不同G蛋白耦联受体激酶对β-肾上腺素受体诱导心肌钙/钙调蛋白依赖性蛋白激酶Ⅱ活化的影响  

Effect of G protein-coupled receptor kinases on β-adrenergic receptor mediated activation of calcium/calmodulin-dependent protein kinase Ⅱ

作　　者：李锐[1] 柴慧娟[1] 屈扬扬[1] 李丹丹[1] 刘艳丽[1] 张玲[1] 

机构地区：[1]天津医科大学基础医学院生理及病生理学系,300070

出　　处：《中国心血管杂志》2014年第4期291-295,共5页Chinese Journal of Cardiovascular Medicine

基　　金：教育部留学回国人员科研启动基金资助项目;天津医科大学科学研究基金(2011ky33)~~

摘　　要：目的探讨不同G蛋白耦联受体激酶（GRK）对β-肾上腺素受体（β-AR）诱导的心肌钙/钙调蛋白依赖性蛋白激酶Ⅱ（CaMKⅡ）活化的影响。方法雄性小鼠（体质量20～28 g）分为野生对照组（WT：SPF级C57BL/6小鼠,作为阳性对照）、GRK2,3,5,6基因敲除组（GRK2KO、GRK3KO、GRK5KO、GRK6KO）和β1、2-AR突变组[GRK（-）：β-AR缺乏GRK磷酸化位点,作为阴性对照]。各组小鼠分别给予异丙基肾上腺素（ISO）刺激或等量生理盐水2周,断颈处死动物,切取左心室并剥离粘连组织,经液态氮处理后贮存于-80℃。Western blot用于检测有活性的CaMKⅡ（p-CaMKⅡThr286/T-CaMKⅡ）的表达变化;采用ELISA方法检测心肌匀浆CaMKⅡ活性;HE染色检测心肌细胞组织学变化。结果给予ISO刺激后,与阳性对照WT小鼠变化相同,GRK2KO,GRK3KO和GRK6KO小鼠心肌组织中p-CaMKⅡ表达水平明显增加（ISO刺激与非刺激小鼠比较,均为P〈0.05）,而GRK5KO与阴性对照GRK（-）小鼠变化相同,ISO刺激组与非刺激组相比心肌组织中p-CaMKⅡ表达无明显增加;ELISA检测也发现,ISO刺激后WT及GRK2KO,GRK3KO,GRK6KO小鼠心肌组织匀浆的CaMKⅡ活性显著增加（ISO刺激与非刺激小鼠比较,均为P〈0.05）,而GRK5KO及GRK（-）小鼠ISO的CaMKⅡ活性无明显增加。HE染色发现,WT小鼠ISO刺激与非刺激组相比心肌横截面积明显增大[（1388.2±270.3）μm^2比（825.5±414.9）μm^2,P〈0.05],GRK5KO小鼠ISO刺激组与非刺激组比较,心肌横截面积无明显差异[（837.1±112.8）μm^2比（883.5±235.9）μm^2,P〉0.05]。结论 GRK5对β-AR诱导的CaMKⅡ激活是必要的;GRK5基因敲除可能通过引起CaMKⅡ表达改变而改善β-AR持久兴奋诱导的心肌肥厚。Objective To investigate in vivo whether a certain subset of G protein-coupled receptor kinases（ GRKs） is required for β-adrenergic receptor（ β-AR） activation of calcium /calmodulin-dependent protein kinase Ⅱ（ CaMK Ⅱ） in heart. Methods Male mice（ body weight 20-28 g） WT（ positive control）, GRK2, 3, 5, 6 knockout and GRK（-） mice（ transgenic mice with cardiac-specific overexpression of a mutant β1,2-AR that lacks GRK phosphorylation sites,as negative control） were used in this experiment. Mice were administered with isoproterenol（ β-AR agonist,ISO） or saline with the same volume. After sacrificing the mice,left ventricle of the heart were excised and immediately frozen in liquid nitrogen and stored at- 80℃ for later biochemical assays. Western Blot was used to detect the expression of CaMK Ⅱ,phospho-CaMK Ⅱ（ Thr286）. CaMK Ⅱ activity of ventricular homogenates was measured by a CaMK Ⅱ assay kit based on ELISA principle. HE staining was used to detect the histological change.Results After ISO administration,the levels of phosphorylated CaMK Ⅱ（ pCaMK Ⅱ; the active form of CaMK Ⅱ） were markedly increased in hearts of WT,GRK2,GRK3 and GRK6 knockout mice（ ISOstimulated mice vs. NS mice,all P〈0. 05）,whereas ISO-mediated CaMK Ⅱ phosphorylation was markedly blunted in hearts from GRK5 knockout mice,as well as in hearts from GRK（-） mice. Furthermore,CaMK Ⅱ activity detected by ELISA method in the heart extracts from WT,GRK2,GRK3 and GRK6 knockout mice were significantly increased followed by ISO stimulation（ ISO-stimulated mice vs. NS mice,all P〈0. 05）,whereas no obvious increase of CaMK Ⅱ activity was observed in hearts from GRK5 knockout and GRK（-） mice. The myocyte cross-sectional area were significantly increased by ISO stimulation in WT mice [WT-ISO（ 1 388. 2 ± 270. 3） μm^2vs. WT-NS（ 825. 5 ± 414. 9） μm^2,P〈0.05],but not in GRK5 knockout mice[GRK5KO-ISO（ 883. 5 ± 235. 9） μm^2vs. GRK5KO-NS（ 837. 1 ± 112. 8） �

关 键 词：G蛋白耦联受体激酶 Β-肾上腺素受体 钙 钙调蛋白依赖性蛋白激酶 心肌肥厚 

分 类 号：R363[医药卫生—病理学]