钙蛋白酶调节致心肌细胞肥大的信号转导

目的： 探讨钙敏感的calpain对血管紧张素Ⅱ（AngⅡ）介导的致心肌细胞肥大的钙调神经磷酸酶（CaN）信号通路的调节。方法: 原代培养的乳鼠心肌细胞为模型，AngⅡ刺激细胞Ca2+内流和（或）内贮细胞Ca2+释放， ［3H］-Leu掺入法反映心肌细胞蛋白合成速率，Fura-2/AM比率荧光成像分析细胞内钙信号变化，免疫印迹检测心肌细胞μ-calpain、m-calpain、CaN磷酸化及蛋白表达。结果: Ang Ⅱ呈浓度依赖性（10^-8-10^-5 mol·L^-1）促心肌细胞［Ca2+］i增加,各刺激组［Ca2+］i与对照组差异显著(P<0.01或P<0.05)。AngⅡ（10-7 mol·L^-1）剌激心肌细胞15 min后, μ-calpain的磷酸化明显增加，但其蛋白表达量无显著改变（P>0.05）, m-calpain蛋白表达及磷酸化量显著差异，CaN的蛋白表达量无显著改变，但其磷酸化增强, 环孢素A（CsA）抑制CaN的磷酸化。AngⅡ可使无血清培养新生大鼠心肌细胞［3H］-Leu掺入率随时间增加而增加，与对照组差异显著(P<0.01或P<0.05)，且在10^-8-10^-5 mol·L^-1范围内呈量效依赖关系。结论: AngⅡ剌激细胞外Ca2+跨膜内流和（或）内贮Ca2+的释放导致μ-calpain的激活，进一步激活钙敏感的CaN信号通路，在心肌肥厚的病理过程中起重要作用。

Calpain regulates signal transduction contributing to cardiomyocyte hypertrophy

AIM: To investigate the regulation of calcium sensitive calpain on calciuneruin (CaN) signal pathway contributing to myocyte hypertrophy by angiotensionⅡ (AngⅡ). METHODS: Cardiomyocytes from fetal rat were cultivated primarily and were irritated with AngⅡ to cause either calcium inflow or release. ［3H］-Leu incorporations were used to indicate the velocity of protein syntheses. Fura-2/AM ratio imaging analysis was applied to detect the changing of intracellular calcium signal. Western blotting was used for observing protein expression and phosphorylation of μ-calpain, m-calpain, calpastatin and CaN. RESULTS: AngⅡ caused a concentration-dependent (10^-8-10^-5 mol·L) increase in intracellular free calcium concentration (［Ca2+］i) with marked differences between every experimental group and control group. After stimulated by AngⅡ for 15 min, enhanced μ-calpain phosphorylation was found in myocytes with no changes in its protein expression and both of m-calpain phosphorylation and protein expression showed no change. The phosphorylation of CaN was increased with no change of its protein expression and its phosphorylation was inhibited by cyclosporin A (CsA). AngⅡ induced a propotional enhancement of ［3H］-Leu incoporation into cells in the serum-free medium, depending on AngⅡ concentration ranged from 10^-8-10^-5 mol·L. CONCLUSION: AngⅡ causes a concentration-dependent increase in ［Ca2+］i due to more Ca2+ inflow and release in cardiomyocytes, by which μ-calpain and CaN signal pathways are activated. This may play an important role in the pathogenesis of myocardial hypertrophy.