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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

何铭垣 葛俊峰 黄玲 王世祥 燕翼

何铭垣, 葛俊峰, 黄玲, 王世祥, 燕翼. 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[J]. 中华全科医学, 2021, 19(8): 1257-1261, 1402. doi: 10.16766/j.cnki.issn.1674-4152.002034
引用本文: 何铭垣, 葛俊峰, 黄玲, 王世祥, 燕翼. 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[J]. 中华全科医学, 2021, 19(8): 1257-1261, 1402. doi: 10.16766/j.cnki.issn.1674-4152.002034
HE Mingyuan, GE Junfeng, HUANG Ling, WANG Shixiang, YAN Yi. Effect of myosin light chain regulated by cardiac myosin light chain kinase on cardiac hypertrophy[J]. Chinese Journal of General Practice, 2021, 19(8): 1257-1261, 1402. doi: 10.16766/j.cnki.issn.1674-4152.002034
Citation: HE Mingyuan, GE Junfeng, HUANG Ling, WANG Shixiang, YAN Yi. Effect of myosin light chain regulated by cardiac myosin light chain kinase on cardiac hypertrophy[J]. Chinese Journal of General Practice, 2021, 19(8): 1257-1261, 1402. doi: 10.16766/j.cnki.issn.1674-4152.002034

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

doi: 10.16766/j.cnki.issn.1674-4152.002034
基金项目: 

国家自然科学基金项目 81870320

国家自然科学基金项目 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

详细信息
    通讯作者:

    燕翼,E-mail:yy8308@163.com

  • 中图分类号: R549.2  R446.62

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

  • 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[(1 787.0±142.6)μm2 vs.(2 458.0±211.3)μm2P < 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[(2 527.0±116.4)μm2 vs.(1 775.0±88.5)μm2P < 0.001];敲除N端可使cMLCK的保护作用丧失[(1 775.0±88.5)μm2 vs.(2 613.0±118.4)μm2P < 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。

     

  • 图  1  cMLCK对MLC2v磷酸化的影响

    注:A为cMLCK磷酸化MLC2v的体外激酶反应体系验证(IB:MLC2v);B为可磷酸化MLC2v的最适cMLCK浓度验证(IB: MLC2v)。

    图  2  敲除氨基端对cMLCK活化MLC2v能力的影响

    注:A为Flag-WT-cMLCK及Flag-ΔNT-cMLCK腺病毒感染心肌细胞后,扩增蛋白的表达情况(IB:Flag);B为图A样品对应的凝胶银染;C为样品体外激酶-底物反应情况(IB: MLC2v)。

    图  3  血管紧张素Ⅱ处理对原代心肌细胞cMLCK活性的影响

    注:A为免疫荧光检测AngⅡ处理后心肌细胞面积的差异(×200);B为AngⅡ(1 μmol/L)处理心肌细胞后,cMLCK、MLC2v及p-MLC2的表达情况;与对照组比较,aP < 0.05。

    图  4  WT-cMLCK与ΔNT-cMLCK对血管紧张素Ⅱ诱导的病理性心肌肥大的影响

    注:A为阴性对照、Flag-WT-cMLCK-EGFP及Flag-ΔNT-cMLCK-EGFP腺病毒感染的心肌细胞,以及用血管紧张素Ⅱ处理后的形态改变(免疫组化染色,×200);B为图A的心肌细胞面积定量分析。与对照组比较,aP < 0.001;与WT-cMLCK+血管紧张素Ⅱ组比较,bP < 0.001。

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出版历程
  • 收稿日期:  2021-01-16
  • 网络出版日期:  2022-02-16

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