CAJ | 학술논문

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-CaMKⅡ Thr286/T-CaMKⅡ)的表达变化；采用ELISA方法检测心肌匀浆 CaMK Ⅱ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 CaMK Ⅱ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)μm2比(825.5±414.9)μm2,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)μm2比(883.5±235.9)μm2,P﹥0.05]。结论 GRK5对β-AR诱导的CaMK Ⅱ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。
목적：탐토불동G단백우련수체격매( GRK)대β-신상선소수체(β-AR)유도적심기개/개조단백의뢰성단백격매Ⅱ( CaMKⅡ)활화적영향。방법웅성소서(체질량20~28 g)분위야생대조조( WT：SPF 급 C57BL/6소서,작위양성대조)、GRK2,3,5,6기인고제조( GRK2KO、GRK3KO、GRK5KO、GRK6KO)화β1、2-AR돌변조[GRK(-)：β-AR결핍GRK린산화위점,작위음성대조]。각조소서분별급여이병기신상선소( ISO)자격혹등량생리염수2주,단경처사동물,절취좌심실병박리점련조직,경액태담처리후저존우-80℃。 Western blot 용우검측유활성적 CaMKⅡ( p-CaMKⅡ Thr286/T-CaMKⅡ)적표체변화；채용ELISA방법검측심기균장 CaMK Ⅱ활성；HE염색검측심기세포조직학변화。결과급여 ISO 자격후,여양성대조 WT 소서변화상동, GRK2KO, GRK3KO화GRK6KO소서심기조직중p-CaMKⅡ표체수평명현증가(ISO자격여비자격소서비교,균위P﹤0.05),이GRK5KO여음성대조GRK(-)소서변화상동,ISO자격조여비자격조상비심기조직중p-CaMKⅡ표체무명현증가；ELISA검측야발현,ISO자격후WT급GRK2KO, GRK3KO, GRK6KO소서심기조직균장적 CaMK Ⅱ활성현저증가( ISO 자격여비자격소서비교,균위 P ﹤0.05),이GRK5KO급GRK(-)소서ISO적CaMKⅡ활성무명현증가。 HE염색발현,WT소서ISO자격여비자격조상비심기횡절면적명현증대[(1388.2±270.3)μm2비(825.5±414.9)μm2,P﹤0.05],GRK5KO소서 ISO 자격조여비자격조비교,심기횡절면적무명현차이[(837.1±112.8)μm2비(883.5±235.9)μm2,P﹥0.05]。결론 GRK5대β-AR유도적CaMK Ⅱ격활시필요적；GRK5기인고제가능통과인기CaMKⅡ표체개변이개선β-AR지구흥강유도적심기비후。
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 ( ISO-stimulated mice vs. NS mice, all P﹤0. 05) , whereas ISO-mediated CaMKⅡphosphorylation 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 CaMKⅡactivity 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)μm2 vs. WT-NS (825. 5 ± 414. 9)μm2, P﹤0. 05], but not in GRK5 knockout mice[GRK5KO-ISO(883. 5 ± 235. 9)μm2 vs. GRK5KO-NS(837. 1 ± 112. 8 )μm2,P﹥0. 05]. Conclusions GRK5 provides a specialized function in modulating β-AR stimulated, β-arrestin-mediated CaMK Ⅱ activity, as well as in developing cardiac hypertrophy in mice heart.