipofectamine 3000 TM transfection reagent to knock down the expression of AKAP5.Hypoxia and reoxygenation: H9c2 cardiomyocytes were exposed to hypoxia(95% N2,4% CO2,1% O2)for 3 hours and reoxygenation(5% CO2,37 ℃)for 24 hours.The experiment consisted of normal group(Con),model group(Model),empty vector group(NC),si RNA-AKAP5 group,which the later three group were treated with hypoxia and reoxygenation.After that,the normal culture medium was replaced and continued to be cultured in the incubator(5% CO2,37 ℃)for 24 hours.The proteins PKA/CAN/NFATc3/p-NFATc3/ANP/BNP/β-MHCwere detected by Western blot,the area of cytoskeleton was detected by phalloidin staining,the relationship among AKAP5,PKA and CAN was detected by immunofluorescence and immunoprecipitation.PKA inhibition was used to observe whether the protein of downstream hypertrophy pathway CAN / NFATc3 / p-NFATc3 was inhibited;CAN inhibitor was used to observe whether the expression of hypertrophy related protein ANP / BNP / β-MHC was inhibited after hypoxia reoxygenation.Results: 1:The results of RT-PCR showed that sirna1,sirna2 and sirna3 could make the expression of AKAP5 down regulated,and sirna3 was the most effective base sequence for knockdown,which could reduce the m RNA of AKAP5 by about65%(P < 0.05).2:Compared with the Con,the result of ANP,BNP,β-MHC and area of cells were increased in the model and the NC(P < 0.05);compared with the model,the expressions of ANP,BNP,β-MHC and cell area were increased in the si RNAAKAP group(P<0.05).Compared with the normal group,the expression of myocardial cell area in the model group and empty vector group was increased(P<0.05),and compared with the model group,the cell area in the si RNA-AKAP5 group was obviously increased(P < 0.05).3:Compared with the Con,the expression of PKA and nfatc3 in model group,empty vector group and si RNA-AKAP5 group had no significant difference.However compared with the normal group,p-PKA/ PKA,CAN protein expression increased,p-nfatc3 / nfatc3 decreased(P < 0.05);compared with the model group,p-PKA /PKA,CAN protein expression apparently increased,p-NFATc3 / NFATc3 evidently decreased in si RNA-AKAP5 group(P<0.05).4: Immunofluorescence suggested that: in the cytoplasm,akap5 showed red,PKA and can showed green,and when they fused respectively,they showed yellow.We found that akap5,PKA and CAN were Co located in the cell.5:Protein coprecipitation showed that there was interaction among AKAP5,PKA and CAN.6:Results: compared with the normal group,the p-NFATc3 / NFATc3 ratio in the model group was decreased,and the expression of CAN was visibly increased(P <0.05);compared with the model group,the p-NFATc3 / NFATc3 percentage in the CAN inhibitor group was increased,and the expression of CAN was decreased(P <0.05).The results showed that can could partially reverse the hypertrophy of cardiomyocytes induced by low expression of akap5,suggesting that CAN is one of the key proteins in the hypertrophy pathway of AKAP5.7:PKA inhibitor results:compared with the normal group,the rate of p-NFATc3 / NFATc3 in the model group was distinctly decreased,and the expression of CAN was significantly increased(P < 0.05);compared with the model group,the proportion of p-NFATc3 /NFATc3 in the PKA inhibitor group was increased,and the expression of CAN was reduced(P < 0.05);the results showed that PKA may be one of the upstream proteins regulating cellular hypertrophy pathway.Conclusion: After hypoxia reoxygenation in H9c2 cardiomyocytes,the downregulation of AKAP5 may accelerate the dephosphorylation of nfatc3 into the nucleus by increasing the phosphorylation of PKA.It may also directly lead to the activation of CAN,and then jointly activate the expression of hypertrophic gene and accelerate the remodeling of cardiomyocytes.