The Effect And Mechanism Of A-Kinase Anchoring Protein 5 Anchoring Protein Kinase A On Inflammation And Hypertrophy After OGD/R In H9C2 Cells

Objective: This study aimed to investigate the role and specific mechanisms of A-kinase anchoring protein 5(AKAP5)and its anchoring complex in the inflammatory and hypertrophic processes after OGD/R in H9C2 cardiomyocytes through the oxygen glucose deprivation/reperfusion(OGD/R)model.Methods:(1)The OGD/R model was constructed by culturing rat H9C2 cardiomyocytes in vitro.(2)AKAP5 was downregulated and overexpressed by si RNA-AKAP5 and plasmid pc DNA3.1-AKAP5,respectively;before OGD/R modeling,forskolin(20 μM),St-Ht31(50μM),and CE3F4(20 μM)were co-incubated with cells for 30 min.(3)Cardiomyocyte viability was detected by the CCK-8 kit after OGD/R modeling.(4)Intracellular c AMP levels were detected by the cyclic adenosine monophosphate(c AMP)assay kit.(5)Immunofluorescence and immunoprecipitation for cellular localization and interaction of AKAP5 with PKA,adenylyl cyclase 5/6(AC5/6),and c AMP-regulated guanine nucleotide exchange factor 1(Epac1).(6)Immunoprecipitation for Rap1 activity(Rap1-GTP).(7)Phalloidin staining for cell surface area.(8)Flow cytometry and fluorescence microscopy for intracellular calcium content.(9)Western blot assay for AKAP5/PKA/AC5/6/Epac1/NFk Bp65 and TNF-α/IL-6/ANP/BNP related protein expression.Results:(1)After OGD/R in rat H9C2 cells,AKAP5 protein expression level was significantly reduced and cell viability was significantly decreased,while intracellular c AMP content and Epac1 protein expression were significantly increased,Rap1 activity was increased,NFk B-p65 intranuclear translocation was increased,and intracellular calcium content was increased,accompanied by TNF-α,IL-6,ANP,BNP,and other related proteins and increased cell surface area;down-regulation of AKAP5 further aggravated these manifestations,while this trend was partially reversed by up-regulation of AKAP5.(2)Immunofluorescence and immunoprecipitation results showed that AKAP5 could localize and interact with PKA,AC5/6,and Epac1 intracellularly;after OGD/R and si RNA-AKAP5 transfection,the interaction of AKAP5 with PKA,AC5/6,and Epac1 was attenuated as the expression of AKAP5 decreased.(3)After interfering with AKAP5 binding to PKA with StHt31,intracellular c AMP content and Epac1 protein expression were significantly increased,Rap1 activity was significantly increased,and TNF-α,IL-6,ANP,and BNP protein expression were significantly increased compared with the OGD/R group.(4)c AMP agonist Forskolin pretreatment of cells resulted in a significant increase in intracellular c AMP content,a significant increase in Rap1 activity,and a significant increase in TNF-α,IL-6,ANP,and BNP protein expression compared with the OGD/R group.(5)Epac1 inhibitor CE3F4 partially inhibited Rap1 activity and intracellular inflammation and hypertrophyassociated protein expression after OGD/R,Forskolin,and St-Ht31 treatment,while attenuating intracellular calcium accumulation and nuclear translocation of NFk B-p65 due to AKAP5 downregulation to some extent.Conclusion: Downregulation of AKAP5 and interference with AKAP5 anchoring to PKA exacerbated OGD/R-induced inflammatory injury and cellular hypertrophy in cardiomyocytes,whereas upregulation of AKAP5 improved this manifestation,where the mechanism was that AKAP5-anchored PKA inhibited the AC5/6/Epac1 pathway in H9C2 cardiomyocytes after OGD/R.