心肌型肌球蛋白轻链激酶调控肌球蛋白轻链对心肌肥大的影响  

Effect of myosin light chain regulated by cardiac myosin light chain kinase on cardiac hypertrophy

作　　者：何铭垣 葛俊峰 黄玲[1,3] 王世祥[1] 燕翼 HE Ming-yuan;GE Jun-feng;HUANG Ling;WANG Shi-xiang;YAN Yi(Department of Cardiology,the Third Affiliated Hospital,Guangzhou Medical University,Guangzhou,Guangdong 510150,China;不详)

机构地区：[1]广州医科大学附属第三医院心血管内科,广东广州510150 [2]南方医院器官衰竭防治国家重点实验室,广东广州510515 [3]广州医科大学3D打印与再生医学中心,广东广州510150

出　　处：《中华全科医学》2021年第8期1257-1261,1402,共6页Chinese Journal of General Practice

基　　金：国家自然科学基金项目(81870320,81600349);南方医院国家器官衰竭防治重点实验室公开课题(201803);广州市科技创新委员会基金项目(201804010008);广州市卫健委临床特色技术专项基金项目(TS20190337);2018年度广东大学生科技创新培育专项基金重点项目(pdjha0413)。

摘　　要：目的探讨心肌型肌球蛋白轻链激酶(cardiac myosin light chain kinase,cMLCK)对AngⅡ诱导的心肌肥大的作用。方法纯化cMLCK作为激酶,体外激酶反应实验探索cMLCK活性位点。制作野生型cMLCK(WT-cMLCK)及敲除了氨基端(N端)的变异体(ΔNT-cMLCK)腺病毒,感染原代心肌细胞,并用随机数法随机分为6组:对照组、AngⅡ组、WT-cMLCK组、WT-cMLCK+AngⅡ组、ΔNT-cMLCK组、ΔNT-cMLCK+AngⅡ组,每组3个样本。RT-PCR检测心衰因子表达;Western blotting检测cMLCK及底物的表达;免疫荧光检测细胞表面积。结果WT-cMLCK在体外体系能对其底物MLC2v进行磷酸化;而ΔNT-cMLCK对MLC2v的磷酸化作用消失;使用AngⅡ处理后,可见细胞面积增大[(1787.0±142.6)μm^(2)vs.(2458.0±211.3)μm^(2),P<0.001],ANP、β-MHC表达上调(均P<0.05);cMLCK及p-MLC2v表达上调(均P<0.05);过表达WT-cMLCK可逆转心肌肥大[(2527.0±116.4)μm^(2)vs.(1775.0±88.5)μm^(2),P<0.001];敲除N端可使cMLCK的保护作用丧失[(1775.0±88.5)μm^(2)vs.(2613.0±118.4)μm^(2),P<0.001]。结论过表达cMLCK可改善AngⅡ诱导的心肌肥大,该作用依赖于其N端。Objective To investigate the effect of cardiac myosin light chain kinase(cMLCK)on the pathological cardiac hypertrophy induced by AngⅡ.Methods The cMLCK was purified and the active sites of cMLCK were explored in vitro.Wild type cMLCK(WT cMLCK)and its N-terminal knockout variants(△NT-cMLCK)adenovirus were prepared.The primary cardiomyocytes were infected and randomly divided into 6 groups:control group,AngⅡgroup,WT cMLCK group,WT cMLCK+AngⅡgroup,△NT cMLCK group,A NT cMLCK+AngⅡgroup,3 samples in each group.The expression of ANP and p-MHC,which are the heart failure relative factor,was detected by RT-PCR.The expression of cMLCK and MLC2v were detected by western blot.The area of cardiomyocytes was detected by immunofluorescence.Results WT-cMLCK could phosphorylated MLC2v effectively,which was abrogated in△NT-cMLCK.After treated with AngⅡ,the cell area[(1787.0±142.6)μm^(2)vs.(2458.0±211.3)μm^(2),P<0.001]and expression of ANP and p-MHC in neonatal rat cardiomyocyte(NRCM)increased(all P<0.05).The expression of cMLCK and p-MLC2v were upregulated(all P<0.05).However,the cardiac hypertrophy induced by AngⅡwas reversed by WT-cMLCK overexpression[(2527.0±116.4)μm^(2)vs.(1775.0±88.5)μm^(2),P<0.001],while△NT-cMLCK overexpression group lost its protective effect[(1775.0±88.5)μm^(2)vs.(2613.0±118.4)μm^(2),P<0.001].Conclusion The protective effect of cMLCK to pathological cardiac hypertrophy induced by Ang D depends on its N terminal.

关 键 词：心肌型肌球蛋白轻链激酶 心肌型肌球蛋白轻链 心肌肥大 

分 类 号：R549.2[医药卫生—心血管疾病] R446.62[医药卫生—内科学]