PRKAG2基因G100S新突变对心肌细胞钙稳态和糖原含量的影响  

Influence of PRKAG2 gene G100S novel mutation on glycogen storage and calcium homeostasis in cardiomyocytes

作　　者：陈长源[1] 郑兴[1] 秦永文[1] 徐荣良[1] 胡建强[1] 

机构地区：[1]第二军医大学长海医院心血管内科,上海200433

出　　处：《第二军医大学学报》2010年第11期1165-1168,共4页Academic Journal of Second Military Medical University

摘　　要：目的探讨首次在国人WPW综合征(PRKAG2心脏综合征)家系中发现的一个新的错义突变PRKAG2 G100S引起心肌肥厚的可能机制。方法使用Invitrogen的GatewayTM重组系统构建含有人全长PRKAG2基因新突变体(PRKAG2 G100S)的重组腺病毒载体;以增强型绿色荧光蛋白(EGFP)为报告基因,将病毒分别感染H9c2大鼠胚胎心肌细胞和SD乳鼠心肌细胞后采用蛋白质印迹方法检测PRKAG2蛋白的表达。重组腺病毒感染H9c2大鼠胚胎心肌细胞48h前后以Rohd-2/AM孵育并测定细胞内游离Ca2+浓度;感染乳鼠心肌细胞48～72h后以过碘酸-雪夫(PAS)法测定细胞内糖原含量。结果重组腺病毒分别感染H9c2大鼠胚胎心肌细胞和SD乳鼠心肌细胞48h后荧光显微镜下可观察到绿色荧光,用PRKAG2单抗能检测到靶蛋白。与野生型PRKAG2和EGFP组比较,重组腺病毒感染H9c2大鼠胚胎心肌细胞48h后突变体PRKAG2 G100S组细胞内游离Ca2+浓度无明显变化;重组腺病毒感染H9c2大鼠胚胎心肌细胞48h前后的细胞内游离Ca2+浓度无明显变化。在重组腺病毒感染乳鼠心肌细胞48h后,PRKAG2 G100S组能检测到明显的心肌细胞内糖原累积。结论 PKRAG2G100S新突变导致心肌肥厚的主要发病机制可能是细胞内糖原累积,Ca2+及其介导的细胞内信号转导途径可能未参与心肌肥厚的发生。Objective To investigate the possible mechanism by which PRKAG2 G100S,a novel mutation recently found in a Chinese family with Wolff-Parkinson-White（WPW） syndrome,induces cardiac hypertrophy.Methods The recombinant adenovirus containing human PRKAG2 G100S was constructed using Invitrogen’s Gateway TM system and was used to infect rat embryonic H9c2 cardiomyocytes and cardiomyocytes of neonatal SD rats using EGFP as the reporter gene;Western blotting analysis was used to determine PRKAG2 protein expression.The intracellular free Ca2 ＋ was determined in the H9c2 cells before and 48 h after infected with Ad-EGFP,Ad-PRKAG2 or Ad-PRKAG2 G100S by incubating with Rohd-2/AM.The glycogen contents in neonatal SD rat cardiomyocytes were determined by PAS 48-72 h after infection.Results Bright green fluorescence was observed in the cultured H9c2 cells and neonatal SD rat cardiomyocytes 48 h after infected with Ad-PRKAG2 G100S,and PRKAG2 protein expression was identified with PRKAG2 monoclonal antibody.Compared with wild-type PRKAG2 and EGFP groups,intercellular free Ca2 ＋ concentration in H9c2 cells of Ad PRKAG2 G100S group had no noticeable changes 48 h after infection,and the concentrations were not significantly different in Ad PRKAG2 G100S group before and 48 h after infection.PAS revealed evident glycogen storage in the neonatal SD rat cardiomyocytes in PRKAG2 G100S group 48 h after infection.Conclusion Our findings indicate that myocardial glycogenosis might contribute to the pathogenesis of cardiac hypertrophy in patients with PRKAG2 G100S mutation,and the imbalance of calcium homeostasis in cardiomyocytes might not be involved in the pathogenesis.

关 键 词：PRKAG2综合征 基因 突变 钙 糖原 

分 类 号：R541.1[医药卫生—心血管疾病]