R59022调节心肌细胞自噬促进ET-1诱导的乳鼠心肌细胞肥大

R59022 promotes ET-1-induced cardiac hypertrophy in neonatal rat cardiomyocytes via regulating autophagy

目的研究甘油二酯激酶(DGK)的抑制剂R59022对心肌细胞自噬及内皮素1(ET-1)诱导的心肌肥大的影响,并探讨其可能机制。方法原代培养乳鼠心肌细胞,ET-1诱导乳鼠心肌细胞肥大;Western blot法检测自噬相关蛋白微管相关蛋白1轻链3(LC3)、beclin-1、p62的表达以及Akt的磷酸化及非磷酸化蛋白表达;RT-PCR技术检测心肌肥大基因脑钠肽(BNP)、β-肌球蛋白重链(β-MHC)的mRNA表达水平;细胞免疫荧光检测心肌细胞表面积。结果 ET-1作用乳鼠心肌细胞24 h明显促进心肌细胞肥大,并诱导自噬相关蛋白LC3-Ⅱ/Ⅰ、beclin-1的表达增强,p62表达减弱。自噬抑制剂氯喹(CQ)、3-甲基腺嘌呤(3-MIA)减小心肌细胞表面积,下调肥大基因BNP、β-MHC的mRNA表达,改善ET-1诱导的心肌细胞肥大,而自噬激动剂雷帕霉素(RAPA)则促进ET-1诱导心肌细胞肥大;R59022预处理增加ET-1诱导的LC3-Ⅱ/Ⅰ、beclin-1的表达,增强ET-1诱导的心肌细胞自噬,同时进一步促进ET-1诱导的心肌细胞表面积的增大,BNP、β-MHC的mRNA表达水平的增加,促进ET-1诱导的心肌细胞肥大;对Akt表达的结果显示,ET-1明显下调Akt的磷酸化水平,R59022对此有促进作用。结论 R59022增强ET-1诱导的心肌细胞自噬,促进心肌细胞肥大,其机制可能与抑制Akt的激活,从而抑制mTOR通路的活化有关。

Aim To investigate the effects of DGK inhibitor R59022 on ET-1-induced myocardial hypertrophy and autophagy,and explore the possible mechanisms.Methods Myocardial hypertrophy was induced by ET-1 in cultured rat neonatal cardiomyocytes.Western blot was used to detect the expression of microtubule-associate protein 1 light chain 3(LC3),beclin-1,p62,p-Akt and Akt.mRNA expression of brain natriuretic peptide(BNP) and beta mysion heavy chain(β-MHC) and the cell size of cardiomyocytes were detected by RT-PCR and immunofluorescence,respectively.Results Treatment cardiomyocytes with ET-1(10^(-7) mol·L^(-1)) for 24 h induced the myocardial hypertrophy in cultured neonatal rat cardiomyocytes with the activation of autophagy as evidenced by the increased expression of autophagy-related proteins LC3-Ⅱ/Ⅰ and beclin-1,as well as the increased p62 degradation.While,myocardial hypertrophy induced by ET-1,including the increased myocardial cell size and the mRNA expression of fetal gene BNP and β-MHC,could be reversed by autophagy inhibitor 3-methyl adenine(3-MA) and chloroquine(CQ),Jaut promoted by autophagy agonist rapamycin(RAPA).Pretreatment cardiomyocytes with R59022,an inhibitor of DGK,enhanced ET-1-induced myocardial hypertrophy by enhancing autophagy in cardiomyocytes.Furthermore,ET-1 treatment inhibited the activation of Akt by the downregulation of the Akt phosphorylation,and R59022 enhanced the effect of ET-1 on the activation of Akt.Conclusions Enhanced autophagy contributes to cardiomyocyte hypertrophy.R59022 deteriorate ET-1-induced myocardial hypertrophy by activating autophagy.The possible mechanism may be related to the inhibition of activation of mTOR signaling pathway by inhibiting the activation of Akt.