Dystroglycan缺陷肌营养不良相关性心肌病发病的细胞与分子机制研究
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摘要
研究背景与目的
一百五十年前人们首次认识到肌营养不良病人合并相关的心脏病,至今临床上仍难以治愈。具有抗肌萎缩蛋白糖蛋白复合物(Dystrophin GlycoproteinComplex,DGC)遗传性缺陷的肌营养不良病人当中会有70—90%的人合并心血管疾病,并且多达20%的病人最终死于心力衰竭。
研究表明DGC是维持心肌和骨骼肌正常结构与功能的关键性复合体,具有双重的机械性的与非机械性的膜稳定与信号转导作用,与肌营养不良症相关性心肌病的发病关系密切。DGC是一种多缘嵌合体,包含抗肌萎缩蛋白(Dystrophin)、肌营养不良蛋白聚糖(Dystroglycan,DG)和一个肌聚多糖复合物(Sarcoglycan(SG)/Sarcospan)。其中,DG是DGC的中心蛋白,DG产生于一种前体肽,这种前体肽可裂解成一种位于细胞外的高度糖基化的αDG蛋白(α-DG)和一种跨膜的βDG蛋白(β-DG)。细胞外高度糖基化的蛋白α-DG通过其高亲和力与细胞外基质蛋白如层黏连蛋白(Lamininα_2)结合从而介导了细胞骨架和基底膜之间的连接。跨膜蛋白β-DG位于细胞内的一端结合于Dystrophin,后者又结合于肌动蛋白(Actin);位于细胞外的另一端则结合于α-DG。
上述这些环节当中的任一元素突变,都会产生临床上表现各异即不同表型的肌营养不良。例如,dystrophin绝对或相对缺乏或不足导致杜兴氏肌营养不良(Duchenne Muscular Dystrophy,DMD)或贝克氏肌营养不良(Becker MuscularDystrophy,BMD),前者常伴有心肌受累和智能障碍,后者进展相对较缓呈相对良性病程表现。编码γ-、α-、β-与δ-SG亚单位的的基因突变将分别产生相应的肢带型肌营养不良(Limb Girdle Muscular Dystrophy,LGMD)的相关亚型LGDM-2C、2D、2E、2F。DG是已知的和推想的糖基化酶蛋白的分子靶子,参与α-DG糖基化的酶蛋白的突变会导致DG的异常糖基化,致使DG作为骨骼肌细胞外基质受体的这一重要功能遭到破坏,与骨骼肌中的配体的结合活性丢失,引发相关的先天性肌营养不良(Congenital Muscular Dystrophy,CMD)。在这些已经确认的疾病当中,同时合并心肌病变是其常见且严重的并发症。因此,在DG的糖基化和功能方面的缺陷是几种人类肌营养不良相关性心肌病的起因。
蛋白糖基化途径常常混杂交错,所以有学者作出假设:其他蛋白可能是此酶学途径的靶点并与疾病的表型有关?由此便产生了一个逻辑上的问题,DG基因的遗传性消除能否在小鼠身上重复出许多甚至所有的疾病表型?DG敲除小鼠是胚胎致死性的,原因是对啮齿类动物来说这是特异性致死性的发育缺陷。因此,目前尚没有一种遗传性疾病的相关描述涉及到α-或β-DG的自身突变,也就是说目前尚未能在任一遗传性疾病当中确认以DG自身突变为主的病变。
本文研究者利用Cre-LoxP技术获得了心肌组织特异性DG缺失的基因突变小鼠M1c2vCre,使得有关DG在心肌这一特定组织中的结构与功能研究成为可能。首先,将确认DG缺失是否能够充分且必要地解释蛋白糖基化缺陷肌营养不良相关性心肌病的发生;其次,将探讨心肌细胞DG基因突变在肌营养不良相关性心肌病的发病中所起的结构性和/或功能性的作用及其确切的作用机制;再次,要研究与此相关的心肌细胞存活信号转导途径上可能参与的酶的磷酸化水平以及其他可能参与其中的蛋白水解酶系统,以积极寻求潜在的临床治疗的新策略。
综上所述,患有DMD,BMD,与SG相关的退行性LGMD的病人,以及mdx小鼠和β-、δ-和γ-SG基因敲除LGMD小鼠模型常常发展为心肌病,然而,肌营养不良相关性心肌病的发病机制尚未完全为我们所了解。目前,DG与DGC在引起肌营养不良相关性心肌病中所起的作用及其机制也仍不明了,可能的原因分析如下:首先,DGC蛋白复合物在心肌组织系统表达分布和功能比较复杂,心肌中DGC破坏可使得心肌细胞易感于膜损伤;其次,是否每一种蛋白在DGC中主要地起到结构性的或是功能性的作用远未确定下来;再者,虽然具有骨骼肌蛋白糖基化异常的病人可发展为心肌病,但在心肌病本身DG的作用及作用机制尚未确立。本文将采用靶基因敲除和靶基因转导小鼠模型,使用在体和离体的研究方法,着力阐释DG在心肌组织系统的结构与功能,探讨DG缺陷导致的与肌营养不良相关的心肌病的发病机制,为肌营养不良相关性心肌病提供一种确定机制和治疗靶向的手段。
实验材料与方法
一、基因缺陷及转基因小鼠的获得及种系的维系:
应用Cre-LoxP技术来组织选择性地敲除靶基因DG。本文所用的M1c2vCre(Cre+/floxed DG)是心肌组织特异性DG敲除小鼠,其对照为相应的统一在floxed/floxed背景下的野生型小鼠,基因型为Cre-/floxed DG。
利用转基因技术获得过表达钙蛋白酶抑制蛋白(Calpastatin,CAST)的转基因小鼠(CAST Tg),再与floxed DG基因型背景的小鼠交配,获得目标转基因小鼠(CAST Tg;Cre+/floxed DG)。
PCR行基因型测定及动物筛选。
二、细胞裂解物或心肌组织匀浆的制备与SDS-PAGE及WesternBlotting:
拟收集的培养细胞用PBS洗掉培养基,加入等量的细胞裂解液;拟裂解的心肌组织加入10倍于其体积的组织裂解液,进行研磨并裂解。使用超声震碎仪将细胞或组织进一步超声震碎10秒钟形成细胞或组织匀浆,14000 rpm高速离心2分钟以沉淀残片,Dc蛋白分析法对细胞或组织匀浆行蛋白浓度定量测定。
将制备好的匀浆样本加样至3-15%梯度凝胶胶孔中行电泳分离蛋白带(SDS-PAGE),然后将分离后的蛋白转印至PVDF膜上拟行免疫印迹(Westernblot),5%脱脂干奶粉封闭1小时后分别使用相应的一抗和辣根过氧化物酶标记的二抗进行孵育各约1小时,TBS-T洗膜后用增强的化学发光法(ECL)的底物显影并摄片。
三、DG蛋白纯化与laminin结合分析:
使用琼脂糖麦胚凝集素(agarose Wheat Germ Agglutinin,agarose-WGA)与N-Acetylglucosaminyl Glycoprotein(NAG)对心肌组织匀浆进行DG蛋白纯化,对纯化后的蛋白进行(1)Western Blot;(2)Laminin膜相层叠分析,程序类似与Western blot;(3)Laminin固相结合实验,使用96孔板,以50μl纯化蛋白样品铺底,再分别添加浓度梯度为0,0.02,0.1,1,2,10,20 nM的laminin孵育1.5小时,每个浓度于每种样品需重复3次,洗孔后再分别添加抗laminin的一抗和相应的二抗孵育,最后加以显色剂及2 M的浓硫酸终止颜色反应,放入色谱仪中读取数据。
四、心肌组织标本冰冻切片的制备与组织化学染色与免疫荧光染色:
心脏组织置于软木塞上承载,置于-120℃的二甲基丁烷(即异戊烷)30秒,再置于-196℃的液氮中进一步冷冻固定,最后置于-80℃的冰箱保存。使用冰冻切片机(Cryostat)在横断面上对组织进行切片制备,留待组织化学和免疫荧光染色。
按照成型的组织化学染色组套程序进行HE(Haematoxylin & Eosin)、SR(Sirius Red)染色以及FG(Fast Green)反染。免疫荧光染色分别使用一抗和相应的荧光标记的二抗,免疫荧光显微镜观察并照相。
五、测定成年小鼠心肌细胞内的PI3K-AKT信号转导途径:
PI3激酶信号转导途径于在体水平测定可分别通过对整体心脏组织匀浆进行Western Blot免疫印记的方法来获得。分别使用抗磷酸化AKT和抗磷酸化GSK3β以及抗总AKT和抗总GSK3β的相应的一抗和二抗。
为确定是否DG与配体之间的相互作用为AKT激活所必需,行成年小鼠心肌细胞原代培养,并导入外源性laminnin,孵育24小时后收集细胞,裂解并超声震碎,然后行Western Blot,抗体及方法同前,分别测定导入外源性laminin之前之后的AKT磷酸化及GSK3β磷酸化水平。
六、离体实验——成年及新生小鼠心肌细胞的原代培养:
1、成年小鼠心肌细胞的原代培养:
本实验使用的成年小鼠心肌细胞培养方法及病毒转导方法按我们已发表文献进行。通过升主动脉套管固定悬挂小鼠心脏于改进的Langendorff心脏灌流系统上行酶学消化,消化后取下心脏小心去除心房和主动脉后对心室肌进行心肌细胞的分离并终止酶学消化。显微手术镊子于镜下轻柔分离心肌细胞。反复离心重悬获得的心肌细胞,最后以含有5%胎牛血清的被覆培养基以悬浮并铺被细胞至事先以56 nM的laminin包被半小时以上的培养皿盖玻片的表面,密度约20000个细胞/片。2小时后,将被覆培养基换成常规培养基。常规培养基每天更换一次。
CAST腺病毒由AdEasy XL腺病毒载体系统产生,使用2637 bp小鼠CASTcDNA,重组腺病毒(AdCAST)在HEK293细胞系中得以扩增并提纯。使用噬菌斑分析来确定病毒的滴度为1-2.4x10~9 pfu/ml。细胞贴附2小时后,凭借200μl培养基向心肌细胞转导200-480 MOI AdCAST孵育1小时,然后转为常规培养基进行细胞培养。24-48小时后收集培养的心肌细胞,裂解后SDS-PAGE蛋白电泳及Western Blot免疫印迹。
2、新生小鼠心肌细胞的原代培养:
出生后一天的新生小鼠直接断头开胸取心脏,0.25%的Trypsin-EDTA消化裂解心肌组织、分离心肌细胞,每次10分钟,反复2-3次,直至无明显组织块残留,等量培养基终止消化,心肌细胞悬液以1000g离心4分钟,弃上清,重新悬浮细胞,分置于7 nM纯化的laminin包被的培养皿中,放入37℃与5%CO_2的温箱中进行培养,24小时后PBS小心冲洗并取出附着细胞用的盖玻片,进行免疫荧光染色,免疫荧光显微镜观察细胞贴附和基质在细胞表面组装的情况。
七、在体实验——小鼠心肌急、慢性负荷效应模型建立:
为了评价急、慢性机械张力对成年DG缺陷小鼠心肌产生的效应,对月龄相当的同系伙伴野生型与突变型小鼠进行平板运动负荷试验来激发负荷对心肌的急性期效应,运动后5小时左右杀鼠取心脏,部分标本用以制备组织标本冰冻切片,使用相应的心肌标志物抗体行免疫荧光染色,部分心肌组织标本被裂解留待Western blot;而关于获得慢性增加的心室压力负荷则行腹主动脉缩窄术(AAB),野生型与突变型小鼠都设假手术组,术后4周杀鼠取心肌组织制备冰冻切片待行HE、SR及FG反染等组化染色。
八、蛋白分析及统计学分析软件与方法:
使用Alpha Innotech提供的蛋白分析软件对Western blot所获得的蛋白带结果进行定量分析;使用Prism 5.0统计学软件进行数据处理,结果以均值±标准误表示,二组组间比较采用t-检验,多组组间比较采用Anova方差分析,采用线性回归制作Dc蛋白定量分析中的蛋白浓度计算方程,使用非线性回归方程制作laminin固相结合实验的曲线图形。
实验结果
1、M1c2vCre小鼠意味着在小鼠心肌组织特异性地DG表达丢失,抗α-DG(IIH6)抗体行免疫荧光染色,结果显示野生型小鼠心肌细胞边界清晰连贯,而突变型小鼠心肌细胞边界则模糊混杂似补丁形。
2、DG蛋白纯化后的Western Blot以及与laminin的结合活性曲线显示DG在组织特异性DG敲除小鼠中几乎缺如,α-DG与细胞外基质蛋白配体laminin的结合性近乎丧失。另行DG与老化的关系研究发现老龄小鼠DG与laminin的结合活性下降,但这种差异尚未达到统计学差异。
3、新生幼鼠心肌细胞培养发现组织特异性DG缺陷小鼠的心肌细胞与laminin包被的表面的贴附力较野生鼠显著减弱,基质组装几近丧失。而在成年小鼠这种差异并不显著。
4、心肌组织细胞内的PI3K-AKT细胞存活信号转导途径检测显示DG缺失突变型小鼠AKT和GSK3β(活性AKT的下游)磷酸化水平较对照低,提示DG具有参与细胞存活信号转导途径这一重要功能。导入外源性laminin之后对照组野生型小鼠的心肌细胞出现较高水平的磷酸化AKT与磷酸化GSK3β,而对DG敲除小鼠的心肌细胞则没有此作用,提示laminin是通过DG将细胞存活信号传递给心肌细胞的,DG与配体之间的相互作用是AKT活化所必需,DG是不可或缺的极为重要的信号转导中介。
5、小鼠急、慢性心肌负荷试验显示心肌细胞对心肌负荷的敏感性增加而易损。AAB术后4周小鼠心肌组织切片HE染色显示心肌组织学改变以及慢性炎症反应改变,DG缺失突变型小鼠心肌组织肌纤维结构紊乱,部分肌纤维连续性中断,肌浆染色不均匀,肌间隙大小不一,炎症细胞多成片状浸润;SR染色及FG反染显示DG缺失突变型小鼠心肌组织胶原沉淀提示心肌局灶性组织损伤与重构。平板运动负荷试验后5小时小鼠心肌组织冰冻切片免疫荧光染色显示基因突变型小鼠的细胞膜破损,cTnI与cTnT弥散分布在受损的心肌细胞及细胞间隙。
6、平板运动负荷实验后几种常见的蛋白水解酶钙蛋白酶(Calpain,CAPN)的底物蛋白如cTnI、cTnT、Talin有不同程度的降解,其中,cTnI的降解率在突变型小鼠为13.60%,显著高于对照野生型小鼠6.81%的降解率,P＜0.01;cTnT的降解率在突变型小鼠为21.20%,显著高于对照野生型小鼠10.55%的降解率,P＜0.01;talin的降解率在突变型小鼠为3.01%,高于对照野生型小鼠2.28%的降解率,但尚未达到统计学差异。
7、AdCAST转染心肌细胞原代培养蛋白表达的结果显示AdCAST转染组的CAPN底物蛋白cTnI、cTnT、Talin的表达量均显著高于非转染对照组,P＜0.05;而CAPN的表达量则显著降低,P＜0.05。
8、Western blot与Zymogram胶分析结果显示转基因小鼠能够高表达人钙调蛋白抑制蛋白(hCAST),且高度抑制CAPN活性,而在非转基因小鼠几乎未能检测到完整片段hCAST,CAPN活性正常。重复平板运动负荷试验,显示CAPN表达在非转基因突变组于急性负荷后显著增加,而在转基因组对照与突变小鼠均受到显著抑制,后二者比较无统计学差异。转基因后对照与突变小鼠比较,CAPN底物蛋白cTnI、cTnT、talin等未显示明显降解,各组蛋白表达量无显著差异。
结论
1、与肌营养不良相关的复合物DGC当中的中心蛋白DG在心肌组织特异性缺乏的动物模型的成功建立,使得直接研究DG在肌营养不良相关的心肌病的发病中的结构性和功能性作用及其相应的细胞分子机制成为可能。
2、DG至少在幼鼠时期的细胞基质组装中起重要作用。
3、DG参与PI3K/AKT细胞存活信号转导,是不可或缺的极为重要的信号转导中介。
4、心肌细胞组织特异性DG缺失导致DGC破坏使得心肌细胞对心肌负荷的敏感性增加而更易感于膜的损伤。
5、钙蛋白酶系统参与了组织特异性DG缺失的心肌细胞对心肌负荷的损伤反应过程。
6、高水平的钙蛋白酶抑制蛋白表达可有效地降低钙蛋白酶的活性及表达,尤其有效地抑制了在DG缺陷时异常激活的钙蛋白酶,提高心肌损伤的阈值,某种程度上对心肌起到保护作用,为DG缺陷肌营养不良相关性心肌病的基因与药物治疗提供有益的临床线索和治疗新策略。
Background and Specific Aims
Heart disease was first identified in human muscular dystrophy one hundred and fifty years ago,there has been no cure so far.70-90%of muscular dystrophy patients, that have genetic defects in the dystrophin glycoprotein complex(DGC),have cardiovascular disease,and about 20%succumb to heart failure.
The DGC is a critical complex for the maintenance of the structure and the function of normal cardiac and skeletal muscle,as a mechanosignaling complex with dual mechanical and nonmechanical membrane stabilizing functions,playing a pathogenic role in the cardiomyopathy associated with muscular dystrophy.DGC is a multimeric transmembrane protein complex,containing dystrophin,dystroglycan(DG) and a sarcoglycan(SG) / sarcospan complex.DG is the central protein in DGC.DG is generated from a propeptide,which is cleaved into a heavily glycosylated extracellularαDG protein(α-DG) and a transmembraneβDG protein(β-DG).α-DG is heavily glocosylated and completes the link from the cytoskeleton to the basal lamina by binding with high affinity to extracellular matrix proteins,such as laminin.β-DG binds intracellularly to dystrophin,which binds the actin cytoskeleton,and binds extracellularly toα-DG.
Mutations in any one of the above components result in variable phenotypes of muscular dystrophy.For example,absolute or relative deficiency in dystrophin leads to Duchenne Muscular Dystrophy(DMD) or Becker Muscular Dystrophy(BMD).DMD usually complicates with cardiac muscle problem and mental retardation,BDM has a slower down progression.The mutations of the genes encodingγ-,α-,β-,andδ-SG subunits generate Limb Girdle Muscular Dystrophy(LGDM) -2C,2D,2E and 2F, respectively.DG is the molecular target for mutations in known and putative glycosylation enzymes that lead to abnormal glycosylation of DG,and its dysfunction as an extracellular matrix receptor,and the loss of binding with the ligand in skeleton muscle,which is responsible for severe congenital muscular dystrophies.The common and severe complication of these disorders is the associated cardiomyopathy.Therefore, the defects in glycosylation and function of DG are a cause of several human muscular dystrophies and associated cardiomyopathies.
Because the glycosylation pathways are often promiscuous,several groups have hypothesized that other proteins may be targets for this enzymatic pathway and responsible for disease phenotypes.One logistic question,does genetic ablation of DG gene recapitulate many or all of the disease phenotypes in mice? The DG knockout mouse is embryonic lethal due to a developmental defect specific to rodents.Therefore, currently,there's no description about mutations ofα- orβ-DG itself involved in genetic disorders.
Using Cre-LoxP technology to specifically delete the DG gene in heart to obtain cardiac muscle tissue-specific DG knockout mice will allow us to study the structural and functional roles of DG in cardiovascular system.First,it will be identified that whether the loss of DG is sufficient and necessary to explain the occurrence of the cardiomyopathy associated with muscular dystrophy due to abnormal glycosylation. Second,it will be explored that what role the mutation of DG plays in associated cardiomyopathy,structural or functional,and what the mechanism is.Third,it will be investigated that whether DG is involved in cell survival signaling,and what the phosphorylated levels of the associated enzymes are,and is there any other possible proteolysis system involved in,so that the novel potential strategy for clinical treatment will be sought out.
As alluded to above,patients with DMD,BMD,and SG associated LGMD,mdx mice and knockout mouse models ofβ-,δ- andγ-SG associated LGMD often develop cardiomyopathy.The mechanisms of cardiomyopathy in muscular dystrophies have not been fully understood.Currently,the mechanisms by which DG and DGC cause cardiomyopathies associated with muscular dystrophy still remain elusive.The possible reasons are,first,the expression distribution and function of DGC in cardiovascular system is complicated,the disruption of DGC may render cardiac myocytes sensitive to membrane damage.Second,whether each protein primarily plays a structural or a functional role within DGC is far from defined.Third,although patients with abnormal glycosylation in skeleton muscle can develop cardimyopathy,the mechanic role of DG in cardiomyopathy has not been established.Using combinations of mutant and genetarget mouse models,and in vivo and in vitro approaches,the research is uniquely suited to address the questions:what the roles DG play in cardiovascular system are, structural or functional or both,what the mechanism by which DG defects cause the cardiomyopathy associated with muscular dystrophy is,providing a means by which the mechanism and target for treatment of cardiomyopathy associated muscular dystrophy can be identified.
Materials and Methods
1.Generation and analysis of Cradiac tissue-specific DG knockout mice(Mlc2vCre) and calpastatin transgene(CAST Tg) mice and strains' maintenance.
Generate cardiac tissue-specific DG knockout mice Mlc2vCre using Cre-LoxP technology.The mutant mouse is Cre+/floxed DG,the control wild type is Cre-/floxed DG.
Generate calpastatin transgene mice using transgene technology.Then cross the CAST Tg mice with Mlc2vCre mice to generate target mouse model whose genotype is CAST Tg;Cre/floxed DG.
Genotyping and screening by PCR.
2.Lysate preparation from cultured cells or cardiac muscle tissue and SDS-PAGE and Western blotting.
Cells were washed with PBS and scraped in equal amounts of a lysis buffer. Cardiac muscle tissue was homogenized in 10 volumes of sample size's lysisi buffer. The samples were sonicated for 10 sec and centrifuged at 14000 rpm for 2 min to pellet cell or tissue debris.All of the resulting cell lysates was loaded or volumes were normalized to the total protein content determined using Dc Protein Assay.Proteins were separated by a 3-15%gradient SDS-PAGE and then transferred to a polyvinylidene fluoridine membrane.The membranes were blocked with 5%nonfat milk in TBS(150 mM Nacl,20 mM Tris pH 7.5) followed by incubation with primary antibody and secondary antibody conjugated to horseradish peroxidase,respectively. The membranes were developed using an enhanced chemiluminiscence assay.
3.DG protein purification and laminin binding assay.
To obtain purified DG protein using agarose wheat germ agglutinin(agarose-WGA) and N-acetylglucosaminyl glycoprotein(NAG).Western Blot and laminin Biniding assay(both membrane phase and solid phase) were performed on the purified DG protein,the rational and procedure of the memebrane phase assay are similar to Western Blot's.The solid phase was performed in 96-well microplate coated with gradient concentrations of laminin,0,0.02,0.1,1,2,10,20 nM,incubating for 1.5 hours,triplicate for each sample,incubated with anti-laminin and responding secondary antibody incubation,then washed,finally added developing reagent and 2M H_2SO_4 to stop the color reaction,plating the microplate to the reader to read data.
4.Frozen tissue specimen preparation and slicing and histochemical staining and immunosatining.
Heart tissue was placed onto the cork,dipped into -120℃isopentane for 30 seconds,then stayed in -196℃liquid nitrogen to be frozen,stored in -80℃freezer. Using cryostat did the slicing with transection for later histochemical staining and immunostaining.
Follow the protocol to work on the histochemical staining,such as HE,SR staining and FG counterstaining.Immunostaining was performed using primary antibody and responding secondary antibody with immunofluroresence label.
5.Assessment of PI3k-ARK signaling in adult cardiac myocytes.
The activation of PI3 kinase signaling pathway in hearts in vivo will be assessed in whole heart homogenates by Western blotting using anti-phospho-AKT and antiphospho -GSK3βas well as the overall levels by anti-AKT and anti-GSK3β.
To determine if the interaction of DG with ligands is necessary for the activation of AKT,adult cardiac myocytes will be isolated from the same group of animals and phosphorylations of AKT and GSK3βwill be assessed prior to and after the addition of exogenous laminin as the above procedures.
6.Mouse cardiac myocytes primary Culture _ in vitro study.
(1) Adult mouse cardiac myocytes primary culture.
Adult mouse cardiac myocytes primary culture was performed by protocol worked out based on our previous studies.The heart was cannulated via the ascending aorta and mounted on a modified Langendorff perfusion apparatus.The heart was perfused with Myocyte Buffer and Enzyme Solution.The atria and aorta were removed;the ventricles were cut in two pieces,disaggregated gently with Dumont #5/45 forceps.Cells were pelleted and resuspended for a couple of times.Finally,cells were resuspended in Plating Media and plated into glass coverslips pre-coated with 56 nM laminin for over 30min.The plating density was 20,000 cells/coverslip.After 2 h,serum-containing Plating Media was switched to Culture Media.Culture Media was changed daily.
Recombinant adenovirus preparation and transduction.The calpastatin adenoviruses were generated with the AdEasy XL adenoviral vector system using a 2637-bp mouse calpastatin cDNA.Recombinant virus was amplified in HEK293 cells and purified using a cesium chloride gradient as described.The titer of the virus was assessed by a plaque assay and was 1-2.4×10~9 plaque forming units(pfu) per 1 ml. Following 2 h of attachment,cardiac myocytes were transduced with 200-480 MOI AdCAST in 200μl culture Media for 1h.After 24-48 hours,collect cells which were lysed by lysis buffer.The SDS-PAGE and Western Blot were performed on Lysate.
(2) Newborn mouse cardiac myocytes primary culture.
Newborn pups at age of 1 day old was decapitated and the heart was removed from chest cavity and dissociated by 0.25%Trysin-EDTA and the cells were centrifuged at speed of 1000g for 4min and resuspended in media and plated into glass coverslips pre-coated with 7 nM purified laminin for about 120min,and placed in 37℃, 5%CO_2.After 24 hour,the coverslips were removed for immunostaining,watching cells attachment and matrix assembly and picture-taking under the immunofluorescence microscope.
7.The establishment of Acute and Chronic heart mechanic strain overloads mouse model _ In vivo study.
In order to assess the effects of acute and chronic mechanic strain on adult DG deficient myocardium,treadmill exercise stress will be performed on mutant Mlc2vCre-DGnull mice compared to littermate controls.After 5 hours,the subjected mice were killed and the heart tissue was partly frozen for slicing and immunostaining thereafter,and was partly lysed for later Wetsern blotting.Chronic increases in ventricular pressure can be accomplishment by suprarenal abdominal aortic banding (AAB).Mutants and controls were both divided into operation and sham-operation groups.After 4 weeks,the mice were killed and the heart tissue was frozen and slicing for later HE,SR staining and FG counterstaining.
8.Protein analysis and statistics software and method.
Quantified protein acquired by western blot using software provided by Alpha Innotech.Statistics software-Prism 5.0 was used for data analysis such as t-test,Anova, linear regression and nonlinear regression,data were shown as mean±se.
Results
1.Mlc2vCre is the heart tissue-specific DG knockout mouse.Frozen sections were stained with an antibody againstα-DG(ⅡH6).The mutants displayed the indistinct, discontinuous,patched borders of the cardiac myocytes compared to controls with distinct,continuous borders.
2.Western blotting and laminin binding assay(solid phase) for purified DG showed that there is almost no DG expression or loss of ability ofα-DG to bind laminin. Furthermore,the additional study on the relationship between DG and aging showed that the activity ofα-DG binding to laminin decreased,but no significant difference.
3.Neonatal mice cardiac myocytes primary culture showed reduced cells attachment and lack of matrix assembly in Mlc2vCre lacking DG compared to controls but no defect in assembly in adult cardiac myocytes.
4.Assessment of PI3k-ARK signaling in adult myocytes showed that lower levels of phosphorylated AKT and GSK3β(down stream of active AKT) was obtained in Mlc2vCre-DGnull mice whole heart homogenates compared to littermate controls, which indicates DG is involved in this cell survival signaling.Higher levels of phospho-AKT and phospho-GSK3βwere found after laminin addition to control myocytes,but had no effect on DG null cardiac myocytes,which indicates that laminin confers a cell survival signal to cardiac myocytes through DG and the interaction of DG with ligands is necessary for the activation of AKT,DG is indispensable to mediate this critical signaling transduction.
5.Assessment of the effects of acute and chronic mechanic strain on adult DG deficient myocardium showed that cardiac myocytes lacking DG was more sensitive to membrane damage due to overload.In Mlc2vCre mice 4 weeks after AAB,HE staining showed the disorders of cardiac myofibers,part of which discontinued,presenting uneven cytoplasmic staining,varied in intercellular space,infiltration of inflammatory corpuscle.SR staining and FG counterstaining showed that focal areas of collagen deposition suggesting focal regions of damage and remodeling in mutants,was performed on mutant Mlc2vCre-DGnull mice compared to littermate controls.In Mlc2vCre mice 5 hours after treadmill exercise stress,immunostainings showed that membranes of the cardiac myocytes were damaged severely and some cytoplasmic proteins such as cTnI and cTnT that leaked out through the disrupted membranes.
6.Western blotting after treadmill exercising showed that the common substrates of Calpain(CAPN) such as cTnI,cTnT,and talin were degraded to some degrees.The mutant's degradation percentages compared to the control's are cTnI 13.6%vs 6.81% with p＜0.01,cTnT 21.2%vs 10.55%with p＜0.01,talin 3.01%vs 2.28%without significant defference.
7.Assement of the effects of AdCAST transfection on proteins expression in cardiac myocytes primary culture showed that the expression of the CAPN substrates such as cTnI,cTnT,and talin were significantly higher in AdCAST transfection group compared to no transfection group,p＜0.05,on the contrary,the expression of CAPN significantly lower in transfection group,p＜0.05.
8.The Western blotting and Zymogram analysis showed that Tg mice have the much higher expression of hCAST by which CAPN activity was highly inhibited, whereas there is almost no detection of intact hCAST but CAPN was normally activated.Repeating treadmill exercising showed that CAPN expression in CAST nontransgene(NTg) group the mutant subgroup increased significantly.In CAST Tg group either mutant or control mice CAPN was inhibited signicantly,there is no significant difference between these two subgroups.Futhermore,there are no differences in CAPN substrates such as cTnI,cTnT,and talin between these two subgroups.
Conclusions
1.The establishment of cardiac tissue-specific DG knockout mouse model renders it possible to directly study the structural and functional roles that DG,the central protein in DGC associated with muscular dystrophy,plays in cardiomyopathy associated with muscular dystrophy,and the associated cellular and molecular mechanism.
2.DG at least plays critical role in matrix assembly in neonatal mice.
3.DG is involved in the PI3K/ART cell survival signaling,and is indispensable to mediate this critical signaling transduction.
4.Cardiac tissue-specific DG deficient leading to the disruption of DGC renders cardiac myocytes sensitive to membrane damage due to heart overload.
5.Calpain protease system is involved in the development of the injury responding to mechanical strain overload on DG deficient heart.
6.Overexpression of calpastatin can effectively decrease calpain activity and expression,especially in DG deficient heart with highly inhibition of abnormally activated Calpain,to some degree,playing a role in cardiac protection and providing a beneficial clue and a novel strategy for gene therapy and clinic medication of muscular dystrophy and associated cardiomyopathy due to DG deficient.
一百五十年前人们首次认识到肌营养不良病人合并相关的心脏病,至今临床上仍难以治愈。具有抗肌萎缩蛋白糖蛋白复合物(Dystrophin GlycoproteinComplex,DGC)遗传性缺陷的肌营养不良病人当中会有70—90%的人合并心血管疾病,并且多达20%的病人最终死于心力衰竭。
研究表明DGC是维持心肌和骨骼肌正常结构与功能的关键性复合体,具有双重的机械性的与非机械性的膜稳定与信号转导作用,与肌营养不良症相关性心肌病的发病关系密切。DGC是一种多缘嵌合体,包含抗肌萎缩蛋白(Dystrophin)、肌营养不良蛋白聚糖(Dystroglycan,DG)和一个肌聚多糖复合物(Sarcoglycan(SG)/Sarcospan)。其中,DG是DGC的中心蛋白,DG产生于一种前体肽,这种前体肽可裂解成一种位于细胞外的高度糖基化的αDG蛋白(α-DG)和一种跨膜的βDG蛋白(β-DG)。细胞外高度糖基化的蛋白α-DG通过其高亲和力与细胞外基质蛋白如层黏连蛋白(Lamininα_2)结合从而介导了细胞骨架和基底膜之间的连接。跨膜蛋白β-DG位于细胞内的一端结合于Dystrophin,后者又结合于肌动蛋白(Actin);位于细胞外的另一端则结合于α-DG。
上述这些环节当中的任一元素突变,都会产生临床上表现各异即不同表型的肌营养不良。例如,dystrophin绝对或相对缺乏或不足导致杜兴氏肌营养不良(Duchenne Muscular Dystrophy,DMD)或贝克氏肌营养不良(Becker MuscularDystrophy,BMD),前者常伴有心肌受累和智能障碍,后者进展相对较缓呈相对良性病程表现。编码γ-、α-、β-与δ-SG亚单位的的基因突变将分别产生相应的肢带型肌营养不良(Limb Girdle Muscular Dystrophy,LGMD)的相关亚型LGDM-2C、2D、2E、2F。DG是已知的和推想的糖基化酶蛋白的分子靶子,参与α-DG糖基化的酶蛋白的突变会导致DG的异常糖基化,致使DG作为骨骼肌细胞外基质受体的这一重要功能遭到破坏,与骨骼肌中的配体的结合活性丢失,引发相关的先天性肌营养不良(Congenital Muscular Dystrophy,CMD)。在这些已经确认的疾病当中,同时合并心肌病变是其常见且严重的并发症。因此,在DG的糖基化和功能方面的缺陷是几种人类肌营养不良相关性心肌病的起因。
蛋白糖基化途径常常混杂交错,所以有学者作出假设:其他蛋白可能是此酶学途径的靶点并与疾病的表型有关?由此便产生了一个逻辑上的问题,DG基因的遗传性消除能否在小鼠身上重复出许多甚至所有的疾病表型?DG敲除小鼠是胚胎致死性的,原因是对啮齿类动物来说这是特异性致死性的发育缺陷。因此,目前尚没有一种遗传性疾病的相关描述涉及到α-或β-DG的自身突变,也就是说目前尚未能在任一遗传性疾病当中确认以DG自身突变为主的病变。
本文研究者利用Cre-LoxP技术获得了心肌组织特异性DG缺失的基因突变小鼠M1c2vCre,使得有关DG在心肌这一特定组织中的结构与功能研究成为可能。首先,将确认DG缺失是否能够充分且必要地解释蛋白糖基化缺陷肌营养不良相关性心肌病的发生;其次,将探讨心肌细胞DG基因突变在肌营养不良相关性心肌病的发病中所起的结构性和/或功能性的作用及其确切的作用机制;再次,要研究与此相关的心肌细胞存活信号转导途径上可能参与的酶的磷酸化水平以及其他可能参与其中的蛋白水解酶系统,以积极寻求潜在的临床治疗的新策略。
综上所述,患有DMD,BMD,与SG相关的退行性LGMD的病人,以及mdx小鼠和β-、δ-和γ-SG基因敲除LGMD小鼠模型常常发展为心肌病,然而,肌营养不良相关性心肌病的发病机制尚未完全为我们所了解。目前,DG与DGC在引起肌营养不良相关性心肌病中所起的作用及其机制也仍不明了,可能的原因分析如下:首先,DGC蛋白复合物在心肌组织系统表达分布和功能比较复杂,心肌中DGC破坏可使得心肌细胞易感于膜损伤;其次,是否每一种蛋白在DGC中主要地起到结构性的或是功能性的作用远未确定下来;再者,虽然具有骨骼肌蛋白糖基化异常的病人可发展为心肌病,但在心肌病本身DG的作用及作用机制尚未确立。本文将采用靶基因敲除和靶基因转导小鼠模型,使用在体和离体的研究方法,着力阐释DG在心肌组织系统的结构与功能,探讨DG缺陷导致的与肌营养不良相关的心肌病的发病机制,为肌营养不良相关性心肌病提供一种确定机制和治疗靶向的手段。
实验材料与方法
一、基因缺陷及转基因小鼠的获得及种系的维系:
应用Cre-LoxP技术来组织选择性地敲除靶基因DG。本文所用的M1c2vCre(Cre+/floxed DG)是心肌组织特异性DG敲除小鼠,其对照为相应的统一在floxed/floxed背景下的野生型小鼠,基因型为Cre-/floxed DG。
利用转基因技术获得过表达钙蛋白酶抑制蛋白(Calpastatin,CAST)的转基因小鼠(CAST Tg),再与floxed DG基因型背景的小鼠交配,获得目标转基因小鼠(CAST Tg;Cre+/floxed DG)。
PCR行基因型测定及动物筛选。
二、细胞裂解物或心肌组织匀浆的制备与SDS-PAGE及WesternBlotting:
拟收集的培养细胞用PBS洗掉培养基,加入等量的细胞裂解液;拟裂解的心肌组织加入10倍于其体积的组织裂解液,进行研磨并裂解。使用超声震碎仪将细胞或组织进一步超声震碎10秒钟形成细胞或组织匀浆,14000 rpm高速离心2分钟以沉淀残片,Dc蛋白分析法对细胞或组织匀浆行蛋白浓度定量测定。
将制备好的匀浆样本加样至3-15%梯度凝胶胶孔中行电泳分离蛋白带(SDS-PAGE),然后将分离后的蛋白转印至PVDF膜上拟行免疫印迹(Westernblot),5%脱脂干奶粉封闭1小时后分别使用相应的一抗和辣根过氧化物酶标记的二抗进行孵育各约1小时,TBS-T洗膜后用增强的化学发光法(ECL)的底物显影并摄片。
三、DG蛋白纯化与laminin结合分析:
使用琼脂糖麦胚凝集素(agarose Wheat Germ Agglutinin,agarose-WGA)与N-Acetylglucosaminyl Glycoprotein(NAG)对心肌组织匀浆进行DG蛋白纯化,对纯化后的蛋白进行(1)Western Blot;(2)Laminin膜相层叠分析,程序类似与Western blot;(3)Laminin固相结合实验,使用96孔板,以50μl纯化蛋白样品铺底,再分别添加浓度梯度为0,0.02,0.1,1,2,10,20 nM的laminin孵育1.5小时,每个浓度于每种样品需重复3次,洗孔后再分别添加抗laminin的一抗和相应的二抗孵育,最后加以显色剂及2 M的浓硫酸终止颜色反应,放入色谱仪中读取数据。
四、心肌组织标本冰冻切片的制备与组织化学染色与免疫荧光染色:
心脏组织置于软木塞上承载,置于-120℃的二甲基丁烷(即异戊烷)30秒,再置于-196℃的液氮中进一步冷冻固定,最后置于-80℃的冰箱保存。使用冰冻切片机(Cryostat)在横断面上对组织进行切片制备,留待组织化学和免疫荧光染色。
按照成型的组织化学染色组套程序进行HE(Haematoxylin & Eosin)、SR(Sirius Red)染色以及FG(Fast Green)反染。免疫荧光染色分别使用一抗和相应的荧光标记的二抗,免疫荧光显微镜观察并照相。
五、测定成年小鼠心肌细胞内的PI3K-AKT信号转导途径:
PI3激酶信号转导途径于在体水平测定可分别通过对整体心脏组织匀浆进行Western Blot免疫印记的方法来获得。分别使用抗磷酸化AKT和抗磷酸化GSK3β以及抗总AKT和抗总GSK3β的相应的一抗和二抗。
为确定是否DG与配体之间的相互作用为AKT激活所必需,行成年小鼠心肌细胞原代培养,并导入外源性laminnin,孵育24小时后收集细胞,裂解并超声震碎,然后行Western Blot,抗体及方法同前,分别测定导入外源性laminin之前之后的AKT磷酸化及GSK3β磷酸化水平。
六、离体实验——成年及新生小鼠心肌细胞的原代培养:
1、成年小鼠心肌细胞的原代培养:
本实验使用的成年小鼠心肌细胞培养方法及病毒转导方法按我们已发表文献进行。通过升主动脉套管固定悬挂小鼠心脏于改进的Langendorff心脏灌流系统上行酶学消化,消化后取下心脏小心去除心房和主动脉后对心室肌进行心肌细胞的分离并终止酶学消化。显微手术镊子于镜下轻柔分离心肌细胞。反复离心重悬获得的心肌细胞,最后以含有5%胎牛血清的被覆培养基以悬浮并铺被细胞至事先以56 nM的laminin包被半小时以上的培养皿盖玻片的表面,密度约20000个细胞/片。2小时后,将被覆培养基换成常规培养基。常规培养基每天更换一次。
CAST腺病毒由AdEasy XL腺病毒载体系统产生,使用2637 bp小鼠CASTcDNA,重组腺病毒(AdCAST)在HEK293细胞系中得以扩增并提纯。使用噬菌斑分析来确定病毒的滴度为1-2.4x10~9 pfu/ml。细胞贴附2小时后,凭借200μl培养基向心肌细胞转导200-480 MOI AdCAST孵育1小时,然后转为常规培养基进行细胞培养。24-48小时后收集培养的心肌细胞,裂解后SDS-PAGE蛋白电泳及Western Blot免疫印迹。
2、新生小鼠心肌细胞的原代培养:
出生后一天的新生小鼠直接断头开胸取心脏,0.25%的Trypsin-EDTA消化裂解心肌组织、分离心肌细胞,每次10分钟,反复2-3次,直至无明显组织块残留,等量培养基终止消化,心肌细胞悬液以1000g离心4分钟,弃上清,重新悬浮细胞,分置于7 nM纯化的laminin包被的培养皿中,放入37℃与5%CO_2的温箱中进行培养,24小时后PBS小心冲洗并取出附着细胞用的盖玻片,进行免疫荧光染色,免疫荧光显微镜观察细胞贴附和基质在细胞表面组装的情况。
七、在体实验——小鼠心肌急、慢性负荷效应模型建立:
为了评价急、慢性机械张力对成年DG缺陷小鼠心肌产生的效应,对月龄相当的同系伙伴野生型与突变型小鼠进行平板运动负荷试验来激发负荷对心肌的急性期效应,运动后5小时左右杀鼠取心脏,部分标本用以制备组织标本冰冻切片,使用相应的心肌标志物抗体行免疫荧光染色,部分心肌组织标本被裂解留待Western blot;而关于获得慢性增加的心室压力负荷则行腹主动脉缩窄术(AAB),野生型与突变型小鼠都设假手术组,术后4周杀鼠取心肌组织制备冰冻切片待行HE、SR及FG反染等组化染色。
八、蛋白分析及统计学分析软件与方法:
使用Alpha Innotech提供的蛋白分析软件对Western blot所获得的蛋白带结果进行定量分析;使用Prism 5.0统计学软件进行数据处理,结果以均值±标准误表示,二组组间比较采用t-检验,多组组间比较采用Anova方差分析,采用线性回归制作Dc蛋白定量分析中的蛋白浓度计算方程,使用非线性回归方程制作laminin固相结合实验的曲线图形。
实验结果
1、M1c2vCre小鼠意味着在小鼠心肌组织特异性地DG表达丢失,抗α-DG(IIH6)抗体行免疫荧光染色,结果显示野生型小鼠心肌细胞边界清晰连贯,而突变型小鼠心肌细胞边界则模糊混杂似补丁形。
2、DG蛋白纯化后的Western Blot以及与laminin的结合活性曲线显示DG在组织特异性DG敲除小鼠中几乎缺如,α-DG与细胞外基质蛋白配体laminin的结合性近乎丧失。另行DG与老化的关系研究发现老龄小鼠DG与laminin的结合活性下降,但这种差异尚未达到统计学差异。
3、新生幼鼠心肌细胞培养发现组织特异性DG缺陷小鼠的心肌细胞与laminin包被的表面的贴附力较野生鼠显著减弱,基质组装几近丧失。而在成年小鼠这种差异并不显著。
4、心肌组织细胞内的PI3K-AKT细胞存活信号转导途径检测显示DG缺失突变型小鼠AKT和GSK3β(活性AKT的下游)磷酸化水平较对照低,提示DG具有参与细胞存活信号转导途径这一重要功能。导入外源性laminin之后对照组野生型小鼠的心肌细胞出现较高水平的磷酸化AKT与磷酸化GSK3β,而对DG敲除小鼠的心肌细胞则没有此作用,提示laminin是通过DG将细胞存活信号传递给心肌细胞的,DG与配体之间的相互作用是AKT活化所必需,DG是不可或缺的极为重要的信号转导中介。
5、小鼠急、慢性心肌负荷试验显示心肌细胞对心肌负荷的敏感性增加而易损。AAB术后4周小鼠心肌组织切片HE染色显示心肌组织学改变以及慢性炎症反应改变,DG缺失突变型小鼠心肌组织肌纤维结构紊乱,部分肌纤维连续性中断,肌浆染色不均匀,肌间隙大小不一,炎症细胞多成片状浸润;SR染色及FG反染显示DG缺失突变型小鼠心肌组织胶原沉淀提示心肌局灶性组织损伤与重构。平板运动负荷试验后5小时小鼠心肌组织冰冻切片免疫荧光染色显示基因突变型小鼠的细胞膜破损,cTnI与cTnT弥散分布在受损的心肌细胞及细胞间隙。
6、平板运动负荷实验后几种常见的蛋白水解酶钙蛋白酶(Calpain,CAPN)的底物蛋白如cTnI、cTnT、Talin有不同程度的降解,其中,cTnI的降解率在突变型小鼠为13.60%,显著高于对照野生型小鼠6.81%的降解率,P＜0.01;cTnT的降解率在突变型小鼠为21.20%,显著高于对照野生型小鼠10.55%的降解率,P＜0.01;talin的降解率在突变型小鼠为3.01%,高于对照野生型小鼠2.28%的降解率,但尚未达到统计学差异。
7、AdCAST转染心肌细胞原代培养蛋白表达的结果显示AdCAST转染组的CAPN底物蛋白cTnI、cTnT、Talin的表达量均显著高于非转染对照组,P＜0.05;而CAPN的表达量则显著降低,P＜0.05。
8、Western blot与Zymogram胶分析结果显示转基因小鼠能够高表达人钙调蛋白抑制蛋白(hCAST),且高度抑制CAPN活性,而在非转基因小鼠几乎未能检测到完整片段hCAST,CAPN活性正常。重复平板运动负荷试验,显示CAPN表达在非转基因突变组于急性负荷后显著增加,而在转基因组对照与突变小鼠均受到显著抑制,后二者比较无统计学差异。转基因后对照与突变小鼠比较,CAPN底物蛋白cTnI、cTnT、talin等未显示明显降解,各组蛋白表达量无显著差异。
结论
1、与肌营养不良相关的复合物DGC当中的中心蛋白DG在心肌组织特异性缺乏的动物模型的成功建立,使得直接研究DG在肌营养不良相关的心肌病的发病中的结构性和功能性作用及其相应的细胞分子机制成为可能。
2、DG至少在幼鼠时期的细胞基质组装中起重要作用。
3、DG参与PI3K/AKT细胞存活信号转导,是不可或缺的极为重要的信号转导中介。
4、心肌细胞组织特异性DG缺失导致DGC破坏使得心肌细胞对心肌负荷的敏感性增加而更易感于膜的损伤。
5、钙蛋白酶系统参与了组织特异性DG缺失的心肌细胞对心肌负荷的损伤反应过程。
6、高水平的钙蛋白酶抑制蛋白表达可有效地降低钙蛋白酶的活性及表达,尤其有效地抑制了在DG缺陷时异常激活的钙蛋白酶,提高心肌损伤的阈值,某种程度上对心肌起到保护作用,为DG缺陷肌营养不良相关性心肌病的基因与药物治疗提供有益的临床线索和治疗新策略。
Background and Specific Aims
Heart disease was first identified in human muscular dystrophy one hundred and fifty years ago,there has been no cure so far.70-90%of muscular dystrophy patients, that have genetic defects in the dystrophin glycoprotein complex(DGC),have cardiovascular disease,and about 20%succumb to heart failure.
The DGC is a critical complex for the maintenance of the structure and the function of normal cardiac and skeletal muscle,as a mechanosignaling complex with dual mechanical and nonmechanical membrane stabilizing functions,playing a pathogenic role in the cardiomyopathy associated with muscular dystrophy.DGC is a multimeric transmembrane protein complex,containing dystrophin,dystroglycan(DG) and a sarcoglycan(SG) / sarcospan complex.DG is the central protein in DGC.DG is generated from a propeptide,which is cleaved into a heavily glycosylated extracellularαDG protein(α-DG) and a transmembraneβDG protein(β-DG).α-DG is heavily glocosylated and completes the link from the cytoskeleton to the basal lamina by binding with high affinity to extracellular matrix proteins,such as laminin.β-DG binds intracellularly to dystrophin,which binds the actin cytoskeleton,and binds extracellularly toα-DG.
Mutations in any one of the above components result in variable phenotypes of muscular dystrophy.For example,absolute or relative deficiency in dystrophin leads to Duchenne Muscular Dystrophy(DMD) or Becker Muscular Dystrophy(BMD).DMD usually complicates with cardiac muscle problem and mental retardation,BDM has a slower down progression.The mutations of the genes encodingγ-,α-,β-,andδ-SG subunits generate Limb Girdle Muscular Dystrophy(LGDM) -2C,2D,2E and 2F, respectively.DG is the molecular target for mutations in known and putative glycosylation enzymes that lead to abnormal glycosylation of DG,and its dysfunction as an extracellular matrix receptor,and the loss of binding with the ligand in skeleton muscle,which is responsible for severe congenital muscular dystrophies.The common and severe complication of these disorders is the associated cardiomyopathy.Therefore, the defects in glycosylation and function of DG are a cause of several human muscular dystrophies and associated cardiomyopathies.
Because the glycosylation pathways are often promiscuous,several groups have hypothesized that other proteins may be targets for this enzymatic pathway and responsible for disease phenotypes.One logistic question,does genetic ablation of DG gene recapitulate many or all of the disease phenotypes in mice? The DG knockout mouse is embryonic lethal due to a developmental defect specific to rodents.Therefore, currently,there's no description about mutations ofα- orβ-DG itself involved in genetic disorders.
Using Cre-LoxP technology to specifically delete the DG gene in heart to obtain cardiac muscle tissue-specific DG knockout mice will allow us to study the structural and functional roles of DG in cardiovascular system.First,it will be identified that whether the loss of DG is sufficient and necessary to explain the occurrence of the cardiomyopathy associated with muscular dystrophy due to abnormal glycosylation. Second,it will be explored that what role the mutation of DG plays in associated cardiomyopathy,structural or functional,and what the mechanism is.Third,it will be investigated that whether DG is involved in cell survival signaling,and what the phosphorylated levels of the associated enzymes are,and is there any other possible proteolysis system involved in,so that the novel potential strategy for clinical treatment will be sought out.
As alluded to above,patients with DMD,BMD,and SG associated LGMD,mdx mice and knockout mouse models ofβ-,δ- andγ-SG associated LGMD often develop cardiomyopathy.The mechanisms of cardiomyopathy in muscular dystrophies have not been fully understood.Currently,the mechanisms by which DG and DGC cause cardiomyopathies associated with muscular dystrophy still remain elusive.The possible reasons are,first,the expression distribution and function of DGC in cardiovascular system is complicated,the disruption of DGC may render cardiac myocytes sensitive to membrane damage.Second,whether each protein primarily plays a structural or a functional role within DGC is far from defined.Third,although patients with abnormal glycosylation in skeleton muscle can develop cardimyopathy,the mechanic role of DG in cardiomyopathy has not been established.Using combinations of mutant and genetarget mouse models,and in vivo and in vitro approaches,the research is uniquely suited to address the questions:what the roles DG play in cardiovascular system are, structural or functional or both,what the mechanism by which DG defects cause the cardiomyopathy associated with muscular dystrophy is,providing a means by which the mechanism and target for treatment of cardiomyopathy associated muscular dystrophy can be identified.
Materials and Methods
1.Generation and analysis of Cradiac tissue-specific DG knockout mice(Mlc2vCre) and calpastatin transgene(CAST Tg) mice and strains' maintenance.
Generate cardiac tissue-specific DG knockout mice Mlc2vCre using Cre-LoxP technology.The mutant mouse is Cre+/floxed DG,the control wild type is Cre-/floxed DG.
Generate calpastatin transgene mice using transgene technology.Then cross the CAST Tg mice with Mlc2vCre mice to generate target mouse model whose genotype is CAST Tg;Cre/floxed DG.
Genotyping and screening by PCR.
2.Lysate preparation from cultured cells or cardiac muscle tissue and SDS-PAGE and Western blotting.
Cells were washed with PBS and scraped in equal amounts of a lysis buffer. Cardiac muscle tissue was homogenized in 10 volumes of sample size's lysisi buffer. The samples were sonicated for 10 sec and centrifuged at 14000 rpm for 2 min to pellet cell or tissue debris.All of the resulting cell lysates was loaded or volumes were normalized to the total protein content determined using Dc Protein Assay.Proteins were separated by a 3-15%gradient SDS-PAGE and then transferred to a polyvinylidene fluoridine membrane.The membranes were blocked with 5%nonfat milk in TBS(150 mM Nacl,20 mM Tris pH 7.5) followed by incubation with primary antibody and secondary antibody conjugated to horseradish peroxidase,respectively. The membranes were developed using an enhanced chemiluminiscence assay.
3.DG protein purification and laminin binding assay.
To obtain purified DG protein using agarose wheat germ agglutinin(agarose-WGA) and N-acetylglucosaminyl glycoprotein(NAG).Western Blot and laminin Biniding assay(both membrane phase and solid phase) were performed on the purified DG protein,the rational and procedure of the memebrane phase assay are similar to Western Blot's.The solid phase was performed in 96-well microplate coated with gradient concentrations of laminin,0,0.02,0.1,1,2,10,20 nM,incubating for 1.5 hours,triplicate for each sample,incubated with anti-laminin and responding secondary antibody incubation,then washed,finally added developing reagent and 2M H_2SO_4 to stop the color reaction,plating the microplate to the reader to read data.
4.Frozen tissue specimen preparation and slicing and histochemical staining and immunosatining.
Heart tissue was placed onto the cork,dipped into -120℃isopentane for 30 seconds,then stayed in -196℃liquid nitrogen to be frozen,stored in -80℃freezer. Using cryostat did the slicing with transection for later histochemical staining and immunostaining.
Follow the protocol to work on the histochemical staining,such as HE,SR staining and FG counterstaining.Immunostaining was performed using primary antibody and responding secondary antibody with immunofluroresence label.
5.Assessment of PI3k-ARK signaling in adult cardiac myocytes.
The activation of PI3 kinase signaling pathway in hearts in vivo will be assessed in whole heart homogenates by Western blotting using anti-phospho-AKT and antiphospho -GSK3βas well as the overall levels by anti-AKT and anti-GSK3β.
To determine if the interaction of DG with ligands is necessary for the activation of AKT,adult cardiac myocytes will be isolated from the same group of animals and phosphorylations of AKT and GSK3βwill be assessed prior to and after the addition of exogenous laminin as the above procedures.
6.Mouse cardiac myocytes primary Culture _ in vitro study.
(1) Adult mouse cardiac myocytes primary culture.
Adult mouse cardiac myocytes primary culture was performed by protocol worked out based on our previous studies.The heart was cannulated via the ascending aorta and mounted on a modified Langendorff perfusion apparatus.The heart was perfused with Myocyte Buffer and Enzyme Solution.The atria and aorta were removed;the ventricles were cut in two pieces,disaggregated gently with Dumont #5/45 forceps.Cells were pelleted and resuspended for a couple of times.Finally,cells were resuspended in Plating Media and plated into glass coverslips pre-coated with 56 nM laminin for over 30min.The plating density was 20,000 cells/coverslip.After 2 h,serum-containing Plating Media was switched to Culture Media.Culture Media was changed daily.
Recombinant adenovirus preparation and transduction.The calpastatin adenoviruses were generated with the AdEasy XL adenoviral vector system using a 2637-bp mouse calpastatin cDNA.Recombinant virus was amplified in HEK293 cells and purified using a cesium chloride gradient as described.The titer of the virus was assessed by a plaque assay and was 1-2.4×10~9 plaque forming units(pfu) per 1 ml. Following 2 h of attachment,cardiac myocytes were transduced with 200-480 MOI AdCAST in 200μl culture Media for 1h.After 24-48 hours,collect cells which were lysed by lysis buffer.The SDS-PAGE and Western Blot were performed on Lysate.
(2) Newborn mouse cardiac myocytes primary culture.
Newborn pups at age of 1 day old was decapitated and the heart was removed from chest cavity and dissociated by 0.25%Trysin-EDTA and the cells were centrifuged at speed of 1000g for 4min and resuspended in media and plated into glass coverslips pre-coated with 7 nM purified laminin for about 120min,and placed in 37℃, 5%CO_2.After 24 hour,the coverslips were removed for immunostaining,watching cells attachment and matrix assembly and picture-taking under the immunofluorescence microscope.
7.The establishment of Acute and Chronic heart mechanic strain overloads mouse model _ In vivo study.
In order to assess the effects of acute and chronic mechanic strain on adult DG deficient myocardium,treadmill exercise stress will be performed on mutant Mlc2vCre-DGnull mice compared to littermate controls.After 5 hours,the subjected mice were killed and the heart tissue was partly frozen for slicing and immunostaining thereafter,and was partly lysed for later Wetsern blotting.Chronic increases in ventricular pressure can be accomplishment by suprarenal abdominal aortic banding (AAB).Mutants and controls were both divided into operation and sham-operation groups.After 4 weeks,the mice were killed and the heart tissue was frozen and slicing for later HE,SR staining and FG counterstaining.
8.Protein analysis and statistics software and method.
Quantified protein acquired by western blot using software provided by Alpha Innotech.Statistics software-Prism 5.0 was used for data analysis such as t-test,Anova, linear regression and nonlinear regression,data were shown as mean±se.
Results
1.Mlc2vCre is the heart tissue-specific DG knockout mouse.Frozen sections were stained with an antibody againstα-DG(ⅡH6).The mutants displayed the indistinct, discontinuous,patched borders of the cardiac myocytes compared to controls with distinct,continuous borders.
2.Western blotting and laminin binding assay(solid phase) for purified DG showed that there is almost no DG expression or loss of ability ofα-DG to bind laminin. Furthermore,the additional study on the relationship between DG and aging showed that the activity ofα-DG binding to laminin decreased,but no significant difference.
3.Neonatal mice cardiac myocytes primary culture showed reduced cells attachment and lack of matrix assembly in Mlc2vCre lacking DG compared to controls but no defect in assembly in adult cardiac myocytes.
4.Assessment of PI3k-ARK signaling in adult myocytes showed that lower levels of phosphorylated AKT and GSK3β(down stream of active AKT) was obtained in Mlc2vCre-DGnull mice whole heart homogenates compared to littermate controls, which indicates DG is involved in this cell survival signaling.Higher levels of phospho-AKT and phospho-GSK3βwere found after laminin addition to control myocytes,but had no effect on DG null cardiac myocytes,which indicates that laminin confers a cell survival signal to cardiac myocytes through DG and the interaction of DG with ligands is necessary for the activation of AKT,DG is indispensable to mediate this critical signaling transduction.
5.Assessment of the effects of acute and chronic mechanic strain on adult DG deficient myocardium showed that cardiac myocytes lacking DG was more sensitive to membrane damage due to overload.In Mlc2vCre mice 4 weeks after AAB,HE staining showed the disorders of cardiac myofibers,part of which discontinued,presenting uneven cytoplasmic staining,varied in intercellular space,infiltration of inflammatory corpuscle.SR staining and FG counterstaining showed that focal areas of collagen deposition suggesting focal regions of damage and remodeling in mutants,was performed on mutant Mlc2vCre-DGnull mice compared to littermate controls.In Mlc2vCre mice 5 hours after treadmill exercise stress,immunostainings showed that membranes of the cardiac myocytes were damaged severely and some cytoplasmic proteins such as cTnI and cTnT that leaked out through the disrupted membranes.
6.Western blotting after treadmill exercising showed that the common substrates of Calpain(CAPN) such as cTnI,cTnT,and talin were degraded to some degrees.The mutant's degradation percentages compared to the control's are cTnI 13.6%vs 6.81% with p＜0.01,cTnT 21.2%vs 10.55%with p＜0.01,talin 3.01%vs 2.28%without significant defference.
7.Assement of the effects of AdCAST transfection on proteins expression in cardiac myocytes primary culture showed that the expression of the CAPN substrates such as cTnI,cTnT,and talin were significantly higher in AdCAST transfection group compared to no transfection group,p＜0.05,on the contrary,the expression of CAPN significantly lower in transfection group,p＜0.05.
8.The Western blotting and Zymogram analysis showed that Tg mice have the much higher expression of hCAST by which CAPN activity was highly inhibited, whereas there is almost no detection of intact hCAST but CAPN was normally activated.Repeating treadmill exercising showed that CAPN expression in CAST nontransgene(NTg) group the mutant subgroup increased significantly.In CAST Tg group either mutant or control mice CAPN was inhibited signicantly,there is no significant difference between these two subgroups.Futhermore,there are no differences in CAPN substrates such as cTnI,cTnT,and talin between these two subgroups.
Conclusions
1.The establishment of cardiac tissue-specific DG knockout mouse model renders it possible to directly study the structural and functional roles that DG,the central protein in DGC associated with muscular dystrophy,plays in cardiomyopathy associated with muscular dystrophy,and the associated cellular and molecular mechanism.
2.DG at least plays critical role in matrix assembly in neonatal mice.
3.DG is involved in the PI3K/ART cell survival signaling,and is indispensable to mediate this critical signaling transduction.
4.Cardiac tissue-specific DG deficient leading to the disruption of DGC renders cardiac myocytes sensitive to membrane damage due to heart overload.
5.Calpain protease system is involved in the development of the injury responding to mechanical strain overload on DG deficient heart.
6.Overexpression of calpastatin can effectively decrease calpain activity and expression,especially in DG deficient heart with highly inhibition of abnormally activated Calpain,to some degree,playing a role in cardiac protection and providing a beneficial clue and a novel strategy for gene therapy and clinic medication of muscular dystrophy and associated cardiomyopathy due to DG deficient.
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