Aim Previous studies showed that the inhibition of proteasome activity could significantly improve cardiac hypertrophy, but its mechanism is not clear.Increased glycogen synthase kinase3 (GSK3) activity can also improve cardiac hypertrophy.However, the relationship between proteasome and GSK3 has not been reported in cardiomyocyte In this study, we will investigate the effect of proteasome inhibition on cardiomyocyte hypertrophy,GSK3 activity and the underlying mechanism.Methods Primary neonatal rat cardiomyocytes were divided into 4groups: Control, AngⅡ (100nmol· L1, 48 h), AngⅡ (100 nmol· L1) + MG132 (0.05 μmol· L1),MG132 (0.05 μmol · L1) ,AngⅡ (100 nmol · L1) + MG132 + LiCl (10 mmol · L1), LiCl.Proteasome activitiy was detected by fluorescent peptide substrate.Cardiomyocyte surface area, ANF mRNA expression, and the rate of protein synthesis were observed as myocardial hypertrophy index.GSK3, Akt, AMPKα, and Histone3(H3) were detected by Western Blot.The expression of GATA4 in the cytoplasm and nucleus was observed by immunofluorescence.Results (1) Compared with the control group, myocardial ANF mRNA expression, the rate of protein synthesis and cell surface area were all increased in Ang Ⅱ group.The chymotrypsinlike, trypsinlike and caspaselike activities of proteasome were all increased significantly.The phosphorylated level of both GSK3α(pGSK3 α) (Ser21) and GSK3 β (pGSK3 β) (Ser9) increased, i.e they were inactivated.(2) Compared with the Ang Ⅱ group, myocardial ANF mRNA expression, the rate of protein synthesis and cell surface area were all decreased after proteasome inhibition.And pGSK3 (Ser21) and pGSK3 β (Ser9) was respondingly decreased,(3) Proteasome inhibition also resulted in the decrease of pAkt (Ser473) and pAMPKa (Thr172), which increased in cardiomyocyte hypertrophy.Immunofluorescence showed that GATA4 was mainly distributed in the nucleus after Ang Ⅱ treatment, while it was obviously increased in the cytoplasm after proteasome inhibition.Mter the GSK3 inhibitorLiCl was given, the above indicators were reversed.(4) pHistone3 was also increased in cardiomyocyte hypertrophy and MG132 reduced its level, but LiCl treatment had no significant effect on its level.Conclusion Proteasome inhibition reduces cardiomyocyte hypertrophy through increase of GSK3a/b activity, which may be related with the decrease of Akt and AMPKa activities, and the decrease of nucleus location of GATA4, but phistone3 is not involved.