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

  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。 

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

  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.