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

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

何铭垣^{1, 2, 3},
葛俊峰^{1, 3},
黄玲^{1, 3},
王世祥¹,
燕翼^{1, 2, 3, ,}

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

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
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- 文章访问数: 485
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- 被引次数: 0
出版历程
- 收稿日期: 2021-01-16
- 网络出版日期: 2022-02-16

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

HE Mingyuan^{1, 2, 3},
GE Junfeng^{1, 3},
HUANG Ling^{1, 3},
WANG Shixiang¹,
YAN Yi^{1, 2, 3
, ,}

1.
Department of Cardiology, the Third Affiliated Hospital, Guangzhou Medical University, Guangzhou, Guangdong 510150, China

摘要

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)μm²，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[(2 527.0±116.4)μm² vs.(1 775.0±88.5)μm²，P < 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)μm²，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。
- 心肌型肌球蛋白轻链激酶 /
- 心肌型肌球蛋白轻链 /
- 心肌肥大
Abstract: 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 (ΔNT-cMLCK) adenovirus were prepared. The primary cardiomyocytes were infected and randomly divided into 6 groups: control group, AngⅡ group, WT cMLCK group, WT cMLCK+AngⅡ group, Δ NT cMLCK group, Δ NT cMLCK+AngⅡ group, 3 samples in each group. The expression of ANP and β-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 ΔNT-cMLCK. After treated with AngⅡ, the cell area [(1 787.0±142.6) μm² vs. (2 458.0±211.3) μm², P < 0.001] and expression of ANP and β-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 AngⅡ was reversed by WT-cMLCK overexpression [(2 527.0±116.4) μm² vs. (1 775.0±88.5) μm², P < 0.001], while ΔNT-cMLCK overexpression group lost its protective effect [(1 775.0±88.5) μm² vs. (2 613.0±118.4) μm², P < 0.001]. Conclusion The protective effect of cMLCK to pathological cardiac hypertrophy induced by AngⅡ depends on its N terminal.
- Cardiac myosin light chain kinase /
- Cardiac myosin light chain /
- Cardiac hypertrophy

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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)。

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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)。

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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。

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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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