钙敏感受体对血管紧张素Ⅱ诱导大鼠心肌细胞肥大的作用

Effect of calcium-sensing receptor in cardiac hypertrophy induced by angiotensin Ⅱ in cultured neonatal rat cardiomyocytes

目的观察钙敏感受体（CaSR）在血管紧张素Ⅱ（AngⅡ）诱导心肌细胞肥大中的作用和可能机制。方法用AngⅡ处理原代新生大鼠心室肌细胞复制心肌肥大细胞模型；用CaSR激动剂氯化钆（GdCl3），GdCl3＋蛋白激酶C（PKC）通路阻断剂（Ro318220）处理AngⅡ诱导的肥大心肌细胞分别作为GdCl3、Ro318220组。通过苏木素-伊红染色（HE）法测定细胞直径，考马斯亮蓝蛋白试剂盒测定蛋白含量来评价细胞肥大的情况；利用激光共聚焦显微镜检测心肌细胞内钙浓度（[Ca^2＋]i）；Western blot法检测CaSR和PKC通路的蛋白表达。结果①与对照组（0．1263±0．0443）比较，AnglI组（0．1963±0．0375）和GdCl3组（0．2778±0．0564）CaSR蛋白表达明显增加（P均〈0．05），且GdCl3组明显高于AngⅡ组（P〈0．05）。②与对照组（222．70±22．09）比较，AngⅡ组（392．16±36．85）和GdCl3组（502．60±44．21）心肌细胞内[Ca^2＋]i显著增加（P均〈0．05）；与AngⅡ组比较，GdCl3组[Ca^2＋]。显著增加（P〈0．05）。③与对照组比较，AngⅡ可诱导心肌细胞肥大，GdCl3可促进AngⅡ的诱导作用，而Ro318220可抑制GdCl3的作用；（少与对照组（0．27±0．07、0．69±0．06、0．87±0．04）比较，AngⅡ组PKCα、PKCε和PKCδ蛋白表达明显增加（0．60±0．16、1．02±0．13、1．20±0．18，P均〈0．05），GdCl3组PKCα、PKCε蛋白表达明显增加（0．82±0．16、1．34±O．12，P均〈0．05）；与AngⅡ组比较，GdCl3组PKCα、PKCε蛋白表达明显增加（P均〈0．05）；与GdCl3组比较，Ro318220组PKCα、PKCε蛋白表达（0．41±0．10、0．85±0．14）明显减少（P均〈0．05）。结论PKC通路参与CaSR激活促进心肌细胞肥大的信号转导。

Objective To explore the roles and possible mechanism of calcium-sensing receptor（CaSR） in cell cardiac hypertrophy model using angiotensin Ⅱ （Ang Ⅱ ）. Methods The cultured neonatal rat ventricular myocytes were treated with Ang Ⅱ as cell cardiac hypertrophy model. Hypertrophic neonatal rat cardiomyocytes were treated with GdCl3 （a specific agonist of CaSR） and/or with Ro318220 （a specific inhibitor of PKC pathway）. To evaluate the status of cardiac hypertrophy, cell diameter was observed by HE dyeing, and protein content was determined through coomassie brilliant blue protein kit. The intracellular calcium concentration （[ Ca^2＋ ]i） was determined by laser scanning confocal microscope. The protein expression of CaSR and PKC pathway were analyzed using Western blotting. Results ①Compared to the control group（0.1263 ± 0.0443）, the protein expression of CaSR was increased in AngⅡ group and in GdC13 group（0.1963±0.0375, 0.2778 ± 0.0564, all P 〈 0.05）. Moreover, compared with Ang U alone, the increase was significant in GdCl3 group （P 〈 0.05）. ②Compared to control group （222.70 ±22.09）, AngⅡ group（392.16 ± 36.85） remarkably increased [Ca^2＋]i（P 〈 0.05）, and this increase of [Ca^2＋]i was further enhanced in GdCl3 group （502.60± 44.21 ） versus Ang Ⅱ group （P 〈 0.05）. ③Compared to control group, Ang Ⅱ could induce cardiomyocyte hypertrophy, and GdCl3 enhanced the effect. Moreover, this enhancement was attenuated by Ro318220. ④Compared to control group（0.27± 0.07,0.69 ±0.06,0.87 ±0.04）, the protein expression of PKCα, PKCε and PKCδ was increased in Ang Ⅱ group（0.60 ±0.16,1.02 ± 0.13,1.20 ＋ 0.18, all P 〈 0.05） and the protein expression of PKCα, PKCε was increased in GdC13 group（0.82 ± 0.16,1.34 ±0.12, all P 〈 0.05）. Moreover, compared with Ang Ⅱ group ,the protein expression of PKCα, PKCε was obviously increased in GdCl3 group（all P 〈 0.05）; compared with GdCl3 group, the protein expression of PKCα, PKCε（0.41 ± 0.10, 0.85 ±0.14） was obviously decreased in Ro318220 group （all P 〈 0.05 ）. Conclusions CaSR is involved in cardiac hypertrophy induced by Ang Ⅱ through PKC pathway in cultured neonatal rat cardiomyocytes.