运动改善高血压肠系膜动脉LTCC和BK_(Ca)通道功能的表观遗传调控机制  

Epigenetic Regulation of Exercise-improved LTCC and BK_(Ca) Channels Function in Hypertension Mesenteric Arteries

作　　者：张严焱 徐召霞 陈渝[1] 李珊珊[1] 石丽君[1] ZHANG Yan-yan;XU Zhao-xia;CHEN Yu;LI Shan-shan;SHI Li-jun(Beijing Sport University,Beijing 100084,China)

机构地区：[1]北京体育大学运动生理教研室,北京100084

出　　处：《体育科学》2018年第11期39-49,共11页China Sport Science

基　　金：国家自然科学基金资助项目(31771312);北京市自然科学基金资助项目(5172023);中央高校基本科研业务费专项资金资助(2018XS004)

摘　　要：目的:探讨有氧运动改善自发性高血压大鼠肠系膜动脉平滑肌L型电压门控钙通道(voltage-gated L-type Ca^(2+)channel,LTCC)和大电导钙激活钾通道(large-conductance Ca^(2+)-activated K^+channel,BK_(Ca))功能的表观遗传机制。方法:12周龄雄性自发性高血压大鼠(Spontaneously hypertensive rat,SHR)及正常血压大鼠(Wistar-Kyoto,WKY),随机分为正常血压安静组(WKY-SED)、正常血压运动组(WKY-EX)、高血压安静组(SHR-SED)和高血压运动组(SHR-EX),运动组进行中等强度跑台运动训练。12周后,取肠系膜动脉,分别采用膜片钳,钙成像、免疫蛋白印迹、qPCR、亚硫酸氢盐测序技术观察血管平滑肌LTCC、BK_(Ca)通道全细胞电流,BK_(Ca)单通道门控特性,钙火花,LTCCα_(1c)、BK_(Ca)α和β1亚基mRNA、蛋白表达,α_(1c)、β1基因启动子区甲基化水平,miR-328表达。离体实验,进行血管平滑肌原代培养,转染miR-328 mimic和miR-328 inhibitor使之过表达或沉默,干扰48 h后检测α_(1c)亚基m RNA、蛋白表达变化。结果:1)有氧运动后,WKY和SHR运动组收缩压均分别显著低于各自安静对照组;2)运动显著减弱SHR肠系膜动脉平滑肌LTCC和BK_(Ca)全细胞电流密度的上调;3)运动降低高血压BK_(Ca)通道开放概率(P_o)及钙火花幅值的升高;4)运动显著抑制了SHR肠系膜动脉BK_(Ca)β1亚基mRNA、蛋白表达上调;高血压时β1基因启动子区呈去甲基化,经有氧运动后甲基化水平升高;5)LTCCα_(1c)蛋白表达在SHR-SED组显著上调,SHR-EX组α_(1c)表达下调;肠系膜动脉miR-328表达与α_(1c)亚基蛋白表达呈高度负相关;6)转染miR-328 mimic 48 h后,α_(1c)亚基蛋白表达显著下降;miR-328 inhibitor组,α_(1c)亚基表达稍上调无显著差异。结论:规律有氧运动可有效降低SHR血压,引起β1亚基启动子区甲基化,调控miR-328在转录后靶向抑制α_(1c)亚基表达,是运动改善高血压肠系膜动脉BK_(Ca)和LTCC通道功能重构的表观遗传机制之一。Objective:To investigate the epigenetic mechanism of the voltage-gated L-type Ca2+channel(LTCC)and the large-conductance Ca2+-activated K+channel(BKCa)function in mesenteric arterial myocytes improved by regular aerobic exercise in hypertension.Methods:12-week-old male SHR and WKY rats were randomly assigned to sedentary and exercise training groups,respectively.Exercise groups were performed a moderate-intensity treadmill running.After 12 weeks,patch clamp study,Ca2+image,Western blot,qPCR,bisulfite sequencing PCR were used to detect the LTCC and BKCa channel currents,BKCa single channel gating properties,Ca2+spark,mRNA and protein expression of LTCCα1c together with BKCaαandβ1 subunits,DNA methylation levels ofα1c andβ1 gene promoter region,miR-328 expression.In vitro experiment,miR-328 mimic and miR-328 inhibitor were transfected into cultured arterial myocytes to make miR-328 overexpressing or silencing,the mRNA and protein levels ofα1c subunits were determined after 48 h transfection.Results:1)After aerobic exercise,SBP in both exercise groups of WKY and SHR were significantly lower than that of their sedentary counterparts.2)Exercise normalized the increased LTCC and BKCa current density of mesenteric arterial myocytes in SHR.3)Exercise attenuated the increased single BKCa channel open probability(Po)and the amplitude of Ca2+spark in hypertension.4)Exercise inhibited the upregulated mRNA and protein expression of BKCaβ1 subunit in mesenteric arteries from SHR;β1 gene promoter was demethylation in hypertension,exercise increased the methylation level atβ1 gene promoter of SHR.5)The protein expression of LTCCα1c subunit was significantly increased in SHR,while decreased by exercise;the expression of miR-328 in mesenteric arteries was highly negative correlation withα1c subunit.6)The miR-328 overexpression by transfecting miR-328 mimic decreasedα1c subunit protein level significantly,while miR-328 inhibitor madeα1c subunit a slight increase.Conclusions:Regular aerobic exercise efficiently reduces

关 键 词：运动 离子通道 DNA甲基化 微小RNA 

分 类 号：G804.7[文化科学—运动人体科学]