心肌肥厚小鼠钙激活氯通道表达与功能研究
首发时间:2014-11-15
摘要:钙激活氯通道存在于很多物种和组织中,主要由TMEM16A或TMEM16B编码。本课题组曾报道小鼠心肌细胞存在TMEM16A编码的钙激活氯通道,但其表达及功能在病理性心肌重构中的作用还未见报道。本实验采用主动脉缩窄的方法建立小鼠压力负荷型心肌肥厚模型,以假手术小鼠作为对照,3周后通过心重/体重比、心重/胫骨长度比确定模型建立成功;采用RT-PCR技术检测小鼠心脏组织中TMEM16家族基因的表达;急性分离肥厚模型小鼠和假手术小鼠的心肌细胞,采用蛋白印迹技术检测TMEM16A的蛋白表达情况;采用全细胞膜片钳技术检测心肌细胞钙激活氯电流(ICl.Ca)密度的变化。RT-PCR结果显示,在小鼠心脏组织中除TMEM16B、TMEM16G及TMEM16J外,其他TMEM16成员均表达;蛋白印迹结果表明心肌肥厚小鼠的心肌细胞中TMEM16A的蛋白表达水平上调;全细胞膜片钳结果显示肥厚小鼠心肌细胞ICl.Ca电流密度与对照组小鼠的相比显著升高,且升高的ICl.Ca可被TMEM16A特异性抑制剂T16Ainh-A01显著抑制。所以在压力负荷型心肌肥厚小鼠的心肌细胞中,TMEM16A 编码的CaCC的功能增强。因此,CaCC可能参与病理性心肌重构。
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Expression and function of the calcium-activated chloride currents in myocardial hypertrophy mouse
Abstract:Calcium-activated chloride channel (CaCC) encoded by TMEM16A or TMEM16B exists in variety tissues of many species. Our previous study has demonstrated that TMEM16A confers the CaCC in mouse cardiomyocytes. But its function in the pathological changes of the heart remodeling has not been studied. To investigate its potential role in cardiac hypertrophy, the expression of TMEM16A and the function of TMEM16A-encoded CaCC were primarily investigated in a pressure-overloaded myocardial hypertrophy mice model. A mouse model of cardiac hypertrophy was established by constriction of thoracic aorta. Three weeks later, heart weight/body weight and heart weight/tibia length ratios were evaluated to validate whether the model was successfully established. RT-PCR technique was performed to determine the expression profile of TMEM16 family in mouse heart. Western blotting and whole cell patch-clamp analysis combined with pharmacological approaches were used to determine the expression level of TMEM16A protein and the amplitude of calcium activated chloride currents (ICl.Ca) in mouse ventricular cardiomyocytes.RT-PCR results demonstrated, except TMEM16B, TMEM16G and TMEM16J, all the members of TMEM16 family were expressed in mouse cardiac tissue. Western blotting showed that the expression level of TMEM16A protein was up-regulated in myocardial hypertrophy mouse. And whole cell patch-clamp analysis revealed that the activity of TMEM16A ICl.Ca were significantly increased in the cardiomyocytes isolated from myocardial hypertrophy mouse, because the enhanced currents were significantly inhibited by a specific TMEM16A inhibitor, T16Ainh-A01. We demonstrate that the activity of CaCC encoded by TMEM16A was up-regulated in the cardiac myocytes isolated from myocardial hypertrophy mouse. Therefore, CaCC may play a potential role in regulating the pathological cardiac remodeling.
Keywords: hypertrophy CaCC TMEM16A
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No.4615644101276914****
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