【作者】 陈长源；

【导师】 郑兴； 秦永文；

【作者基本信息】 第二军医大学 ， 内科学， 2010， 博士

【摘要】 研究背景：PRKAG2 G100S是我科张静博士于2007年在一个中国人心肌肥厚合并传导异常及心室预激家系中发现的一个新的PRKAG2基因错义突变。该家系患者临床表现有心肌肥厚(56%)、预激综合征(46%)、窦房结功能不良或高度房室传导阻滞、猝死,有的还伴有房颤、房扑等房性心律失常,符合国外PRKAG2心脏综合征家系报道的共性。经过对该家系成员进行PRKAG2基因测序,发现PRKAG2基因第3外显子上出现第100位甘氨酸突变为丝氨酸(PRKAG2 G100S),考虑其可能是导致该家系患者心肌肥厚及传导系统异常的致病基因,因此我们认为PRKAG2 G100S可能导致中国人PRKAG2心脏综合征的发生。2001年Gollob等提出PRKAG2心脏综合征是一种心脏特异的糖原综合征的假说,认为心肌肥厚及心室预激等临床表型可能与心肌细胞内的过度糖原累积有关。此后,有研究认为PRKAG2基因可能参与心脏的发育过程并导致心脏旁道的形成,因此研究人员认为应从心脏发育和能量代谢异常的视角了解PRKAG2突变的致病机制。然而,最近研究发现心肌糖原累积并不能完全说明心肌肥厚的发病机制,AMPK的异常活化可能影响细胞内信号转导途径从而导致心肌肥厚。PRKAG2 G100S是首次发现的一个导致中国人PRKAG2心脏综合征的新突变,在国内外均未见报道,其引起家系患者心肌肥厚的致病机制还不清楚。为明确PRKAG2 G100S突变对心肌细胞糖代谢和心肌肥厚标志基因表达的影响,深入认识PRKAG2心脏综合征的疾病本质,本课题通过在SD乳鼠心肌细胞及H9c2胚胎心肌细胞中过表达PRKAG2 G100S突变基因,研究该突变基因是否引起心肌细胞内糖原累积、Ca2+稳态失衡、心肌肥厚标志基因表达增加,初步阐明PRKAG2 G100S新突变引起中国人PRKAG2心脏综合征的致病机制。目的：研究PRKAG2 G100S新突变对心肌细胞内糖代谢、Ca2+稳态以及心肌肥厚标志基因表达的影响。方法：(1)携带野生型及突变型PRKAG2基因的重组腺病毒载体构建：利用Invitrogen的GatewayTM技术,通过重叠PCR技术将目的基因突变体PRKAG2 G100S-IRES-EGFP及野生型PRKAG2-IRES-EGFP定向BP重组至入门载体pDONR221上,然后与腺病毒载体pAd/CMV/V5-DEST进行LR重组反应,形成共表达目的基因及增强型绿色荧光蛋白(EGFP)的腺病毒表达克隆。经筛选阳性表达克隆并测序验证后,Pad酶切、包装、扩增、纯化获得携带EGFP及PRKAG2基因的重组体腺病毒。病毒PCR鉴定并确定病毒滴度。(2)乳鼠心肌细胞的培养及重组腺病毒感染心肌细胞：培养乳鼠心肌细胞,用重组腺病毒感染并使用Western blot技术鉴定PRKAG2蛋白是否在心肌细胞内正确表达。(3)重组腺病毒感染H9c2胚胎心肌细胞及细胞内Ca2+检测：用构建好的重组腺病毒感染H9c2胚胎心肌细胞,使用Western blot技术鉴定PRKAG2蛋白是否在心肌细胞内正确表达；以Roth-2/AM为钙指示剂,使用流式细胞荧光定量技术检测细胞内Ca2+浓度。(4)重组腺病毒感染乳鼠心肌细胞及细胞内糖原含量、ANP、α-MHC、β-MHC基因水平检测：用构建好的重组腺病毒感染SD乳鼠心肌细胞,使用periodic acid-schiff (PAS)染色检测心肌细胞内糖原含量以及使用Real-time qPCR技术检测心房利钠肽(atrial natriuretic factor,ANP)、β-肌球蛋白重链(β-myosin heavy chain,β-MHC)、α-肌球蛋白重链(α-myosin heavy chain,α-MHC) mRNA的表达水平。结果：(1)成功构建了携带EGFP基因的PRKAG2突变体重组腺病毒及野生型重组腺病毒,PCR鉴定结果表明PRKAG2基因正确插入腺病毒载体。病毒的滴度为：Ad-PRKAG2-IRES-EGFP 7.8×108ifu/ml, Ad-PRKAG2 G100S-IRES-EGFP 8.4×108ifu/ml。(2)成功培养了SD乳鼠心肌细胞并建立了过表达正常或突变PRKAG2基因的乳鼠心肌细胞模型；荧光显微镜下可见突变体及野生型重组腺病毒感染后的SD乳鼠心肌细胞表达EGFP而发出绿色荧光；Western blot证明PRKAG2蛋白成功在乳鼠心肌细胞中过表达。(3)在H9c2胚胎心肌细胞成功过表达正常或突变的PRKAG2基因,Western blot证明PRKAG2蛋白过表达成功；心肌细胞内Ca2+检测显示重组腺病毒感染H9c2胚胎心肌细胞24-48小时,突变(GS)组(12.72±7.68)细胞浆内Ca2+浓度与阴性对照(PK)组(17.28±7.79)、空白对照(GFP)组(19.33±5.50)组间差异无统计学意义(P>0.05)；重组腺病毒感染H9c2胚胎心肌细胞48-72小时,突变(GS)组(24.18±2.43)细胞浆内Ca2+浓度与阴性对照(PK)组(26.24±4.77)、空白对照(GFP)组(26.04±5.71)组间差异无统计学意义(P>0.05)；重组腺病毒感染H9c2胚胎心肌细胞48小时前后,突变(GS)组、阴性对照(PK)组及空白对照(GFP)组的细胞浆内Ca2+浓度差异无统计学意义(P>0.05)。(4)重组腺病毒感染乳鼠心肌细胞后,检测乳鼠心肌细胞糖原含量并检测细胞内ANP、α-MHC、β-MHC的基因表达水平。PRKAG2G100S突变使乳鼠心肌细胞质内有明显糖原累积,而未突变组(阴性对照(PK)组、空白对照(GFP)组)糖原累积较少。重组腺病毒感染乳鼠心肌细胞48-72小时,突变(GS)组(1.00±0.04)ANP mRNA水平与阴性对照(PK)组(1.00±0.10)、空白对照(GFP)组(1.00±0.06)间差异无统计学意义(P>0.05)；α-MHC mRNA水平,突变(GS)组(1.00±0.13)与阴性对照(PK)组(1.00±0.00)、空白对照(GFP)组(1.05±0.38)组间差异无统计学意义(P>0.05)；β-MHC mRNA水平,突变(GS)组(1.04±0.33)与阴性对照(PK)组(1.00±0.04)、空白对照(GFP)组(1.09±0.53)三组间差异无统计学意义(P>0.05)。结论：(1)PRKAG2 G100S未能引起心肌细胞内钙稳态失衡,表明Ca2+及其依赖的信号转导途径可能未参与心肌肥厚的发病过程；同时表明PRKAG2 G100S导致的AMPK活性异常改变可能对心肌细胞钙稳态的影响较小。(2)PRKAG2 G100S未能引起心肌细胞内ANP、α-MHC及β-MHC mRNA水平明显改变,表明其对ANP、α-MHC及β-MHC基因的表达水平可能无明显影响。(3)PRKAG2 G100S引起心肌细胞内糖原累积,表明PRKAG2 G100S导致心肌肥厚的致病机制可能主要是心肌细胞内糖原累积。更多还原

【Abstract】 Background:PRKAG2 G100S which was identified by Doctor Jing Zhang in 2007 is a novel PRKAG2 gene missense mutation and it was responsible for familial cardiac hypertrophy with conduction abnormity and ventricular preexcitation of Chinese. The patients presented with cardiac hypertrophy (56%), preexcitation syndrome (46%), sinus node dysfunction, advanced atrioventricular block, even sudden death. Atrial arrhythmia, such as atrial fibrillation and atrial flutter also coud been observed in some family members. The clinical phenotype in this Chinese family with PRKAG2 cardiac syndrome corresponded to general characters of the foreign families. After direct DNA sequencing was used, glycolamine (100) to serine, in exon 3 of PRKAG2 was identified and shown to be present in the living affected family members. The mutation results from guanine (G) substituted for adenine (A) at nucleotide 298. Therefore, we believe that PRKAG2 G100S could be the cause of PRKAG2 cardiac syndrome in Chinese people. In 2001, Gollob et al proposed hypothesis that PRKAG2 cardiac syndrome could be a novel cardiac glycogenosis syndrome, and they believed that genetic defects in PRKAG2 may lead to a phenotype of hypertrophic cardiomyopathy with ventricular pre-excitation by virtue of the massive accumulation of glycogen. Some studies suggest that PRKAG2 gene may be involved in the heart of the development process and lead to the formation of the heart bypass, so the identification of the PRKAG2 genetic defect should lead to an understanding of the pathogenesis of accessory atrioventricular connections in relate to the developing heart and of the pathogenesis of cardiac hypertrophy in relate to imbalance of energy metabolism. However, recent studies found that pathogenesis of cardiac hypertrophy could not be fully ascribed to accumulation of glycogen in myocardial cells. Abnormal activation of AMPK could lead to cardiac hypertrophy by affecting intracellular signal transduction pathways. PRKAG2 G100S is a novel mutation which was identified the first time, so pathogenesis of PRKAG2 cardiac syndrome in Chinese people is worth studying. To understand the effect of PRKAG2 G100S mutation on glucose metabolism and cardiac myocyte hypertrophy marker gene expression, we over-expressed PRKAG2 G100S in neonatal rat cardiomyocytes and H9c2 cells in order to define whether PRKAG2 G100S would result in diversification in intracellular ANP, a-MHC,β-MHC gene level and glycogen content and to clatify the pathogenesis of PRKAG2 cardiac syndrome in Chinese people.Objective:To investigated the effect of PRKAG2 G100S mutation on glucose metabolism in myocardial cells, Ca2+homeostasis and cardiac hypertrophy marker gene level such as ANP,α-MHC,β-MHC. Methods:(1) To construct recombinant adenovirus vector carrying wide type and mutants of human PRKAG2 gene:PRKAG2 G100S-IRES-EGFP and PRKAG2-IRES-EGFP were acquired by the overlapping PCR, and were cloned directly into entry vector pDONR221 by using Invitrogen’s GatewayTM technology. Then, along with the BP and LR recombination reactions finished, the recombinant adenovirus vector containing human PRKAG2 G100S gene is constructed. The sequencing sequence was right. The pAd-PRKAG2 G100S-IRES-EGFP and pAd-PRKAG2-IRES-EGFP were digested by Pac I, packaging, amplification and purified. PCR technique was applied to detect the target gene. The titre of Ad-PRKAG2-EGFP and Ad-PRKAG2 G100S-EGFP was measured with the aid of enhanced green fluorescence protein (EGFP) expression. (2) Neonatal rat cardiomyocytes was cultured and was infected with recombinant adenovirus vector carrying wide type and mutants of human PRKAG2 gene:Neonatal rat cardiomyocytes was cultured and was infected with recombinant adenovirus vector carrying wide type and mutants of human PRKAG2 gene. Western Blot technique was applied to verify the expression of PRKAG2 protein. (3) Determine of intracellular free Ca2+:H9c2 cells was cultured and was infected with recombinant adenovirus vector carrying wide type and mutants of human PRKAG2 gene. It was determined after the cells incubated with Rohd-2/AM that Rohd-2 ratio in H9C2 cells before and after transfected 48 hours with Ad-EGFP, Ad-PRKAG2 and Ad-PRKAG2 G100S. (4)Determine of intracellular ANP,α-MHC,β-MHC gene level and glycogen content:Neonatal rat cardiomyocytes was cultured and was infected with recombinant adenovirus vector. Intracellular ANP,α-MHC,β-MHC gene level were determined by real-time qPCR technique. At last, glycogen contents in neonatal rat cardiomyocytes were detected by PAS.Results:(1) Restriction enzyme digestion analysis and the sequence analysis confirmed that PRKAG2 G100S gene was successfully inserted into the adenovirus vector. Myocardial cells which were transfected with Ad-PRKAG2 G100S-EGFP gave off strikingly bright green fluorescence, which implicated PRKAG2 protein was over-expressed. The titre of purified recombinant adenovirus Ad-PRKAG2-EGFP was 7.8×108 ifu/ml and the titre of Ad-PRKAG2 G100S-IRES-EGFP was 8.4×108ifu/ml. (2) Neonatal rat cardiomyocytes were successfully cultured and normal or mutant PRKAG2 gene was over-expressed in neonatal rat cardiac cells. Green fluorescence was seen in the cells infected with different recombinant adenovirus vectors. PRKAG2 protein was verified to be expressed successfully in neonatal rat cardiac myocytes by Western Blot. (3)Normal or mutant PRKAG2 gene was over-expressed in H9c2 cells and PRKAG2 protein was verified to be expressed successfully by Western Blot. Intracellular free Ca2+ assay using Rohd-2/AM showed that intracellular free Ca2+concentration ([Ca2+]i) in H9c2 cells which were transfected with recombinant adenovirus vectors in 24-48 hours in mutation (GS) group (12.72±7.68) was no significant difference with wild-type(PK)group (17.28±7.79) and GFP groups (19.33±5.50) (P> 0.05). In 48-72 hours, intracellular free Ca2+concentration ([Ca2+]i) in H9c2 cells which were transfected with recombinant adenovirus vectors in mutation (GS) group (24.18±2.43) was no significant difference with wild-type(PK)group (26.24±4.77) and GFP groups (26.04±5.71) (P> 0.05). (4) When neonatal rat cardiomyocytes was infected with recombinant adenovirus vectors in 48-72 hours, ANP mRNA level in mutation (GS) group (1.00±0.04) was no significant difference with wild-type(PK)group (1.00±0.10) and blank control (GFP) group (1.00±0.06)(P>0.05). In addition,α-MHC mRNA level in mutation (GS) group (1.00±0.13) was no significant difference with wild-type (PK) group (1.00±0.00) and GFP groups (1.05±0.38) (P>0.05). As toβ-MHC mRNA level, no significant difference was also found in GS group (1.04±0.33)and wild-type(PK) group (1.00±0.04) and blank control (GFP) group (1.09±0.53) (P>0.05). PAS using dyeing kit revealed that more glycogen was diffusely distributed in the cytoplasm in mutant group, however, less glycogen in WT and blank control group.Conclusions:(1) The recombinant adenovirus containing human PRKAG2 G100S gene is successfully constructed and also expressed in myocardial cells. It will be helpful for the further study on PRKAG2 gene mutation. (2)PRKAG2 G100S mutation resulted in intracellular glycogen accumulation, but it failed to result in significant change in intracellular Ca2+, ANP, andα-MHC,β-MHC mRNA level. This implied that accumulation of intracellular glycogen is probably the key of pathogenesis of PRKAG2 cardiac syndrome in Chinese people. Furthermore, inappropriate activation of AMPK secondary to the G100S PRKAG2 mutation couldn’t be associated with imbalance of calcium homeostasis. (3)PRKAG2 G100S could mainly result in disorder of myocardial cellular energy metabolism, and it had probably few disturbances in ANP,α-MHC andβ-MHC mRNA level.更多还原