Profilin-1在自发性高血压大鼠心肌肥大中的作用及机制研究
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摘要
第一部分自发性高血压大鼠心肌肥大的定量比较蛋白质组学研究
研究背景
心肌肥大是心肌细胞和细胞外间质对血流动力学超负荷和神经体液失调等多种病理刺激的基本应答和代偿反应,是许多心血管疾病(如高血压、心脏瓣膜病、急性心肌梗塞和先天性心脏病)发生发展过程中共有的病理变化。临床流行病学资料显示,心肌肥大导致心源性猝死和心力衰竭的发病率明显增高,严重影响患者的预后,已被列为影响心血管疾病发病率和死亡率的独立危险因子。因此,对心肌肥大发生机制的探讨并寻找有效的预防和治疗措施,具有十分深远的临床意义和科研价值。
蛋白质组学是以蛋白质组为研究对象,探究细胞内所有蛋白质及其动态变化规律的科学,在细胞和生命有机体的整体水平上阐明生命现象的本质和活动规律。同位素标记的相对和绝对定量技术(isobaric tags for relative and absolute quantitation, iTRAQ)是近年来开发的一种新的比较蛋白质组学定量研究技术。iTRAQ技术可对一个基因组表达的全部蛋白质进行精确定量和鉴定,寻找差异表达蛋白,并分析其蛋白功能。与传统的依赖凝胶电泳的定量技术相比,iTRAQ是基于高度敏感性和精确性的串联质谱方法；可同时对4种甚至8种不同来源的样品进行绝对和相对定量研究；不仅可以发现胞浆蛋白,还可发现膜蛋白、核蛋白甚至胞外蛋白；可以检测出低丰度蛋白、强碱性蛋白、小于10kD或大于200kD的蛋白。iTRAQ技术的发展为探讨心肌肥大的发生机制,寻找关键的作用蛋白提供了高效可靠的研究方法。
自发性高血压大鼠(Spontaneous Hypertension Rat,SHR)是1959年由日本京都大学Okamoto和Aoki培育而成的遗传性高血压动物模型。SHR在遗传特性、发病机制以及高血压心血管并发症等方面均与人类原发性高血压十分相似,是目前国际公认最接近于人类原发性高血压的动物模型。心肌肥大是SHR重要特征之一,高血压性心肌肥大在幼年SHR已出现,并存在于高血压整个自然病程中。
因此,本研究选用特定年龄阶段的SHR大鼠为研究对象,采用定量比较蛋白质组学iTRAQ技术,着力于发现SHR大鼠早期心肌肥大发生发展过程中差异表达的功能性蛋白或蛋白群,并利用生物信息学方法分析差异蛋白的分子功能、蛋白分类及生物学过程,筛选并鉴定在高血压性心肌肥大发病过程中起关键作用的蛋白分子,为高血压心肌肥大的防治提供有效靶点。
研究目的
1.研究自发性高血压大鼠心肌病变的特点,观察5周龄和17周龄SHR大鼠心肌组织的病理学和超微结构变化；
2.应用iTRAQ蛋白质组学技术研究SHR大鼠肥大心肌与同周龄WKY大鼠正常心肌组织的差异表达蛋白,从整体水平上揭示高血压心肌肥大病变的病理生理机制,并筛选关键靶蛋白；
研究方法
4周龄雄性SHR大鼠8只,WKY大鼠8只,16周龄雄性SHR大鼠8只,WKY大鼠8只,适应性饲养1周后(即5周龄和17周龄时)进入实验：
1.大鼠尾动脉收缩压测定：处死前通过无创血压测量系统检测各组大鼠尾动脉收缩压；
2.心脏质量指数测定：大鼠处死后,称量心脏重和左室重,测量主动脉瓣至心尖的距离为左心室长轴。以左室重/心脏重和左心室长轴作为反应心室肥大的指标；
3.心肌组织病理学检测：HE染色观察心肌组织病理变化,天狼星红染色测定胶原纤维面积百分比；
4.心肌组织超微结构观察：透射电镜技术观察心肌肌丝、肌节、线粒体及细胞核等亚细胞结构；
5.蛋白质组学(iTRAQ)分析：5周龄WKY大鼠(WKY-5)、5周龄SHR大鼠(SHR-5)、17周龄WKY大鼠(WKY-17)和17周龄SHR大鼠(SHR-17)各8只,提取左心室总蛋白。采用胰酶消化蛋白得到肽段,按照ABI公司说明书操作标记肽段,114标记WKY-5组,115标记SHR-5组,116标记WKY-17组,117标记SHR-17组。将各组已标记的肽段混合后用强阳离子交换柱和C18柱进行分离,然后采用MALDI-TOF/TOF和MicroQ-TOF进行质谱鉴定。不同报告基团离子的强度代表其所标记的多肽的相对丰度；
6.差异蛋白质功能分析：利用Data Analysis4.0软件分析质谱数据,用Mascot软件搜索与质谱匹配的蛋白。采用Panther软件(http://www.pantherdb.org/)对差异蛋白进行GO分析,包括分子功能,生物过程和细胞组成三个部分；
7.验证profilin-1的表达：采用实时定量聚合酶链反应(qPCR)技术检测各组大鼠心肌组织中profilin-1的mRNA水平,蛋白质免疫印迹方法(Westernblot)检测profilin-1的蛋白表达水平,以验证蛋白质组学的可靠性。
研究结果
1.大鼠尾动脉收缩压：5周龄WKY大鼠收缩压为111.2±7.6mmHg,5周龄SHR大鼠收缩压为143.1±10.5mmHg,已显著升高(P<0.001),17周龄WKY大鼠收缩压为117.7±8.6mmHg,17周龄SHR大鼠收缩压为216.3±12.1mmHg,收缩压的差异更为显著(P<0.001)。
2.大鼠心脏肥大指数：5周龄SHR大鼠的平均LVW/HW比值为0.55±0.02,5周龄WKY大鼠的平均LVW/HW比值为0.54±0.02,两者无显著差异；17周龄SHR大鼠的平均LVW/HW比值为0.77±0.06,同周龄WKY大鼠的平均LVW/HW比值为0.60±0.03,SHR大鼠较WKY大鼠升高28.33%。5周龄SHR大鼠的左室长轴平均长度为6.45±0.33mm,同周龄WKY大鼠的左室长轴平均长度为6.42±0.36mm,两者无显著差异；17周龄SHR大鼠的左室长轴平均长度为13.31±0.67mm,同周龄WKY大鼠的左室长轴平均长度为9.97±0.60mm,SHR大鼠较WKY大鼠升高33.50%。
3.大鼠心肌病理学分析
3.1心肌HE染色：5周龄及17周龄的WKY大鼠心肌细胞形态正常,排列整齐、致密,肌纤维无断裂,横纹、闰盘及细胞核染色清晰；5周龄SHR大鼠心肌细胞形态、结构及排列基本正常；17周龄SHR大鼠心肌细胞肿胀、内容物成颗粒状,部分细胞间排列稀疏、间隙不清,心肌纤维断裂、融合呈波浪状改变,多数横纹、闰盘尚清晰,细胞核肥大或固缩,个别视野心肌间质纤维化；横截面上可见心肌细胞直径增宽,单个心肌细胞面积明显增加。
3.2心肌天狼星红染色：WKY大鼠心肌间质几乎无胶原纤维分布,对40个随机视野分析后得到其天狼星红染色阳性区域约占视野下总面积的0.13±0.01%(5周龄)和0.18±0.02%(17周龄)；5周龄SHR大鼠心肌胶原纤维占视野下总面积的0.15±0.01%,与同周龄WKY大鼠相比无显著差异；17周龄SHR大鼠心肌胶原分布较同周龄WKY大鼠明显增多,约占总面积的2.93±0.26%(P<0.001)。
4.大鼠心肌超微结构观察：电镜下可见5周龄和17周龄WKY大鼠心肌组织中,肌原纤维含量丰富,排列规整,Z线和明暗带清晰；闰盘结构正常；线粒体沿肌原纤维长轴平行排列,可见均匀的基质颗粒和完整致密的线粒体嵴；细胞核为圆形或椭圆形,核染色质均匀,核仁清晰,核膜平滑完整。5周龄SHR大鼠心肌细胞超微结构尚未出现典型的心肌肥大表现,但肌原纤维排列已出现紊乱,部分区域肌丝溶解,肌小节结构破坏,Z线断裂成波浪状；线粒体数量略增多,排布出现不规整；细胞核及其他细胞器形态基本正常。17周龄SHR大鼠心肌细胞内肌原纤维排列紊乱,肌小节结构破坏,Z线断裂成波浪状,部分区域肌丝溶解消失而被增生的线粒体取代；线粒体形态异常,数量增多且排布紊乱,部分线粒体肿胀,因基质颗粒的缺失出现了电子透亮区,线粒体嵴模糊不清；细胞核肥大、畸形,核膜呈不规则凹陷,染色质聚集成小团块,在核膜内侧尤为显著,核仁较为明显；粗面内质网和高尔基体发达,提示合成分泌功能旺盛,符合心肌肥厚表现。
5.质谱分析结果：实验共鉴定出大鼠心肌蛋白506个,5周龄SHR大鼠与WKY大鼠相比差异表达蛋白共7个,其中上调3个,下调4个；17周龄SHR大鼠与WKY大鼠相比差异表达蛋白28个,其中上调17个,下调11个。进一步分析发现,5周龄和17周龄WKY与SHR两组中共同的差异蛋白有3个,分别是：Profilin-1、Myosin light chain kinase2和Mitochondrial NADP+-isocitrate dehydrogenase。
6.SHR大鼠肥大心肌差异蛋白的功能分析：根据分子功能注释,差异蛋白主要执行结构性分子功能(36.7%),结合功能(30.0%)和催化功能(20.0%)。根据生物过程注释,差异蛋白主要参与细胞过程(18.0%)、代谢过程(16.0%)、生长发育(14.0%)及生物合成(14.0%)4种生物过程。根据细胞成分注释,主要包含细胞组成(47.6%)、细胞器(42.9%)、细胞膜(4.8%)及细胞连接(4.8%)四部分。Panther蛋白种类分析显示差异蛋白主要包含细胞骨架蛋白(33.3%)、酶调节蛋白(13.3%)、结构蛋白(10.0%)和氧化还原蛋白(6.7%)等,细胞骨架蛋白占据的高比例(三分之一)与前面结构性分子功能(36.7%)相呼应,可见细胞骨架蛋白在SHR大鼠心肌肥大病理过程中占据重要地位。
7.实时定量PCR检钡Profilin-1的mRNA表达：5周龄SHR大鼠profilin-1mRNA表达水平为同周龄WKY大鼠的1.86倍,表达明显上调(P<0.001)；17周龄SHR大鼠profilin-1mRNA表达水平为同周龄WKY大鼠的2.83倍,表达上调具有显著的统计学差异(P<0.001)。实时定量PCR结果与iTRAQ研究结果一致。
8. Western Blot检测Profilin-1的蛋白表达：与同周龄WKY大鼠相比,5周龄SHR大鼠心肌组织profilin-1的蛋白水平上调了约23.33%,差异具有统计学意义(P<0.05)；17周龄SHR大鼠心肌组织profilin-1的蛋白水平较同周龄WKY大鼠上调了约64.10%,差异具有统计学意义(P<0.01)。Westem-blot结果与比较蛋白质组学研究结果一致。
结论
1.心脏质量指数、心肌病理学分析以及超微结构观察结果均证实17周龄SHR大鼠心肌肥大模型已构建成功,5周龄SHR大鼠心肌出现早期病理改变,尚未发生心肌肥大。
2.作为定量比较蛋白质组学的新技术iTRAQ具有较好的精确性、重复性和定量效果,可用于研究SHR大鼠心肌肥大的分子机制,找到该发病过程的关键蛋白及通路。
3.质谱鉴定17周龄SHR大鼠肥大心肌与同周龄WKY大鼠正常心肌的差异表达蛋白28个,其中上调17个,下调11个,经G0分析后发现细胞骨架蛋白在SHR大鼠心肌肥大病理过程中占据重要地位。
4.实时定量PCR及Western blot实验结果均证实SHR大鼠心肌组织中profilin-1的表达水平明显上调,这一方面验证了iTRAQ技术的可靠性,同时为深入研究高血压诱导的心肌肥大的发病机制提供了关键的候选靶点。第二部分profilin-1在高血压大鼠心肌肥大中的作用及机制研究
研究背景高血压是心肌肥大的首要致病因素,患者的心脏处于血流动力学超负荷状态时,心肌细胞可感知该种牵张刺激,产生、释放多种细胞因子,这些细胞因子与心肌细胞膜上的相应受体结合后激活多种肥大相关信号级联反应,随后诱导核内原癌基因的转录活化,启动心肌细胞的肥大反应过程。胞膜的功能蛋白、细胞骨架系统和细胞因子间的相互作用共同参与了这一病理生理过程,其中细胞骨架系统是感知力学刺激、将机械应力转化为生化信号的关键组件,研究细胞骨架,尤其是肌动蛋白微丝系统,是深入认识血流动力学超负荷诱导下心肌肥大发生机制的重要切入点。
Profilin-1是一种重要的肌动蛋白结合蛋白,可受细胞外信号影响而调节肌动蛋白细胞骨架的动态组装。课题组前期研究提示profilin-1在哇巴因高血压大鼠心肌肥大早期发挥关键作用,且第一部分的蛋白质组学结果揭示自发性高血压大鼠早期肥大心肌中profilin-1的表达较对照组显著升高。因此,本课题将利用自发性高血压大鼠心肌肥大模型,构建腺病毒表达载体,诱导心肌细胞中profilin-1基因的过表达和沉默,综合运用各种病理学组织学染色技术和分子生物学技术研究profilin-1在高血压心肌肥厚病变发生发展中的作用及其可能机制。这些结果将为高血压心肌肥大患者的早期防治提供新靶点,具有重要的学术理论意义和潜在的临床应用价值。
研究目的
1.自发性高血压心肌肥大模型大鼠体内转染高表达腺病毒载体pAd-profilin-1-IRES-EGFP或干扰腺病毒载体pAd-miR-profilin-1,观察对心肌肥大病变的影响：
2.在组织病理学水平、细胞分子水平和超微结构水平研究profilin-1参与高血压心肌肥大发生发展的机制;
研究方法
1.5周龄自发性高血压大鼠60只,随机分为SHR-C组、SHR-I组和SHR-H组；5周龄健康WKY大鼠20只作为对照组WKY-C组.SHR-C组与WKY-C组大鼠经尾静脉注射阴性对照腺病毒载体3×109PFU/只,SHR-I组大鼠经尾静脉注射干扰腺病毒载体pAd-miR-profilin-13×109PFU/只,SHR-H组大鼠经尾静脉注射高表达腺病毒载体pAd-profilin-1-IRES-EGFP3×109PFU/只。6周后补充注射一次,剂量同第一次,第二次腺病毒注射6周后处死动物留取标本;
2.大鼠尾动脉收缩压监测：实验开始前(5周龄)及腺病毒转染后每周末通过大鼠尾动脉血压测量仪监测大鼠尾动脉收缩压,观察profilin-1过表达和沉默后对自发性高血压大鼠血压的影响；
3.心脏质量指数测定：大鼠处死后,称量心脏重和左室重,测量主动脉瓣至心尖的距离为左心室长轴。左室与心脏的重量比和左心室长轴均可作为反应心室肥大的指标；
4.腺病毒转染效率检测：第二次注射腺病毒后10天,取心肌标本切冰冻切片,荧光显微镜下观察EGFP绿色荧光并拍照,利用Image-Pro Plus6.0软件检测每个视野下EGFP绿色荧光细胞占所有心肌细胞的比例,该比值反应腺病毒转染效率；
5.病理学检测：对大鼠心肌组织分别进行HE染色和天狼星红染色,观察腺病毒转染后各组大鼠心肌组织病理改变;
6.心肌组织超微结构观察：透射电镜技术观察心肌肌丝、肌节、线粒体及小窝等亚细胞结构；
7.免疫组织化学染色和免疫荧光染色：应用免疫组织化学染色观察profilin-1在心肌中的定位,免疫荧光染色观察小窝蛋白-3与肌动蛋白微丝的共定位；
8.大鼠血清和心肌组织中NO含量测定：应用N0测定试剂盒检测大鼠血清和心肌组织匀浆中NO的含量；
9.实时定量PCR检测：取大鼠心肌组织,Trizol法提取RNA,实时荧光定量RT-PCR技术检钡profilin-1、eNOS、小窝蛋白-3mRNA相对表达量；
10. Western blot检测：取大鼠心肌组织,提取蛋白,Western blot检测profilin-1,eNOS,p-eNOS和小窝蛋白-3的蛋白表达量。
研究结果
1.各组大鼠血压变化：整个实验过程中(5周龄-17周龄)SHR大鼠血压随周龄增加而升高,且显著高于同周龄WKY大鼠血压；实验末,与SHR-C组相比,SHR-I组血压略下降,差异无统计学意义；与SHR-C组相比,SHR-H组血压略升高,差异无统计学意义。提示profilin-1过表达和沉默对SHR大鼠血压无明显影响。
2.心脏质量指数测定：与WKY-C组大鼠相比,SHR-C组大鼠左室重量／心脏重量、左室长轴均明显增加,差异具有统计学意义(P<0.01)；与SHR-C组大鼠相比,SHR-I组大鼠左室重量／心脏重量(P<0.05)、左室长轴(P<0.01)均明显减小,而SHR-H组大鼠左室重量/心脏重量、左室长轴均明显增加(P<0.05)。profilin-1过表达加重SHR大鼠心肌肥大,同时profilin-1基因沉默显著减轻了SHR大鼠心肌肥大。
3.腺病毒转染效率检测：采用天空蓝染色屏蔽自发荧光后,各组大鼠心肌组织中绿色荧光细胞占细胞总数的30%-35%,注射生理盐水的WKY大鼠和SHR大鼠心肌组织中检测不到绿色荧光细胞。
4.心肌组织病理学分析
4.1心肌HE染色：与WKY大鼠相比,SHR大鼠心肌细胞肿胀,部分细胞间排列稀疏、间隙不清,心肌纤维断裂、融合呈波浪状改变；与SHR-C组大鼠相比,SHR-H组大鼠的心肌细胞肿胀更加明显,部分心肌呈灶状与片状坏死,伴有脂肪变性和炎性细胞浸润。相反,SHR-I组大鼠心肌细胞的病理损伤明显减轻。
4.2心肌天狼星红染色：与WKY-C组大鼠相比,SHR-C组大鼠心肌胶原分布明显增多,差异具有统计学意义(P<0.001)；与SHR-C组大鼠相比,SHR-H组大鼠心肌胶原分布明显增多(P<0.001),而SHR-I组大鼠心肌胶原分布明显减少(P<0.001)。这些结果表明profilin-1基因的高表达促进高血压诱导的心肌纤维化,而profilin-1基因沉默则有助于减轻心肌纤维化程度。
5.心肌超微结构观察：与WKY-C组大鼠相比,SHR-C组大鼠心肌细胞内肌原纤维排列紊乱,肌小节结构浪状破坏,Z线断裂成波,线粒体形态异常,数量增多且排布紊乱,线粒体嵴模糊不清,粗面内质网和高尔基体发达,提示合成分泌功能旺盛,符合心肌肥厚表现；与SHR-C组相比,SHR-H组大鼠心肌超微结构破坏更为严重,大片区域肌原纤维皱缩或崩解,闰盘及Z线不清,线粒体严重肿胀,线粒体嵴断裂甚至消失,内质网扩大,胞核呈早期凋亡形态,大量的胶原纤维增生,可见其将肥大的心肌细胞包绕分隔成束；而SHR-I组大鼠心肌细胞肌原纤维含量较SHR-C组大鼠明显增加,肌节较规则,线粒体轻度肿胀,线粒体内空泡结构较少,心肌细胞核仁清晰,染色质分布均匀,边集现象不明显,内质网扩张不明显,偶有点状胶原纤维增生。
6.心肌中肌动蛋白细胞骨架的改变：WKY-C组大鼠心肌中鬼笔环肽荧光染色丰富,而SHR-C组大鼠中染色细弱,提示肌动蛋白细胞骨架含量下降；与SHR-C组大鼠相比,降低profilin-1的表达有助于肌动蛋白微丝结构和含量的保存,而profilin-1高表达则进一步减低肌动蛋白微丝的含量。
7.心肌细胞膜上小窝丰度的检测：WKY-C组大鼠心肌中小窝的数量是SHR-C组大鼠的3倍,统计学差异显著(P<0.001),与SHR-C组相比,SHR-H组心肌纤维膜上小窝的数量进一步减低(P<0.001),而profilin-1的沉默则有助于保存SHR-I组小窝的丰度(P<0.001)。实时定量PCR和western blot的结果显示SHR-H组大鼠心肌小窝蛋白-3的mRNA含量是SHR-C组大鼠的1.66±0.33倍(P<0.05),其蛋白含量是SHR-C组大鼠的1.71±0.21倍(P<0.001)；而SHR-I组大鼠心肌小窝蛋白-3的mRNA含量是SHR-C组大鼠的42.5±2.6%(P<0.01),其蛋白含量是SHR-C组大鼠的33.6±3.2%(P<0.001),小窝蛋白-3的mRNA和蛋白表达水平与肌浆膜上小窝的数量相一致。
8.大鼠心肌组织中profilin-1的表达水平及细胞定位：实时定量PCR结果显示,SHR-C组大鼠肥大心肌中profilin-1的mRNA水平较WKY-C组明显升高,差异具有统计学意义(P<0.01),SHR-H组大鼠心肌组织中profilin-1的mRNA水平较SHR-C组上调了38.10%(P<0.05),而SHR-I组profilin-1的mRNA表达水平较SHR-C组降低了44.29%(P<0.05)；Western blot结果显示,SHR-C组大鼠肥大心肌中profilin-1的蛋白水平较WKY-C组明显升高,差异具有统计学意义(P<0.05),与SHR-C组相比,SHR-H组大鼠心肌profilin-1蛋白水平上调了约35.29%(P<0.05),而SHR-I组profilin-1蛋白表达下调了38.24%(P<0.01)；免疫组化图片显示profilin-1蛋白表达在心肌细胞浆内,尤其在核周分布,在血管平滑肌细胞中也见到profilin-1微弱的表达,profilin-1在各组大鼠心肌细胞中的表达水平与western blot结果一致。
9.大鼠血清和心肌组织中N0的含量：与WKY-C组大鼠相比,SHR-C组大鼠肥大心肌中NO含量明显降低,差异具有统计学意义(P<0.05),SHR-H组心肌组织中NO含量明显低于SHR-C组(P<0.01),而SHR-I组心肌组织中NO含量则明显高于对照组(P<0.05)。同时,我们检测了血清中NO水平,结果显示SHR-I组、SHR-C组和SHR-H组大鼠血清中NO水平无显著差异,这提示profilin-1的高表达和沉默所引起的NO水平的变化是心肌组织内的局部变化而非系统性反应。
10.大鼠心肌组织中eNOS表达水平和活性检测：实时定量PCRwestern blot结果显示四组大鼠心肌中eNOS的表达水平无显著差异。进而检测eNOS在Ser1177位点上的磷酸化水平以观察eNOS的活性,结果显示,与WKY-C组大鼠相比,SHR-C组大鼠心肌中eNOS的活性明显减低；与SHR-C组大鼠相比,SHR-H组大鼠心肌中磷酸化eNOS的表达明显降低而SHR-I组的磷酸化eNOS的表达明显升高,差异具有统计学意义(P<0.05)。
结论
1.自发性高血压大鼠肥大心肌组织中,profilin-1的表达上调,伴随心肌细胞肥大、胶原纤维增生和肌动蛋白细胞骨架受损。
2. Profilin-1的过表达通过影响肌动蛋白细胞骨架的组装,干扰心肌纤维膜上小窝的形成,抑制eNOS/N0信号通路的活性而促进高血压诱导的心肌肥大,是高血压心肌肥大的重要发病机制。
3. Profilin-1基因沉默主要通过增加心肌组织内肌动蛋白微丝含量,提高肌浆膜上小窝数量,恢复eNOS活性和NO的含量,从分子水平有效抑制心肌肥大的发生发展。
PART ONE COMPARATIVE PROTEOMICS ANALYSIS OF CARDIAC HYPERTROPHY IN SPONTANEOUS HYPERTENSIVE RATS
Background
Myocardial hypertrophy is a compensatory response of cardiomyocytes and extracellular matrix to the hemodynamic overload and neurohumoral disorders. It is also a common pathological process of many cardiovascular diseases, such as high blood pressure, heart valve disease, acute myocardial infarction and congenital heart disease. Clinical epidemiological data showed that myocardial hypertrophy results in significantly increased incidence of sudden cardiac death and heart failure, causing poor prognosis of patients. Myocardial hypertrophy has been listed as an independent risk factor for cardiovascular morbidity and mortality. Therefore, to explore the mechanisms of myocardial hypertrophy and find effective measures of prevention and treatment of myocardial hypertrophy is of great clinical significance and research value.
Proteomics is a science with the whole proteome as research object. The aim of proteomics is to explore all the proteins in cells and their dynamic changes and to reveal the essence of life and the rule of activity in the overall level of cell and living organisms. The isobaric tags for relative and absolute quantitative techniques (iTRAQ) is a new quantitative study of comparative proteomics technology developed in recent years. iTRAQ technology can supply accurate quantitation and identification of proteins from a whole genome, search for differentially expressed proteins, and analyzes the protein function. Compared with the traditional quantitative technique dependent on gel electrophoresis, iTRAQ is of high sensitivity and accuracy based on tandem mass spectrometry method; It can do absolute and relative quantitative analysis of four and even eight different samples at the same time; Not only cytoplasm protein but also membrane protein, nucleoprotein and extracellular protein can be detected; The proteins of low abundance, strong alkaline, less than10kD or greater than200kD can all be detected by iTRAQ. The development of iTRAQ technology provides an effective and reliable method to study the mechanism of myocardial hypertrophy and look for the key proteins.
Spontaneously hypertensive rats (SHR) is a genetic hypertensive animal model founded by Okamoto and Aoki of Japan's Kyoto university in1959. SHR provide an excellent model system to investigate hypertension-mediated cardiac hypertrophy because of their similarities to human hypertension and natural development of the pathophysiological alterations also happened in patients with essential hypertension. Cardiac hypertrophy is one of the important characteristics of SHR. Hypertensive myocardial hypertrophy occurs in juvenile SHR and exists during the whole course of high blood pressure.
Therefore, in this study we choosed SHR of specific ages as research object and tried to find the differentially expressed proteins in the early development of myocardial hypertrophy by using iTRAQ technology. Then we analyzed the molecular function, classification and biological processes of the differentially expressed proteins using bioinformatics methods, selected and identified the key proteins playing critical effects on the pathogenesis of hypertensive myocardial hypertrophy, and provided effective target for prevention and treatment of hypertensive myocardial hypertrophy.
Objectives
1. To study the characteristics of myocardial leasion in SHR and observe the pathologic and ultrastructural changes of myocardial tissue in SHR of5and17weeks old;
2. To study the differentially expressed proteins between hypertrophic myocardium of SHR and normal myocardium of WKY by using iTRAQ technology, reveal the pathophysiological mechanisms of hypertensive myocardial hypertrophy on the overall level, and screen the key proteins playing critical roles;
Methods
Male SHR (n=8,4weeks old), male WKY (n=8,4weeks old), male SHR (n=8,16weeks old) and male WKY (n=8,16weeks old) were kept under observation for1week (i.e.,5weeks old and17weeks old) prior to the start of experiments:
1. Blood pressure measurement:Systolic blood pressure (SBP) was measured using the noninvasive blood pressure measurement system before the euthanasia of rats.
2. Cardiac mass index measurement:Rats were euthanized under anesthesia and hearts were removed and weighed. Then we dissected the left ventricule and measured the left ventricular weight-to-heart weight ratio (LVW/HW Ratio). The distance from the atrial valve to the apex, called the left ventricular long axis, was measured as another reflection of left ventricular enlargement.
3. Pathological analysis:The morphological changes of myocardium were examined by light microscope following hematoxylin and eosin staining. Sirius red staining was carried out to determine the extent of fibrosis. Image-Pro Plus6.0software was used to determine the collagen content, which was expressed as fraction of the total myocardial cross-sectional area.
4. Ultrastructure observation:The ultrastructure of myocyte was observed under an H-800transmission electron microscope.
5. Proteomics (iTRAQ) analysis:Protein was extracted and digested by trypsin. The peptides were labeled with iTRAQ reagents following the manufacturer's instructions (Applied Biosystems,114for WKY-5group,115for SHR-5group,116for WKY-17group and117for SHR-17group). The labeled peptides were separated by Strong Cation Exchange chromatography and desalted by a C18column. MALDI-TOF/TOF and MicroQ-TOF mass spectrometry was used for the identification of peptides. The different ionic strength of reporter group represents the different abundance of its labeled polypeptides.
6. Gene Ontology (GO) analysis of the differentially expressed proteins:Mass spectrum data was analyzed using Data Analysis software4.0and the matched proteins were searched with Mascot software. GO analysis of the differencially expressed proteins, including molecular function, biological process and cell composition, was performed using Panther software (http://www.pantherdb.org/);
7. Profilin-1expression:Real-time quantitative polymerase chain reaction (qPCR) and western blot technology were used to detect the mRNA and protein level of profilin-1in each group.
Results
1. SBP of rats:The SBP of SHR-5group (143.1±10.5mmHg) is significantly higher than that of WKY-5group (111.2±7.6mmHg, P<0.001). The SBP of SHR-17group (216.3±12.1mmHg) is significantly higher than that of WKY-17group (117.7±8.6mmHg, P<0.001).
2. Cardiac mass index measurement:The LVW/HW Ratio of WKY-5group is0.54±.02while the LVW/HW Ratio of SHR-5group is0.55±0.02.No significant differences between the two groups were found. The LVW/HW Ratio of SHR-17group (0.77±0.66) is28.3%higher than that of WKY-17group (0.60±0.03), which is significantly different. The left ventricular long axis of WKY-5group is6.42±0.36mm while the left ventricular long axis of SHR-5group is6.45±0.33mm. No significant differences between the two groups were found. The left ventricular long axis of SHR-17group (13.31±0.67) is33.5%higher than that of WKY-17group (9.97±0.60), which is significantly different.
3. Pathological analysis:The myocardial cells of WKY rats and5-week-old SHR showed normal morphology. The arrangement of myocardial cells was neat and dense, with no fiber fracture. The staining of intercalated disc and nuclei are clear. SHR-17rats showed myocardial cell swelling, sparse arrangement, myocardial fiber rupture and increased interstitial fibrosis. Sirius red staining showed that fibrillar collagen in hearts of SHR-17group (2.93±0.26%) was much more evident than that in SHR-5group (0.15±0.01%), WKY-5group(0.13±0.01%) and WKY-17group (0.18±0.02%, P<0.001).
4. Ultrastructural examination:The ultrastructure of myocardium in WKY-5and WKY-17group was normal. Although no typical hypertrophic changes were found in SHR-5rats, the cytoskeleton of myocardial cells started to show changes: the parallel and linear arrangement of the Z-discs in SHR-5rats was lost, the Z-discs appeared wavy, the mitochondria swelled and showed electron-lucent areas because of the loss of matrix granules. The nuclei and other organelles showed normal morphology. Myocytes of the SHR-17group showed total disruption of the contractile system and complete disorganization of myofibrils. In some areas, myofibrils disappeared and were replaced by bizarre mitochondria. Mitochondrial swelling was more serious and displayed vacuolation. The mitochondria lost the normal morphology and distributed as clusters, with over-dense matrix and blurred cristae. Deformation in nuclear shape was observed. Nuclei appeared elongated and bizarre and the nuclear membranes were found convoluted. Condensed chromatin was found in nuclei as small blocks and thin cords. Rough endoplsmic reticulum and Golgi apparatus is well developed, suggesting that the synthesis and secretion is active in myocardial cells.The ultrastructral morphology of SHR-17group is consistent with that of cardiac hypertrophy.
5. Mass Spectrometry Identify the Differentially Expressed Proteins We identified506proteins in this study. Seven proteins were shown to have significantly different abundance between SHR-5group and WKY-5group, of which3were upregulated and4were downregulated. Twenty eight proteins were shown to have significantly different abundance between SHR-17group and WKY-17group, of which17were upregulated and11were downregulated. There are three mutual differentially expressed proteins:Profilin-1, Myosin light chain kinase2and Mitochondrial NADP+-isocitrate dehydrogenase.
6. GO analysis of differentially expressed proteins:According to the molecular functional annotation, the differentially expressed proteins are mainly involved in structural molecule function (36.7%), binding function (30.0%) and catalytic activity (20.0%). According to the biological processes, proteins are mainly involved in cellular processes (18.0%), metabolic processes (16.0%), biogenesis (14.0%) and developmental process (14.0%). According to the cellular components,47.6%in cytoplasm,42.9%in organelles,4.8%on cell membrane and4.8%in the cellular junction. Panther protein class analysis showed that the differentially expressed proteins mainly include cytoskeletal proteins (33.3%), enzyme regulatory proteins (13.3%), structural proteins (10.0%) and calcium binding proteins (6.7%) etc. The results showed that cytoskeletal proteins play critical roles in the development of myocardial hypertrophy in SHR rats.
7. Profilin-1expression levels:Real-time PCR results showed that the mRNA level of profilin-1in SHR is significantly higher than that in WKY rats (P<0.001). Western blot showed that the protein expression level of profilin-1in SHR is significantly higher than that in WKY rats (P<0.001). Abundance of profilin-1protein shows a similar trend as that iTRAQ.
Conclusion
1. The myocardial hypertrophy model of17-week-old SHR was successfully built, which was confirmed by cardiac mass index measurement, myocardial pathology analysis and ultrastructural observations.
2. As a new technology of quantitative comparative proteomic, iTRAQ has high accuracy, repeatability and quantitative effect. It can be used to explore the molecular mechanisms of myocardial hypertrophy and look for the key proteins and pathways.
3. Mass spectrometry identifed28differentially expressed proteins between 17-week-old SHR rats with hypertrophic myocardium and17-week-old WKY rats with normal myocardium. GO analysis showed that the cytoskeleton proteins in SHR rats play critical roles in the pathological process of myocardial hypertrophy.4. Real-time quantitative PCR and Western blot examination confirmed the high expression level of profilin-lin myocardial tissues of SHR rats, which verified the reliability of iTRAQ technology and provided key candidate targets for further research on the pathogenesis of myocardial hypertrophy.
Part Two
The Effects and Mechanism of Profilin-1in Hypertension Induced Cardiac Hypertrophy
Background
High blood pressure is the first cause of myocardial hypertrophy. When the heart is under hemodynamic overload, myocardial cells can perceive the tension and generate and release a variety of cytokines. These cytokines will combine with corresponding receptors on the cell membrane and activate a variety of hypertrophy signaling cascade. Finally the nuclear oncogene transcription is activated and myocyte hypertrophic process starts. Functional membrane proteins, cytoskeleton system as well as mutual interaction among cytokines involve in the above pathophysiological process. Cytoskeleton system is the key component to perceive the mechanical stimulation and convert mechanical stress into biochemical signals. The cytoskeleton, especially actin microfilament system, is an important point to study the mechanisem of hypertension induced cardiac hypertrophy
Profilin-1is an important actin binding protein. It can modulate the dynamic assembly of actin cytoskeleton according to extracellular signals. Our previous research suggested that profilin-1plays a critical role in the early myocardial hypertrophy caused by ouabain. And the proteomics analysis mentioned in part one reveals a significant upregulation of profilin-1expression in early hypertrophic myocardium of spontaneously hypertensive rats compared with the control group. In this project, we constructed adenovirus vectors carrying profilin-1mRNA and siRNA separately, and injected them into spontaneously hypertensive rat models to induce the overexpression and silence of profilin-1gene. By this way, we studied the effects of profilin-1in the development of hypertension-induced myocardial hypertrophy and its possible mechanisms using various pathological and histologic technologies as well as molecular biology. These results will provide a new target for early prevention and treatment of hypertensive myocardial hypertrophy and is of great academic significance and potential clinical value.
Objectives
1. To investigate the improvement/deterioration of hypertension-induced myocardial hypertrophy by transfection of interference adeno virus vector pAd-miR-profilin-1/overexpression adeno virus vector pAd-profilin-1-IRES-EGFP into spontaneously hypertensive rats;
2. To explore the mechanism by which profilin-1silencing/overexpression would ameliorate/deteriorate hypertension-induced myocardial hypertrophy on molecular, histological and ultrastructural levels.
Methods
1. Five-week-old boy SHR and WKY rats were randomly assigned to four groups (n=20per group):WKY-C (WKY control rats treated with negative control adenovirus); SHR-C (SHR control rats treated with negative control adenovirus); SHR-I (SHR treated with adenovirus vector pAd-miR-profilin-1to knockdown profilin-1expression); and SHR-H (SHR treated with adenovirus vector Adprofilin-1-IRES-enhanced green fluorescent protein (EGFP) to overexpress profilin-1). The adenoviruses were injected twice into tail veins of rats at a dosage of3×109infectious units per rat with an interval of6weeks between injections. Six weeks after the second administration of adenovirus, subsets of animals were randomly selected and used for cardiac mass index measurement, histopathologic and ultrastructural observation, or biochemical analysis.
2. Blood pressure measurement:SBP was measured with the Heart Rate and BP Recorder for Rats, using the tail-cuff method before the start of treatment and weekly during treatment.
3. Cardiac mass index measurement:Rats were weighed and euthanized under anesthesia at17weeks of age, and hearts were removed and weighed. Atria were cut away and right ventricular free walls were carefully dissected from the left. The intraventricular septum was included in the left ventricular weight. The distance from the atrial valve to the apex, called the left ventricular long axis, was measured as a reflection of left ventricular enlargement.
4. Transfection efficiency detection:Ten days after the second injection of adenovirus, five rats in each group were euthanized and their hearts removed to determine transfection efficiency. EGFP expression in the cardiac tissues was observed with a fluorescence microscope to determine transfection efficiency. For each sample, EGFP expression and transfection efficiency were evaluated in six randomly chosen fields per section and quantitated using Image-Pro Plus6.0software.
5. Pathological analysis:The morphologicalchanges of myocardium were examined by light microscope following hematoxylin and eosin staining. Sirius red staining was carried out to determine the extent of fibrosis. Image-Pro Plus6.0software was used to determine the collagen content, which was expressed as fraction of the total myocardial cross-sectional area.
6.Ultrastructure observation:The ultrastructure of myocyte was observed under an H-800transmission electron microscope. Individual myocyte membranes (n=15for each group) were imaged at×40000magnifications, and the number of caveolae per micrometer plasmalemmal membrane (/μm) were counted by blinded investigators.
7. Immunohistochemical and immunofluorescence staining:The expression of profilin-1in myocardium was viewed by immunohistochemical staining under a light microscope. The colocalization of caveolin-3and actin microfilament was confirmed using immunofluorescence staining.
8. Measurement of nitric oxide production in serum and left ventricle:A serous and cardiac NO assay was performed using the NO assay kit. The homogenates of left ventricular samples and serum were centrifuged and the supernatant was taken for NO assay. With this method, the nitrate present in the sample was converted into nitrite by the enzyme nitrate reductase.The nitrite reacted with sulphanilamide and N-(L-naphthyl)-ethylenediamine dihydrochloride to give a red-violet diazo dye. Then the diazo dye was measured at the absorbance of550nm and the content of nitrite was obtained to reflect the production of NO. Results were expressed as μmol/g protein.
9. Real-time quantitative RT-PCR:Total RNA of the rat myocardium tissues was extracted by Trizol. The mRNA expression levels of profilin-1, eNOS and caveolin-3in the left ventricle of rats were examined by real-time PCR.
10. Western blot analysis:The protien expression levels of profilin-1, eNOS, p-eNOS and caveolin-3in the left ventricle of rats were examined by western blot analysis.
Results
1. Effect of profilin-1on SBP of spontaneous hypertensive rats:The effect of adenovirus-mediated profilin-1gene overexpression and knockdown on SBP was monitored in SHR and WKY before injection and every week afterward. There were significant differences in SBP between SHR and WKY throughout the study (P<0.001). However, no significant differences were observed among the three SHR groups throughout the experiment.
2. Cardiac mass index measurement:Compared with WKY-C group, SHR-C group showed a27%increase in left ventricular weight-to-heart weight ratio (LVW/HW Ratio) and a29%increase in the left ventricular long axis measurement. The SHR-I group displayed a significantly decreased LVW/HW Ratio when compared with the SHR-C group (P<0.05), indicating that lowering profilin-1expression reversed cardiac hypertrophy. The left ventricular long axis, another measurement of left ventricular enlargement, was also significantly reduced in SHR-I group compared with control SHR (P<0.01).
3. Detection of adenovirus transfection efficiency:The high transfection efficiency was confirmed by observing EGFP expression under the fluorescence microscope. In all four groups, the green fluorescent cells accounted for a proportion of30%-35%. Meanwhile, it's hard to see any EGFP expression in myocardial tissue of rats injected with saline.
4. Pathological analysis:Compared with WKY-C rats, SHR rats showed myocardial cell swelling, sparse arrangement, myocardial fiber rupture and increased interstitial fibrosis. To further confirm the effects of profilin-1on cardiac hypertrophy and fibrosis, we overexpressed profilin-1in the cardiac tissue of SHR using an adenovirus carrying the profilin-1gene.The myocardium from the SHR-H group developed significant hypertrophy with cardiomyoliposis. Sirius red staining showed that fibrillar collagen in hearts of SHR-H group was much more evident than that in SHR-C group (P<0.001). These results demonstrate that high levels of profilin-1promote cardiac hypertrophy and fibrosis.
5. Ultrastructural examination:Compared with WKY-C group, the parallel and linear arrangement of the Z-discs in SHR rats was lost, the Z-discs appeared wavy, the mitochondria swelled and showed electron-lucent areas because of the loss of matrix granules. Deformation in nuclear shape was observed. Nuclei appeared elongated and bizarre and the nuclear membranes were found convoluted. Condensed chromatin was found in nuclei as small blocks and thin cords. Myocytes of the SHR-H group showed total disruption of the contractile system and complete disorganization of myofibrils. In some areas, myofibrils disappeared and were replaced by bizarre mitochondria. Mitochondrial swelling was more serious and displayed vacuolation. The mitochondria lost the normal morphology and distributed as clusters, with over-dense matrix and blurred cristae. Distortion of nuclear shape was persistent and chromatin condense became more serious. The chromatin constituted a thick layer in the inner boundary of the nuclear membrane.
6. Effects of profilin-1on actin cytoskeleton:Phalloidin staining in the WKY-C group was abundant but became weaker and thinner in SHR-C group. Downregulation of profilin-1expression helps to conserve the abundance of actin filaments in SHR, whereas overexpression of profilin-1further reduced actin filaments organization.
7. Profilin-1overexpression decreased the caveolar abundance:Transmission electron microscopy was performed to quantify caveolar abundance. Caveolae were three-fold more abundant in WKY-C group compared with SHR-C group (P<0.001). Compared with the control SHR, transgenic overexpression of profilin-1in SHR caused a further significant reduction of sarcolemmal caveolae (P<0.001). In contrast, downregulation of profilin-1expression helps to conserve the abundance of caveolae in SHR (P<0.001). This result was consistent with the abundance of caveolin-3mRNA and protein in cardium. Both mRNA and protein expression of caveolin-3were lower in hearts of SHR compared with those in WKY. The reduction in caveolin-3expression was more severe in the SHR-H group with profilin-1overexpression. In contrast, caveolin-3expression was restored in the SHR-Ⅰ group following profilin-1knockdown.
8. Profilin-1expression levels and cellular localization in the myocardial tissue of rats:Real-time PCR results showed that the mRNA level of profilin-1in SHR-H group is significantly higher than that in SHR-C group (P<0.05) while the mRNA level of profilin-1in SHR-Ⅰ group is significantly lower than that in SHR-C group (P<0.05). Western blot showed that the protein expression level of profilin-1in four groups is similar as that of mRNA. The immunohistochemical analysis of profilin-1in the heart of SHR and WKY showed that profilin-1protein was expressed in the cytoplasm of cardiomyocytes, especially around the nucleus. Abundance of profilin-1protein shows a similar trend as that in western blot analysis. We also observed mild profilin-1expression in vascular smooth cells.
9. Content of NO in serum and myocardial tissue:NO secretion was lower in hearts of SHR compared with WKY. We investigated production of NO in left ventricular and found a significant decrease in SHR-H group (P<0.01) and a significant increase in SHR-I group (P<0.05) when compared with SHR-C group. We also tested the NO levels in the serum of rats and no significant differences were found among the SHR-C group, SHR-I group, and SHR-H group, indicating that the changes of NO levels is a local phenomenon rather than a systemic reaction.
10. Expression and activity of eNOS in rat myocardial tissue:No significant difference of eNOS expression was found among the four treatment groups. eNOS activity and NO secretion were lower in hearts of SHR compared with WKY. Our results showed a significant decrease of phospho-S1177eNOS in SHR-H group and a significant elevation in SHR-I group, when compared with SHR-C group (P<0.05).
Conclusion
1. Profilin-1expression is upregulated in hypertrophic myocardium of spontaneously hypertensive rats, accompanying with cardiomyocyte hypertrophy, hyperplasia of collagen fibers and actin cytoskeleton damage.
2. Overexpression of profilin-1interfered with the assembly of actin cytoskeleton and the formation of caveolae on myocardial membrane, then inhibited the activity of the eNOS/NO signaling pathways and finally promoted hypertension-induced myocardial hypertrophy. This might be one of the most important pathogenesis of myocardial hypertrophy.
3. Profilin-1gene silencing could effectively reverse myocardial hypertrophy by increasing the content of myocardial actin microfilament, increasing the caveolar number and restoring eNOS activity and the product of NO.
研究背景
心肌肥大是心肌细胞和细胞外间质对血流动力学超负荷和神经体液失调等多种病理刺激的基本应答和代偿反应,是许多心血管疾病(如高血压、心脏瓣膜病、急性心肌梗塞和先天性心脏病)发生发展过程中共有的病理变化。临床流行病学资料显示,心肌肥大导致心源性猝死和心力衰竭的发病率明显增高,严重影响患者的预后,已被列为影响心血管疾病发病率和死亡率的独立危险因子。因此,对心肌肥大发生机制的探讨并寻找有效的预防和治疗措施,具有十分深远的临床意义和科研价值。
蛋白质组学是以蛋白质组为研究对象,探究细胞内所有蛋白质及其动态变化规律的科学,在细胞和生命有机体的整体水平上阐明生命现象的本质和活动规律。同位素标记的相对和绝对定量技术(isobaric tags for relative and absolute quantitation, iTRAQ)是近年来开发的一种新的比较蛋白质组学定量研究技术。iTRAQ技术可对一个基因组表达的全部蛋白质进行精确定量和鉴定,寻找差异表达蛋白,并分析其蛋白功能。与传统的依赖凝胶电泳的定量技术相比,iTRAQ是基于高度敏感性和精确性的串联质谱方法；可同时对4种甚至8种不同来源的样品进行绝对和相对定量研究；不仅可以发现胞浆蛋白,还可发现膜蛋白、核蛋白甚至胞外蛋白；可以检测出低丰度蛋白、强碱性蛋白、小于10kD或大于200kD的蛋白。iTRAQ技术的发展为探讨心肌肥大的发生机制,寻找关键的作用蛋白提供了高效可靠的研究方法。
自发性高血压大鼠(Spontaneous Hypertension Rat,SHR)是1959年由日本京都大学Okamoto和Aoki培育而成的遗传性高血压动物模型。SHR在遗传特性、发病机制以及高血压心血管并发症等方面均与人类原发性高血压十分相似,是目前国际公认最接近于人类原发性高血压的动物模型。心肌肥大是SHR重要特征之一,高血压性心肌肥大在幼年SHR已出现,并存在于高血压整个自然病程中。
因此,本研究选用特定年龄阶段的SHR大鼠为研究对象,采用定量比较蛋白质组学iTRAQ技术,着力于发现SHR大鼠早期心肌肥大发生发展过程中差异表达的功能性蛋白或蛋白群,并利用生物信息学方法分析差异蛋白的分子功能、蛋白分类及生物学过程,筛选并鉴定在高血压性心肌肥大发病过程中起关键作用的蛋白分子,为高血压心肌肥大的防治提供有效靶点。
研究目的
1.研究自发性高血压大鼠心肌病变的特点,观察5周龄和17周龄SHR大鼠心肌组织的病理学和超微结构变化；
2.应用iTRAQ蛋白质组学技术研究SHR大鼠肥大心肌与同周龄WKY大鼠正常心肌组织的差异表达蛋白,从整体水平上揭示高血压心肌肥大病变的病理生理机制,并筛选关键靶蛋白；
研究方法
4周龄雄性SHR大鼠8只,WKY大鼠8只,16周龄雄性SHR大鼠8只,WKY大鼠8只,适应性饲养1周后(即5周龄和17周龄时)进入实验：
1.大鼠尾动脉收缩压测定：处死前通过无创血压测量系统检测各组大鼠尾动脉收缩压；
2.心脏质量指数测定：大鼠处死后,称量心脏重和左室重,测量主动脉瓣至心尖的距离为左心室长轴。以左室重/心脏重和左心室长轴作为反应心室肥大的指标；
3.心肌组织病理学检测：HE染色观察心肌组织病理变化,天狼星红染色测定胶原纤维面积百分比；
4.心肌组织超微结构观察：透射电镜技术观察心肌肌丝、肌节、线粒体及细胞核等亚细胞结构；
5.蛋白质组学(iTRAQ)分析：5周龄WKY大鼠(WKY-5)、5周龄SHR大鼠(SHR-5)、17周龄WKY大鼠(WKY-17)和17周龄SHR大鼠(SHR-17)各8只,提取左心室总蛋白。采用胰酶消化蛋白得到肽段,按照ABI公司说明书操作标记肽段,114标记WKY-5组,115标记SHR-5组,116标记WKY-17组,117标记SHR-17组。将各组已标记的肽段混合后用强阳离子交换柱和C18柱进行分离,然后采用MALDI-TOF/TOF和MicroQ-TOF进行质谱鉴定。不同报告基团离子的强度代表其所标记的多肽的相对丰度；
6.差异蛋白质功能分析：利用Data Analysis4.0软件分析质谱数据,用Mascot软件搜索与质谱匹配的蛋白。采用Panther软件(http://www.pantherdb.org/)对差异蛋白进行GO分析,包括分子功能,生物过程和细胞组成三个部分；
7.验证profilin-1的表达：采用实时定量聚合酶链反应(qPCR)技术检测各组大鼠心肌组织中profilin-1的mRNA水平,蛋白质免疫印迹方法(Westernblot)检测profilin-1的蛋白表达水平,以验证蛋白质组学的可靠性。
研究结果
1.大鼠尾动脉收缩压：5周龄WKY大鼠收缩压为111.2±7.6mmHg,5周龄SHR大鼠收缩压为143.1±10.5mmHg,已显著升高(P<0.001),17周龄WKY大鼠收缩压为117.7±8.6mmHg,17周龄SHR大鼠收缩压为216.3±12.1mmHg,收缩压的差异更为显著(P<0.001)。
2.大鼠心脏肥大指数：5周龄SHR大鼠的平均LVW/HW比值为0.55±0.02,5周龄WKY大鼠的平均LVW/HW比值为0.54±0.02,两者无显著差异；17周龄SHR大鼠的平均LVW/HW比值为0.77±0.06,同周龄WKY大鼠的平均LVW/HW比值为0.60±0.03,SHR大鼠较WKY大鼠升高28.33%。5周龄SHR大鼠的左室长轴平均长度为6.45±0.33mm,同周龄WKY大鼠的左室长轴平均长度为6.42±0.36mm,两者无显著差异；17周龄SHR大鼠的左室长轴平均长度为13.31±0.67mm,同周龄WKY大鼠的左室长轴平均长度为9.97±0.60mm,SHR大鼠较WKY大鼠升高33.50%。
3.大鼠心肌病理学分析
3.1心肌HE染色：5周龄及17周龄的WKY大鼠心肌细胞形态正常,排列整齐、致密,肌纤维无断裂,横纹、闰盘及细胞核染色清晰；5周龄SHR大鼠心肌细胞形态、结构及排列基本正常；17周龄SHR大鼠心肌细胞肿胀、内容物成颗粒状,部分细胞间排列稀疏、间隙不清,心肌纤维断裂、融合呈波浪状改变,多数横纹、闰盘尚清晰,细胞核肥大或固缩,个别视野心肌间质纤维化；横截面上可见心肌细胞直径增宽,单个心肌细胞面积明显增加。
3.2心肌天狼星红染色：WKY大鼠心肌间质几乎无胶原纤维分布,对40个随机视野分析后得到其天狼星红染色阳性区域约占视野下总面积的0.13±0.01%(5周龄)和0.18±0.02%(17周龄)；5周龄SHR大鼠心肌胶原纤维占视野下总面积的0.15±0.01%,与同周龄WKY大鼠相比无显著差异；17周龄SHR大鼠心肌胶原分布较同周龄WKY大鼠明显增多,约占总面积的2.93±0.26%(P<0.001)。
4.大鼠心肌超微结构观察：电镜下可见5周龄和17周龄WKY大鼠心肌组织中,肌原纤维含量丰富,排列规整,Z线和明暗带清晰；闰盘结构正常；线粒体沿肌原纤维长轴平行排列,可见均匀的基质颗粒和完整致密的线粒体嵴；细胞核为圆形或椭圆形,核染色质均匀,核仁清晰,核膜平滑完整。5周龄SHR大鼠心肌细胞超微结构尚未出现典型的心肌肥大表现,但肌原纤维排列已出现紊乱,部分区域肌丝溶解,肌小节结构破坏,Z线断裂成波浪状；线粒体数量略增多,排布出现不规整；细胞核及其他细胞器形态基本正常。17周龄SHR大鼠心肌细胞内肌原纤维排列紊乱,肌小节结构破坏,Z线断裂成波浪状,部分区域肌丝溶解消失而被增生的线粒体取代；线粒体形态异常,数量增多且排布紊乱,部分线粒体肿胀,因基质颗粒的缺失出现了电子透亮区,线粒体嵴模糊不清；细胞核肥大、畸形,核膜呈不规则凹陷,染色质聚集成小团块,在核膜内侧尤为显著,核仁较为明显；粗面内质网和高尔基体发达,提示合成分泌功能旺盛,符合心肌肥厚表现。
5.质谱分析结果：实验共鉴定出大鼠心肌蛋白506个,5周龄SHR大鼠与WKY大鼠相比差异表达蛋白共7个,其中上调3个,下调4个；17周龄SHR大鼠与WKY大鼠相比差异表达蛋白28个,其中上调17个,下调11个。进一步分析发现,5周龄和17周龄WKY与SHR两组中共同的差异蛋白有3个,分别是：Profilin-1、Myosin light chain kinase2和Mitochondrial NADP+-isocitrate dehydrogenase。
6.SHR大鼠肥大心肌差异蛋白的功能分析：根据分子功能注释,差异蛋白主要执行结构性分子功能(36.7%),结合功能(30.0%)和催化功能(20.0%)。根据生物过程注释,差异蛋白主要参与细胞过程(18.0%)、代谢过程(16.0%)、生长发育(14.0%)及生物合成(14.0%)4种生物过程。根据细胞成分注释,主要包含细胞组成(47.6%)、细胞器(42.9%)、细胞膜(4.8%)及细胞连接(4.8%)四部分。Panther蛋白种类分析显示差异蛋白主要包含细胞骨架蛋白(33.3%)、酶调节蛋白(13.3%)、结构蛋白(10.0%)和氧化还原蛋白(6.7%)等,细胞骨架蛋白占据的高比例(三分之一)与前面结构性分子功能(36.7%)相呼应,可见细胞骨架蛋白在SHR大鼠心肌肥大病理过程中占据重要地位。
7.实时定量PCR检钡Profilin-1的mRNA表达：5周龄SHR大鼠profilin-1mRNA表达水平为同周龄WKY大鼠的1.86倍,表达明显上调(P<0.001)；17周龄SHR大鼠profilin-1mRNA表达水平为同周龄WKY大鼠的2.83倍,表达上调具有显著的统计学差异(P<0.001)。实时定量PCR结果与iTRAQ研究结果一致。
8. Western Blot检测Profilin-1的蛋白表达：与同周龄WKY大鼠相比,5周龄SHR大鼠心肌组织profilin-1的蛋白水平上调了约23.33%,差异具有统计学意义(P<0.05)；17周龄SHR大鼠心肌组织profilin-1的蛋白水平较同周龄WKY大鼠上调了约64.10%,差异具有统计学意义(P<0.01)。Westem-blot结果与比较蛋白质组学研究结果一致。
结论
1.心脏质量指数、心肌病理学分析以及超微结构观察结果均证实17周龄SHR大鼠心肌肥大模型已构建成功,5周龄SHR大鼠心肌出现早期病理改变,尚未发生心肌肥大。
2.作为定量比较蛋白质组学的新技术iTRAQ具有较好的精确性、重复性和定量效果,可用于研究SHR大鼠心肌肥大的分子机制,找到该发病过程的关键蛋白及通路。
3.质谱鉴定17周龄SHR大鼠肥大心肌与同周龄WKY大鼠正常心肌的差异表达蛋白28个,其中上调17个,下调11个,经G0分析后发现细胞骨架蛋白在SHR大鼠心肌肥大病理过程中占据重要地位。
4.实时定量PCR及Western blot实验结果均证实SHR大鼠心肌组织中profilin-1的表达水平明显上调,这一方面验证了iTRAQ技术的可靠性,同时为深入研究高血压诱导的心肌肥大的发病机制提供了关键的候选靶点。第二部分profilin-1在高血压大鼠心肌肥大中的作用及机制研究
研究背景高血压是心肌肥大的首要致病因素,患者的心脏处于血流动力学超负荷状态时,心肌细胞可感知该种牵张刺激,产生、释放多种细胞因子,这些细胞因子与心肌细胞膜上的相应受体结合后激活多种肥大相关信号级联反应,随后诱导核内原癌基因的转录活化,启动心肌细胞的肥大反应过程。胞膜的功能蛋白、细胞骨架系统和细胞因子间的相互作用共同参与了这一病理生理过程,其中细胞骨架系统是感知力学刺激、将机械应力转化为生化信号的关键组件,研究细胞骨架,尤其是肌动蛋白微丝系统,是深入认识血流动力学超负荷诱导下心肌肥大发生机制的重要切入点。
Profilin-1是一种重要的肌动蛋白结合蛋白,可受细胞外信号影响而调节肌动蛋白细胞骨架的动态组装。课题组前期研究提示profilin-1在哇巴因高血压大鼠心肌肥大早期发挥关键作用,且第一部分的蛋白质组学结果揭示自发性高血压大鼠早期肥大心肌中profilin-1的表达较对照组显著升高。因此,本课题将利用自发性高血压大鼠心肌肥大模型,构建腺病毒表达载体,诱导心肌细胞中profilin-1基因的过表达和沉默,综合运用各种病理学组织学染色技术和分子生物学技术研究profilin-1在高血压心肌肥厚病变发生发展中的作用及其可能机制。这些结果将为高血压心肌肥大患者的早期防治提供新靶点,具有重要的学术理论意义和潜在的临床应用价值。
研究目的
1.自发性高血压心肌肥大模型大鼠体内转染高表达腺病毒载体pAd-profilin-1-IRES-EGFP或干扰腺病毒载体pAd-miR-profilin-1,观察对心肌肥大病变的影响：
2.在组织病理学水平、细胞分子水平和超微结构水平研究profilin-1参与高血压心肌肥大发生发展的机制;
研究方法
1.5周龄自发性高血压大鼠60只,随机分为SHR-C组、SHR-I组和SHR-H组；5周龄健康WKY大鼠20只作为对照组WKY-C组.SHR-C组与WKY-C组大鼠经尾静脉注射阴性对照腺病毒载体3×109PFU/只,SHR-I组大鼠经尾静脉注射干扰腺病毒载体pAd-miR-profilin-13×109PFU/只,SHR-H组大鼠经尾静脉注射高表达腺病毒载体pAd-profilin-1-IRES-EGFP3×109PFU/只。6周后补充注射一次,剂量同第一次,第二次腺病毒注射6周后处死动物留取标本;
2.大鼠尾动脉收缩压监测：实验开始前(5周龄)及腺病毒转染后每周末通过大鼠尾动脉血压测量仪监测大鼠尾动脉收缩压,观察profilin-1过表达和沉默后对自发性高血压大鼠血压的影响；
3.心脏质量指数测定：大鼠处死后,称量心脏重和左室重,测量主动脉瓣至心尖的距离为左心室长轴。左室与心脏的重量比和左心室长轴均可作为反应心室肥大的指标；
4.腺病毒转染效率检测：第二次注射腺病毒后10天,取心肌标本切冰冻切片,荧光显微镜下观察EGFP绿色荧光并拍照,利用Image-Pro Plus6.0软件检测每个视野下EGFP绿色荧光细胞占所有心肌细胞的比例,该比值反应腺病毒转染效率；
5.病理学检测：对大鼠心肌组织分别进行HE染色和天狼星红染色,观察腺病毒转染后各组大鼠心肌组织病理改变;
6.心肌组织超微结构观察：透射电镜技术观察心肌肌丝、肌节、线粒体及小窝等亚细胞结构；
7.免疫组织化学染色和免疫荧光染色：应用免疫组织化学染色观察profilin-1在心肌中的定位,免疫荧光染色观察小窝蛋白-3与肌动蛋白微丝的共定位；
8.大鼠血清和心肌组织中NO含量测定：应用N0测定试剂盒检测大鼠血清和心肌组织匀浆中NO的含量；
9.实时定量PCR检测：取大鼠心肌组织,Trizol法提取RNA,实时荧光定量RT-PCR技术检钡profilin-1、eNOS、小窝蛋白-3mRNA相对表达量；
10. Western blot检测：取大鼠心肌组织,提取蛋白,Western blot检测profilin-1,eNOS,p-eNOS和小窝蛋白-3的蛋白表达量。
研究结果
1.各组大鼠血压变化：整个实验过程中(5周龄-17周龄)SHR大鼠血压随周龄增加而升高,且显著高于同周龄WKY大鼠血压；实验末,与SHR-C组相比,SHR-I组血压略下降,差异无统计学意义；与SHR-C组相比,SHR-H组血压略升高,差异无统计学意义。提示profilin-1过表达和沉默对SHR大鼠血压无明显影响。
2.心脏质量指数测定：与WKY-C组大鼠相比,SHR-C组大鼠左室重量／心脏重量、左室长轴均明显增加,差异具有统计学意义(P<0.01)；与SHR-C组大鼠相比,SHR-I组大鼠左室重量／心脏重量(P<0.05)、左室长轴(P<0.01)均明显减小,而SHR-H组大鼠左室重量/心脏重量、左室长轴均明显增加(P<0.05)。profilin-1过表达加重SHR大鼠心肌肥大,同时profilin-1基因沉默显著减轻了SHR大鼠心肌肥大。
3.腺病毒转染效率检测：采用天空蓝染色屏蔽自发荧光后,各组大鼠心肌组织中绿色荧光细胞占细胞总数的30%-35%,注射生理盐水的WKY大鼠和SHR大鼠心肌组织中检测不到绿色荧光细胞。
4.心肌组织病理学分析
4.1心肌HE染色：与WKY大鼠相比,SHR大鼠心肌细胞肿胀,部分细胞间排列稀疏、间隙不清,心肌纤维断裂、融合呈波浪状改变；与SHR-C组大鼠相比,SHR-H组大鼠的心肌细胞肿胀更加明显,部分心肌呈灶状与片状坏死,伴有脂肪变性和炎性细胞浸润。相反,SHR-I组大鼠心肌细胞的病理损伤明显减轻。
4.2心肌天狼星红染色：与WKY-C组大鼠相比,SHR-C组大鼠心肌胶原分布明显增多,差异具有统计学意义(P<0.001)；与SHR-C组大鼠相比,SHR-H组大鼠心肌胶原分布明显增多(P<0.001),而SHR-I组大鼠心肌胶原分布明显减少(P<0.001)。这些结果表明profilin-1基因的高表达促进高血压诱导的心肌纤维化,而profilin-1基因沉默则有助于减轻心肌纤维化程度。
5.心肌超微结构观察：与WKY-C组大鼠相比,SHR-C组大鼠心肌细胞内肌原纤维排列紊乱,肌小节结构浪状破坏,Z线断裂成波,线粒体形态异常,数量增多且排布紊乱,线粒体嵴模糊不清,粗面内质网和高尔基体发达,提示合成分泌功能旺盛,符合心肌肥厚表现；与SHR-C组相比,SHR-H组大鼠心肌超微结构破坏更为严重,大片区域肌原纤维皱缩或崩解,闰盘及Z线不清,线粒体严重肿胀,线粒体嵴断裂甚至消失,内质网扩大,胞核呈早期凋亡形态,大量的胶原纤维增生,可见其将肥大的心肌细胞包绕分隔成束；而SHR-I组大鼠心肌细胞肌原纤维含量较SHR-C组大鼠明显增加,肌节较规则,线粒体轻度肿胀,线粒体内空泡结构较少,心肌细胞核仁清晰,染色质分布均匀,边集现象不明显,内质网扩张不明显,偶有点状胶原纤维增生。
6.心肌中肌动蛋白细胞骨架的改变：WKY-C组大鼠心肌中鬼笔环肽荧光染色丰富,而SHR-C组大鼠中染色细弱,提示肌动蛋白细胞骨架含量下降；与SHR-C组大鼠相比,降低profilin-1的表达有助于肌动蛋白微丝结构和含量的保存,而profilin-1高表达则进一步减低肌动蛋白微丝的含量。
7.心肌细胞膜上小窝丰度的检测：WKY-C组大鼠心肌中小窝的数量是SHR-C组大鼠的3倍,统计学差异显著(P<0.001),与SHR-C组相比,SHR-H组心肌纤维膜上小窝的数量进一步减低(P<0.001),而profilin-1的沉默则有助于保存SHR-I组小窝的丰度(P<0.001)。实时定量PCR和western blot的结果显示SHR-H组大鼠心肌小窝蛋白-3的mRNA含量是SHR-C组大鼠的1.66±0.33倍(P<0.05),其蛋白含量是SHR-C组大鼠的1.71±0.21倍(P<0.001)；而SHR-I组大鼠心肌小窝蛋白-3的mRNA含量是SHR-C组大鼠的42.5±2.6%(P<0.01),其蛋白含量是SHR-C组大鼠的33.6±3.2%(P<0.001),小窝蛋白-3的mRNA和蛋白表达水平与肌浆膜上小窝的数量相一致。
8.大鼠心肌组织中profilin-1的表达水平及细胞定位：实时定量PCR结果显示,SHR-C组大鼠肥大心肌中profilin-1的mRNA水平较WKY-C组明显升高,差异具有统计学意义(P<0.01),SHR-H组大鼠心肌组织中profilin-1的mRNA水平较SHR-C组上调了38.10%(P<0.05),而SHR-I组profilin-1的mRNA表达水平较SHR-C组降低了44.29%(P<0.05)；Western blot结果显示,SHR-C组大鼠肥大心肌中profilin-1的蛋白水平较WKY-C组明显升高,差异具有统计学意义(P<0.05),与SHR-C组相比,SHR-H组大鼠心肌profilin-1蛋白水平上调了约35.29%(P<0.05),而SHR-I组profilin-1蛋白表达下调了38.24%(P<0.01)；免疫组化图片显示profilin-1蛋白表达在心肌细胞浆内,尤其在核周分布,在血管平滑肌细胞中也见到profilin-1微弱的表达,profilin-1在各组大鼠心肌细胞中的表达水平与western blot结果一致。
9.大鼠血清和心肌组织中N0的含量：与WKY-C组大鼠相比,SHR-C组大鼠肥大心肌中NO含量明显降低,差异具有统计学意义(P<0.05),SHR-H组心肌组织中NO含量明显低于SHR-C组(P<0.01),而SHR-I组心肌组织中NO含量则明显高于对照组(P<0.05)。同时,我们检测了血清中NO水平,结果显示SHR-I组、SHR-C组和SHR-H组大鼠血清中NO水平无显著差异,这提示profilin-1的高表达和沉默所引起的NO水平的变化是心肌组织内的局部变化而非系统性反应。
10.大鼠心肌组织中eNOS表达水平和活性检测：实时定量PCRwestern blot结果显示四组大鼠心肌中eNOS的表达水平无显著差异。进而检测eNOS在Ser1177位点上的磷酸化水平以观察eNOS的活性,结果显示,与WKY-C组大鼠相比,SHR-C组大鼠心肌中eNOS的活性明显减低；与SHR-C组大鼠相比,SHR-H组大鼠心肌中磷酸化eNOS的表达明显降低而SHR-I组的磷酸化eNOS的表达明显升高,差异具有统计学意义(P<0.05)。
结论
1.自发性高血压大鼠肥大心肌组织中,profilin-1的表达上调,伴随心肌细胞肥大、胶原纤维增生和肌动蛋白细胞骨架受损。
2. Profilin-1的过表达通过影响肌动蛋白细胞骨架的组装,干扰心肌纤维膜上小窝的形成,抑制eNOS/N0信号通路的活性而促进高血压诱导的心肌肥大,是高血压心肌肥大的重要发病机制。
3. Profilin-1基因沉默主要通过增加心肌组织内肌动蛋白微丝含量,提高肌浆膜上小窝数量,恢复eNOS活性和NO的含量,从分子水平有效抑制心肌肥大的发生发展。
PART ONE COMPARATIVE PROTEOMICS ANALYSIS OF CARDIAC HYPERTROPHY IN SPONTANEOUS HYPERTENSIVE RATS
Background
Myocardial hypertrophy is a compensatory response of cardiomyocytes and extracellular matrix to the hemodynamic overload and neurohumoral disorders. It is also a common pathological process of many cardiovascular diseases, such as high blood pressure, heart valve disease, acute myocardial infarction and congenital heart disease. Clinical epidemiological data showed that myocardial hypertrophy results in significantly increased incidence of sudden cardiac death and heart failure, causing poor prognosis of patients. Myocardial hypertrophy has been listed as an independent risk factor for cardiovascular morbidity and mortality. Therefore, to explore the mechanisms of myocardial hypertrophy and find effective measures of prevention and treatment of myocardial hypertrophy is of great clinical significance and research value.
Proteomics is a science with the whole proteome as research object. The aim of proteomics is to explore all the proteins in cells and their dynamic changes and to reveal the essence of life and the rule of activity in the overall level of cell and living organisms. The isobaric tags for relative and absolute quantitative techniques (iTRAQ) is a new quantitative study of comparative proteomics technology developed in recent years. iTRAQ technology can supply accurate quantitation and identification of proteins from a whole genome, search for differentially expressed proteins, and analyzes the protein function. Compared with the traditional quantitative technique dependent on gel electrophoresis, iTRAQ is of high sensitivity and accuracy based on tandem mass spectrometry method; It can do absolute and relative quantitative analysis of four and even eight different samples at the same time; Not only cytoplasm protein but also membrane protein, nucleoprotein and extracellular protein can be detected; The proteins of low abundance, strong alkaline, less than10kD or greater than200kD can all be detected by iTRAQ. The development of iTRAQ technology provides an effective and reliable method to study the mechanism of myocardial hypertrophy and look for the key proteins.
Spontaneously hypertensive rats (SHR) is a genetic hypertensive animal model founded by Okamoto and Aoki of Japan's Kyoto university in1959. SHR provide an excellent model system to investigate hypertension-mediated cardiac hypertrophy because of their similarities to human hypertension and natural development of the pathophysiological alterations also happened in patients with essential hypertension. Cardiac hypertrophy is one of the important characteristics of SHR. Hypertensive myocardial hypertrophy occurs in juvenile SHR and exists during the whole course of high blood pressure.
Therefore, in this study we choosed SHR of specific ages as research object and tried to find the differentially expressed proteins in the early development of myocardial hypertrophy by using iTRAQ technology. Then we analyzed the molecular function, classification and biological processes of the differentially expressed proteins using bioinformatics methods, selected and identified the key proteins playing critical effects on the pathogenesis of hypertensive myocardial hypertrophy, and provided effective target for prevention and treatment of hypertensive myocardial hypertrophy.
Objectives
1. To study the characteristics of myocardial leasion in SHR and observe the pathologic and ultrastructural changes of myocardial tissue in SHR of5and17weeks old;
2. To study the differentially expressed proteins between hypertrophic myocardium of SHR and normal myocardium of WKY by using iTRAQ technology, reveal the pathophysiological mechanisms of hypertensive myocardial hypertrophy on the overall level, and screen the key proteins playing critical roles;
Methods
Male SHR (n=8,4weeks old), male WKY (n=8,4weeks old), male SHR (n=8,16weeks old) and male WKY (n=8,16weeks old) were kept under observation for1week (i.e.,5weeks old and17weeks old) prior to the start of experiments:
1. Blood pressure measurement:Systolic blood pressure (SBP) was measured using the noninvasive blood pressure measurement system before the euthanasia of rats.
2. Cardiac mass index measurement:Rats were euthanized under anesthesia and hearts were removed and weighed. Then we dissected the left ventricule and measured the left ventricular weight-to-heart weight ratio (LVW/HW Ratio). The distance from the atrial valve to the apex, called the left ventricular long axis, was measured as another reflection of left ventricular enlargement.
3. Pathological analysis:The morphological changes of myocardium were examined by light microscope following hematoxylin and eosin staining. Sirius red staining was carried out to determine the extent of fibrosis. Image-Pro Plus6.0software was used to determine the collagen content, which was expressed as fraction of the total myocardial cross-sectional area.
4. Ultrastructure observation:The ultrastructure of myocyte was observed under an H-800transmission electron microscope.
5. Proteomics (iTRAQ) analysis:Protein was extracted and digested by trypsin. The peptides were labeled with iTRAQ reagents following the manufacturer's instructions (Applied Biosystems,114for WKY-5group,115for SHR-5group,116for WKY-17group and117for SHR-17group). The labeled peptides were separated by Strong Cation Exchange chromatography and desalted by a C18column. MALDI-TOF/TOF and MicroQ-TOF mass spectrometry was used for the identification of peptides. The different ionic strength of reporter group represents the different abundance of its labeled polypeptides.
6. Gene Ontology (GO) analysis of the differentially expressed proteins:Mass spectrum data was analyzed using Data Analysis software4.0and the matched proteins were searched with Mascot software. GO analysis of the differencially expressed proteins, including molecular function, biological process and cell composition, was performed using Panther software (http://www.pantherdb.org/);
7. Profilin-1expression:Real-time quantitative polymerase chain reaction (qPCR) and western blot technology were used to detect the mRNA and protein level of profilin-1in each group.
Results
1. SBP of rats:The SBP of SHR-5group (143.1±10.5mmHg) is significantly higher than that of WKY-5group (111.2±7.6mmHg, P<0.001). The SBP of SHR-17group (216.3±12.1mmHg) is significantly higher than that of WKY-17group (117.7±8.6mmHg, P<0.001).
2. Cardiac mass index measurement:The LVW/HW Ratio of WKY-5group is0.54±.02while the LVW/HW Ratio of SHR-5group is0.55±0.02.No significant differences between the two groups were found. The LVW/HW Ratio of SHR-17group (0.77±0.66) is28.3%higher than that of WKY-17group (0.60±0.03), which is significantly different. The left ventricular long axis of WKY-5group is6.42±0.36mm while the left ventricular long axis of SHR-5group is6.45±0.33mm. No significant differences between the two groups were found. The left ventricular long axis of SHR-17group (13.31±0.67) is33.5%higher than that of WKY-17group (9.97±0.60), which is significantly different.
3. Pathological analysis:The myocardial cells of WKY rats and5-week-old SHR showed normal morphology. The arrangement of myocardial cells was neat and dense, with no fiber fracture. The staining of intercalated disc and nuclei are clear. SHR-17rats showed myocardial cell swelling, sparse arrangement, myocardial fiber rupture and increased interstitial fibrosis. Sirius red staining showed that fibrillar collagen in hearts of SHR-17group (2.93±0.26%) was much more evident than that in SHR-5group (0.15±0.01%), WKY-5group(0.13±0.01%) and WKY-17group (0.18±0.02%, P<0.001).
4. Ultrastructural examination:The ultrastructure of myocardium in WKY-5and WKY-17group was normal. Although no typical hypertrophic changes were found in SHR-5rats, the cytoskeleton of myocardial cells started to show changes: the parallel and linear arrangement of the Z-discs in SHR-5rats was lost, the Z-discs appeared wavy, the mitochondria swelled and showed electron-lucent areas because of the loss of matrix granules. The nuclei and other organelles showed normal morphology. Myocytes of the SHR-17group showed total disruption of the contractile system and complete disorganization of myofibrils. In some areas, myofibrils disappeared and were replaced by bizarre mitochondria. Mitochondrial swelling was more serious and displayed vacuolation. The mitochondria lost the normal morphology and distributed as clusters, with over-dense matrix and blurred cristae. Deformation in nuclear shape was observed. Nuclei appeared elongated and bizarre and the nuclear membranes were found convoluted. Condensed chromatin was found in nuclei as small blocks and thin cords. Rough endoplsmic reticulum and Golgi apparatus is well developed, suggesting that the synthesis and secretion is active in myocardial cells.The ultrastructral morphology of SHR-17group is consistent with that of cardiac hypertrophy.
5. Mass Spectrometry Identify the Differentially Expressed Proteins We identified506proteins in this study. Seven proteins were shown to have significantly different abundance between SHR-5group and WKY-5group, of which3were upregulated and4were downregulated. Twenty eight proteins were shown to have significantly different abundance between SHR-17group and WKY-17group, of which17were upregulated and11were downregulated. There are three mutual differentially expressed proteins:Profilin-1, Myosin light chain kinase2and Mitochondrial NADP+-isocitrate dehydrogenase.
6. GO analysis of differentially expressed proteins:According to the molecular functional annotation, the differentially expressed proteins are mainly involved in structural molecule function (36.7%), binding function (30.0%) and catalytic activity (20.0%). According to the biological processes, proteins are mainly involved in cellular processes (18.0%), metabolic processes (16.0%), biogenesis (14.0%) and developmental process (14.0%). According to the cellular components,47.6%in cytoplasm,42.9%in organelles,4.8%on cell membrane and4.8%in the cellular junction. Panther protein class analysis showed that the differentially expressed proteins mainly include cytoskeletal proteins (33.3%), enzyme regulatory proteins (13.3%), structural proteins (10.0%) and calcium binding proteins (6.7%) etc. The results showed that cytoskeletal proteins play critical roles in the development of myocardial hypertrophy in SHR rats.
7. Profilin-1expression levels:Real-time PCR results showed that the mRNA level of profilin-1in SHR is significantly higher than that in WKY rats (P<0.001). Western blot showed that the protein expression level of profilin-1in SHR is significantly higher than that in WKY rats (P<0.001). Abundance of profilin-1protein shows a similar trend as that iTRAQ.
Conclusion
1. The myocardial hypertrophy model of17-week-old SHR was successfully built, which was confirmed by cardiac mass index measurement, myocardial pathology analysis and ultrastructural observations.
2. As a new technology of quantitative comparative proteomic, iTRAQ has high accuracy, repeatability and quantitative effect. It can be used to explore the molecular mechanisms of myocardial hypertrophy and look for the key proteins and pathways.
3. Mass spectrometry identifed28differentially expressed proteins between 17-week-old SHR rats with hypertrophic myocardium and17-week-old WKY rats with normal myocardium. GO analysis showed that the cytoskeleton proteins in SHR rats play critical roles in the pathological process of myocardial hypertrophy.4. Real-time quantitative PCR and Western blot examination confirmed the high expression level of profilin-lin myocardial tissues of SHR rats, which verified the reliability of iTRAQ technology and provided key candidate targets for further research on the pathogenesis of myocardial hypertrophy.
Part Two
The Effects and Mechanism of Profilin-1in Hypertension Induced Cardiac Hypertrophy
Background
High blood pressure is the first cause of myocardial hypertrophy. When the heart is under hemodynamic overload, myocardial cells can perceive the tension and generate and release a variety of cytokines. These cytokines will combine with corresponding receptors on the cell membrane and activate a variety of hypertrophy signaling cascade. Finally the nuclear oncogene transcription is activated and myocyte hypertrophic process starts. Functional membrane proteins, cytoskeleton system as well as mutual interaction among cytokines involve in the above pathophysiological process. Cytoskeleton system is the key component to perceive the mechanical stimulation and convert mechanical stress into biochemical signals. The cytoskeleton, especially actin microfilament system, is an important point to study the mechanisem of hypertension induced cardiac hypertrophy
Profilin-1is an important actin binding protein. It can modulate the dynamic assembly of actin cytoskeleton according to extracellular signals. Our previous research suggested that profilin-1plays a critical role in the early myocardial hypertrophy caused by ouabain. And the proteomics analysis mentioned in part one reveals a significant upregulation of profilin-1expression in early hypertrophic myocardium of spontaneously hypertensive rats compared with the control group. In this project, we constructed adenovirus vectors carrying profilin-1mRNA and siRNA separately, and injected them into spontaneously hypertensive rat models to induce the overexpression and silence of profilin-1gene. By this way, we studied the effects of profilin-1in the development of hypertension-induced myocardial hypertrophy and its possible mechanisms using various pathological and histologic technologies as well as molecular biology. These results will provide a new target for early prevention and treatment of hypertensive myocardial hypertrophy and is of great academic significance and potential clinical value.
Objectives
1. To investigate the improvement/deterioration of hypertension-induced myocardial hypertrophy by transfection of interference adeno virus vector pAd-miR-profilin-1/overexpression adeno virus vector pAd-profilin-1-IRES-EGFP into spontaneously hypertensive rats;
2. To explore the mechanism by which profilin-1silencing/overexpression would ameliorate/deteriorate hypertension-induced myocardial hypertrophy on molecular, histological and ultrastructural levels.
Methods
1. Five-week-old boy SHR and WKY rats were randomly assigned to four groups (n=20per group):WKY-C (WKY control rats treated with negative control adenovirus); SHR-C (SHR control rats treated with negative control adenovirus); SHR-I (SHR treated with adenovirus vector pAd-miR-profilin-1to knockdown profilin-1expression); and SHR-H (SHR treated with adenovirus vector Adprofilin-1-IRES-enhanced green fluorescent protein (EGFP) to overexpress profilin-1). The adenoviruses were injected twice into tail veins of rats at a dosage of3×109infectious units per rat with an interval of6weeks between injections. Six weeks after the second administration of adenovirus, subsets of animals were randomly selected and used for cardiac mass index measurement, histopathologic and ultrastructural observation, or biochemical analysis.
2. Blood pressure measurement:SBP was measured with the Heart Rate and BP Recorder for Rats, using the tail-cuff method before the start of treatment and weekly during treatment.
3. Cardiac mass index measurement:Rats were weighed and euthanized under anesthesia at17weeks of age, and hearts were removed and weighed. Atria were cut away and right ventricular free walls were carefully dissected from the left. The intraventricular septum was included in the left ventricular weight. The distance from the atrial valve to the apex, called the left ventricular long axis, was measured as a reflection of left ventricular enlargement.
4. Transfection efficiency detection:Ten days after the second injection of adenovirus, five rats in each group were euthanized and their hearts removed to determine transfection efficiency. EGFP expression in the cardiac tissues was observed with a fluorescence microscope to determine transfection efficiency. For each sample, EGFP expression and transfection efficiency were evaluated in six randomly chosen fields per section and quantitated using Image-Pro Plus6.0software.
5. Pathological analysis:The morphologicalchanges of myocardium were examined by light microscope following hematoxylin and eosin staining. Sirius red staining was carried out to determine the extent of fibrosis. Image-Pro Plus6.0software was used to determine the collagen content, which was expressed as fraction of the total myocardial cross-sectional area.
6.Ultrastructure observation:The ultrastructure of myocyte was observed under an H-800transmission electron microscope. Individual myocyte membranes (n=15for each group) were imaged at×40000magnifications, and the number of caveolae per micrometer plasmalemmal membrane (/μm) were counted by blinded investigators.
7. Immunohistochemical and immunofluorescence staining:The expression of profilin-1in myocardium was viewed by immunohistochemical staining under a light microscope. The colocalization of caveolin-3and actin microfilament was confirmed using immunofluorescence staining.
8. Measurement of nitric oxide production in serum and left ventricle:A serous and cardiac NO assay was performed using the NO assay kit. The homogenates of left ventricular samples and serum were centrifuged and the supernatant was taken for NO assay. With this method, the nitrate present in the sample was converted into nitrite by the enzyme nitrate reductase.The nitrite reacted with sulphanilamide and N-(L-naphthyl)-ethylenediamine dihydrochloride to give a red-violet diazo dye. Then the diazo dye was measured at the absorbance of550nm and the content of nitrite was obtained to reflect the production of NO. Results were expressed as μmol/g protein.
9. Real-time quantitative RT-PCR:Total RNA of the rat myocardium tissues was extracted by Trizol. The mRNA expression levels of profilin-1, eNOS and caveolin-3in the left ventricle of rats were examined by real-time PCR.
10. Western blot analysis:The protien expression levels of profilin-1, eNOS, p-eNOS and caveolin-3in the left ventricle of rats were examined by western blot analysis.
Results
1. Effect of profilin-1on SBP of spontaneous hypertensive rats:The effect of adenovirus-mediated profilin-1gene overexpression and knockdown on SBP was monitored in SHR and WKY before injection and every week afterward. There were significant differences in SBP between SHR and WKY throughout the study (P<0.001). However, no significant differences were observed among the three SHR groups throughout the experiment.
2. Cardiac mass index measurement:Compared with WKY-C group, SHR-C group showed a27%increase in left ventricular weight-to-heart weight ratio (LVW/HW Ratio) and a29%increase in the left ventricular long axis measurement. The SHR-I group displayed a significantly decreased LVW/HW Ratio when compared with the SHR-C group (P<0.05), indicating that lowering profilin-1expression reversed cardiac hypertrophy. The left ventricular long axis, another measurement of left ventricular enlargement, was also significantly reduced in SHR-I group compared with control SHR (P<0.01).
3. Detection of adenovirus transfection efficiency:The high transfection efficiency was confirmed by observing EGFP expression under the fluorescence microscope. In all four groups, the green fluorescent cells accounted for a proportion of30%-35%. Meanwhile, it's hard to see any EGFP expression in myocardial tissue of rats injected with saline.
4. Pathological analysis:Compared with WKY-C rats, SHR rats showed myocardial cell swelling, sparse arrangement, myocardial fiber rupture and increased interstitial fibrosis. To further confirm the effects of profilin-1on cardiac hypertrophy and fibrosis, we overexpressed profilin-1in the cardiac tissue of SHR using an adenovirus carrying the profilin-1gene.The myocardium from the SHR-H group developed significant hypertrophy with cardiomyoliposis. Sirius red staining showed that fibrillar collagen in hearts of SHR-H group was much more evident than that in SHR-C group (P<0.001). These results demonstrate that high levels of profilin-1promote cardiac hypertrophy and fibrosis.
5. Ultrastructural examination:Compared with WKY-C group, the parallel and linear arrangement of the Z-discs in SHR rats was lost, the Z-discs appeared wavy, the mitochondria swelled and showed electron-lucent areas because of the loss of matrix granules. Deformation in nuclear shape was observed. Nuclei appeared elongated and bizarre and the nuclear membranes were found convoluted. Condensed chromatin was found in nuclei as small blocks and thin cords. Myocytes of the SHR-H group showed total disruption of the contractile system and complete disorganization of myofibrils. In some areas, myofibrils disappeared and were replaced by bizarre mitochondria. Mitochondrial swelling was more serious and displayed vacuolation. The mitochondria lost the normal morphology and distributed as clusters, with over-dense matrix and blurred cristae. Distortion of nuclear shape was persistent and chromatin condense became more serious. The chromatin constituted a thick layer in the inner boundary of the nuclear membrane.
6. Effects of profilin-1on actin cytoskeleton:Phalloidin staining in the WKY-C group was abundant but became weaker and thinner in SHR-C group. Downregulation of profilin-1expression helps to conserve the abundance of actin filaments in SHR, whereas overexpression of profilin-1further reduced actin filaments organization.
7. Profilin-1overexpression decreased the caveolar abundance:Transmission electron microscopy was performed to quantify caveolar abundance. Caveolae were three-fold more abundant in WKY-C group compared with SHR-C group (P<0.001). Compared with the control SHR, transgenic overexpression of profilin-1in SHR caused a further significant reduction of sarcolemmal caveolae (P<0.001). In contrast, downregulation of profilin-1expression helps to conserve the abundance of caveolae in SHR (P<0.001). This result was consistent with the abundance of caveolin-3mRNA and protein in cardium. Both mRNA and protein expression of caveolin-3were lower in hearts of SHR compared with those in WKY. The reduction in caveolin-3expression was more severe in the SHR-H group with profilin-1overexpression. In contrast, caveolin-3expression was restored in the SHR-Ⅰ group following profilin-1knockdown.
8. Profilin-1expression levels and cellular localization in the myocardial tissue of rats:Real-time PCR results showed that the mRNA level of profilin-1in SHR-H group is significantly higher than that in SHR-C group (P<0.05) while the mRNA level of profilin-1in SHR-Ⅰ group is significantly lower than that in SHR-C group (P<0.05). Western blot showed that the protein expression level of profilin-1in four groups is similar as that of mRNA. The immunohistochemical analysis of profilin-1in the heart of SHR and WKY showed that profilin-1protein was expressed in the cytoplasm of cardiomyocytes, especially around the nucleus. Abundance of profilin-1protein shows a similar trend as that in western blot analysis. We also observed mild profilin-1expression in vascular smooth cells.
9. Content of NO in serum and myocardial tissue:NO secretion was lower in hearts of SHR compared with WKY. We investigated production of NO in left ventricular and found a significant decrease in SHR-H group (P<0.01) and a significant increase in SHR-I group (P<0.05) when compared with SHR-C group. We also tested the NO levels in the serum of rats and no significant differences were found among the SHR-C group, SHR-I group, and SHR-H group, indicating that the changes of NO levels is a local phenomenon rather than a systemic reaction.
10. Expression and activity of eNOS in rat myocardial tissue:No significant difference of eNOS expression was found among the four treatment groups. eNOS activity and NO secretion were lower in hearts of SHR compared with WKY. Our results showed a significant decrease of phospho-S1177eNOS in SHR-H group and a significant elevation in SHR-I group, when compared with SHR-C group (P<0.05).
Conclusion
1. Profilin-1expression is upregulated in hypertrophic myocardium of spontaneously hypertensive rats, accompanying with cardiomyocyte hypertrophy, hyperplasia of collagen fibers and actin cytoskeleton damage.
2. Overexpression of profilin-1interfered with the assembly of actin cytoskeleton and the formation of caveolae on myocardial membrane, then inhibited the activity of the eNOS/NO signaling pathways and finally promoted hypertension-induced myocardial hypertrophy. This might be one of the most important pathogenesis of myocardial hypertrophy.
3. Profilin-1gene silencing could effectively reverse myocardial hypertrophy by increasing the content of myocardial actin microfilament, increasing the caveolar number and restoring eNOS activity and the product of NO.
引文
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