血管紧张素Ⅱ对心肌细胞缝隙连接蛋白Cx43表达的时相性调控

目的研究血管紧张素Ⅱ(AngⅡ)诱导心肌细胞肥大后缝隙连接蛋白Cx43表达的变化规律和机制。方法分离培养大鼠心肌细胞后分为正常对照组、AngⅡ组和缬沙坦组,用Westernblot和免疫荧光法观察不同时间(12、24、48、72h)和不同浓度(1.0×10-9～1.0×10-5mol/L)下心肌细胞Cx43表达的变化,采用免疫荧光法检测3组心肌细胞24和72hCx43的表达。结果AngⅡ组心肌细胞较正常对照组明显肥大,蛋白含量增加。AngⅡ组心肌细胞在24～48hCx43表达上调,72h则明显下调,较正常对照组减少30%,而且在AngⅡ作用下呈浓度依赖性下调,24hAngⅡ组荧光阳性细胞数较正常对照组和缬沙坦组明显上调,72h则较其他组明显下调。缬沙坦可拮抗AngⅡ对Cx43表达的作用。结论AngⅡ诱导心肌细胞肥大过程中Cx43的表达出现一定时相性变化,并和AngⅡ呈明显的量效关系,提示AngⅡ可能通过AT1受体调控Cx43的表达而参与心肌细胞缝隙连接重构。

Effect of angiotensin Ⅱ on expression of connexin 43 in cultured neonatal rat ventricular myocytes

Objective To study the effects of angiotensin Ⅱ(AngⅡ),as a mediator of cardiac hypertrophy,on expression of connexin43(Cx43)in cultured neonatal rat ventricular myocytes.Methods Cardiomyocytes were isolated from newborn Wistar rats and divided randomly into the normal control group,the group treated with Ang Ⅱ(1.0×10-6mol/L)and the group treated with AngⅡ(1.0×10-6mol/L)plus valsartan(1.0×10-6mol/L).After the rat ventricular myocytes were treated with AngⅡ for different periods of time(12,24,48 and 72 h)and at different concentrations(1.0×10-9-1.0×10-5 mol/L),the changes of total amount of Cx43 protein in cultures exposed to AngⅡwere determined.Total protein was separated from control and treated cultures by SDS-PAGE and analyzed by immunoblotting.Meanwhile,the effect of valsartan on expression of Cx43 protein in cultures exposed to AngⅡ was observed by immunofluorescence technique.Results Immunofluorescence and immunoblotting analyses revealed that there were up-regulation of Cx43 protein in cultured neonatal rat ventricular myocytes treated with AngⅡ for 12 or 24 h and down-regulation of Cx43 protein in cultured cardiomyocytes treated with AngⅡ for 48 or 72 h.These changes were blocked by valsartan.The cultured neonatal rat cardiomyocytes exposed to increasing concentrations of AngⅡ(1.0×10-9-1.0×10-6 mol/L)for 72 h showed significant concentration-dependent decrease in Cx43 expression.Conclusion AngⅡ up-regulates expression of Cx43 protein in cultured neonatal rat ventricular myocytes treated for 12 or 24 h,and down-regulates Cx43 content after treated for 48-72 hours.These changes were concentration-dependent and can be blocked by valsartan,which can initiate remodeling of gap junctions.