α-烯醇化酶在内皮素-1诱导的肥大心肌细胞中的表达和调控通路

目的 观察内皮素-1(ET-1)诱导的心肌细胞肥大模型中细胞外信号调节激酶(ERK1/2)、磷酸化细胞外信号调节激酶(p-ERK1/2)、低氧诱导因子(HIF-1α),α-烯醇化酶(α-enolase)蛋白表达情况,探讨肥大心肌细胞α-enolase高表达的调控机制. 方法 建立ET-1诱导的心肌细胞肥大模型,从细胞表面积、细胞蛋白合成速率和肌原纤维的重排3方面进行验证;将原代培养心肌细胞随机分为4组:(1)对照组;(2)PD98059干预组;(3)ET-1刺激组;(4)PD98059+ET-1刺激组.免疫印迹方法 检测ERK1/2、p-ERK1/2、HIF-1α、α-enolase的蛋白表达. 结果 ET-1刺激后心肌细胞表面积为(1350.7±107.5)μm2,较对照组(896.1±70.2)μm2增加(P<0.05);ET-1刺激后心肌细胞3H]亮氨酸掺入量较对照组增加分别为(1387.9±14.8)dpm和(787.7±10.2)dpm,P<0.013;肌原纤维染色可见ET-1刺激屙心肌细胞肌原纤维排列较对照组紧密、染色浓,表明ET-1能诱导心肌细胞肥大.ERK1/2抑制剂PD98059预处理的心肌细胞在ET-1刺激后细胞表面积和细胞的3H]亮氨酸掺入量均较ET-1刺激组减少细胞表面积分别为(907.0±92.5)μm2和(1350.7±107.5)μm2,P<0.05;3H]亮氨酸掺入量:(841.5±10.5)dpm和(1387.9±14.8)dpm,P<0.05].ET-1刺激后心肌细胞有p-ERK1/2表达,其抑制剂PD98059在抑制ERK1/2活化的同时也部分抑制了HIF-1α、α-enolase蛋白的表达. 结论 ERK1/2的活化与ET-1诱导的细胞肥大关系密切,在ET-1促心肌细胞肥大过程中,从MAPK/ERK1/2至HlF-1α及α-enolase这条信号通路町能参与α-enolase高表达的调控.

The expression of α-enolase in hypertrophic cardiomyocytes induced by endothelin-1(ET-1)and regulation mechanism for the expression

Objective To investigate the protein expression of ERK1/2,p-ERK1/2,HIF-1α and α-enolase in hypertrophic cardiomyocytes induced by ET-1 and explore the regulation mechanism of overexpression of α-enolase in hypertrophic cardiomyocytes. Methods ET-1-induced abnormal cardiomyocytes were used as model of cardiac hypertrophy. Cell surface area, <'3>H]-leucine incorporation and the actin staining were measured to determine the extent of hypertrophy. Cultured cardiomyocytes were divided into 4 groups at random, control group, PD98059 treated group, ET-1 treated group and PD980594- ET-1 treated group. The protein expressions of ERK1/2, p-ERK1/2,HIF-1α and α-enolase were detected by immunoblotting analysis. Results Compared with the control group , cell surface area and <'3>H] leucine incorporation were increased in ET-1 treated group ((1350.7±107.5)μtm<'2> vs. (896.1±70. 2)μtm<'2> , P<0.05; (1387.9±14.8) dpm vs. (787.7±10.2)dpm,P<0.013. Actin staining showed that ET-1-treated cardiomyoeytes had more intense actin staining and clear cross-striations than did control group, which suggested that myocardial cell hypertrophy could be induced by ET-1 in WKY neonatal cardiomyocytes. After MEK 1/2 inhibitor PD98059 was used, the cell surface area and <'3>H] leucine incorporation were decreased in PD980594-ET-1 treated group compared with ET-1 treated group(907.0±92.5)μm<'2> vs. (1350.7±107.5)μm<'2>;(841.5±10. 5)dpm vs. (1387.9±14.8)dpm, both P<0.05], which suggested that myocardial cell hypertrophy could be regulated by ERK1/2 signal pathway. Immunoblotting analysis showed that the protein expressions of p-ERK1/2, HIF-1α and α-enolase increased after ET-1 treatment, while PD98059 as an inhibitor of the upstream kinase of ERK1/2 was used, the protein expressions of HIF-1α and α-enolase were partially inhibited. Conclusions ET-1 induces hypertrophic cardiomyocytes through ERK1/2 phosphorylation in cultured neonatal rat cardiac myocytes. ERK1/2 and HIF-1α signal pathway may play an important role in the overexpression of α-enolase in the hypertrophic cardiomyocytes.