抗β_1肾上腺素受体细胞外第二环自身抗体导致大鼠多脏器损伤及其相关机制
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摘要
研究背景和目的
β肾上腺素受体(β-adrenoceptor,β-AR)是交感神经系统的重要成员,通过介导体内儿茶酚胺类物质的生理效应,在调节心脏活动中发挥着重要作用。β-AR属于G蛋白偶联受体家族,目前已确认的β1、β2和β3AR三个亚型具有相同的结构特征,即含有由7个22~28个疏水性氨基酸残基组成的跨膜区,连接这些跨膜区的亲水性氨基酸片段构成了3个细胞内环和3个细胞外环。在这三种β-AR亚型中,β1AR在心脏的分布密度最高。当该受体受神经、体液因素调节激活时,通过兴奋型G蛋白-腺苷酸环化酶-环磷酸腺苷系统,使心脏产生正性变时、正性变力、正性变传导效应,以适应机体对血液供应的需求。
1987年Wallukat和Wollenberger在原发型扩张性心肌病(IDCM)患者血清中发现针对β1-AR细胞外第二环(the second extracellular loop ofβ1-adrenoceptor,β1-AR-ECII,人鼠同源性为100%)的自身抗体。随后的研究发现,该抗体具有可以使培养的乳鼠心肌细胞跳动频率加快;使正常心房肌收缩增强;可以抑制放射性配体结合,同时增强受体介导的信号转导等作用,因而推断抗β1-AR-ECII自身抗体具有类激动剂样作用。随着研究的深入,国外学者及我们均发现,抗β1-AR-ECII自身抗体并非IDCM独有,Chagasic病(1994)、原发性电紊乱病(1995)、高血压性心脏病、风湿性心脏病(1999)等患者血清中均有高水平的抗β1-AR-ECII自身抗体,从而推测该抗体可能与各种伴有心肌结构和功能改变的心脏疾患有关。
2000年,我们用缩窄腹主动脉和注射阿霉素制备大鼠心肌重构模型的方法观察抗β1-AR自身抗体的生成情况,结果发现:在两种心肌重构模型的建立过程中,大鼠体内抗β1-AR-ECII自身抗体均由处理前的阴性大多数转为阳性,其阳性率分别为87.5%(缩窄腹主动脉组)和79.2%(注射阿霉素组),而且该抗体滴度随心衰的发展呈现升高、维持和减退的现象,提示不同机制引起的心肌重构/心衰都可以导致抗β1-AR-ECII自身抗体的产生。为进一步探讨抗β1-AR-ECII自身抗体对心脏结构和功能的影响,我们用人工合成的β1-AR-ECII抗原肽段对大鼠进行了18个月的主动免疫,观察到免疫大鼠心肌组织发生类扩张性心肌病样改变和心功能下降。以上结果提示,心脏结构的改变可以诱导机体产生抗β1-AR-ECII抗体,后者反过来又可以加重心肌负荷,二者互为因果,最终加重心肌损伤。
在我们对大鼠进行18个月主动免疫的实验中,抗β1-AR-ECII抗体诱导的扩心病模型存在心肌细胞丢失的现象引起了我们的关注。一般认为,细胞丢失的主要形式有凋亡和坏死,凋亡是一种基因控制的主动过程,抑制凋亡更容易进行也更有意义。有研究证明,阻断细胞凋亡的信号转导过程可以显著减少心梗面积,改善心功能。为明确在抗β1-AR-ECII抗体诱导的扩心病模型中出现的细胞丢失中是否有凋亡的存在,我们进行了一种离体实验研究,发现(2002):抗β1-AR-ECII自身抗体可以使培养的乳鼠心肌细胞发生凋亡;2003年,Staudt等的研究结果也表明,抗β1-AR-ECII抗体可使成年大鼠心肌细胞发生凋亡。2004年我们通过主动免疫在体实验进一步证明,抗β1-AR-ECII抗体可在体诱导大鼠心肌细胞发生凋亡。这都提示,细胞凋亡可能在抗β1-AR-ECII抗体对心脏活动产生影响的过程中起重要作用。但是,以上研究采用的动物模型为主动免疫模型,该模型不能排除导致心肌损伤的原因中是否有抗原的参与。此外,抗β1-AR-ECII抗体致凋亡过程中,其信号转导机制如何尚不清楚。对这些问题的研究,有助于明确抗β1-AR-ECII自身抗体的病理意义,同时也是随后寻找关键蛋白作为临床治疗的靶点所必需的。
有研究表明,抗β1-AR-ECⅡ抗体可通过cAMP依赖性的蛋白激酶激活Ca2+通道,使心肌细胞内Ca2+升高,胞内钙超载是诱发细胞凋亡的重要机制之一。在细胞内,钙离子通过和钙调蛋白结合,形成Ca2+/CaM复合物后活化依赖Ca2+/CaM的蛋白激酶(CaMK)。CaMK有4个亚型:Ⅰ、Ⅱ、Ⅲ和Ⅳ。其中CaMKⅡ是一种多功能蛋白激酶,存在于体内多数重要器官中。分子克隆发现四个不同的基因分别转录CaMKⅡ的α、β、γ和δ四个亚基;有研究表明CaMKⅡ的亚基中γ和δ产物表达于心肌细胞。目前已经发现,CaMKⅡδ可引起心肌细胞肥大,且与细胞凋亡关系密切。因此,我们考虑,在抗β1-AR-ECII抗体诱导的扩心病模型中所出现的心肌细胞凋亡,是否通过Ca2+下游的CaMKⅡδ信号通路起作用呢?
心肌重构是心衰发生发展过程中的重要病理生理阶段,这种重构的发生是心衰患者出现心功能改变和心律失常的潜在原因。有研究报道,扩张性心肌病合并心律失常的患者血清中存在的抗β1AR-ECⅡ自身抗体,其阳性率高于其他心脏病患者,并且证实该抗体与心律失常的发生相关。另有研究表明:抗β1AR-ECⅡ抗体可以增加ICa-L电流,缩短Q-T间期,那么抗β1-AR-ECⅡ抗体是否能直接诱发心律失常还有待进一步研究。此外,心律失常最本质的变化在于心肌细胞生物电活动的改变,为了深入探讨抗β1-AR-ECⅡ抗体引起心律失常的具体机制,我们在本课题中,将进一步观察抗β1-AR-ECⅡ抗体对心肌细胞生物电活动,如:细胞膜静息电位(Resting Potential,RP)、动作电位时程(Action potential duration,APD)、L型钙电流(L-type Calcium current,ICa-L)、瞬时外向钾电流(transient outside potassium current,Ito)、延迟整流钾电流(delayed-rectifier potassium current, Ik1)及钠钙交换电流(Na+/Ca2+ exchange current,INa-Ca)等的直接影响,并利用乳头肌进行离体实验,探讨抗β1-AR-ECⅡ抗体对心肌组织生物电活动的影响。
已有的研究表明,β1-AR主要分布在心脏和肾脏,在肝脏也有少量的分布;以往大量研究证实:抗β1-AR-ECⅡ抗体可以通过与β1-AR结合发挥类激动剂样的作用,从而引起心脏损伤。但与激动剂不同的是,抗β1-AR-ECⅡ自身抗体具有不脱敏样的受体效应。那么循环中产生的抗β1-AR-ECⅡ抗体是否也能通过与肝脏和肾脏细胞表面的β1-AR结合,从而产生一定的作用呢?我们前期在用β1-AR-ECⅡ肽段进行18个月的主动免疫大鼠过程中观察到:有相当数量的大鼠出现了腹水和肝缘变钝的现象,引起我们的极大关注。对于腹水的临床检验结果表明:腹水中细胞数及蛋白水平偏高,葡萄糖水平偏低,同时腹水可以自然凝固,基于以上性质确定该腹水为渗出液,而非漏出液;同时结合大鼠的心功能状况判定,腹水不是由于心衰而导致的,那么形成腹水的原因是什么,尚不得而知;但结合肝缘变钝这一大体现象,我们考虑肝脏病变可能是形成腹水的原因之一,但不能排除肾脏在免疫过程中是否也受到损伤。因此在本课题中,我们也将对这一问题进行初步探索。
综上所述,本课题拟进行以下三方面的研究:①采用被动免疫模型,探讨抗β1-AR-ECⅡ抗体长期存在是否会导致心肌组织CaMKⅡδ表达的改变,同时观察心肌细胞内钙离子水平的变化;观察抗β1-AR-ECⅡ抗体致心律失常的直接作用、长期作用及其可能的机制;②建立主动及被动免疫模型,观察抗β1-AR-ECⅡ抗体长期存在是否会导致肝脏损伤的发生;③建立主动及被动免疫模型,观察抗β1-AR-ECⅡ抗体长期存在是否会导致肾脏损伤的发生。
Ⅰ.抗β1-AR-ECⅡ抗体可导致心肌重构及可能的机制
第一分题:抗β1-AR-ECⅡ抗体可导致心肌结构重构及可能的机制
目的
1.观察抗β1-AR-ECⅡ抗体被动免疫过程中,大鼠心肌形态学及心功能的变化情况;
2.观察被动免疫过程中,细胞内游离钙水平及CaMKⅡδ的表达情况。
方法
(1)分别选取健康成年雄性Wistar大鼠(n=60,体重180~220g),由山西医科大学实验动物中心提供,用ELISA法筛选血清抗β1AR-ECⅡ抗体阴性的动物进行分组。
①抗β1AR-ECⅡ抗体组(β1AAb group,n=32):将主动免疫收集的抗β1-AR-ECⅡ抗体阳性血清进行纯化,得到血清中总的IgG,BCA蛋白定量后,以0.7μg/g的剂量通过尾静脉给入大鼠体内,每2周加强免疫一次,共免疫40周;
②阴性血清IgG组(Negative sera group,n=28):将收集到的抗β1-AR-ECⅡ抗体阴性血清进行纯化,得到血清中总的IgG,BCA蛋白定量后,以0.7μg/g的剂量通过尾静脉给入大鼠体内,每2周加强免疫一次,共免疫40周。
(2)肽段合成:抗原肽段由吉尔生化上海有限公司合成,相当于人β1-AR细胞外第二环氨基酸序列的特异性抗原决定簇( 197-223位, HWW RAESDEARRCY NDPKCCDFVT NRA),合成肽段纯度为95%(见表1)。合成的肽段储存于-20℃备用。
(3)通过大鼠尾静脉定期给入抗体建立被动免疫模型,用酶联免疫吸附法(ELISA法)检测免疫过程中大鼠血清中抗β1-AR-ECII抗体水平的变化;
(4)分别用体视学指标、Masson三色染色及心重体重比的测量来反映被动免疫过程中大鼠心脏结构的变化;
(5)经右侧颈总动脉插管进入左心室,监测心率(Heart rates, HR)、左心室收缩压(Left ventricular systolic pressure, LVSP)、左心室舒张压(Left ventricular diastolic pressure, LVDP)和左心室压力变化最大速率(±dp/dtmax)等心功能指标;
(6)分别采用原位末端标记法(TUNEL)和Caspase-3、8、9活性测定法检测大鼠心肌组织中心肌细胞凋亡发生情况;
(7)用Fluo-3-AM探针标记,激光共聚焦测定被动免疫末期大鼠单个心室肌细胞内游离钙离子的水平;
(8)用Western-blot及免疫组化法检测大鼠心肌组织中CaMKⅡδ蛋白的表达水平。
结果
1.抗β1-AR-ECⅡ抗体可导致心肌结构重构
1.1免疫过程中抗β1-AR-ECⅡ抗体水平的变化
被动免疫组大鼠(其中因麻醉死亡2只,不明原因死亡2只)血清中抗β1-AR-ECⅡ抗体OD值由免疫前的0.09±0.030,逐步升高为免疫后4周的0.21±0.029,明显高于对照组的0.08±0.040(P<0.001),此后抗体一直维持在该水平至实验结束(见Fig.1)。在整个免疫过程中,血清中抗β1-AR-ECⅡ抗体水平波动不大,组内比较无统计学差异,但和阴性血清IgG组相比有显著差异(P<0.001)。
1.2抗β1-AR-ECⅡ抗体长期存在可导致心肌重构的发生
由于心肌结构重构的概念是:“心脏大小、形状、重量的变化以及心肌细胞形态、数量变化和胶原纤维沉积等改变”,所以我们用心重/体重比(heart weight to body weight ratio, HW/BW)、心脏体视学指标以及心肌胶原纤维含量等,作为抗β1AR-ECⅡ抗体长期存在下,是否可以导致心肌重构发生的指标。
1.2.1心重/体重比(HW/BW):HW/BW在抗β1AR-ECⅡ抗体被动免疫24周开始明显
下降为2.10±0.09,低于对照组的2.72±0.04(P<0.01);免疫36周时该比值为2.35±0.05,仍低于对照组的2.68±0.05(P<0.01,见Fig.2C)。
1.2.2体视学指标:心脏在免疫晚期出现心腔变大,心壁变薄等扩心病样的病理改变(见Fig.2B)。
1.2.3 Masson三色染色:免疫36周的心肌Masson三色染色显示:在心肌间质中有大量胶原纤维的分布(见Fig.3)。
1.3抗β1-AR-ECⅡ抗体长期存在可导致心功能的下降
随着被动免疫时间的延长,大鼠的心功能逐渐降低(见Fig.4)。心率在整个免疫过程中变化不明显,仅在免疫36周时出现心率的略微下降311±20.3次/分,低于对照组的360±23.0次/分(P<0.05,见Fig.4A)。室内压上升的最大速率(+dp/dtmax)在免疫24周开始出现明显下降(213±31.1 Kpa/s),显著低于对照组的510±29.0 Kpa/s(P<0.01);免疫36周时为289±39.6 Kpa/s,低于对照组的505±30.0 Kpa/s(P<0.01,见Fig.4B)。室内压下降的最大速率(-dp/dtmax)也在免疫24周开始出现明显下降(-233±34.7 Kpa/s),显著低于对照组的-415±31.0 Kpa/s(P<0.01);免疫36周时为-283±29.6 Kpa/s,低于对照组的-421±29.0 Kpa/s(P<0.01,见Fig.4C)。左室收缩压(LVSP)和左室舒张压(LVDP)在整个免疫过程中变化不明显。
以上结果提示:抗β1-AR-ECⅡ抗体长期被动免疫可导致大鼠发生心肌重构,同时心功能明显下降。
2.抗β1-AR-ECⅡ抗体致心肌结构重构的可能机制
2.1抗β1-AR-ECⅡ抗体长期存在可导致心室肌细胞凋亡增加
2.1.1抗β1-AR-ECⅡ抗体长期存在可导致心室肌细胞Caspase-3、8、9活性增加
被动免疫过程中Caspase-3、8、9的测定使用荧光定量法来进行,用AFC标品制作标准曲线(见Fig.5),通过各点拟合后的直线斜率为0.998。关于Caspase-3、8、9荧光底物作用最佳时间点的估计见Fig.6。底物和不同样品孵育30分钟时,阴性对照的荧光强度接近峰值,而待测品的荧光强度相对较低;孵育60分钟时,阴性对照的荧光强度刚接近曲线底部,而待测品的荧光强度还在继续增加过程中;孵育90分钟时,阴性对照的荧光强度维持在曲线底部,而待测品的荧光强度还在继续增加过程中,但已达较高水平;孵育120分钟时,阴性对照的荧光强度又开始逐渐升高,待测品的荧光强度还在继续增加过程中。根据选择最佳时间点的要求,即选择表现出最高样本读数和最低阴性对照读数的时间点,最后确定孵育的时间为90分钟。
在整个被动免疫过程中,Caspase-3、8、9的活性表现为随免疫时间的增加而增加(见Fig.7)。其中Caspase-8在免疫16周开始增加至13.41±6.14 pmol/h/mg,显著高于对照组的3.45±0.97 pmol/h/mg( P<0.01);此后Caspase-8继续升高,在免疫36周时达高峰34.1±10.09 pmol/h/mg,远高于对照组的4.15±1.09 pmol/h/mg(P<0.01,见Fig.7A)。Caspase-9在免疫16周开始增加至7.47±0.94 pmol/h/mg,高于对照组的1.27±0.18 pmol/h/mg(P<0.01);此后Caspase-9继续升高,在免疫36周时达高峰16.24±3.31 pmol/h/mg,远高于对照组的1.38±0.37 pmol/h/mg(P<0.01,见Fig.7B)。Caspase-3在免疫16周开始增加至18.84±7.23 pmol/h/mg,显著高于对照组的1.49±0.18 pmol/h/mg(P<0.01);此后Caspase-3继续升高,在免疫36周时达高峰25.06±6.80 pmol/h/mg,远高于对照组的1.83±0.60 pmol/h/mg (P<0.01,见Fig.7C)。
2.1.2抗β1-AR-ECⅡ抗体长期存在可导致心肌凋亡细胞数目的增加
用TUNEL法检测被动免疫36周大鼠凋亡心肌细胞的数目,发现:在免疫36周时,平均有7.86±0.43%的心肌细胞发生凋亡,其比例远高于对照组的0.86±0.29 %(P<0.01,见Fig.8G)。凋亡心肌细胞典型图见Fig.8A-8F。
以上结果提示,抗β1-AR-ECⅡ抗体被动免疫大鼠心肌细胞凋亡明显增加,但导致凋亡细胞增加的机制还有待进一步研究。
2.2抗β1-AR-ECⅡ抗体长期存在可导致心室肌细胞内游离钙离子水平升高
在抗β1-AR-ECⅡ抗体被动免疫末期,即免疫36周时,检测心室肌细胞内游离钙离子水平,发现抗β1-AR-ECⅡ抗体被动免疫组大鼠单个心室肌细胞内游离钙离子水平为456.34±35.47,远高于对照组的51.96±1.18(P<0.001,见Fig.9)。
以上结果提示:抗β1-AR-ECⅡ抗体长期作用可导致细胞内游离钙离子水平的升高,但升高的钙离子如何导致心肌细胞凋亡,即其下游的信号通路还有待进一步研究。
2.3抗β1-AR-ECⅡ抗体长期存在可导致心室肌CaMKⅡδ的表达增加
2.3.1 Western blot结果显示抗β1-AR-ECⅡ抗体长期存在可导致心室肌CaMKⅡδ的表达增加
在抗β1-AR-ECⅡ抗体被动免疫过程中,随免疫时间的延长CaMKⅡδ的表达逐渐增加(见Fig.10)。被动免疫16周时,CaMKⅡδ的表达开始升高达1.19±0.38,高于对照组的0.39±0.22 (P<0.05);此后CaMKⅡδ的表达持续升高,在免疫36周时达高峰(1.94±0.77),高于对照组的0.46±0.27(P<0.05)。
2.3.2免疫组化结果显示抗β1-AR-ECⅡ抗体长期存在可导致心肌CaMKⅡδ的表达增加在抗β1-AR-ECⅡ抗体被动免疫36周时,免疫组化测定心肌细胞内CaMKⅡδ的表达明显增加(见Fig.11)。
以上结果提示,抗β1-AR-ECⅡ抗体被动免疫大鼠心肌中CaMKⅡδ表达的升高可能与细胞凋亡的增加有关。
小结:
1.抗β1-AR-ECⅡ抗体长期存在可导致心肌结构重构的发生;
2.抗β1-AR-ECⅡ抗体长期被动免疫过程中,心功能逐渐下降;
3.抗β1-AR-ECⅡ抗体长期被动免疫可导致凋亡心肌细胞的增加;
4.抗β1-AR-ECⅡ抗体长期存在可导致心肌细胞内游离钙离子水平的升高;
5.抗β1-AR-ECⅡ抗体长期存在可导致心肌CaMKⅡδ的表达增加。
第二分题:抗β1-AR-ECⅡ抗体导致心电重构及其可能的机制
目的
1.观察抗β1-AR-ECⅡ抗体是否具有直接致心律失常的作用;
2.在组织和细胞水平,探讨抗β1-AR-ECⅡ抗体致心律失常的可能机制,以求阐明抗
β1-AR-ECⅡ抗体是否可致心电重构及其可能的机制,为临床抗β1-AR-ECⅡ抗体阳性心律失常患者的治疗提供理论依据。
方法
(1)分别选取健康成年雄性Wistar大鼠(n=172)(其中因麻醉死亡5只,不明原因死亡5只),体重180~220g,由山西医科大学实验动物中心提供,用ELISA法筛选血清抗β1AR-ECⅡ抗体阴性的动物进行分组。
①β1AR肽段免疫组(β1AR group,n=64):将抗原和免疫佐剂的混合物按0.4μg/g剂量注入动物背部皮内,每两周加强免疫一次,共免疫32周;
②免疫佐剂组(Vehicle group,n=48):将生理盐水和免疫佐剂的混合物以一定比例注入动物背部皮内,每两周加强免疫一次,共免疫32周;
③抗β1AR-ECⅡ抗体组(β1AAb group,n=32):将主动免疫收集的抗β1-AR-ECⅡ抗体阳性血清进行纯化,得到血清中总的IgG,BCA蛋白定量后,以0.7μg/g的剂量通过尾静脉给入大鼠体内,每2周加强免疫一次,共免疫40周;
④阴性血清IgG组(Negative sera group,n=28):将收集到的抗β1-AR-ECⅡ抗体阴性血清进行纯化,得到血清中总的IgG,BCA蛋白定量后,以0.7μg/g的剂量通过尾静脉给入大鼠体内,每2周加强免疫一次,共免疫40周。
(2)通过在体心功能及心电测定方法监测给入抗β1-AR-ECⅡ抗体后正常大鼠心功能及心电图的变化情况;
(3)通过微电极记录动作电位的方法,观察在组织水平抗β1-AR-ECⅡ抗体对乳头肌动作电位的影响,同时分析动作电位时程(APD)的变化情况;
(4)通过建立主动及被动免疫模型,观察抗β1-AR-ECⅡ抗体长期存在对大鼠心电活动的影响,同时分析QT间期的变化;
(5)通过膜片钳技术及胞内钙荧光测定技术,观察抗β1-AR-ECⅡ抗体长期存在对大鼠心肌细胞膜电流及胞内钙的影响。
结果
1.抗β1-AR-ECⅡ抗体可致心电重构
1.1抗β1-AR-ECⅡ抗体可致正常大鼠发生心律失常
1.1.1抗β1-AR-ECⅡ抗体可直接诱导正常大鼠发生心律失常
68μM抗β1-AR-ECⅡ抗体(0.7μg/g)可诱导约66.7%(6/9)的正常雄性大鼠(体重:180-220g)发生心律失常,其阳性率远高于阴性血清组(12.5%, 1/8)和生理盐水组(11.1%, 1/9)。抗β1-AR-ECⅡ抗体致心律失常的种类以室性早搏为主(见Fig.12A),同时还存在室上性早搏(见Fig.12B)。观察抗β1-AR-ECⅡ抗体从动脉给入一小时内大鼠心脏产生异常心律的次数发现,抗β1-AR-ECⅡ抗体引起室早和室上性早搏的次数为16±10.28(次/小时),高于生理盐水组的4±1.00(次/小时,P<0.01)和阴性血清组的5±0.82(次/小时,P<0.01,见Fig.12C)。
1.1.2抗β1-AR-ECⅡ抗体可诱导豚鼠乳头肌发生触发激动和延迟后除极(delayed after-depolarization, DAD)
0.1μM抗β1-AR-ECⅡ抗体作用于豚鼠乳头肌,在串刺激的诱导下几乎可以使所有分离的乳头肌发生触发激动(见Fig.13B),且该作用长时间持续不易衰减(最长观测时间2小时),这与异丙肾上腺素(ISO,1μM)的作用(见Fig.13A)很类似。同时,抗β1-AR-ECⅡ抗体还可诱发DAD(见Fig.13C);抗体和ISO的作用可被β1-AR选择性阻断剂美托洛尔(10μM)完全阻断(见Fig.13D)。同时,0.1μM该抗体可使豚鼠乳头肌的动作电位时程延长为355.5±32.98 ms,高于生理盐水组的306±18 ms(P<0.05)和阴性血清组的277.2±27.30 ms(P<0.01);美托洛尔(10μM)在一定程度上可减弱该抗体对动作电位时程的延长作用(P<0.001,见Fig.13E)。
1.1.3抗β1-AR-ECⅡ抗体可诱导大鼠乳头肌发生触发激动及后除极
1μM抗β1-AR-ECⅡ抗体作用于大鼠乳头肌,在串刺激的诱导下几乎可以使多数分离的乳头肌发生触发激动(60%,9/15,见Fig.14A)。部分乳头肌还可观察到早后除极(early after-depolarization,EAD)的发生(46.7%,7/15,Fig.14B)。少量乳头肌可观察到延迟后除极(delayed after-depolarization,DAD)现象(26.7%,4/15,Fig.14C)。在ISO组(1μM)EAD,DAD和触发激动均未被观察到。美托洛尔(10μM)可以抑制抗β1-AR-ECⅡ抗体诱发的EAD,DAD和触发激动(Fig.14D)。
以上结果提示:抗β1-AR-ECⅡ抗体可能通过使正常心肌细胞发生触发活动及后除极而导致心律失常的发生,但由于抗β1-AR-ECⅡ抗体在机体内是长期持续存在的,那么抗β1-AR-ECⅡ抗体长期存在时如何引发心律失常的发生,其在细胞水平的机制如何还有待进一步的研究。
1.2抗β1-AR-ECⅡ抗体长期存在可诱导心律失常的发生
1.2.1在用β1-AR-ECⅡ肽段免疫大鼠的不同时期可观察到不同程度及类型的心律失常
1.2.1.1主动免疫过程中,抗β1-AR-ECⅡ抗体水平呈现一个自然产生和消退的过程
在免疫4周时,抗β1-AR-ECⅡ抗体测定的OD值为0.4±0.21,高于对照组的0.10±0.05(P<0.05);此后抗体水平逐渐升高,在免疫8周时达高峰(2.10±0.18),高于对照组的0.15±0.09(P<0.001);此后抗体水平逐渐下降,免疫28周时抗体水平为0.73±0.21,仍高于对照组的0.15±0.10(P<0.01,见Fig22)。
1.2.1.2随着免疫时间的延长,心律失常的出现频率有所增加,阳性率也有一定程度增加
在主动免疫的0、4、8、12、16、20、24、28、32周,出现心律失常的动物数和总动物数的比例分别为0/6、0/6、1/7、1/8、1/6、0/6、3/3、6/7、8/8。观察到的心律失常类型主要有室性早搏(见Fig.15A)、室上性早搏(见Fig.15B)、室速(见Fig.15C)及房室传导阻滞等,其中以室性早搏和室上性早搏比较多见。其中主动免疫28周时心律失常的发生频率为220±25.17(次/小时),远高于对照组的10±0.65(次/小时,P<0.01);在主动免疫32周时,心律失常的发生频率为1509±40.88(次/小时),远高于对照组的15±0.75(次/小时,P<0.001,见Fig.15D-F)。
1.2.1.3 Q-T间期延长出现于β1-AR-ECⅡ肽段免疫大鼠的不同时期
在用β1AR-ECⅡ肽段免疫大鼠的不同时期,心电图监测发现:在主动免疫的12周Q-T间期为65.0±2.50 ms,远远长于对照组的55.0±0.86 ms(P<0.05);主免28周时Q-T间期为84.0±2.50 ms,远长于对照组的58.9±0.95 ms(P<0.05,见Fig.16C)。同时考虑到心率对Q-T间期的影响,根据Bazett公式计算QTc(校正QT间期),主动免疫12周时QTc为0.36±0.002,远长于对照组的0.34±0.007(P<0.05);主免28周时QTc为0.46±0.007,远长于对照组的0.32±0.009(P<0.05,见Fig.16D)。
以上结果提示:β1-AR-ECⅡ抗原肽段长期主动免疫可导致大鼠发生不同程度及类型的心律失常,同时Q-T间期也明显延长;但由于实验中不能完全排除抗原可能的致病作用,因此需通过直接给入抗体的被动免疫模型来进一步证实:免疫过程中心律失常的发生是抗原还是抗体导致的。
1.2.2抗β1-AR-ECⅡ抗体长期被动免疫也可诱导大鼠心律失常的发生
1.2.2.1被动免疫过程中血清抗β1-AR-ECⅡ抗体水平的变化
被动免疫组大鼠血清中抗β1-AR-ECⅡ抗体OD值由免疫前的0.09±0.030,逐步升高为免疫后4周的0.21±0.029,明显高于对照组的0.08±0.040(P<0.001)此后抗体一直维持在该水平至实验结束(见Fig.1)。在整个免疫过程中,血清中抗β1-AR-ECⅡ抗体水平波动不大,组内比较无统计学差异,但和阴性血清IgG组相比有显著差异(P<0.001)。
以上结果提示:被动免疫模型可使大鼠血清中产生抗β1-AR-ECⅡ抗体,同时抗体维持在一个相对稳定的低水平状态,这与临床病人筛查的结果相一致;和主动免疫模型过程中的抗体水平相比,其抗体水平的波动较小。
1.2.2.2抗β1-AR-ECⅡ抗体长期存在可诱导正常大鼠出现不同种类的心律失常
抗β1-AR-ECⅡ抗体被动免疫大鼠过程中,大鼠出现了不同程度及类型的心律失常(见Fig.17A-17F)。其中最多见的为室性早搏,也可见到一定数量的室上性早搏,同时还有室性心动过速(室速)及一些无法确定其类型的心律失常。心律失常的出现时间集中于免疫24周以后。
1.2.2.3抗β1-AR-ECⅡ抗体长期存在可使心律失常持续时间延长
在抗β1-AR-ECⅡ抗体被动免疫大鼠过程中,对不同时间点大鼠心律失常持续时间的分析发现:随着免疫时间的延长,发生心律失常的大鼠其心律失常持续时间有延长的趋势(见Fig.17G)。免疫24周时,心律失常持续时间为90±34.6 s,远高于对照组的0 s(P<0.01);免疫36周,心律失常持续时间为170±124.2 s,远高于对照组的0 s(P<0.01)。抗β1AR-ECⅡ抗体免疫组组内不同时间点比较结果为:免疫24周和36周分别和免疫前及免疫12周相比有统计学差异(P<0.01);免疫24周和36周的结果相比无统计学差异(P>0.05)。
1.2.2.4抗β1-AR-ECⅡ抗体长期存在可导致Q-T间期延长
在抗β1-AR-ECⅡ抗体被动免疫大鼠过程中,对不同时间点QTc的监测结果进行分析后得出:抗β1-AR-ECⅡ抗体被动免疫大鼠可导致QTc的延长(见Fig.18C)。从数据可以看到:抗β1-AR-ECⅡ抗体被动免疫24周开始,QTc开始明显升高达0.46±0.015,高于对照组的0.32±0.019(P<0.01);免疫36周时,QTc继续升高为0.51±0.015,明显高于对照组的0.33±0.018(P<0.01)。
以上研究结果提示:被动免疫过程中,抗β1-AR-ECⅡ抗体的长期存在可导致大鼠发生不同程度及类型的心律失常,同时心电图Q-T间期明显延长,这与主动免疫过程中观察到的现象一致,说明在免疫过程中抗β1-AR-ECⅡ抗体是导致心律失常发生的主要原因,但抗β1-AR-ECⅡ抗体导致心律失常发生的机制未知,有待进一步研究。
2.抗β1-AR-ECⅡ抗体致心电重构的可能机制
2.1抗β1-AR-ECⅡ抗体长期存在可导致细胞膜静息电位的减小和动作电位时程的延长
在抗β1-AR-ECⅡ抗体被动免疫末期,即免疫36周时,检测心室肌细胞膜静息电位发现:抗β1-AR-ECⅡ抗体被动免疫组大鼠心室肌细胞膜的静息电位为-62.37±0.69 mv,远小于对照组的-81.27±0.91 mv(P<0.05);同时被动免疫组大鼠心室肌细胞动作电位时程(Action potential duration, APD)测定为51.09±2.66 ms,长于对照组的33.26±2.58 ms(P<0.05,见Table 2,Fig.19)。
2.2抗β1-AR-ECⅡ抗体长期存在可导致心肌细胞膜延迟整流钾电流(Ik1)和瞬时外向钾电流(Ito)的减弱
在抗β1-AR-ECⅡ抗体被动免疫末期,即免疫36周时,检测心室肌细胞膜Ik1电流,发现:抗β1-AR-ECⅡ抗体被动免疫组大鼠心室肌细胞膜的Ik1电流减小,和对照组相比有统计学差异(P<0.05,见Fig.20A)。
在抗β1-AR-ECⅡ抗体被动免疫末期,即免疫36周时,检测心室肌细胞膜Ito电流,发现:抗β1-AR-ECⅡ抗体被动免疫组大鼠心室肌细胞的Ito电流明显弱于对照组的Ito电流(P<0.05,见Fig.20B)。
2.3抗β1-AR-ECⅡ抗体长期存在可导致心肌细胞膜L型钙电流(ICa-L)的增强及钠钙交换电流(INa-Ca)的减弱
在抗β1-AR-ECⅡ抗体被动免疫末期,即免疫36周时,检测心室肌细胞膜ICa-L电流,发现:抗β1-AR-ECⅡ抗体被动免疫组大鼠心室肌细胞的ICa-L电流在0mv-+40 mv的变化过程中明显强于对照组的ICa-L电流(P<0.05,见Fig.21A)。
同时对INa-Ca交换电流的测定也发现:抗β1-AR-ECⅡ抗体被动免疫组大鼠心室肌细胞的INa-Ca电流明显弱于对照组的INa-Ca电流(P<0.05,见Fig.21B-21E)。
2.4抗β1-AR-ECⅡ抗体长期存在可导致心肌细胞内游离钙离子水平的升高
在抗β1-AR-ECⅡ抗体被动免疫末期,即免疫36周时,检测心室肌细胞膜内游离钙离子水平,发现抗β1-AR-ECⅡ抗体被动免疫组大鼠单个心室肌细胞内游离钙离子水平为456.34±35.47,远高于对照组的51.96±1.18(P<0.001,见Fig.9)
以上研究结果提示:抗β1-AR-ECⅡ抗体长期被动免疫可导致心肌细胞内游离钙离子水平的升高,这一改变可能是由于该抗体抑制了INa-Ca,同时增强了ICa-L的结果;同时抗β1-AR-ECⅡ抗体长期存在可导致APD的延长,这一改变可能是由于该抗体抑制了Ik1和Ito电流,同时增强了ICa-L的结果;且该抗体长期作用可减小静息电位。以上这一些列改变,包括静息电位减小、APD延长及细胞内钙离子水平升高都是抗β1-AR-ECⅡ抗体致心律失常发生的可能机制。
小结
1.抗β1-AR-ECⅡ抗体可直接诱导正常大鼠在体发生心律失常,同时抗β1-AR-ECⅡ抗体可诱导豚鼠及大鼠乳头肌发生触发激动及后除极;
2.主动及被动免疫均可诱导大鼠发生不同程度及类型的心律失常;随着免疫时间的延长,心律失常的发生频率和发生时间都有不同程度的增加;同时长期主动及被动免疫均可导致大鼠心电图Q-T间期的延长;
3.抗β1-AR-ECⅡ抗体长期被动免疫可导致心肌细胞静息电位的减小和动作电位时程的延长;抗β1-AR-ECⅡ抗体长期作用可导致ICa-L电流的增强,Ik1、Ito及INa-Ca电流的减弱;抗β1-AR-ECⅡ抗体长期作用可导致心肌细胞内游离钙离子水平升高。
结论:
1.抗β1-AR-ECⅡ抗体长期存在可导致大鼠心肌重构及心功能障碍的发生,这可能与心肌细胞中游离钙离子水平升高导致CaMKⅡδ的表达增加,进而诱导心肌细胞凋亡增加有关。
2.抗β1-AR-ECⅡ抗体可导致心律失常的发生,其机制可能与细胞膜静息电位减小,Ik1、Ito及INa-Ca电流的减弱和ICa-L电流的增强导致的胞内钙离子水平升高和动作电位时程延长有关。
Ⅱ.抗β1-AR-ECⅡ抗体长期存在可导致大鼠肝脏损伤
目的
证实抗β1-AR-ECⅡ抗体长期存在是否会对大鼠肝脏的结构和功能产生影响。
方法
(1)建立主动及被动免疫模型,用ELISA法检测大鼠血清中抗β1-AR-ECⅡ抗体水平的变化;
(2)用HE和Masson三色染色,观察抗β1-AR-ECⅡ抗体长期存在对肝脏结构的影响;
(3)通过检测反映肝功能的血清学指标,来证实抗β1-AR-ECⅡ抗体长期存在对肝功能的影响。
结果
1.免疫模型建立过程中抗β1-AR-ECⅡ抗体水平的变化
1.1主动免疫模型建立过程中血清中抗β1-AR-ECⅡ抗体水平的变化
用β1-AR-ECⅡ肽段主动免疫过程中,血清中抗β1-AR-ECⅡ抗体呈现一个自然产生和衰退的过程(见Fig.22)。在免疫4周时,血清中抗β1-AR-ECⅡ抗体开始升高,在免疫8周时达高峰,用ELISA法检测血清抗体的OD值为0.65±0.11,远高于对照组的0.10±0.03(P<0.01);此后血清中抗β1-AR-ECⅡ抗体的水平开始逐渐下降,免疫28周时,抗β1-AR-ECⅡ抗体的OD值为0.15±0.03,仍高于对照组的0.11±0.04(P<0.01)。同时免疫过程中采集的所有血清进行了抗β2-AR-ECⅡ抗体的筛查,未发现抗β2-AR-ECⅡ抗体阳性的血清标本。
1.2被动免疫模型建立过程中血清中抗β1-AR-ECⅡ抗体水平的变化结果同第一部分。
以上提示:主动及被动免疫过程中,血清中均可产生高水平的抗β1AR-ECⅡ抗体。
2.β1-AR-ECⅡ抗原肽段主动免疫可导致肝脏损伤
2.1体视学指标
在主动免疫过程中,大鼠的肝脏出现了不同程度、肉眼可分辨的肝缘变钝现象(见Fig.23B)。本实验共免疫大鼠112只,其中β1-AR-ECⅡ肽段免疫组和免疫佐剂组分别为64和48只,免疫过程中大鼠不明原因死亡4只;其中β1-AR-ECⅡ肽段免疫组有24只大鼠出现肝缘变钝现象,占到总免疫动物数的37.5%(24/64),出现肝缘变钝的大鼠多集中于免疫20周以后;而免疫佐剂组大鼠在免疫过程中未见明显肉眼可见的肝缘变钝现象。
2.2形态学指标
2.2.1 HE染色:在β1-AR-ECⅡ肽段主动免疫过程末期,有3只大鼠(3/6)肝脏HE染色显示在肝脏边缘有大量脂肪细胞的聚集(见Fig.24D/E/F)。
2.2.2 Masson三色染色:在β1-AR-ECⅡ肽段主动免疫过程中,部分(21/64)大鼠的肝脏Masson三色染色显示在肝窦周围有大量胶原纤维沉积(见Fig.25D/E/F)。
以上结果提示:在主动免疫过程中,部分大鼠出现了肝脏结构的损伤。
2.3血清学指标
2.3.1转氨酶测定
用β1-AR-ECⅡ肽段主动免疫过程大鼠血清学指标显示:大鼠肝功能受到一定损伤。在用β1-AR-ECⅡ肽段主动免疫大鼠4周时,血清中谷草转氨酶(AST)开始升高,8周AST水平达高峰(281.4±68.64 U/L),远高于对照组的84.1±4.10 U/L(P<0.001);此后AST逐渐下降,免疫28周时,AST降为126.3±15.75 U/L,但仍高于对照组的84.6±4.89 U/L(P<0.001,见Fig.26A)。同样,谷丙转氨酶(ALT)也在免疫4周开始升高,8周达高峰(121.7±40.34 U/L),显著高于对照组的42.0±2.95 U/L(P<0.05);此后ALT水平开始下降,该变化一直持续至免疫28周实验结束时(见Fig.26A)。AST/ALT的比值在免疫4周时急剧升高达高峰(3.24±0.42),远高于对照组的1.93±0.076(P<0.01);此后该比值逐渐下降,免疫28周时,该比值降为2.03±0.030,和对照组(1.94±0.043)相比无统计学差异(见Fig.26B)。
2.3.2血清蛋白测定
血清总蛋白在用β1-AR-ECⅡ肽段主动免疫4周时开始降低达57.7±2.33 g/L,低于免疫佐剂对照组的68.5±2.12 g/L(P<0.01),此后总蛋白水平基本稳定于低水平,该变化一直持续至免疫28周时,血清总蛋白水平为58.9±0.88 g/L,低于免疫佐剂对照组的69.5±1.00 g/L(P<0.01)。β1-AR-ECⅡ肽段免疫组大鼠血清中免疫球蛋白水平在实验过程中基本维持相对恒定,波动不明显,和对照组相比无统计学差异。β1-AR-ECⅡ肽段免疫组大鼠血清中白蛋白在免疫的4周开始下降为12.3±1.20 g/L,明显低于对照组的17.1±0.35 g/L(P<0.01);此后白蛋白水平基本维持稳定于该水平至免疫28周时,血清总蛋白水平为13.0±1.15 g/L,低于免疫佐剂对照组的17.1±0.36 g/L(P<0.01,见Fig.26C)。同时白球比(A/G)在免疫4周时开始下降,8周时明显降低为0.28±0.010,明显低于对照组的0.35±0.011(P<0.01),该变化一直持续至免疫28周时的0.29±0.020,仍低于对照组的0.35±0.009(P<0.01,见Fig.26D)。
以上结果提示:在主动免疫过程中,大鼠肝功能也受到不同程度的损伤,证实了我们在前期实验中的推测;但由于该模型无法排除抗原可能的肝脏毒性作用,因此需通过进一步的被动免疫来观察相同指标在不同模型中的变化情况,以此对模型的优劣进行评价。
3.抗β1-AR-ECⅡ抗体被动免疫也可导致肝脏损伤的发生
3.1体视学指标
在抗β1-AR-ECⅡ抗体被动免疫过程中,大鼠的肝脏出现了肉眼可分辨的肝缘变钝现象(见Fig.27B)。本实验共免疫大鼠60只,其中抗β1-AR-ECⅡ抗体免疫组和阴性血清抗体组分别为32和28只,免疫过程中大鼠不明原因死亡3只;其中抗β1-AR-ECⅡ抗体免疫组有8只大鼠出现肝缘变钝现象,占到总免疫动物数的25%(8/32),出现肝缘变钝的大鼠多集中于免疫24周以后;而阴性血清抗体组大鼠在免疫过程中未见明显肉眼可见的肝缘变钝现象。
3.2形态学指标
3.2.1 HE染色:在抗β1-AR-ECⅡ抗体被动免疫晚期,有7只大鼠(7/12)肝脏HE染色显示在肝窦周围有大量淋巴细胞浸润(见Fig.28D/28E/28F)。
3.2.2 Masson三色染色:在抗β1-AR-ECⅡ抗体被动免疫晚期,多数大鼠(5/12)的肝脏Masson三色染色显示肝窦周围有大量胶原纤维的沉积(见Fig.29D/29E/29F)。
以上结果提示:抗β1-AR-ECⅡ抗体长期被动免疫也对大鼠肝脏结构造成一定损伤。
3.3血清学指标:
3.3.1转氨酶测定
用抗β1-AR-ECⅡ抗体被动免疫过程中,大鼠血清学指标显示:大鼠肝功能受到一定损伤。在用抗β1-AR-ECⅡ抗体被动免疫24周时,大鼠血清中谷草转氨酶(AST)明显升高至240.0±111.59 U/L,远高于对照组的81.6±4.43 U/L(P<0.01);此后血清中AST水平逐渐下降,免疫36周时,AST降为169.3±14.03 U/L,但仍高于对照组的82.0±4.87 U/L (P<0.01,见Fig.30A)。同样,谷丙转氨酶(ALT)也在被免24周开始升高至75.0±14.79 U/L,高于对照组的45.0±5.90 U/L(P<0.01);该变化一直持续至免疫36周,免疫组ALT水平高达99.8±7.63 U/L,远高于对照组的46.0±8.90 U/L(P<0.01,见Fig.30A)。AST/ALT的比值在免疫24周时急剧升高达2.86±0.22,远高于对照组的1.96±0.09(P<0.01);免疫36周时,该比值降为1.74±0.22,和对照组相比无统计学差异,但有略微下降的趋势(见Fig.30B)。
3.3.2血清蛋白测定
血清总蛋白在用抗β1-AR-ECⅡ抗体被动免疫12周时略微升高达71.8±0.59 g/L,高于对照组的66±0.85g/L(P<0.05);此后血清总蛋白水平逐步下降,在免疫24周时达58.0±2.80g/L,36周时为56.3±0.95g/L,均显著低于对照组的67.1±0.97g/L(P<0.01)和67.9±1.73g/L(P<0.01,见Fig.30C)。
抗β1-AR-ECⅡ抗体被动免疫过程中,血清中免疫球蛋白的水平基本维持相对恒定,波动不明显,组内比较无统计学差异,和对照组相比亦无统计学差异(见Fig.30C)。
抗β1-AR-ECⅡ抗体被动免疫大鼠过程中,血清中白蛋白在免疫的24周下降为12.5±0.29 g/L,明显低于对照组的17.43±0.29 g/L(P<0.01);36周时白蛋白水平继续降低为10.00±0.48 g/L,明显低于对照组的17.69±0.49 g/L(P<0.01,见Fig.30C)。
同时白球比在抗β1-AR-ECⅡ抗体免疫24周时降低为0.28±0.019,明显低于对照组的0.35±0.01(P<0.01),该变化加剧至免疫36周时的0.22±0.030,明显低于对照组的0.36±0.01(P<0.01,见Fig.30D)。
3.3.3血糖测定
被动免疫过程中大鼠血糖监测发现:在用抗β1-AR-ECⅡ抗体被动免疫24周时,血糖水平升高达高峰24.7±7.42mmol/L,远高于对照组的6.7±0.30 mmol/L(P<0.01);免疫36周时,血糖水平回落至19.5±2.06 mmol/L,仍高于对照组的6.73±0.18 mmol/L(P<0.01,见Fig.31)。
以上结果提示:抗β1-AR-ECⅡ抗体长期被动免疫可导致大鼠肝功能的损伤。同时血糖监测的结果发现,在被动免疫过程中大鼠血糖水平不断升高,这提示我们抗β1-AR-ECⅡ抗体长期存在还有可能对机体的糖代谢产生一定影响,但该方面的具体作用还需进一步研究证实。
小结
1.长期主动及被动免疫均可导致大鼠肝功能的下降;
2.长期主动及被动免疫均可导致部分大鼠肝脏结构的损伤。
结论:抗β1-AR-ECⅡ抗体长期存在可导致肝脏结构和功能的损伤,因此,血清中存在高水平抗β1-AR-ECⅡ抗体时,应密切关注患者肝功能的变化。
Ⅲ.抗β1-AR-ECⅡ抗体长期存在可导致大鼠肾脏损伤
目的
观察抗β1-AR-ECⅡ抗体长期存在能否导致大鼠肾脏结构和功能的损伤。
方法
(1)建立主动及被动免疫模型,用ELISA法检测大鼠血清中抗β1-AR-ECⅡ抗体水平的变化;
(2)用HE和Masson三色染色,观察抗β1-AR-ECⅡ抗体长期存在对肾脏结构的影响;
(3)通过检测反映肾功能的血清学指标,来证实抗β1-AR-ECⅡ抗体长期存在对肾功能的影响。
结果
1.免疫模型建立过程中抗β1-AR-ECⅡ抗体水平的变化结果同第二部分。
2.抗β1-AR-ECⅡ抗体长期作用可导致肾脏损伤
2.1形态学指标
2.1.1 HE染色:在主动免疫末期,HE染色显示β1-AR-ECⅡ肽段免疫组大鼠(13/16)在肾小球及肾血管周围出现大量淋巴细胞浸润(见Fig.32D、32E、32F),免疫佐剂组未见明显改变。
2.1.2 Masson三色染色:在抗原肽段主动免疫末期,β1-AR-ECⅡ肽段免疫组大鼠(10/16)大鼠肾小管周围出现大量胶原纤维的沉积(见Fig.33D、33E、33F)。以上结果提示:用β1-AR-ECⅡ肽段长期主动免疫可导致大鼠一定程度肾脏结构的损伤。
2.2血清学指标:
2.2.1血清尿素氮(Blood urea nitrogen,BUN)测定
在用β1-AR-ECⅡ肽段主动免疫过程中,血清BUN水平呈现升高趋势(见Fig.34A)。血清中BUN水平在免疫4周开始升高为8.9±0.95 mmol/L,高于对照组的6.7±0.35mmol/L(P<0.05);此后,BUN水平开始下降,8周时为7.74±0.58 mmol/L,此后BUN一直维持在该水平,至28周时为8.05±0.25 mmol/L,和对照组(6.90±0.33 mmol/L)相比有统计学意义(P<0.05)。
2.2.2血清肌酐(Creatinine,CR)测定
在用β1-AR-ECⅡ肽段主动免疫过程中,血清CR水平呈现升高趋势(见Fig.34B)。血清中CR水平在免疫4周开始升高为66.0±2.08 mmol/L,高于对照组的40.9±1.38mmol/L(P<0.01);8周时达高峰为70.0±3.85 mmol/L,此后CR水平略有下降,28周时为59.7±2.73 mmol/L,和对照组(42.7±2.63 mmol/L)相比有统计学意义(P<0.01)。
2.2.3血清尿酸(Uric acid,UA)测定
在用β1-AR-ECⅡ肽段主动免疫过程中,血清UA水平也呈现升高趋势(见Fig.34C)。血清中UA水平在免疫4周开始升高为198.0±6.93 mmol/L,高于对照组的89.5±9.80 mmol/L(P<0.01);8周时达高峰为235.6±16.43 mmol/L,此后CR水平开始下降,28周时为116.3±9.94 mmol/L,和对照组(93.8±11.64 mmol/L)相比无统计学差异(P>0.05)。
2.2.4尿素氮和肌酐比值(BUN/Cr)测定
主动免疫8周时,血清尿素氮和肌酐之比开始明显下降,此时该比值为0.11±0.008,远低于对照组的0.16±0.008(P<0.01)。随后,该比值继续降低,在免疫12周时达到最低值,为0.09±0.006,该比值显著低于对照组水平(0.17±0.004,P<0.01)。此后该比值开始逐渐升高,在免疫20周时升高达0.13±0.008,仍显著低于对照组的0.15±0.006(P<0.05)。免疫28周时,该比值已接近正常水平。(见Fig.34D)
以上结果提示:用β1-AR-ECⅡ抗原肽段长期主动免疫也可导致一定程度肾脏功能的损伤。
3.抗β1-AR-ECⅡ抗体长期被动免疫亦可导致肾脏损伤
3.1形态学指标
3.1.1 HE染色:被动免疫末期,抗β1-AR-ECⅡ抗体免疫组大鼠(5/6)肾小球及肾血管周围出现大量淋巴细胞浸润(见Fig.35D、35E、35F),对照组未见明显肾脏结构改变。
3.1.2 Masson三色染色:被动免疫末期,抗β1-AR-ECⅡ抗体免疫组大鼠(4/6)大量胶原纤维沉积在肾小管周围(见Fig.36D、36E、36F),对照组未见明显胶原纤维沉积现象。以上结果提示:用抗β1-AR-ECⅡ抗体长期被动免疫也可导致大鼠一定程度肾脏结构的损伤。
3.2血清学指标:
3.2.1血清尿素氮(BUN)测定
在用抗β1-AR-ECⅡ抗体被动免疫24周时,血清BUN水平升高为9.
BACKGROUND AND OBJECTIVE
β-Adrenoceptor (β-AR) is the important member of sympathetic nervous system.β-AR plays an important role on regulating the heart function by mediating the physiological effect of catecholamine.β-AR belongs to G protein–coupled receptor family.β-AR includesβ1、β2 andβ3-AR three types by now. They have the same structural features which are composed with 7 transmembrane domains, 3 intracellular loops and 3 extracellular loops. Among them, the distribution ofβ1-AR in heart is predominant.β1-AR can activate adenyl cyclase (AC) and cyclic adenosine monophosphate (cAMP) system by stimulating G protein (Gs) and displaying positive chronotropic, inotropic and dromotropic actions.
In 1987, Wallukat and Wollenberger found the antibodies against the second extracellular loop ofβ1-AR (anti-β1-AR-ECⅡ, Homology of human and rat is 100%) in the sera of patients with idiopathic dilated cardiomyopathy (IDCM). The following studies indicated that the autoantibodies displayed an analogous agonist effect which includes the inhibition of radioligand binding and enhanced signal transduction mediated byβ1-AR. With the further studies, other researches and ours all found that anti-β1-AR-ECⅡnot only existed in the patients with IDCM, but also existed in the patients with Chagasic disease (1994), primarily electrical derangement (1995), hypertensive heart disease and rheumatic heart disease (1999) et al. From the above results, we presumed that there may be a close relationship between anti-β1-AR-ECⅡand many kinds of heart diseases with the changes of cardiac structure and function.
In 2000, we observed the formation of anti-β1-AR-ECⅡin the cardiac remodeling models induced by abdominal aorta contraction and Adriamycin. The results indicated that high positive frequencies of anti-β1-AR-ECⅡwere found in abdominal aorta contraction group (87.5%) and Adriamycin group (79.2%). Moreover, with the development of heart failure, the titers of anti-β1-AR-ECⅡdisplayed an increasing, maintaining and decreasing process which suggested that cardiac remodeling or heart failure induced by different reasons all could induce the formation of anti-β1-AR-ECⅡ. In order to study the influence of anti-β1-AR-ECⅡon the cardiac structure and function, we immunized rats with the synthetic peptides according to the sequences of humanβ1-AR-ECⅡfor 18 months. In the late stage of immunization, analogous changes of dilated cardiomyopathy and reduced heart function were observed which suggested that the changes of cardiac structure could induce the formation of anti-β1-AR-ECⅡwhich could aggravate heart injury in return. At last, aggravated heart injury was displayed.
The phenomenon of cardiomyocytes lose provoked our attention in the process of 18 months active immunization. Cell lose has two types: apoptosis and necrosis in generally. Apoptosis is an active programmed procedure controlled by genes which is easy to be controlled. There is a research demonstrated that blocking the signal pathway of apoptosis could decrease the area of myocardial infarction significantly and improve heart function. In order to make it clear whether apoptosis existed in the process of DCM induced by anti-β1-AR-ECⅡ, we did an experiment in vitro and found that anti-β1-AR-ECⅡcould induce apoptosis of cultured neonatal cardiomyocytes (2002). In 2003, Staudt et al also demonstrated that the apoptosis of cultured adult cardiomyocytes could be induced by anti-β1-AR-ECⅡ. In 2004, we use active immunization model to demonstrate that anti-β1-AR-ECⅡcould induce apoptosis of cardiomyocytes in vivo. The above results all suggested that apoptosis played an important role in the changes of cardiac structure and function induced by anti-β1-AR-ECⅡ. However, the above researches all used the active immunization model in which we could not exclude the possibly toxic role of antigen peptides. Moreover, the exact mechanism of anti-β1-AR-ECⅡinducing apoptosis was not clear yet. Therefore, making it clear was helpful to determine the pathophysiologic significance of anti-β1-AR-ECⅡand was necessary for the clinical treatment.
Some researches demonstrated that anti-β1-AR-ECⅡcould enhance the current of L-type calcium by activating cAMP-PKA pathway. Calcium overload is the important mechanism of apoptosis. In many kinds of cells, calcium can bind with calmodulin (CaM). The compounds of Ca2+/CaM can activate protease depending on Ca2+/CaM. CaMK has four subtypes:Ⅰ,Ⅱ,ⅢandⅣ. Among them, CaMKⅡis a kind of multifunctional protease which is expressed in many important organs. CaMKⅡis composed with four subunits:α,β,γandδ. Among them, the products ofγandδexpress in cardiomyocytes. Up to now, we have found that CaMKⅡδcould induce hypertrophy of cardiomyocytes and also intimately related with apoptosis. So if there is a lot of apoptosis existing in the DCM model induced by anti-β1-AR-ECⅡ, then anti-β1-AR-ECⅡinduce apoptosis by the CaMKⅡδsignal pathway at the dowm stream of calcium?
Cardiac remodeling is an important stage in the process of heart failure which may be the possible reason of reduced cardiac function and arrhythmia. Some researches indicated that high level of anti-β1-AR-ECⅡwas detected in the sera of patients with DCM complicating with ventricular arrhythmia and conduction blockade, moreover there was a close relation between anti-β1-AR-ECⅡand arrhythmia. Other researches indicated that anti-β1-AR-ECⅡcould enhance the current of L-type calcium. Then whether anti-β1-AR-ECⅡcan induce arrhythmia directly need further study. The essential mechanisms of arrhythmia are the changes of bioelectricity of cardiomyocytes. In order to investigate the exact mechanism of anti-β1-AR-ECⅡinducing arrhythmia, we will observe the long-term role of anti-β1-AR-ECⅡon resting potential (RP), action potential duration (APD), L-type Calcium current (ICa-L), transient outside potassium current (Ito), delayed-rectifier potassium current (Ik1) and Na+/Ca2+ exchange current (INa-Ca) in cardiomyocytes. Moreover we will observe the acute role of anti-β1AR-ECⅡon bioelectricity of papillary muscles.
Researches indicated that the distribution ofβ1-AR is mainly on heart and kidney, and slightly on liver. A lot of researches in the past demonstrated that anti-β1AR-ECⅡcould induce the injury of heart by binding withβ1-AR. Different with agonist; anti-β1-AR-ECⅡhad the character of non-desensitization. Then whether anti-β1-AR-ECⅡexisting in the circulation system could bind withβ1-AR in liver and kidney is not clear. Our previous experiment of long-term active immunization of 18 months with synthetic peptides according to humanβ1-AR-ECⅡon rats, the phenomena of a lot of ascitic fluid and blunt liver edge were observed. In the ascitic fluid, high in protein, more cells and a low glucose level were found by us. Moreover, the isolated ascitic fluid coagulated naturally. In view of the ascitic fluid analysis, the ascitic fluid should be determined as exudate but not transudate. But the reason of ascitic fluid formation was not clear. Considering the phenomenon of blunt liver edge, liver injury may be the possible reason, but we could not exclude the kidney injury. So could anti-β1AR-ECⅡalso damage liver and kidney? In our study, we will do some initial research on the direction.
In summary, we will continue the study from two following portions:①observe the changes of intracellular free calcium and CaMKⅡδexpression in the long-term existence of anti-β1-AR-ECⅡ; set up active and passive immunization model and observe whether long-term existence of anti-β1-AR-ECⅡcan induce arrhythmia; moreover analyze the possible mechanism of anti-β1-AR-ECⅡinducing arrhythmia in order to provide the theoretical direction for clinical treatment;②set up active and passive immunization model and observe whether long-term existence of anti-β1-AR-ECⅡcan induce the injury of liver and kidney.
I. Anti-β1-AR-ECⅡCould Result in Cardiac Remodeling and its Possible Mechanism
PART ONE: Long-term Existence of Autoantibodies against the Second Extracellular Loop ofβ1-Adrenoceptor Could Induce the Increase of Calcium/Calmodulin-Dependent Protein KinaseⅡδin Rat Cardiomyocytes
Objective
1. To observe the changes of cardiac structure and function in the process of passive immunization with anti-β1-AR-ECⅡ;
2. To observe the changes of intracellular free calcium and the expression of CaMKⅡδin the process of passive immunization with anti-β1AR-ECⅡ.
Methods
(1) Select healthy adult Wistar rats (180~220g ,body wt, n=60).The Wistar rats used in the present study were obtained from the Animal Center of Shanxi Medical University, P.R. China. Detect anti-β1-AR-ECⅡin the sera by ELISA. The rats with no anti-β1-AR-ECⅡwere divided into the following two groups: (2 rats died for over dose anesthesia; 2 rats died for unknown reasons)
①β1AAb group(β1-AR-ECⅡantibody group, n=32):Purify the IgG from sera obtained from active immunized rats. Then quantity the total IgG by BCA method. Inject the purified IgG into vena caudalis of rats at the dose of 0.7μg/g, boosting for every 2 weeks. The experiment lasts 40 weeks.
②Negative sera group(n=28):Purify the negative sera, then quantity the total IgG by BCA method. Inject the purified IgG into vena caudalis of rats at the dose of 0.7μg/g, boosting for every 2 weeks. The experiment lasts 40 weeks.
(2) Peptides composition: A peptide (HWWRAESDEARRCYNDPKCCDFVTNRA, 197-223 amino acid residues) corresponding to the sequence of the second extracellular loop of humanβ1-AR was synthesized by GL Biochem (Shanghai) Ltd. The purity is 95% (Table. 1). The synthetic peptides were stored at -20℃for use.
(3) Set up passive immunization model by injecting anti-β1-AR-ECⅡinto vena caudalis of rats regularly. And detect the level of anti-β1AR-ECⅡin the sera;
(4) Use anatomic measurements, Masson staining and heart weight to body weight ratio to reflect the changes of cardiac structure in the process of passive immunization;
(5) Monitor Heart rates (HR), left ventricular systolic pressure (LVSP), and Left ventricular diastolic pressure (LVDP) and±dp/dtmax by putting the arterial cannula into left ventricle by right carotid artery;
(6) Detect the changes of apoptosis in the process of immunization by TUNEL and detection of caspase-3, 8 and 9 activity in cardiomyocytes;
(7) detect the intracellular free calcium in single ventricular myocytes at the late stage of passive immunization labeled with Fluo-3-AM by Confocal microscope;
(8) Detect the expression of CaMKⅡδin cardiomyocytes by Western-blot and immunohistochemical method.
Results
1. Anti-β1-AR-ECⅡcould result in cardiac remodeling of structure
1.1 In the process of passive immunization, anti-β1-AR-ECⅡin the sera still maintained a relative permanent level
The optical density (OD) value of anti-β1-AR-ECⅡin the sera detected by ELISA method ranged from 0.09±0.030 before immunization to 0.21±0.029 at the 4th week after immunization which was higher than 0.08±0.040 (P<0.001) in negative sera group. At the 4th week after immunization, anti-β1-AR-ECⅡkept the high level till to the end of experiment (Fig.1). In the whole immunization process, the level of anti-β1-AR-ECⅡin the sera didn’t fluctuate too much. There was no significant difference among different time points of active immunization (P>0.05).
1.2 Long-term existence of anti-β1-AR-ECⅡcould result in cardiac remodeling
1.2.1 The changes of the ratio of heart weight to body weight in the passive immunization
The ratio of heart weight to body weight (HW/BW) can reflect whether cardiac remodeling took place. HW/BW began to decrease significantly to 2.10±0.09 at the 24th week after passive immunization which was lower than 2.72±0.04 (P<0.01) in the negative sera group. At the 36th week, the ratio decreased to 2.35±0.05 inβ1AAb group which was still lower than 2.68±0.05 (P<0.01) in the negative sera group (Fig.2C).
1.2.2 Anatomic index
At the end stage of passive immunization, some pathological changes such as enlarged chamber heart and thinning heart wall could be observed (Fig.2B).
1.2.3 Masson’s trichromic staining
At the 36th week after passive immunization, a lot of collagen fibers depositing in the stroma of heart could be observed by Masson’s t staining (Fig.3).
1.3 Long-term existence of anti-β1-AR-ECⅡcould result in the descending of heart function
With the time of passive immunization, heart function cut down gradually (Fig.4). In the whole immunization, heart rates didn’t change obviously. Only at the 36th week of immunization, heart rates decreased to 311±20.3 times/min which was lower than 360±23.0 times/min (P<0.05) in negative sera group (Fig.4A). +dp/dtmax significantly descended to 213±31.1 Kpa/s at the 24th week of immunization which was lower than 510±29.0 Kpa/s (P<0.01) in negative sera group. At the 36th week, it was 289±39.6 Kpa/s inβ1AAb group which was lower than 505±30.0 Kpa/s (P<0.01) in negative sera group (Fig.4B). -dp/dtmax began to decrease obviously at the 24th week of immunization. At that time, -dp/dtmax was -233±34.7 Kpa/s which was lower than -415±31.0 Kpa/s (P<0.01) in negative sera group. At the 36th week, -dp/dtmax was -283±29.6 Kpa/s which was also lower than -421±29.0 Kpa/s (P<0.01) in negative sera group (Fig.4C). There was no significant difference of LVSP and LVDP betweenβ1AAb group and negative sera group.
Above results suggested that the long-tem passive immunization with anti-β1-AR-ECⅡcould result in the formation of cardiac remodeling and decreased heart function. But the possible mechanisms of these changes were not clear which need further study.
2. The possible mechanisms of cardiac remodeling induced by anti-β1-AR-ECⅡ
2.1 Long-term existence of anti-β1-AR-ECⅡcould induce the increase of apoptosis in cardiac myocytes
2.1.1 Long-term existence of anti-β1-AR-ECⅡcould induce the increase of caspase-3,8 and 9 activities of cardiomyocytes
In the passive immunization, fluorescent quantitation was used to detect the activity of caspase3, 8 and 9. Use AFC standard to make the standard curve (Fig.5). The slope rate of the straight line was 0.998 by fitting every point. The best incubation time of caspase3, 8 and 9 was determined in Fig.6. When incubate sample and fluorescence substrate for 30min, the fluorescence intensity of negative control was near to the peak, whereas the fluorescence intensity of samples were in low level. When incubating them for 60min, the fluorescence intensity of negative control was near to the bottom of the curve, whereas the fluorescence intensity of samples was increasing. Incubating 90min, the fluorescence intensity of negative control was still at the bottom, whereas the fluorescence intensity of samples was still increasing to a high level. Incubating 120min, the fluorescence intensity of negative control began to increase, whereas the fluorescence intensity of samples was still increasing. According to the standard, we select the time point with high fluorescence intensity of sample and low fluorescence intensity of negative control. So the incubation time was determined at 90min.
In the process of passive immunization, the activity of caspase-3, 8 and 9 increased with the time of immunization (Fig.7). The activity of caspase-8 began to increase at the 16th week. At that time, the activity of caspase-8 was 13.41±6.14 pmol/h/mg which was higher than 3.45±0.97 pmol/h/mg (P<0.01) in the negative sera group. After that, the activity of caspase-8 kept increasing. At the 36th week, it reached the peak (34.1±10.09 pmol/h/mg) which was significantly higher than 4.15±1.09 pmol/h/mg (P<0.01) in the negative sera group (Fig.7A). Caspase-9 had the same variation regularity. At the 16th week, it increased to 7.47±0.94 pmol/h/mg which was higher than 1.27±0.18 pmol/h/mg (P<0.01) in the negative sera group. It kept rising. At the 36th week, it reached the peak 16.24±3.31 pmol/h/mg which was higher than 1.38±0.37 pmol/h/mg (P<0.01) in the negative sera group (Fig.7B). The variation regularity of caspase-3 was the same. At the 16th week, it increased to 18.84±7.23 pmol/h/mg which was significantly higher than 1.49±0.18 pmol/h/mg (P<0.01) in the negative sera group. At the 36th week, it reached the peak (25.06±6.80 pmol/h/mg) which was higher than 1.83±0.60 pmol/h/mg (P<0.01) in the negative sera group (Fig.7C).
2.1.2 Anti-β1-AR-ECⅡexisting for a long time in the sera could induce the increase of apoptosis in cardiomyocytes
At the 36th week after immunization, there was about 7.86±0.43% cardiomyocytes took place apoptosis which was higher than 0.86±0.29 % (P<0.01) in the negative sera group at the same time (Fig.8G). The representative figures were shown in Fig.8A-F. Among them, Fig.8A-C belonged to negative sera group; Fig.8D-F belonged toβ1AAb group.
The above results suggested that anti-β1-AR-ECⅡcould increase apoptosis in cardiomyocytes, but the mechanism of augmented apoptosis need further study.
2.2 Anti-β1-AR-ECⅡexisting for a long time in the sera could induce the increase of intracellular free calcium in rats’ventricular myocytes
At the end stage of passive immunization, the intracellular free calcium in rats’ventricular myocytes was detected by confocal microscope. The data showed that the level of intracellular free calcium was 456.34±35.47 in anti-β1-AR-ECⅡgroup which was significantly higher than 51.96±1.18 (P<0.001) in negative sera group (Fig.9).
The above results suggested that long-term role of anti-β1-AR-ECⅡcould result in the increase of intracellular free calcium. But how the elevated calcium resulted in apoptosis was not clear. In another word, the downstream signal pathway of calcium inducing apoptosis need further study.
2.3 Long-term existence of anti-β1-AR-ECⅡcould result in the increasing expression of CaMKⅡδin cardiomyocytes
2.3.1 Western blot result
In the passive immunization with anti-β1-AR-ECⅡ, the expression of CaMKⅡδincreased with the time (Fig.10). At the 16th week after immunization, the expression of CaMKⅡδincreased to 1.19±0.38 which was higher than 0.39±0.22 (P<0.05) in the negative sera group. After that, the expression of CaMKⅡδcontinued to increase. At the 36th week after immunization, it reached the peak (1.94±0.77) which was higher than 0.46±0.27 (P<0.05) in the negative sera group.
2.3.2 Immunohistochemical result
In the process of passive immunization, the expression of CaMKⅡδincreased gradually. At the 36th week, the expression of CaMKⅡδsignificantly increased which was higher than that in the negative sera group (Fig.11).
The above results suggested that the elevated expression of CaMKⅡδin cardiomyocytes may be related with the increased apoptosis of cardiomyocytes in the passive immunization.
Summary
1. Long-term existence of anti-β1-AR-ECⅡcould result in cardiac remodeling;
2. Heart function decreased gradually in the long-term passive immunization with anti-β1-AR-ECⅡ;
3. Long-term existence of anti-β1-AR-ECⅡcould result in the increase of apoptosis;
4. Long-term existence of anti-β1-AR-ECⅡcould result in the increase of intracellular free calcium;
5. Long-term existence of anti-β1-AR-ECⅡcould result in the increasing expression of CaMKⅡδin cardiomyocytes.
PART TWO: The Autoantibodies against the Second Extracellular Loop ofβ1-Adrenoceptor Could Induce Arrhythmia in vivo and in vitro and the possibly Underlying Mechanism
Objective
1. To observe whether anti-β1-AR-ECⅡcould induce arrhythmia directly;
2. On papillary muscle level and cell level, using microelectrode recording AP and patch clamp to study the possible mechanism of anti-β1-AR-ECⅡinducing arrhythmia which is helpful for the clinical treatment of patients with high level of anti-β1-AR-ECⅡin the sera.
Methods
(1) Select healthy adult Wistar rats (180~220g, body wt,n=172).The Wistar rats used in the present study were obtained from the Animal Center of Shanxi Medical University, P.R. China. Detect anti-β1-AR-ECⅡin the sera by ELISA. The rats with no anti-β1-AR-ECⅡwere divided into the following four groups: (5 rats died for over dose anesthesia; 4 rats died in nubibus reason)
①β1AR group(n=64):inject the mixture of antigen peptides and immunoadjuvant into the dermis of back at the dose of 0.4μg/g, boosting for every 2 weeks. The experiment lasts 40 weeks.
②Vehicle group(n=48):inject the mixture of saline and immunoadjuvant into the dermis of back boosting for every 2 weeks. The experiment lasts 40 weeks.
③β1AAb group(n=32):Purify the IgG from sera obtained from active immunized rats. Then quantity the total IgG by BCA method. Inject the purified IgG into vena caudalis of rats at the dose of 0.7μg/g boosting for every 2 weeks. The experiment lasts 40 weeks.
④Negative sera group(n=28):Purify the negative sera, then quantity the total IgG by BCA method. Inject the purified IgG into vena caudalis of rats at the dose of 0.7μg/g, boosting for every 2 weeks. The experiment lasts 40 weeks.
(2) Observe whether arrhythmia can be induced by anti-β1-AR-ECⅡby the measurement of cardiac function and in vivo;
(3) Observe whether triggered activity can be induced by adding anti-β1-AR-ECⅡto the isolated papillary muscle of guinea pig and analyze the changes of action potential duration (APD) ;
(4) Observe whether arrhythmia can be induced by long-term existence of anti-β1-AR-ECⅡin the active and passive immunization model and analyze the changes of QT interval;
(5) Analyze the possible mechanisms of anti-β1-AR-ECⅡinducing arrhythmia by patch clamp and fluorescent assay of intracellular calcium.
Results
1. Anti-β1-AR-ECⅡcould result in cardiac remodeling of electricity
1.1 Anti-β1-AR-ECⅡcould induce arrhythmia on normal rats
1.1.1 Anti-β1-AR-ECⅡcould directly induce arrhythmia on normal rats in vivo
68μM anti-β1-AR-ECⅡcould induce arrhythmia in 66.7% (6/9) normal rats (weight: 180-220g). The percent was higher than 12.5% (1/8) in negative sera group and 11.1% (1/9) in saline group. Premature ventricular contraction (PVC) was the major type of arrhythmia (Fig.12A). We also could see considerable premature supraventricular contraction (PSVC, Fig.12B). After injecting anti-β1-AR-ECⅡinto artery, we observed the frequency of arrhythmia per hour. We find that the frequency of arrhythmia is 16±10.28 times/hour in anti-β1AR-ECⅡgroup which was higher than 4±1.00 times/hour (P<0.01) in saline group and 5±0.82 times/hour (P<0.01) in the negative sera group (Fig.12C).
1.1.2 Anti-β1-AR-ECⅡcould induce triggered activity on the papillary muscles of guinea pigs
Adding 0.1μM anti-β1-AR-ECⅡto the papillary muscle of guinea pig, almost all isolated papillary muscles took place triggered activity inducing by train (Fig.13B). Moreover its role didn’t attenuate with time (the longest observation time is 2 hours). Its role was similar to Isoprenaline (ISO, 1μM, Fig.13A). But the papillary muscle displayed more sensitive in anti-β1-AR-ECⅡgroup than ISO. The triggered activity induced by anti-β1-AR-ECⅡand ISO could be blocked by metoprolol (MET) which was a selectiveβ1-AR antagonist (Fig.13C). Moreover, anti-β1-AR-ECⅡcould prolong the APD of papillary muscle of guinea pig to 355.5±32.98 ms which was higher than 306±18ms (P<0.05) in saline group and 277.2±27.30 ms (P<0.01) in negative sera group. MET could block the role of anti-β1-AR-ECⅡprolonging APD (P<0.001, Fig.13D).
1.1.3 Anti-β1-AR-ECⅡcould induce after-depolarization and triggered activity on the papillary muscles of rats
Adding 1μM anti-β1-AR-ECⅡto the papillary muscles of rats, most isolated papillary muscles (60%, 9/15) took place triggered activity inducing by train (Fig.14A). EAD could be observed on some other papillary muscles (46.7%, 7/15, Fig.14B). Moreover DAD could be observed on some papillary muscles (26.7%, 4/15, Fig.14C). EAD, DAD and triggered activity could not be observed in ISO (1μM) group. MET (10μM) could inhibit EAD, DAD and triggered activity induced by anti-β1-AR-ECⅡ(Fig.14D).
The above results suggested that anti-β1-AR-ECⅡcould induce arrhythmia by inducing triggered activity and after depolarization on normal cardiomyocytes. Because anti-β1-AR-ECⅡexited in our body for a long time, so what were the exact effects of anti-β1-AR-ECⅡon electrocardio-activity need further study.
1.2 Long-term existence of anti-β1-AR-ECⅡcould induce arrhythmia
1.2.1 In the process of immunization with peptides according to the sequence ofβ1-AR-ECⅡ, different kinds and degrees arrhythmia could be observed in rats; moreover Q-T interval was prolonged obviously
1.2.1.1 In the process of active immunization, the level of anti-β1-AR-ECⅡshowed a naturally generated and extinctive process
At the 4th week after first immunization, the level of anti-β1-AR-ECⅡwas 0.4±0.21 which was higher than 0.10±0.05 (P<0.05) in the vehicle group. After that, the level of anti-β1AR-ECⅡincreased more and more. At the 8th week, it reached the peak. The value was 2.10±0.18 which was higher than 0.15±0.09 in the vehicle group (P<0.001). After that, anti-β1-AR-ECⅡdecreased. And at the end of the experiment, the value of anti-β1-AR-ECⅡwas 0.73±0.21 which was higher than 0.15±0.10 in the vehicle group (P<0.01, Fig.22).
1.2.1.2 with the immunization time, the frequency of arrhythmia increased
At the 4th、8th、12th、16th、20th、24th、28th、32nd week after the first immunization, the ratio of arrhythmia number to total rats number was 0/6、1/7、1/8、1/6、0/6、3/3、6/7、8/8 respectively. The type of arrhythmia we observed includes premature ventricular contraction (PVC, Fig.15A), premature supraventricular contraction (PSVC, Fig.15B), ventricular tachycardia (VF, Fig.15C), and atrial ventricular block (A-V block) et al. Among them, PVC and PSVC were common. At the 28th week, the frequency of arrhythmia per hour was 220±25.17 (times/hour) which was higher than 10±0.65 (times/hour, P<0.01) in the vehicle group. At the 32th week, the frequency of arrhythmia per hour was1509±40.88 (times/hour) which was higher than 15±0.75 (times/hour, P<0.001) in the vehicle group. At the different time point of immunization, the frequency of PVC, PSVC and VF could be seen on Fig. 15D-F.
1.2.1.3 In the process of active immunization, Q-T interval was prolonged
At the 12th week of immunization, the results of ECG showed that Q-T interval was 65.0±0.56ms which was longer than 55.5±0.86ms (P<0.05) in the vehicle group. At the 28th week of immunization, Q-T interval was 84.0±2.42 ms which was longer than 58.9±0.95 ms (P<0.05) in the vehicle group (Fig.16C). Because considering the influence of heart rates, QTc was calculated by formula Bazett. At the 12th week of immunization, QTc was 0.36±0.002 which was longer than 0.34±0.007 (P<0.05) in the vehicle group. At the 28th week of immunization, QTc was 0.46±0.007 which was longer than 0.32±0.009 (P<0.05) in the vehicle group (Fig.16D).
The above results suggested that different kinds and degrees arrhythmia could be induced and Q-T interval was prolonged in the process of active immunization. However we could not exclude the possible role of antigen on cardiac electricity. Therefore we need to do further research in order to find the exact reason of arrhythmia, antibody or antigen?
1.2.2 Long-term passive immunization with anti-β1-AR-ECⅡcould induce arrhythmia
1.2.2.1 In the process of passive immunization, anti-β1-AR-ECⅡin the sera still maintained a relative permanent level
The optical density (O.D.) value of anti-β1-AR-ECⅡin the sera detected by ELISA method ranged from 0.09±0.030 before immunization to 0.21±0.029 at the 4th week after immunization which was higher than 0.08±0.040 (P<0.001) in negative sera group. At the 4th week after immunization, anti-β1-AR-ECⅡkept the high level till to the end of experiment (Fig.1). In the whole immunization process, the level of anti-β1AR-ECⅡin the sera didn’t fluctuate too much. There was no significant difference among different time points of passive immunization (P>0.05).
The above results suggested that anti-β1-AR-ECⅡcould be produced in the sera in the passive immunization model; moreover the level of antibody was relative lower than that in active immunization model. But the level of anti-β1-AR-ECⅡin the passive immunization model was near to that in patients. Comparing the changes of anti-β1-AR-ECⅡin the sera, we found that the fluctuation of anti-β1-AR-ECⅡin the passive immunization model was slender.
1.2.2.2 Different kinds of arrhythmia could be observed in the process of passive immunization
Representative changes of ECG were recorded in Fig. 17A-F. Fig.17A: normal ECG at the 36th week after immunization in anti-β1-AR-ECⅡgroup. Fig.17B-F: different kinds of arrhythmia were observed in the same rat. Among all kinds of arrhythmia, ventricular arrhythmia was predominant. Some supraventricular arrhythmia also could be observed. Moreover there was some arrhythmia which type of arrhythmia couldn’t be determined. The time of arrhythmia occurrence localized after the 24th week of immunization.
1.2.2.3 Long-term existence of anti-β1-AR-ECⅡcould prolong the persistence time of arrhythmia
In the process of passive immunization with anti-β1-AR-ECⅡ, the data of ECG showed that there was a prolongatus tendency of the persistence time of arrhythmia with the time of passive immunization (Fig.17G). The persistence time of arrhythmia at the 24th week after immunization was 90±34.6 s in anti-β1-AR-ECⅡgroup which was higher than 0 s (P<0.01) in the negative sera group; at the 36th week after immunization, the persistence time of arrhythmia was 170±124.2 s in anti-β1-AR-ECⅡgroup which was higher than 0 s (P<0.01) in the negative sera group.
1.2.2.4 Anti-β1-AR-ECⅡexisting for a long time in the sera could induce the prolongation of Q-T interval
Q-T interval prolongation occurred at different time points of passive immunization. Representative Q-T intervals were recorded in Fig.18A and 18B. Fig.18A: ECG at the 36th week after immunization in the negative sera group; Fig.18B: ECG at the 36th weeks after passive immunization in anti-β1AR-ECⅡgroup. Considering the influence of heart rates, QTc was calculated by formula Bazett. At the 24th week of immunization, QTc was 0.46±0.015 in anti-β1-AR-ECⅡgroup (n=8) which was longer than 0.32±0.019 (P<0.01) in the vehicle group. At the 36th week of immunization, QTc was 0.51±0.015 in anti-β1-AR-ECⅡgroup (n=8) which was longer than 0.33±0.018 (P<0.01) in the vehicle group (n=6, Fig.18C).
The above results suggested that different kinds and degrees of arrhythmia could be induced and prolongation of Q-T interval could be observed in the process of passive immunization which was coincident with the findings in active immunization model. The phenomenon indicated that anti-β1-AR-ECⅡwas the main cause of arrhythmia. But how it induced arrhythmia or the exact mechanisms of arrhythmia induced by anti-β1-AR-ECⅡneed further study.
2. The possible mechanisms of anti-β1-AR-ECⅡinducing cardiac remodeling of electricity
2.1 Long-term effects of anti-β1-AR-ECⅡon AP waveforms and RMP in rat ventricular myocytes at the 36th week after passive immunization
Anti-β1-AR-ECⅡexisting for a long time in the sera could induce the attenuation of RMP and prolongation of APD in rats’ventricular myocytes. Anti-β1-AR-ECⅡgroup: immunize rats with antibody againstβ1-AR-ECⅡ(n=6); negative sera group: immunize rats with IgG from negative sera (n=6). Representative APs and RMP were recorded in the negative sera group (black traces) and anti-β1-AR-ECⅡgroup (dash line) in Fig.19. RMP was attenuated to -62.37±0.69 mv in anti-β1-AR-ECⅡgroup which was lower than -81.27±0.91 mv in the negative sera group (P<0.05). Moreover APD was prolonged to 51.09±2.66 ms in anti-β1-AR-ECⅡgroup which was longer than 33.26±2.58 ms in the negative sera group (P<0.05, Table 2).
2.2 Anti-β1-AR-ECⅡexisting for a long time in th
β肾上腺素受体(β-adrenoceptor,β-AR)是交感神经系统的重要成员,通过介导体内儿茶酚胺类物质的生理效应,在调节心脏活动中发挥着重要作用。β-AR属于G蛋白偶联受体家族,目前已确认的β1、β2和β3AR三个亚型具有相同的结构特征,即含有由7个22~28个疏水性氨基酸残基组成的跨膜区,连接这些跨膜区的亲水性氨基酸片段构成了3个细胞内环和3个细胞外环。在这三种β-AR亚型中,β1AR在心脏的分布密度最高。当该受体受神经、体液因素调节激活时,通过兴奋型G蛋白-腺苷酸环化酶-环磷酸腺苷系统,使心脏产生正性变时、正性变力、正性变传导效应,以适应机体对血液供应的需求。
1987年Wallukat和Wollenberger在原发型扩张性心肌病(IDCM)患者血清中发现针对β1-AR细胞外第二环(the second extracellular loop ofβ1-adrenoceptor,β1-AR-ECII,人鼠同源性为100%)的自身抗体。随后的研究发现,该抗体具有可以使培养的乳鼠心肌细胞跳动频率加快;使正常心房肌收缩增强;可以抑制放射性配体结合,同时增强受体介导的信号转导等作用,因而推断抗β1-AR-ECII自身抗体具有类激动剂样作用。随着研究的深入,国外学者及我们均发现,抗β1-AR-ECII自身抗体并非IDCM独有,Chagasic病(1994)、原发性电紊乱病(1995)、高血压性心脏病、风湿性心脏病(1999)等患者血清中均有高水平的抗β1-AR-ECII自身抗体,从而推测该抗体可能与各种伴有心肌结构和功能改变的心脏疾患有关。
2000年,我们用缩窄腹主动脉和注射阿霉素制备大鼠心肌重构模型的方法观察抗β1-AR自身抗体的生成情况,结果发现:在两种心肌重构模型的建立过程中,大鼠体内抗β1-AR-ECII自身抗体均由处理前的阴性大多数转为阳性,其阳性率分别为87.5%(缩窄腹主动脉组)和79.2%(注射阿霉素组),而且该抗体滴度随心衰的发展呈现升高、维持和减退的现象,提示不同机制引起的心肌重构/心衰都可以导致抗β1-AR-ECII自身抗体的产生。为进一步探讨抗β1-AR-ECII自身抗体对心脏结构和功能的影响,我们用人工合成的β1-AR-ECII抗原肽段对大鼠进行了18个月的主动免疫,观察到免疫大鼠心肌组织发生类扩张性心肌病样改变和心功能下降。以上结果提示,心脏结构的改变可以诱导机体产生抗β1-AR-ECII抗体,后者反过来又可以加重心肌负荷,二者互为因果,最终加重心肌损伤。
在我们对大鼠进行18个月主动免疫的实验中,抗β1-AR-ECII抗体诱导的扩心病模型存在心肌细胞丢失的现象引起了我们的关注。一般认为,细胞丢失的主要形式有凋亡和坏死,凋亡是一种基因控制的主动过程,抑制凋亡更容易进行也更有意义。有研究证明,阻断细胞凋亡的信号转导过程可以显著减少心梗面积,改善心功能。为明确在抗β1-AR-ECII抗体诱导的扩心病模型中出现的细胞丢失中是否有凋亡的存在,我们进行了一种离体实验研究,发现(2002):抗β1-AR-ECII自身抗体可以使培养的乳鼠心肌细胞发生凋亡;2003年,Staudt等的研究结果也表明,抗β1-AR-ECII抗体可使成年大鼠心肌细胞发生凋亡。2004年我们通过主动免疫在体实验进一步证明,抗β1-AR-ECII抗体可在体诱导大鼠心肌细胞发生凋亡。这都提示,细胞凋亡可能在抗β1-AR-ECII抗体对心脏活动产生影响的过程中起重要作用。但是,以上研究采用的动物模型为主动免疫模型,该模型不能排除导致心肌损伤的原因中是否有抗原的参与。此外,抗β1-AR-ECII抗体致凋亡过程中,其信号转导机制如何尚不清楚。对这些问题的研究,有助于明确抗β1-AR-ECII自身抗体的病理意义,同时也是随后寻找关键蛋白作为临床治疗的靶点所必需的。
有研究表明,抗β1-AR-ECⅡ抗体可通过cAMP依赖性的蛋白激酶激活Ca2+通道,使心肌细胞内Ca2+升高,胞内钙超载是诱发细胞凋亡的重要机制之一。在细胞内,钙离子通过和钙调蛋白结合,形成Ca2+/CaM复合物后活化依赖Ca2+/CaM的蛋白激酶(CaMK)。CaMK有4个亚型:Ⅰ、Ⅱ、Ⅲ和Ⅳ。其中CaMKⅡ是一种多功能蛋白激酶,存在于体内多数重要器官中。分子克隆发现四个不同的基因分别转录CaMKⅡ的α、β、γ和δ四个亚基;有研究表明CaMKⅡ的亚基中γ和δ产物表达于心肌细胞。目前已经发现,CaMKⅡδ可引起心肌细胞肥大,且与细胞凋亡关系密切。因此,我们考虑,在抗β1-AR-ECII抗体诱导的扩心病模型中所出现的心肌细胞凋亡,是否通过Ca2+下游的CaMKⅡδ信号通路起作用呢?
心肌重构是心衰发生发展过程中的重要病理生理阶段,这种重构的发生是心衰患者出现心功能改变和心律失常的潜在原因。有研究报道,扩张性心肌病合并心律失常的患者血清中存在的抗β1AR-ECⅡ自身抗体,其阳性率高于其他心脏病患者,并且证实该抗体与心律失常的发生相关。另有研究表明:抗β1AR-ECⅡ抗体可以增加ICa-L电流,缩短Q-T间期,那么抗β1-AR-ECⅡ抗体是否能直接诱发心律失常还有待进一步研究。此外,心律失常最本质的变化在于心肌细胞生物电活动的改变,为了深入探讨抗β1-AR-ECⅡ抗体引起心律失常的具体机制,我们在本课题中,将进一步观察抗β1-AR-ECⅡ抗体对心肌细胞生物电活动,如:细胞膜静息电位(Resting Potential,RP)、动作电位时程(Action potential duration,APD)、L型钙电流(L-type Calcium current,ICa-L)、瞬时外向钾电流(transient outside potassium current,Ito)、延迟整流钾电流(delayed-rectifier potassium current, Ik1)及钠钙交换电流(Na+/Ca2+ exchange current,INa-Ca)等的直接影响,并利用乳头肌进行离体实验,探讨抗β1-AR-ECⅡ抗体对心肌组织生物电活动的影响。
已有的研究表明,β1-AR主要分布在心脏和肾脏,在肝脏也有少量的分布;以往大量研究证实:抗β1-AR-ECⅡ抗体可以通过与β1-AR结合发挥类激动剂样的作用,从而引起心脏损伤。但与激动剂不同的是,抗β1-AR-ECⅡ自身抗体具有不脱敏样的受体效应。那么循环中产生的抗β1-AR-ECⅡ抗体是否也能通过与肝脏和肾脏细胞表面的β1-AR结合,从而产生一定的作用呢?我们前期在用β1-AR-ECⅡ肽段进行18个月的主动免疫大鼠过程中观察到:有相当数量的大鼠出现了腹水和肝缘变钝的现象,引起我们的极大关注。对于腹水的临床检验结果表明:腹水中细胞数及蛋白水平偏高,葡萄糖水平偏低,同时腹水可以自然凝固,基于以上性质确定该腹水为渗出液,而非漏出液;同时结合大鼠的心功能状况判定,腹水不是由于心衰而导致的,那么形成腹水的原因是什么,尚不得而知;但结合肝缘变钝这一大体现象,我们考虑肝脏病变可能是形成腹水的原因之一,但不能排除肾脏在免疫过程中是否也受到损伤。因此在本课题中,我们也将对这一问题进行初步探索。
综上所述,本课题拟进行以下三方面的研究:①采用被动免疫模型,探讨抗β1-AR-ECⅡ抗体长期存在是否会导致心肌组织CaMKⅡδ表达的改变,同时观察心肌细胞内钙离子水平的变化;观察抗β1-AR-ECⅡ抗体致心律失常的直接作用、长期作用及其可能的机制;②建立主动及被动免疫模型,观察抗β1-AR-ECⅡ抗体长期存在是否会导致肝脏损伤的发生;③建立主动及被动免疫模型,观察抗β1-AR-ECⅡ抗体长期存在是否会导致肾脏损伤的发生。
Ⅰ.抗β1-AR-ECⅡ抗体可导致心肌重构及可能的机制
第一分题:抗β1-AR-ECⅡ抗体可导致心肌结构重构及可能的机制
目的
1.观察抗β1-AR-ECⅡ抗体被动免疫过程中,大鼠心肌形态学及心功能的变化情况;
2.观察被动免疫过程中,细胞内游离钙水平及CaMKⅡδ的表达情况。
方法
(1)分别选取健康成年雄性Wistar大鼠(n=60,体重180~220g),由山西医科大学实验动物中心提供,用ELISA法筛选血清抗β1AR-ECⅡ抗体阴性的动物进行分组。
①抗β1AR-ECⅡ抗体组(β1AAb group,n=32):将主动免疫收集的抗β1-AR-ECⅡ抗体阳性血清进行纯化,得到血清中总的IgG,BCA蛋白定量后,以0.7μg/g的剂量通过尾静脉给入大鼠体内,每2周加强免疫一次,共免疫40周;
②阴性血清IgG组(Negative sera group,n=28):将收集到的抗β1-AR-ECⅡ抗体阴性血清进行纯化,得到血清中总的IgG,BCA蛋白定量后,以0.7μg/g的剂量通过尾静脉给入大鼠体内,每2周加强免疫一次,共免疫40周。
(2)肽段合成:抗原肽段由吉尔生化上海有限公司合成,相当于人β1-AR细胞外第二环氨基酸序列的特异性抗原决定簇( 197-223位, HWW RAESDEARRCY NDPKCCDFVT NRA),合成肽段纯度为95%(见表1)。合成的肽段储存于-20℃备用。
(3)通过大鼠尾静脉定期给入抗体建立被动免疫模型,用酶联免疫吸附法(ELISA法)检测免疫过程中大鼠血清中抗β1-AR-ECII抗体水平的变化;
(4)分别用体视学指标、Masson三色染色及心重体重比的测量来反映被动免疫过程中大鼠心脏结构的变化;
(5)经右侧颈总动脉插管进入左心室,监测心率(Heart rates, HR)、左心室收缩压(Left ventricular systolic pressure, LVSP)、左心室舒张压(Left ventricular diastolic pressure, LVDP)和左心室压力变化最大速率(±dp/dtmax)等心功能指标;
(6)分别采用原位末端标记法(TUNEL)和Caspase-3、8、9活性测定法检测大鼠心肌组织中心肌细胞凋亡发生情况;
(7)用Fluo-3-AM探针标记,激光共聚焦测定被动免疫末期大鼠单个心室肌细胞内游离钙离子的水平;
(8)用Western-blot及免疫组化法检测大鼠心肌组织中CaMKⅡδ蛋白的表达水平。
结果
1.抗β1-AR-ECⅡ抗体可导致心肌结构重构
1.1免疫过程中抗β1-AR-ECⅡ抗体水平的变化
被动免疫组大鼠(其中因麻醉死亡2只,不明原因死亡2只)血清中抗β1-AR-ECⅡ抗体OD值由免疫前的0.09±0.030,逐步升高为免疫后4周的0.21±0.029,明显高于对照组的0.08±0.040(P<0.001),此后抗体一直维持在该水平至实验结束(见Fig.1)。在整个免疫过程中,血清中抗β1-AR-ECⅡ抗体水平波动不大,组内比较无统计学差异,但和阴性血清IgG组相比有显著差异(P<0.001)。
1.2抗β1-AR-ECⅡ抗体长期存在可导致心肌重构的发生
由于心肌结构重构的概念是:“心脏大小、形状、重量的变化以及心肌细胞形态、数量变化和胶原纤维沉积等改变”,所以我们用心重/体重比(heart weight to body weight ratio, HW/BW)、心脏体视学指标以及心肌胶原纤维含量等,作为抗β1AR-ECⅡ抗体长期存在下,是否可以导致心肌重构发生的指标。
1.2.1心重/体重比(HW/BW):HW/BW在抗β1AR-ECⅡ抗体被动免疫24周开始明显
下降为2.10±0.09,低于对照组的2.72±0.04(P<0.01);免疫36周时该比值为2.35±0.05,仍低于对照组的2.68±0.05(P<0.01,见Fig.2C)。
1.2.2体视学指标:心脏在免疫晚期出现心腔变大,心壁变薄等扩心病样的病理改变(见Fig.2B)。
1.2.3 Masson三色染色:免疫36周的心肌Masson三色染色显示:在心肌间质中有大量胶原纤维的分布(见Fig.3)。
1.3抗β1-AR-ECⅡ抗体长期存在可导致心功能的下降
随着被动免疫时间的延长,大鼠的心功能逐渐降低(见Fig.4)。心率在整个免疫过程中变化不明显,仅在免疫36周时出现心率的略微下降311±20.3次/分,低于对照组的360±23.0次/分(P<0.05,见Fig.4A)。室内压上升的最大速率(+dp/dtmax)在免疫24周开始出现明显下降(213±31.1 Kpa/s),显著低于对照组的510±29.0 Kpa/s(P<0.01);免疫36周时为289±39.6 Kpa/s,低于对照组的505±30.0 Kpa/s(P<0.01,见Fig.4B)。室内压下降的最大速率(-dp/dtmax)也在免疫24周开始出现明显下降(-233±34.7 Kpa/s),显著低于对照组的-415±31.0 Kpa/s(P<0.01);免疫36周时为-283±29.6 Kpa/s,低于对照组的-421±29.0 Kpa/s(P<0.01,见Fig.4C)。左室收缩压(LVSP)和左室舒张压(LVDP)在整个免疫过程中变化不明显。
以上结果提示:抗β1-AR-ECⅡ抗体长期被动免疫可导致大鼠发生心肌重构,同时心功能明显下降。
2.抗β1-AR-ECⅡ抗体致心肌结构重构的可能机制
2.1抗β1-AR-ECⅡ抗体长期存在可导致心室肌细胞凋亡增加
2.1.1抗β1-AR-ECⅡ抗体长期存在可导致心室肌细胞Caspase-3、8、9活性增加
被动免疫过程中Caspase-3、8、9的测定使用荧光定量法来进行,用AFC标品制作标准曲线(见Fig.5),通过各点拟合后的直线斜率为0.998。关于Caspase-3、8、9荧光底物作用最佳时间点的估计见Fig.6。底物和不同样品孵育30分钟时,阴性对照的荧光强度接近峰值,而待测品的荧光强度相对较低;孵育60分钟时,阴性对照的荧光强度刚接近曲线底部,而待测品的荧光强度还在继续增加过程中;孵育90分钟时,阴性对照的荧光强度维持在曲线底部,而待测品的荧光强度还在继续增加过程中,但已达较高水平;孵育120分钟时,阴性对照的荧光强度又开始逐渐升高,待测品的荧光强度还在继续增加过程中。根据选择最佳时间点的要求,即选择表现出最高样本读数和最低阴性对照读数的时间点,最后确定孵育的时间为90分钟。
在整个被动免疫过程中,Caspase-3、8、9的活性表现为随免疫时间的增加而增加(见Fig.7)。其中Caspase-8在免疫16周开始增加至13.41±6.14 pmol/h/mg,显著高于对照组的3.45±0.97 pmol/h/mg( P<0.01);此后Caspase-8继续升高,在免疫36周时达高峰34.1±10.09 pmol/h/mg,远高于对照组的4.15±1.09 pmol/h/mg(P<0.01,见Fig.7A)。Caspase-9在免疫16周开始增加至7.47±0.94 pmol/h/mg,高于对照组的1.27±0.18 pmol/h/mg(P<0.01);此后Caspase-9继续升高,在免疫36周时达高峰16.24±3.31 pmol/h/mg,远高于对照组的1.38±0.37 pmol/h/mg(P<0.01,见Fig.7B)。Caspase-3在免疫16周开始增加至18.84±7.23 pmol/h/mg,显著高于对照组的1.49±0.18 pmol/h/mg(P<0.01);此后Caspase-3继续升高,在免疫36周时达高峰25.06±6.80 pmol/h/mg,远高于对照组的1.83±0.60 pmol/h/mg (P<0.01,见Fig.7C)。
2.1.2抗β1-AR-ECⅡ抗体长期存在可导致心肌凋亡细胞数目的增加
用TUNEL法检测被动免疫36周大鼠凋亡心肌细胞的数目,发现:在免疫36周时,平均有7.86±0.43%的心肌细胞发生凋亡,其比例远高于对照组的0.86±0.29 %(P<0.01,见Fig.8G)。凋亡心肌细胞典型图见Fig.8A-8F。
以上结果提示,抗β1-AR-ECⅡ抗体被动免疫大鼠心肌细胞凋亡明显增加,但导致凋亡细胞增加的机制还有待进一步研究。
2.2抗β1-AR-ECⅡ抗体长期存在可导致心室肌细胞内游离钙离子水平升高
在抗β1-AR-ECⅡ抗体被动免疫末期,即免疫36周时,检测心室肌细胞内游离钙离子水平,发现抗β1-AR-ECⅡ抗体被动免疫组大鼠单个心室肌细胞内游离钙离子水平为456.34±35.47,远高于对照组的51.96±1.18(P<0.001,见Fig.9)。
以上结果提示:抗β1-AR-ECⅡ抗体长期作用可导致细胞内游离钙离子水平的升高,但升高的钙离子如何导致心肌细胞凋亡,即其下游的信号通路还有待进一步研究。
2.3抗β1-AR-ECⅡ抗体长期存在可导致心室肌CaMKⅡδ的表达增加
2.3.1 Western blot结果显示抗β1-AR-ECⅡ抗体长期存在可导致心室肌CaMKⅡδ的表达增加
在抗β1-AR-ECⅡ抗体被动免疫过程中,随免疫时间的延长CaMKⅡδ的表达逐渐增加(见Fig.10)。被动免疫16周时,CaMKⅡδ的表达开始升高达1.19±0.38,高于对照组的0.39±0.22 (P<0.05);此后CaMKⅡδ的表达持续升高,在免疫36周时达高峰(1.94±0.77),高于对照组的0.46±0.27(P<0.05)。
2.3.2免疫组化结果显示抗β1-AR-ECⅡ抗体长期存在可导致心肌CaMKⅡδ的表达增加在抗β1-AR-ECⅡ抗体被动免疫36周时,免疫组化测定心肌细胞内CaMKⅡδ的表达明显增加(见Fig.11)。
以上结果提示,抗β1-AR-ECⅡ抗体被动免疫大鼠心肌中CaMKⅡδ表达的升高可能与细胞凋亡的增加有关。
小结:
1.抗β1-AR-ECⅡ抗体长期存在可导致心肌结构重构的发生;
2.抗β1-AR-ECⅡ抗体长期被动免疫过程中,心功能逐渐下降;
3.抗β1-AR-ECⅡ抗体长期被动免疫可导致凋亡心肌细胞的增加;
4.抗β1-AR-ECⅡ抗体长期存在可导致心肌细胞内游离钙离子水平的升高;
5.抗β1-AR-ECⅡ抗体长期存在可导致心肌CaMKⅡδ的表达增加。
第二分题:抗β1-AR-ECⅡ抗体导致心电重构及其可能的机制
目的
1.观察抗β1-AR-ECⅡ抗体是否具有直接致心律失常的作用;
2.在组织和细胞水平,探讨抗β1-AR-ECⅡ抗体致心律失常的可能机制,以求阐明抗
β1-AR-ECⅡ抗体是否可致心电重构及其可能的机制,为临床抗β1-AR-ECⅡ抗体阳性心律失常患者的治疗提供理论依据。
方法
(1)分别选取健康成年雄性Wistar大鼠(n=172)(其中因麻醉死亡5只,不明原因死亡5只),体重180~220g,由山西医科大学实验动物中心提供,用ELISA法筛选血清抗β1AR-ECⅡ抗体阴性的动物进行分组。
①β1AR肽段免疫组(β1AR group,n=64):将抗原和免疫佐剂的混合物按0.4μg/g剂量注入动物背部皮内,每两周加强免疫一次,共免疫32周;
②免疫佐剂组(Vehicle group,n=48):将生理盐水和免疫佐剂的混合物以一定比例注入动物背部皮内,每两周加强免疫一次,共免疫32周;
③抗β1AR-ECⅡ抗体组(β1AAb group,n=32):将主动免疫收集的抗β1-AR-ECⅡ抗体阳性血清进行纯化,得到血清中总的IgG,BCA蛋白定量后,以0.7μg/g的剂量通过尾静脉给入大鼠体内,每2周加强免疫一次,共免疫40周;
④阴性血清IgG组(Negative sera group,n=28):将收集到的抗β1-AR-ECⅡ抗体阴性血清进行纯化,得到血清中总的IgG,BCA蛋白定量后,以0.7μg/g的剂量通过尾静脉给入大鼠体内,每2周加强免疫一次,共免疫40周。
(2)通过在体心功能及心电测定方法监测给入抗β1-AR-ECⅡ抗体后正常大鼠心功能及心电图的变化情况;
(3)通过微电极记录动作电位的方法,观察在组织水平抗β1-AR-ECⅡ抗体对乳头肌动作电位的影响,同时分析动作电位时程(APD)的变化情况;
(4)通过建立主动及被动免疫模型,观察抗β1-AR-ECⅡ抗体长期存在对大鼠心电活动的影响,同时分析QT间期的变化;
(5)通过膜片钳技术及胞内钙荧光测定技术,观察抗β1-AR-ECⅡ抗体长期存在对大鼠心肌细胞膜电流及胞内钙的影响。
结果
1.抗β1-AR-ECⅡ抗体可致心电重构
1.1抗β1-AR-ECⅡ抗体可致正常大鼠发生心律失常
1.1.1抗β1-AR-ECⅡ抗体可直接诱导正常大鼠发生心律失常
68μM抗β1-AR-ECⅡ抗体(0.7μg/g)可诱导约66.7%(6/9)的正常雄性大鼠(体重:180-220g)发生心律失常,其阳性率远高于阴性血清组(12.5%, 1/8)和生理盐水组(11.1%, 1/9)。抗β1-AR-ECⅡ抗体致心律失常的种类以室性早搏为主(见Fig.12A),同时还存在室上性早搏(见Fig.12B)。观察抗β1-AR-ECⅡ抗体从动脉给入一小时内大鼠心脏产生异常心律的次数发现,抗β1-AR-ECⅡ抗体引起室早和室上性早搏的次数为16±10.28(次/小时),高于生理盐水组的4±1.00(次/小时,P<0.01)和阴性血清组的5±0.82(次/小时,P<0.01,见Fig.12C)。
1.1.2抗β1-AR-ECⅡ抗体可诱导豚鼠乳头肌发生触发激动和延迟后除极(delayed after-depolarization, DAD)
0.1μM抗β1-AR-ECⅡ抗体作用于豚鼠乳头肌,在串刺激的诱导下几乎可以使所有分离的乳头肌发生触发激动(见Fig.13B),且该作用长时间持续不易衰减(最长观测时间2小时),这与异丙肾上腺素(ISO,1μM)的作用(见Fig.13A)很类似。同时,抗β1-AR-ECⅡ抗体还可诱发DAD(见Fig.13C);抗体和ISO的作用可被β1-AR选择性阻断剂美托洛尔(10μM)完全阻断(见Fig.13D)。同时,0.1μM该抗体可使豚鼠乳头肌的动作电位时程延长为355.5±32.98 ms,高于生理盐水组的306±18 ms(P<0.05)和阴性血清组的277.2±27.30 ms(P<0.01);美托洛尔(10μM)在一定程度上可减弱该抗体对动作电位时程的延长作用(P<0.001,见Fig.13E)。
1.1.3抗β1-AR-ECⅡ抗体可诱导大鼠乳头肌发生触发激动及后除极
1μM抗β1-AR-ECⅡ抗体作用于大鼠乳头肌,在串刺激的诱导下几乎可以使多数分离的乳头肌发生触发激动(60%,9/15,见Fig.14A)。部分乳头肌还可观察到早后除极(early after-depolarization,EAD)的发生(46.7%,7/15,Fig.14B)。少量乳头肌可观察到延迟后除极(delayed after-depolarization,DAD)现象(26.7%,4/15,Fig.14C)。在ISO组(1μM)EAD,DAD和触发激动均未被观察到。美托洛尔(10μM)可以抑制抗β1-AR-ECⅡ抗体诱发的EAD,DAD和触发激动(Fig.14D)。
以上结果提示:抗β1-AR-ECⅡ抗体可能通过使正常心肌细胞发生触发活动及后除极而导致心律失常的发生,但由于抗β1-AR-ECⅡ抗体在机体内是长期持续存在的,那么抗β1-AR-ECⅡ抗体长期存在时如何引发心律失常的发生,其在细胞水平的机制如何还有待进一步的研究。
1.2抗β1-AR-ECⅡ抗体长期存在可诱导心律失常的发生
1.2.1在用β1-AR-ECⅡ肽段免疫大鼠的不同时期可观察到不同程度及类型的心律失常
1.2.1.1主动免疫过程中,抗β1-AR-ECⅡ抗体水平呈现一个自然产生和消退的过程
在免疫4周时,抗β1-AR-ECⅡ抗体测定的OD值为0.4±0.21,高于对照组的0.10±0.05(P<0.05);此后抗体水平逐渐升高,在免疫8周时达高峰(2.10±0.18),高于对照组的0.15±0.09(P<0.001);此后抗体水平逐渐下降,免疫28周时抗体水平为0.73±0.21,仍高于对照组的0.15±0.10(P<0.01,见Fig22)。
1.2.1.2随着免疫时间的延长,心律失常的出现频率有所增加,阳性率也有一定程度增加
在主动免疫的0、4、8、12、16、20、24、28、32周,出现心律失常的动物数和总动物数的比例分别为0/6、0/6、1/7、1/8、1/6、0/6、3/3、6/7、8/8。观察到的心律失常类型主要有室性早搏(见Fig.15A)、室上性早搏(见Fig.15B)、室速(见Fig.15C)及房室传导阻滞等,其中以室性早搏和室上性早搏比较多见。其中主动免疫28周时心律失常的发生频率为220±25.17(次/小时),远高于对照组的10±0.65(次/小时,P<0.01);在主动免疫32周时,心律失常的发生频率为1509±40.88(次/小时),远高于对照组的15±0.75(次/小时,P<0.001,见Fig.15D-F)。
1.2.1.3 Q-T间期延长出现于β1-AR-ECⅡ肽段免疫大鼠的不同时期
在用β1AR-ECⅡ肽段免疫大鼠的不同时期,心电图监测发现:在主动免疫的12周Q-T间期为65.0±2.50 ms,远远长于对照组的55.0±0.86 ms(P<0.05);主免28周时Q-T间期为84.0±2.50 ms,远长于对照组的58.9±0.95 ms(P<0.05,见Fig.16C)。同时考虑到心率对Q-T间期的影响,根据Bazett公式计算QTc(校正QT间期),主动免疫12周时QTc为0.36±0.002,远长于对照组的0.34±0.007(P<0.05);主免28周时QTc为0.46±0.007,远长于对照组的0.32±0.009(P<0.05,见Fig.16D)。
以上结果提示:β1-AR-ECⅡ抗原肽段长期主动免疫可导致大鼠发生不同程度及类型的心律失常,同时Q-T间期也明显延长;但由于实验中不能完全排除抗原可能的致病作用,因此需通过直接给入抗体的被动免疫模型来进一步证实:免疫过程中心律失常的发生是抗原还是抗体导致的。
1.2.2抗β1-AR-ECⅡ抗体长期被动免疫也可诱导大鼠心律失常的发生
1.2.2.1被动免疫过程中血清抗β1-AR-ECⅡ抗体水平的变化
被动免疫组大鼠血清中抗β1-AR-ECⅡ抗体OD值由免疫前的0.09±0.030,逐步升高为免疫后4周的0.21±0.029,明显高于对照组的0.08±0.040(P<0.001)此后抗体一直维持在该水平至实验结束(见Fig.1)。在整个免疫过程中,血清中抗β1-AR-ECⅡ抗体水平波动不大,组内比较无统计学差异,但和阴性血清IgG组相比有显著差异(P<0.001)。
以上结果提示:被动免疫模型可使大鼠血清中产生抗β1-AR-ECⅡ抗体,同时抗体维持在一个相对稳定的低水平状态,这与临床病人筛查的结果相一致;和主动免疫模型过程中的抗体水平相比,其抗体水平的波动较小。
1.2.2.2抗β1-AR-ECⅡ抗体长期存在可诱导正常大鼠出现不同种类的心律失常
抗β1-AR-ECⅡ抗体被动免疫大鼠过程中,大鼠出现了不同程度及类型的心律失常(见Fig.17A-17F)。其中最多见的为室性早搏,也可见到一定数量的室上性早搏,同时还有室性心动过速(室速)及一些无法确定其类型的心律失常。心律失常的出现时间集中于免疫24周以后。
1.2.2.3抗β1-AR-ECⅡ抗体长期存在可使心律失常持续时间延长
在抗β1-AR-ECⅡ抗体被动免疫大鼠过程中,对不同时间点大鼠心律失常持续时间的分析发现:随着免疫时间的延长,发生心律失常的大鼠其心律失常持续时间有延长的趋势(见Fig.17G)。免疫24周时,心律失常持续时间为90±34.6 s,远高于对照组的0 s(P<0.01);免疫36周,心律失常持续时间为170±124.2 s,远高于对照组的0 s(P<0.01)。抗β1AR-ECⅡ抗体免疫组组内不同时间点比较结果为:免疫24周和36周分别和免疫前及免疫12周相比有统计学差异(P<0.01);免疫24周和36周的结果相比无统计学差异(P>0.05)。
1.2.2.4抗β1-AR-ECⅡ抗体长期存在可导致Q-T间期延长
在抗β1-AR-ECⅡ抗体被动免疫大鼠过程中,对不同时间点QTc的监测结果进行分析后得出:抗β1-AR-ECⅡ抗体被动免疫大鼠可导致QTc的延长(见Fig.18C)。从数据可以看到:抗β1-AR-ECⅡ抗体被动免疫24周开始,QTc开始明显升高达0.46±0.015,高于对照组的0.32±0.019(P<0.01);免疫36周时,QTc继续升高为0.51±0.015,明显高于对照组的0.33±0.018(P<0.01)。
以上研究结果提示:被动免疫过程中,抗β1-AR-ECⅡ抗体的长期存在可导致大鼠发生不同程度及类型的心律失常,同时心电图Q-T间期明显延长,这与主动免疫过程中观察到的现象一致,说明在免疫过程中抗β1-AR-ECⅡ抗体是导致心律失常发生的主要原因,但抗β1-AR-ECⅡ抗体导致心律失常发生的机制未知,有待进一步研究。
2.抗β1-AR-ECⅡ抗体致心电重构的可能机制
2.1抗β1-AR-ECⅡ抗体长期存在可导致细胞膜静息电位的减小和动作电位时程的延长
在抗β1-AR-ECⅡ抗体被动免疫末期,即免疫36周时,检测心室肌细胞膜静息电位发现:抗β1-AR-ECⅡ抗体被动免疫组大鼠心室肌细胞膜的静息电位为-62.37±0.69 mv,远小于对照组的-81.27±0.91 mv(P<0.05);同时被动免疫组大鼠心室肌细胞动作电位时程(Action potential duration, APD)测定为51.09±2.66 ms,长于对照组的33.26±2.58 ms(P<0.05,见Table 2,Fig.19)。
2.2抗β1-AR-ECⅡ抗体长期存在可导致心肌细胞膜延迟整流钾电流(Ik1)和瞬时外向钾电流(Ito)的减弱
在抗β1-AR-ECⅡ抗体被动免疫末期,即免疫36周时,检测心室肌细胞膜Ik1电流,发现:抗β1-AR-ECⅡ抗体被动免疫组大鼠心室肌细胞膜的Ik1电流减小,和对照组相比有统计学差异(P<0.05,见Fig.20A)。
在抗β1-AR-ECⅡ抗体被动免疫末期,即免疫36周时,检测心室肌细胞膜Ito电流,发现:抗β1-AR-ECⅡ抗体被动免疫组大鼠心室肌细胞的Ito电流明显弱于对照组的Ito电流(P<0.05,见Fig.20B)。
2.3抗β1-AR-ECⅡ抗体长期存在可导致心肌细胞膜L型钙电流(ICa-L)的增强及钠钙交换电流(INa-Ca)的减弱
在抗β1-AR-ECⅡ抗体被动免疫末期,即免疫36周时,检测心室肌细胞膜ICa-L电流,发现:抗β1-AR-ECⅡ抗体被动免疫组大鼠心室肌细胞的ICa-L电流在0mv-+40 mv的变化过程中明显强于对照组的ICa-L电流(P<0.05,见Fig.21A)。
同时对INa-Ca交换电流的测定也发现:抗β1-AR-ECⅡ抗体被动免疫组大鼠心室肌细胞的INa-Ca电流明显弱于对照组的INa-Ca电流(P<0.05,见Fig.21B-21E)。
2.4抗β1-AR-ECⅡ抗体长期存在可导致心肌细胞内游离钙离子水平的升高
在抗β1-AR-ECⅡ抗体被动免疫末期,即免疫36周时,检测心室肌细胞膜内游离钙离子水平,发现抗β1-AR-ECⅡ抗体被动免疫组大鼠单个心室肌细胞内游离钙离子水平为456.34±35.47,远高于对照组的51.96±1.18(P<0.001,见Fig.9)
以上研究结果提示:抗β1-AR-ECⅡ抗体长期被动免疫可导致心肌细胞内游离钙离子水平的升高,这一改变可能是由于该抗体抑制了INa-Ca,同时增强了ICa-L的结果;同时抗β1-AR-ECⅡ抗体长期存在可导致APD的延长,这一改变可能是由于该抗体抑制了Ik1和Ito电流,同时增强了ICa-L的结果;且该抗体长期作用可减小静息电位。以上这一些列改变,包括静息电位减小、APD延长及细胞内钙离子水平升高都是抗β1-AR-ECⅡ抗体致心律失常发生的可能机制。
小结
1.抗β1-AR-ECⅡ抗体可直接诱导正常大鼠在体发生心律失常,同时抗β1-AR-ECⅡ抗体可诱导豚鼠及大鼠乳头肌发生触发激动及后除极;
2.主动及被动免疫均可诱导大鼠发生不同程度及类型的心律失常;随着免疫时间的延长,心律失常的发生频率和发生时间都有不同程度的增加;同时长期主动及被动免疫均可导致大鼠心电图Q-T间期的延长;
3.抗β1-AR-ECⅡ抗体长期被动免疫可导致心肌细胞静息电位的减小和动作电位时程的延长;抗β1-AR-ECⅡ抗体长期作用可导致ICa-L电流的增强,Ik1、Ito及INa-Ca电流的减弱;抗β1-AR-ECⅡ抗体长期作用可导致心肌细胞内游离钙离子水平升高。
结论:
1.抗β1-AR-ECⅡ抗体长期存在可导致大鼠心肌重构及心功能障碍的发生,这可能与心肌细胞中游离钙离子水平升高导致CaMKⅡδ的表达增加,进而诱导心肌细胞凋亡增加有关。
2.抗β1-AR-ECⅡ抗体可导致心律失常的发生,其机制可能与细胞膜静息电位减小,Ik1、Ito及INa-Ca电流的减弱和ICa-L电流的增强导致的胞内钙离子水平升高和动作电位时程延长有关。
Ⅱ.抗β1-AR-ECⅡ抗体长期存在可导致大鼠肝脏损伤
目的
证实抗β1-AR-ECⅡ抗体长期存在是否会对大鼠肝脏的结构和功能产生影响。
方法
(1)建立主动及被动免疫模型,用ELISA法检测大鼠血清中抗β1-AR-ECⅡ抗体水平的变化;
(2)用HE和Masson三色染色,观察抗β1-AR-ECⅡ抗体长期存在对肝脏结构的影响;
(3)通过检测反映肝功能的血清学指标,来证实抗β1-AR-ECⅡ抗体长期存在对肝功能的影响。
结果
1.免疫模型建立过程中抗β1-AR-ECⅡ抗体水平的变化
1.1主动免疫模型建立过程中血清中抗β1-AR-ECⅡ抗体水平的变化
用β1-AR-ECⅡ肽段主动免疫过程中,血清中抗β1-AR-ECⅡ抗体呈现一个自然产生和衰退的过程(见Fig.22)。在免疫4周时,血清中抗β1-AR-ECⅡ抗体开始升高,在免疫8周时达高峰,用ELISA法检测血清抗体的OD值为0.65±0.11,远高于对照组的0.10±0.03(P<0.01);此后血清中抗β1-AR-ECⅡ抗体的水平开始逐渐下降,免疫28周时,抗β1-AR-ECⅡ抗体的OD值为0.15±0.03,仍高于对照组的0.11±0.04(P<0.01)。同时免疫过程中采集的所有血清进行了抗β2-AR-ECⅡ抗体的筛查,未发现抗β2-AR-ECⅡ抗体阳性的血清标本。
1.2被动免疫模型建立过程中血清中抗β1-AR-ECⅡ抗体水平的变化结果同第一部分。
以上提示:主动及被动免疫过程中,血清中均可产生高水平的抗β1AR-ECⅡ抗体。
2.β1-AR-ECⅡ抗原肽段主动免疫可导致肝脏损伤
2.1体视学指标
在主动免疫过程中,大鼠的肝脏出现了不同程度、肉眼可分辨的肝缘变钝现象(见Fig.23B)。本实验共免疫大鼠112只,其中β1-AR-ECⅡ肽段免疫组和免疫佐剂组分别为64和48只,免疫过程中大鼠不明原因死亡4只;其中β1-AR-ECⅡ肽段免疫组有24只大鼠出现肝缘变钝现象,占到总免疫动物数的37.5%(24/64),出现肝缘变钝的大鼠多集中于免疫20周以后;而免疫佐剂组大鼠在免疫过程中未见明显肉眼可见的肝缘变钝现象。
2.2形态学指标
2.2.1 HE染色:在β1-AR-ECⅡ肽段主动免疫过程末期,有3只大鼠(3/6)肝脏HE染色显示在肝脏边缘有大量脂肪细胞的聚集(见Fig.24D/E/F)。
2.2.2 Masson三色染色:在β1-AR-ECⅡ肽段主动免疫过程中,部分(21/64)大鼠的肝脏Masson三色染色显示在肝窦周围有大量胶原纤维沉积(见Fig.25D/E/F)。
以上结果提示:在主动免疫过程中,部分大鼠出现了肝脏结构的损伤。
2.3血清学指标
2.3.1转氨酶测定
用β1-AR-ECⅡ肽段主动免疫过程大鼠血清学指标显示:大鼠肝功能受到一定损伤。在用β1-AR-ECⅡ肽段主动免疫大鼠4周时,血清中谷草转氨酶(AST)开始升高,8周AST水平达高峰(281.4±68.64 U/L),远高于对照组的84.1±4.10 U/L(P<0.001);此后AST逐渐下降,免疫28周时,AST降为126.3±15.75 U/L,但仍高于对照组的84.6±4.89 U/L(P<0.001,见Fig.26A)。同样,谷丙转氨酶(ALT)也在免疫4周开始升高,8周达高峰(121.7±40.34 U/L),显著高于对照组的42.0±2.95 U/L(P<0.05);此后ALT水平开始下降,该变化一直持续至免疫28周实验结束时(见Fig.26A)。AST/ALT的比值在免疫4周时急剧升高达高峰(3.24±0.42),远高于对照组的1.93±0.076(P<0.01);此后该比值逐渐下降,免疫28周时,该比值降为2.03±0.030,和对照组(1.94±0.043)相比无统计学差异(见Fig.26B)。
2.3.2血清蛋白测定
血清总蛋白在用β1-AR-ECⅡ肽段主动免疫4周时开始降低达57.7±2.33 g/L,低于免疫佐剂对照组的68.5±2.12 g/L(P<0.01),此后总蛋白水平基本稳定于低水平,该变化一直持续至免疫28周时,血清总蛋白水平为58.9±0.88 g/L,低于免疫佐剂对照组的69.5±1.00 g/L(P<0.01)。β1-AR-ECⅡ肽段免疫组大鼠血清中免疫球蛋白水平在实验过程中基本维持相对恒定,波动不明显,和对照组相比无统计学差异。β1-AR-ECⅡ肽段免疫组大鼠血清中白蛋白在免疫的4周开始下降为12.3±1.20 g/L,明显低于对照组的17.1±0.35 g/L(P<0.01);此后白蛋白水平基本维持稳定于该水平至免疫28周时,血清总蛋白水平为13.0±1.15 g/L,低于免疫佐剂对照组的17.1±0.36 g/L(P<0.01,见Fig.26C)。同时白球比(A/G)在免疫4周时开始下降,8周时明显降低为0.28±0.010,明显低于对照组的0.35±0.011(P<0.01),该变化一直持续至免疫28周时的0.29±0.020,仍低于对照组的0.35±0.009(P<0.01,见Fig.26D)。
以上结果提示:在主动免疫过程中,大鼠肝功能也受到不同程度的损伤,证实了我们在前期实验中的推测;但由于该模型无法排除抗原可能的肝脏毒性作用,因此需通过进一步的被动免疫来观察相同指标在不同模型中的变化情况,以此对模型的优劣进行评价。
3.抗β1-AR-ECⅡ抗体被动免疫也可导致肝脏损伤的发生
3.1体视学指标
在抗β1-AR-ECⅡ抗体被动免疫过程中,大鼠的肝脏出现了肉眼可分辨的肝缘变钝现象(见Fig.27B)。本实验共免疫大鼠60只,其中抗β1-AR-ECⅡ抗体免疫组和阴性血清抗体组分别为32和28只,免疫过程中大鼠不明原因死亡3只;其中抗β1-AR-ECⅡ抗体免疫组有8只大鼠出现肝缘变钝现象,占到总免疫动物数的25%(8/32),出现肝缘变钝的大鼠多集中于免疫24周以后;而阴性血清抗体组大鼠在免疫过程中未见明显肉眼可见的肝缘变钝现象。
3.2形态学指标
3.2.1 HE染色:在抗β1-AR-ECⅡ抗体被动免疫晚期,有7只大鼠(7/12)肝脏HE染色显示在肝窦周围有大量淋巴细胞浸润(见Fig.28D/28E/28F)。
3.2.2 Masson三色染色:在抗β1-AR-ECⅡ抗体被动免疫晚期,多数大鼠(5/12)的肝脏Masson三色染色显示肝窦周围有大量胶原纤维的沉积(见Fig.29D/29E/29F)。
以上结果提示:抗β1-AR-ECⅡ抗体长期被动免疫也对大鼠肝脏结构造成一定损伤。
3.3血清学指标:
3.3.1转氨酶测定
用抗β1-AR-ECⅡ抗体被动免疫过程中,大鼠血清学指标显示:大鼠肝功能受到一定损伤。在用抗β1-AR-ECⅡ抗体被动免疫24周时,大鼠血清中谷草转氨酶(AST)明显升高至240.0±111.59 U/L,远高于对照组的81.6±4.43 U/L(P<0.01);此后血清中AST水平逐渐下降,免疫36周时,AST降为169.3±14.03 U/L,但仍高于对照组的82.0±4.87 U/L (P<0.01,见Fig.30A)。同样,谷丙转氨酶(ALT)也在被免24周开始升高至75.0±14.79 U/L,高于对照组的45.0±5.90 U/L(P<0.01);该变化一直持续至免疫36周,免疫组ALT水平高达99.8±7.63 U/L,远高于对照组的46.0±8.90 U/L(P<0.01,见Fig.30A)。AST/ALT的比值在免疫24周时急剧升高达2.86±0.22,远高于对照组的1.96±0.09(P<0.01);免疫36周时,该比值降为1.74±0.22,和对照组相比无统计学差异,但有略微下降的趋势(见Fig.30B)。
3.3.2血清蛋白测定
血清总蛋白在用抗β1-AR-ECⅡ抗体被动免疫12周时略微升高达71.8±0.59 g/L,高于对照组的66±0.85g/L(P<0.05);此后血清总蛋白水平逐步下降,在免疫24周时达58.0±2.80g/L,36周时为56.3±0.95g/L,均显著低于对照组的67.1±0.97g/L(P<0.01)和67.9±1.73g/L(P<0.01,见Fig.30C)。
抗β1-AR-ECⅡ抗体被动免疫过程中,血清中免疫球蛋白的水平基本维持相对恒定,波动不明显,组内比较无统计学差异,和对照组相比亦无统计学差异(见Fig.30C)。
抗β1-AR-ECⅡ抗体被动免疫大鼠过程中,血清中白蛋白在免疫的24周下降为12.5±0.29 g/L,明显低于对照组的17.43±0.29 g/L(P<0.01);36周时白蛋白水平继续降低为10.00±0.48 g/L,明显低于对照组的17.69±0.49 g/L(P<0.01,见Fig.30C)。
同时白球比在抗β1-AR-ECⅡ抗体免疫24周时降低为0.28±0.019,明显低于对照组的0.35±0.01(P<0.01),该变化加剧至免疫36周时的0.22±0.030,明显低于对照组的0.36±0.01(P<0.01,见Fig.30D)。
3.3.3血糖测定
被动免疫过程中大鼠血糖监测发现:在用抗β1-AR-ECⅡ抗体被动免疫24周时,血糖水平升高达高峰24.7±7.42mmol/L,远高于对照组的6.7±0.30 mmol/L(P<0.01);免疫36周时,血糖水平回落至19.5±2.06 mmol/L,仍高于对照组的6.73±0.18 mmol/L(P<0.01,见Fig.31)。
以上结果提示:抗β1-AR-ECⅡ抗体长期被动免疫可导致大鼠肝功能的损伤。同时血糖监测的结果发现,在被动免疫过程中大鼠血糖水平不断升高,这提示我们抗β1-AR-ECⅡ抗体长期存在还有可能对机体的糖代谢产生一定影响,但该方面的具体作用还需进一步研究证实。
小结
1.长期主动及被动免疫均可导致大鼠肝功能的下降;
2.长期主动及被动免疫均可导致部分大鼠肝脏结构的损伤。
结论:抗β1-AR-ECⅡ抗体长期存在可导致肝脏结构和功能的损伤,因此,血清中存在高水平抗β1-AR-ECⅡ抗体时,应密切关注患者肝功能的变化。
Ⅲ.抗β1-AR-ECⅡ抗体长期存在可导致大鼠肾脏损伤
目的
观察抗β1-AR-ECⅡ抗体长期存在能否导致大鼠肾脏结构和功能的损伤。
方法
(1)建立主动及被动免疫模型,用ELISA法检测大鼠血清中抗β1-AR-ECⅡ抗体水平的变化;
(2)用HE和Masson三色染色,观察抗β1-AR-ECⅡ抗体长期存在对肾脏结构的影响;
(3)通过检测反映肾功能的血清学指标,来证实抗β1-AR-ECⅡ抗体长期存在对肾功能的影响。
结果
1.免疫模型建立过程中抗β1-AR-ECⅡ抗体水平的变化结果同第二部分。
2.抗β1-AR-ECⅡ抗体长期作用可导致肾脏损伤
2.1形态学指标
2.1.1 HE染色:在主动免疫末期,HE染色显示β1-AR-ECⅡ肽段免疫组大鼠(13/16)在肾小球及肾血管周围出现大量淋巴细胞浸润(见Fig.32D、32E、32F),免疫佐剂组未见明显改变。
2.1.2 Masson三色染色:在抗原肽段主动免疫末期,β1-AR-ECⅡ肽段免疫组大鼠(10/16)大鼠肾小管周围出现大量胶原纤维的沉积(见Fig.33D、33E、33F)。以上结果提示:用β1-AR-ECⅡ肽段长期主动免疫可导致大鼠一定程度肾脏结构的损伤。
2.2血清学指标:
2.2.1血清尿素氮(Blood urea nitrogen,BUN)测定
在用β1-AR-ECⅡ肽段主动免疫过程中,血清BUN水平呈现升高趋势(见Fig.34A)。血清中BUN水平在免疫4周开始升高为8.9±0.95 mmol/L,高于对照组的6.7±0.35mmol/L(P<0.05);此后,BUN水平开始下降,8周时为7.74±0.58 mmol/L,此后BUN一直维持在该水平,至28周时为8.05±0.25 mmol/L,和对照组(6.90±0.33 mmol/L)相比有统计学意义(P<0.05)。
2.2.2血清肌酐(Creatinine,CR)测定
在用β1-AR-ECⅡ肽段主动免疫过程中,血清CR水平呈现升高趋势(见Fig.34B)。血清中CR水平在免疫4周开始升高为66.0±2.08 mmol/L,高于对照组的40.9±1.38mmol/L(P<0.01);8周时达高峰为70.0±3.85 mmol/L,此后CR水平略有下降,28周时为59.7±2.73 mmol/L,和对照组(42.7±2.63 mmol/L)相比有统计学意义(P<0.01)。
2.2.3血清尿酸(Uric acid,UA)测定
在用β1-AR-ECⅡ肽段主动免疫过程中,血清UA水平也呈现升高趋势(见Fig.34C)。血清中UA水平在免疫4周开始升高为198.0±6.93 mmol/L,高于对照组的89.5±9.80 mmol/L(P<0.01);8周时达高峰为235.6±16.43 mmol/L,此后CR水平开始下降,28周时为116.3±9.94 mmol/L,和对照组(93.8±11.64 mmol/L)相比无统计学差异(P>0.05)。
2.2.4尿素氮和肌酐比值(BUN/Cr)测定
主动免疫8周时,血清尿素氮和肌酐之比开始明显下降,此时该比值为0.11±0.008,远低于对照组的0.16±0.008(P<0.01)。随后,该比值继续降低,在免疫12周时达到最低值,为0.09±0.006,该比值显著低于对照组水平(0.17±0.004,P<0.01)。此后该比值开始逐渐升高,在免疫20周时升高达0.13±0.008,仍显著低于对照组的0.15±0.006(P<0.05)。免疫28周时,该比值已接近正常水平。(见Fig.34D)
以上结果提示:用β1-AR-ECⅡ抗原肽段长期主动免疫也可导致一定程度肾脏功能的损伤。
3.抗β1-AR-ECⅡ抗体长期被动免疫亦可导致肾脏损伤
3.1形态学指标
3.1.1 HE染色:被动免疫末期,抗β1-AR-ECⅡ抗体免疫组大鼠(5/6)肾小球及肾血管周围出现大量淋巴细胞浸润(见Fig.35D、35E、35F),对照组未见明显肾脏结构改变。
3.1.2 Masson三色染色:被动免疫末期,抗β1-AR-ECⅡ抗体免疫组大鼠(4/6)大量胶原纤维沉积在肾小管周围(见Fig.36D、36E、36F),对照组未见明显胶原纤维沉积现象。以上结果提示:用抗β1-AR-ECⅡ抗体长期被动免疫也可导致大鼠一定程度肾脏结构的损伤。
3.2血清学指标:
3.2.1血清尿素氮(BUN)测定
在用抗β1-AR-ECⅡ抗体被动免疫24周时,血清BUN水平升高为9.
BACKGROUND AND OBJECTIVE
β-Adrenoceptor (β-AR) is the important member of sympathetic nervous system.β-AR plays an important role on regulating the heart function by mediating the physiological effect of catecholamine.β-AR belongs to G protein–coupled receptor family.β-AR includesβ1、β2 andβ3-AR three types by now. They have the same structural features which are composed with 7 transmembrane domains, 3 intracellular loops and 3 extracellular loops. Among them, the distribution ofβ1-AR in heart is predominant.β1-AR can activate adenyl cyclase (AC) and cyclic adenosine monophosphate (cAMP) system by stimulating G protein (Gs) and displaying positive chronotropic, inotropic and dromotropic actions.
In 1987, Wallukat and Wollenberger found the antibodies against the second extracellular loop ofβ1-AR (anti-β1-AR-ECⅡ, Homology of human and rat is 100%) in the sera of patients with idiopathic dilated cardiomyopathy (IDCM). The following studies indicated that the autoantibodies displayed an analogous agonist effect which includes the inhibition of radioligand binding and enhanced signal transduction mediated byβ1-AR. With the further studies, other researches and ours all found that anti-β1-AR-ECⅡnot only existed in the patients with IDCM, but also existed in the patients with Chagasic disease (1994), primarily electrical derangement (1995), hypertensive heart disease and rheumatic heart disease (1999) et al. From the above results, we presumed that there may be a close relationship between anti-β1-AR-ECⅡand many kinds of heart diseases with the changes of cardiac structure and function.
In 2000, we observed the formation of anti-β1-AR-ECⅡin the cardiac remodeling models induced by abdominal aorta contraction and Adriamycin. The results indicated that high positive frequencies of anti-β1-AR-ECⅡwere found in abdominal aorta contraction group (87.5%) and Adriamycin group (79.2%). Moreover, with the development of heart failure, the titers of anti-β1-AR-ECⅡdisplayed an increasing, maintaining and decreasing process which suggested that cardiac remodeling or heart failure induced by different reasons all could induce the formation of anti-β1-AR-ECⅡ. In order to study the influence of anti-β1-AR-ECⅡon the cardiac structure and function, we immunized rats with the synthetic peptides according to the sequences of humanβ1-AR-ECⅡfor 18 months. In the late stage of immunization, analogous changes of dilated cardiomyopathy and reduced heart function were observed which suggested that the changes of cardiac structure could induce the formation of anti-β1-AR-ECⅡwhich could aggravate heart injury in return. At last, aggravated heart injury was displayed.
The phenomenon of cardiomyocytes lose provoked our attention in the process of 18 months active immunization. Cell lose has two types: apoptosis and necrosis in generally. Apoptosis is an active programmed procedure controlled by genes which is easy to be controlled. There is a research demonstrated that blocking the signal pathway of apoptosis could decrease the area of myocardial infarction significantly and improve heart function. In order to make it clear whether apoptosis existed in the process of DCM induced by anti-β1-AR-ECⅡ, we did an experiment in vitro and found that anti-β1-AR-ECⅡcould induce apoptosis of cultured neonatal cardiomyocytes (2002). In 2003, Staudt et al also demonstrated that the apoptosis of cultured adult cardiomyocytes could be induced by anti-β1-AR-ECⅡ. In 2004, we use active immunization model to demonstrate that anti-β1-AR-ECⅡcould induce apoptosis of cardiomyocytes in vivo. The above results all suggested that apoptosis played an important role in the changes of cardiac structure and function induced by anti-β1-AR-ECⅡ. However, the above researches all used the active immunization model in which we could not exclude the possibly toxic role of antigen peptides. Moreover, the exact mechanism of anti-β1-AR-ECⅡinducing apoptosis was not clear yet. Therefore, making it clear was helpful to determine the pathophysiologic significance of anti-β1-AR-ECⅡand was necessary for the clinical treatment.
Some researches demonstrated that anti-β1-AR-ECⅡcould enhance the current of L-type calcium by activating cAMP-PKA pathway. Calcium overload is the important mechanism of apoptosis. In many kinds of cells, calcium can bind with calmodulin (CaM). The compounds of Ca2+/CaM can activate protease depending on Ca2+/CaM. CaMK has four subtypes:Ⅰ,Ⅱ,ⅢandⅣ. Among them, CaMKⅡis a kind of multifunctional protease which is expressed in many important organs. CaMKⅡis composed with four subunits:α,β,γandδ. Among them, the products ofγandδexpress in cardiomyocytes. Up to now, we have found that CaMKⅡδcould induce hypertrophy of cardiomyocytes and also intimately related with apoptosis. So if there is a lot of apoptosis existing in the DCM model induced by anti-β1-AR-ECⅡ, then anti-β1-AR-ECⅡinduce apoptosis by the CaMKⅡδsignal pathway at the dowm stream of calcium?
Cardiac remodeling is an important stage in the process of heart failure which may be the possible reason of reduced cardiac function and arrhythmia. Some researches indicated that high level of anti-β1-AR-ECⅡwas detected in the sera of patients with DCM complicating with ventricular arrhythmia and conduction blockade, moreover there was a close relation between anti-β1-AR-ECⅡand arrhythmia. Other researches indicated that anti-β1-AR-ECⅡcould enhance the current of L-type calcium. Then whether anti-β1-AR-ECⅡcan induce arrhythmia directly need further study. The essential mechanisms of arrhythmia are the changes of bioelectricity of cardiomyocytes. In order to investigate the exact mechanism of anti-β1-AR-ECⅡinducing arrhythmia, we will observe the long-term role of anti-β1-AR-ECⅡon resting potential (RP), action potential duration (APD), L-type Calcium current (ICa-L), transient outside potassium current (Ito), delayed-rectifier potassium current (Ik1) and Na+/Ca2+ exchange current (INa-Ca) in cardiomyocytes. Moreover we will observe the acute role of anti-β1AR-ECⅡon bioelectricity of papillary muscles.
Researches indicated that the distribution ofβ1-AR is mainly on heart and kidney, and slightly on liver. A lot of researches in the past demonstrated that anti-β1AR-ECⅡcould induce the injury of heart by binding withβ1-AR. Different with agonist; anti-β1-AR-ECⅡhad the character of non-desensitization. Then whether anti-β1-AR-ECⅡexisting in the circulation system could bind withβ1-AR in liver and kidney is not clear. Our previous experiment of long-term active immunization of 18 months with synthetic peptides according to humanβ1-AR-ECⅡon rats, the phenomena of a lot of ascitic fluid and blunt liver edge were observed. In the ascitic fluid, high in protein, more cells and a low glucose level were found by us. Moreover, the isolated ascitic fluid coagulated naturally. In view of the ascitic fluid analysis, the ascitic fluid should be determined as exudate but not transudate. But the reason of ascitic fluid formation was not clear. Considering the phenomenon of blunt liver edge, liver injury may be the possible reason, but we could not exclude the kidney injury. So could anti-β1AR-ECⅡalso damage liver and kidney? In our study, we will do some initial research on the direction.
In summary, we will continue the study from two following portions:①observe the changes of intracellular free calcium and CaMKⅡδexpression in the long-term existence of anti-β1-AR-ECⅡ; set up active and passive immunization model and observe whether long-term existence of anti-β1-AR-ECⅡcan induce arrhythmia; moreover analyze the possible mechanism of anti-β1-AR-ECⅡinducing arrhythmia in order to provide the theoretical direction for clinical treatment;②set up active and passive immunization model and observe whether long-term existence of anti-β1-AR-ECⅡcan induce the injury of liver and kidney.
I. Anti-β1-AR-ECⅡCould Result in Cardiac Remodeling and its Possible Mechanism
PART ONE: Long-term Existence of Autoantibodies against the Second Extracellular Loop ofβ1-Adrenoceptor Could Induce the Increase of Calcium/Calmodulin-Dependent Protein KinaseⅡδin Rat Cardiomyocytes
Objective
1. To observe the changes of cardiac structure and function in the process of passive immunization with anti-β1-AR-ECⅡ;
2. To observe the changes of intracellular free calcium and the expression of CaMKⅡδin the process of passive immunization with anti-β1AR-ECⅡ.
Methods
(1) Select healthy adult Wistar rats (180~220g ,body wt, n=60).The Wistar rats used in the present study were obtained from the Animal Center of Shanxi Medical University, P.R. China. Detect anti-β1-AR-ECⅡin the sera by ELISA. The rats with no anti-β1-AR-ECⅡwere divided into the following two groups: (2 rats died for over dose anesthesia; 2 rats died for unknown reasons)
①β1AAb group(β1-AR-ECⅡantibody group, n=32):Purify the IgG from sera obtained from active immunized rats. Then quantity the total IgG by BCA method. Inject the purified IgG into vena caudalis of rats at the dose of 0.7μg/g, boosting for every 2 weeks. The experiment lasts 40 weeks.
②Negative sera group(n=28):Purify the negative sera, then quantity the total IgG by BCA method. Inject the purified IgG into vena caudalis of rats at the dose of 0.7μg/g, boosting for every 2 weeks. The experiment lasts 40 weeks.
(2) Peptides composition: A peptide (HWWRAESDEARRCYNDPKCCDFVTNRA, 197-223 amino acid residues) corresponding to the sequence of the second extracellular loop of humanβ1-AR was synthesized by GL Biochem (Shanghai) Ltd. The purity is 95% (Table. 1). The synthetic peptides were stored at -20℃for use.
(3) Set up passive immunization model by injecting anti-β1-AR-ECⅡinto vena caudalis of rats regularly. And detect the level of anti-β1AR-ECⅡin the sera;
(4) Use anatomic measurements, Masson staining and heart weight to body weight ratio to reflect the changes of cardiac structure in the process of passive immunization;
(5) Monitor Heart rates (HR), left ventricular systolic pressure (LVSP), and Left ventricular diastolic pressure (LVDP) and±dp/dtmax by putting the arterial cannula into left ventricle by right carotid artery;
(6) Detect the changes of apoptosis in the process of immunization by TUNEL and detection of caspase-3, 8 and 9 activity in cardiomyocytes;
(7) detect the intracellular free calcium in single ventricular myocytes at the late stage of passive immunization labeled with Fluo-3-AM by Confocal microscope;
(8) Detect the expression of CaMKⅡδin cardiomyocytes by Western-blot and immunohistochemical method.
Results
1. Anti-β1-AR-ECⅡcould result in cardiac remodeling of structure
1.1 In the process of passive immunization, anti-β1-AR-ECⅡin the sera still maintained a relative permanent level
The optical density (OD) value of anti-β1-AR-ECⅡin the sera detected by ELISA method ranged from 0.09±0.030 before immunization to 0.21±0.029 at the 4th week after immunization which was higher than 0.08±0.040 (P<0.001) in negative sera group. At the 4th week after immunization, anti-β1-AR-ECⅡkept the high level till to the end of experiment (Fig.1). In the whole immunization process, the level of anti-β1-AR-ECⅡin the sera didn’t fluctuate too much. There was no significant difference among different time points of active immunization (P>0.05).
1.2 Long-term existence of anti-β1-AR-ECⅡcould result in cardiac remodeling
1.2.1 The changes of the ratio of heart weight to body weight in the passive immunization
The ratio of heart weight to body weight (HW/BW) can reflect whether cardiac remodeling took place. HW/BW began to decrease significantly to 2.10±0.09 at the 24th week after passive immunization which was lower than 2.72±0.04 (P<0.01) in the negative sera group. At the 36th week, the ratio decreased to 2.35±0.05 inβ1AAb group which was still lower than 2.68±0.05 (P<0.01) in the negative sera group (Fig.2C).
1.2.2 Anatomic index
At the end stage of passive immunization, some pathological changes such as enlarged chamber heart and thinning heart wall could be observed (Fig.2B).
1.2.3 Masson’s trichromic staining
At the 36th week after passive immunization, a lot of collagen fibers depositing in the stroma of heart could be observed by Masson’s t staining (Fig.3).
1.3 Long-term existence of anti-β1-AR-ECⅡcould result in the descending of heart function
With the time of passive immunization, heart function cut down gradually (Fig.4). In the whole immunization, heart rates didn’t change obviously. Only at the 36th week of immunization, heart rates decreased to 311±20.3 times/min which was lower than 360±23.0 times/min (P<0.05) in negative sera group (Fig.4A). +dp/dtmax significantly descended to 213±31.1 Kpa/s at the 24th week of immunization which was lower than 510±29.0 Kpa/s (P<0.01) in negative sera group. At the 36th week, it was 289±39.6 Kpa/s inβ1AAb group which was lower than 505±30.0 Kpa/s (P<0.01) in negative sera group (Fig.4B). -dp/dtmax began to decrease obviously at the 24th week of immunization. At that time, -dp/dtmax was -233±34.7 Kpa/s which was lower than -415±31.0 Kpa/s (P<0.01) in negative sera group. At the 36th week, -dp/dtmax was -283±29.6 Kpa/s which was also lower than -421±29.0 Kpa/s (P<0.01) in negative sera group (Fig.4C). There was no significant difference of LVSP and LVDP betweenβ1AAb group and negative sera group.
Above results suggested that the long-tem passive immunization with anti-β1-AR-ECⅡcould result in the formation of cardiac remodeling and decreased heart function. But the possible mechanisms of these changes were not clear which need further study.
2. The possible mechanisms of cardiac remodeling induced by anti-β1-AR-ECⅡ
2.1 Long-term existence of anti-β1-AR-ECⅡcould induce the increase of apoptosis in cardiac myocytes
2.1.1 Long-term existence of anti-β1-AR-ECⅡcould induce the increase of caspase-3,8 and 9 activities of cardiomyocytes
In the passive immunization, fluorescent quantitation was used to detect the activity of caspase3, 8 and 9. Use AFC standard to make the standard curve (Fig.5). The slope rate of the straight line was 0.998 by fitting every point. The best incubation time of caspase3, 8 and 9 was determined in Fig.6. When incubate sample and fluorescence substrate for 30min, the fluorescence intensity of negative control was near to the peak, whereas the fluorescence intensity of samples were in low level. When incubating them for 60min, the fluorescence intensity of negative control was near to the bottom of the curve, whereas the fluorescence intensity of samples was increasing. Incubating 90min, the fluorescence intensity of negative control was still at the bottom, whereas the fluorescence intensity of samples was still increasing to a high level. Incubating 120min, the fluorescence intensity of negative control began to increase, whereas the fluorescence intensity of samples was still increasing. According to the standard, we select the time point with high fluorescence intensity of sample and low fluorescence intensity of negative control. So the incubation time was determined at 90min.
In the process of passive immunization, the activity of caspase-3, 8 and 9 increased with the time of immunization (Fig.7). The activity of caspase-8 began to increase at the 16th week. At that time, the activity of caspase-8 was 13.41±6.14 pmol/h/mg which was higher than 3.45±0.97 pmol/h/mg (P<0.01) in the negative sera group. After that, the activity of caspase-8 kept increasing. At the 36th week, it reached the peak (34.1±10.09 pmol/h/mg) which was significantly higher than 4.15±1.09 pmol/h/mg (P<0.01) in the negative sera group (Fig.7A). Caspase-9 had the same variation regularity. At the 16th week, it increased to 7.47±0.94 pmol/h/mg which was higher than 1.27±0.18 pmol/h/mg (P<0.01) in the negative sera group. It kept rising. At the 36th week, it reached the peak 16.24±3.31 pmol/h/mg which was higher than 1.38±0.37 pmol/h/mg (P<0.01) in the negative sera group (Fig.7B). The variation regularity of caspase-3 was the same. At the 16th week, it increased to 18.84±7.23 pmol/h/mg which was significantly higher than 1.49±0.18 pmol/h/mg (P<0.01) in the negative sera group. At the 36th week, it reached the peak (25.06±6.80 pmol/h/mg) which was higher than 1.83±0.60 pmol/h/mg (P<0.01) in the negative sera group (Fig.7C).
2.1.2 Anti-β1-AR-ECⅡexisting for a long time in the sera could induce the increase of apoptosis in cardiomyocytes
At the 36th week after immunization, there was about 7.86±0.43% cardiomyocytes took place apoptosis which was higher than 0.86±0.29 % (P<0.01) in the negative sera group at the same time (Fig.8G). The representative figures were shown in Fig.8A-F. Among them, Fig.8A-C belonged to negative sera group; Fig.8D-F belonged toβ1AAb group.
The above results suggested that anti-β1-AR-ECⅡcould increase apoptosis in cardiomyocytes, but the mechanism of augmented apoptosis need further study.
2.2 Anti-β1-AR-ECⅡexisting for a long time in the sera could induce the increase of intracellular free calcium in rats’ventricular myocytes
At the end stage of passive immunization, the intracellular free calcium in rats’ventricular myocytes was detected by confocal microscope. The data showed that the level of intracellular free calcium was 456.34±35.47 in anti-β1-AR-ECⅡgroup which was significantly higher than 51.96±1.18 (P<0.001) in negative sera group (Fig.9).
The above results suggested that long-term role of anti-β1-AR-ECⅡcould result in the increase of intracellular free calcium. But how the elevated calcium resulted in apoptosis was not clear. In another word, the downstream signal pathway of calcium inducing apoptosis need further study.
2.3 Long-term existence of anti-β1-AR-ECⅡcould result in the increasing expression of CaMKⅡδin cardiomyocytes
2.3.1 Western blot result
In the passive immunization with anti-β1-AR-ECⅡ, the expression of CaMKⅡδincreased with the time (Fig.10). At the 16th week after immunization, the expression of CaMKⅡδincreased to 1.19±0.38 which was higher than 0.39±0.22 (P<0.05) in the negative sera group. After that, the expression of CaMKⅡδcontinued to increase. At the 36th week after immunization, it reached the peak (1.94±0.77) which was higher than 0.46±0.27 (P<0.05) in the negative sera group.
2.3.2 Immunohistochemical result
In the process of passive immunization, the expression of CaMKⅡδincreased gradually. At the 36th week, the expression of CaMKⅡδsignificantly increased which was higher than that in the negative sera group (Fig.11).
The above results suggested that the elevated expression of CaMKⅡδin cardiomyocytes may be related with the increased apoptosis of cardiomyocytes in the passive immunization.
Summary
1. Long-term existence of anti-β1-AR-ECⅡcould result in cardiac remodeling;
2. Heart function decreased gradually in the long-term passive immunization with anti-β1-AR-ECⅡ;
3. Long-term existence of anti-β1-AR-ECⅡcould result in the increase of apoptosis;
4. Long-term existence of anti-β1-AR-ECⅡcould result in the increase of intracellular free calcium;
5. Long-term existence of anti-β1-AR-ECⅡcould result in the increasing expression of CaMKⅡδin cardiomyocytes.
PART TWO: The Autoantibodies against the Second Extracellular Loop ofβ1-Adrenoceptor Could Induce Arrhythmia in vivo and in vitro and the possibly Underlying Mechanism
Objective
1. To observe whether anti-β1-AR-ECⅡcould induce arrhythmia directly;
2. On papillary muscle level and cell level, using microelectrode recording AP and patch clamp to study the possible mechanism of anti-β1-AR-ECⅡinducing arrhythmia which is helpful for the clinical treatment of patients with high level of anti-β1-AR-ECⅡin the sera.
Methods
(1) Select healthy adult Wistar rats (180~220g, body wt,n=172).The Wistar rats used in the present study were obtained from the Animal Center of Shanxi Medical University, P.R. China. Detect anti-β1-AR-ECⅡin the sera by ELISA. The rats with no anti-β1-AR-ECⅡwere divided into the following four groups: (5 rats died for over dose anesthesia; 4 rats died in nubibus reason)
①β1AR group(n=64):inject the mixture of antigen peptides and immunoadjuvant into the dermis of back at the dose of 0.4μg/g, boosting for every 2 weeks. The experiment lasts 40 weeks.
②Vehicle group(n=48):inject the mixture of saline and immunoadjuvant into the dermis of back boosting for every 2 weeks. The experiment lasts 40 weeks.
③β1AAb group(n=32):Purify the IgG from sera obtained from active immunized rats. Then quantity the total IgG by BCA method. Inject the purified IgG into vena caudalis of rats at the dose of 0.7μg/g boosting for every 2 weeks. The experiment lasts 40 weeks.
④Negative sera group(n=28):Purify the negative sera, then quantity the total IgG by BCA method. Inject the purified IgG into vena caudalis of rats at the dose of 0.7μg/g, boosting for every 2 weeks. The experiment lasts 40 weeks.
(2) Observe whether arrhythmia can be induced by anti-β1-AR-ECⅡby the measurement of cardiac function and in vivo;
(3) Observe whether triggered activity can be induced by adding anti-β1-AR-ECⅡto the isolated papillary muscle of guinea pig and analyze the changes of action potential duration (APD) ;
(4) Observe whether arrhythmia can be induced by long-term existence of anti-β1-AR-ECⅡin the active and passive immunization model and analyze the changes of QT interval;
(5) Analyze the possible mechanisms of anti-β1-AR-ECⅡinducing arrhythmia by patch clamp and fluorescent assay of intracellular calcium.
Results
1. Anti-β1-AR-ECⅡcould result in cardiac remodeling of electricity
1.1 Anti-β1-AR-ECⅡcould induce arrhythmia on normal rats
1.1.1 Anti-β1-AR-ECⅡcould directly induce arrhythmia on normal rats in vivo
68μM anti-β1-AR-ECⅡcould induce arrhythmia in 66.7% (6/9) normal rats (weight: 180-220g). The percent was higher than 12.5% (1/8) in negative sera group and 11.1% (1/9) in saline group. Premature ventricular contraction (PVC) was the major type of arrhythmia (Fig.12A). We also could see considerable premature supraventricular contraction (PSVC, Fig.12B). After injecting anti-β1-AR-ECⅡinto artery, we observed the frequency of arrhythmia per hour. We find that the frequency of arrhythmia is 16±10.28 times/hour in anti-β1AR-ECⅡgroup which was higher than 4±1.00 times/hour (P<0.01) in saline group and 5±0.82 times/hour (P<0.01) in the negative sera group (Fig.12C).
1.1.2 Anti-β1-AR-ECⅡcould induce triggered activity on the papillary muscles of guinea pigs
Adding 0.1μM anti-β1-AR-ECⅡto the papillary muscle of guinea pig, almost all isolated papillary muscles took place triggered activity inducing by train (Fig.13B). Moreover its role didn’t attenuate with time (the longest observation time is 2 hours). Its role was similar to Isoprenaline (ISO, 1μM, Fig.13A). But the papillary muscle displayed more sensitive in anti-β1-AR-ECⅡgroup than ISO. The triggered activity induced by anti-β1-AR-ECⅡand ISO could be blocked by metoprolol (MET) which was a selectiveβ1-AR antagonist (Fig.13C). Moreover, anti-β1-AR-ECⅡcould prolong the APD of papillary muscle of guinea pig to 355.5±32.98 ms which was higher than 306±18ms (P<0.05) in saline group and 277.2±27.30 ms (P<0.01) in negative sera group. MET could block the role of anti-β1-AR-ECⅡprolonging APD (P<0.001, Fig.13D).
1.1.3 Anti-β1-AR-ECⅡcould induce after-depolarization and triggered activity on the papillary muscles of rats
Adding 1μM anti-β1-AR-ECⅡto the papillary muscles of rats, most isolated papillary muscles (60%, 9/15) took place triggered activity inducing by train (Fig.14A). EAD could be observed on some other papillary muscles (46.7%, 7/15, Fig.14B). Moreover DAD could be observed on some papillary muscles (26.7%, 4/15, Fig.14C). EAD, DAD and triggered activity could not be observed in ISO (1μM) group. MET (10μM) could inhibit EAD, DAD and triggered activity induced by anti-β1-AR-ECⅡ(Fig.14D).
The above results suggested that anti-β1-AR-ECⅡcould induce arrhythmia by inducing triggered activity and after depolarization on normal cardiomyocytes. Because anti-β1-AR-ECⅡexited in our body for a long time, so what were the exact effects of anti-β1-AR-ECⅡon electrocardio-activity need further study.
1.2 Long-term existence of anti-β1-AR-ECⅡcould induce arrhythmia
1.2.1 In the process of immunization with peptides according to the sequence ofβ1-AR-ECⅡ, different kinds and degrees arrhythmia could be observed in rats; moreover Q-T interval was prolonged obviously
1.2.1.1 In the process of active immunization, the level of anti-β1-AR-ECⅡshowed a naturally generated and extinctive process
At the 4th week after first immunization, the level of anti-β1-AR-ECⅡwas 0.4±0.21 which was higher than 0.10±0.05 (P<0.05) in the vehicle group. After that, the level of anti-β1AR-ECⅡincreased more and more. At the 8th week, it reached the peak. The value was 2.10±0.18 which was higher than 0.15±0.09 in the vehicle group (P<0.001). After that, anti-β1-AR-ECⅡdecreased. And at the end of the experiment, the value of anti-β1-AR-ECⅡwas 0.73±0.21 which was higher than 0.15±0.10 in the vehicle group (P<0.01, Fig.22).
1.2.1.2 with the immunization time, the frequency of arrhythmia increased
At the 4th、8th、12th、16th、20th、24th、28th、32nd week after the first immunization, the ratio of arrhythmia number to total rats number was 0/6、1/7、1/8、1/6、0/6、3/3、6/7、8/8 respectively. The type of arrhythmia we observed includes premature ventricular contraction (PVC, Fig.15A), premature supraventricular contraction (PSVC, Fig.15B), ventricular tachycardia (VF, Fig.15C), and atrial ventricular block (A-V block) et al. Among them, PVC and PSVC were common. At the 28th week, the frequency of arrhythmia per hour was 220±25.17 (times/hour) which was higher than 10±0.65 (times/hour, P<0.01) in the vehicle group. At the 32th week, the frequency of arrhythmia per hour was1509±40.88 (times/hour) which was higher than 15±0.75 (times/hour, P<0.001) in the vehicle group. At the different time point of immunization, the frequency of PVC, PSVC and VF could be seen on Fig. 15D-F.
1.2.1.3 In the process of active immunization, Q-T interval was prolonged
At the 12th week of immunization, the results of ECG showed that Q-T interval was 65.0±0.56ms which was longer than 55.5±0.86ms (P<0.05) in the vehicle group. At the 28th week of immunization, Q-T interval was 84.0±2.42 ms which was longer than 58.9±0.95 ms (P<0.05) in the vehicle group (Fig.16C). Because considering the influence of heart rates, QTc was calculated by formula Bazett. At the 12th week of immunization, QTc was 0.36±0.002 which was longer than 0.34±0.007 (P<0.05) in the vehicle group. At the 28th week of immunization, QTc was 0.46±0.007 which was longer than 0.32±0.009 (P<0.05) in the vehicle group (Fig.16D).
The above results suggested that different kinds and degrees arrhythmia could be induced and Q-T interval was prolonged in the process of active immunization. However we could not exclude the possible role of antigen on cardiac electricity. Therefore we need to do further research in order to find the exact reason of arrhythmia, antibody or antigen?
1.2.2 Long-term passive immunization with anti-β1-AR-ECⅡcould induce arrhythmia
1.2.2.1 In the process of passive immunization, anti-β1-AR-ECⅡin the sera still maintained a relative permanent level
The optical density (O.D.) value of anti-β1-AR-ECⅡin the sera detected by ELISA method ranged from 0.09±0.030 before immunization to 0.21±0.029 at the 4th week after immunization which was higher than 0.08±0.040 (P<0.001) in negative sera group. At the 4th week after immunization, anti-β1-AR-ECⅡkept the high level till to the end of experiment (Fig.1). In the whole immunization process, the level of anti-β1AR-ECⅡin the sera didn’t fluctuate too much. There was no significant difference among different time points of passive immunization (P>0.05).
The above results suggested that anti-β1-AR-ECⅡcould be produced in the sera in the passive immunization model; moreover the level of antibody was relative lower than that in active immunization model. But the level of anti-β1-AR-ECⅡin the passive immunization model was near to that in patients. Comparing the changes of anti-β1-AR-ECⅡin the sera, we found that the fluctuation of anti-β1-AR-ECⅡin the passive immunization model was slender.
1.2.2.2 Different kinds of arrhythmia could be observed in the process of passive immunization
Representative changes of ECG were recorded in Fig. 17A-F. Fig.17A: normal ECG at the 36th week after immunization in anti-β1-AR-ECⅡgroup. Fig.17B-F: different kinds of arrhythmia were observed in the same rat. Among all kinds of arrhythmia, ventricular arrhythmia was predominant. Some supraventricular arrhythmia also could be observed. Moreover there was some arrhythmia which type of arrhythmia couldn’t be determined. The time of arrhythmia occurrence localized after the 24th week of immunization.
1.2.2.3 Long-term existence of anti-β1-AR-ECⅡcould prolong the persistence time of arrhythmia
In the process of passive immunization with anti-β1-AR-ECⅡ, the data of ECG showed that there was a prolongatus tendency of the persistence time of arrhythmia with the time of passive immunization (Fig.17G). The persistence time of arrhythmia at the 24th week after immunization was 90±34.6 s in anti-β1-AR-ECⅡgroup which was higher than 0 s (P<0.01) in the negative sera group; at the 36th week after immunization, the persistence time of arrhythmia was 170±124.2 s in anti-β1-AR-ECⅡgroup which was higher than 0 s (P<0.01) in the negative sera group.
1.2.2.4 Anti-β1-AR-ECⅡexisting for a long time in the sera could induce the prolongation of Q-T interval
Q-T interval prolongation occurred at different time points of passive immunization. Representative Q-T intervals were recorded in Fig.18A and 18B. Fig.18A: ECG at the 36th week after immunization in the negative sera group; Fig.18B: ECG at the 36th weeks after passive immunization in anti-β1AR-ECⅡgroup. Considering the influence of heart rates, QTc was calculated by formula Bazett. At the 24th week of immunization, QTc was 0.46±0.015 in anti-β1-AR-ECⅡgroup (n=8) which was longer than 0.32±0.019 (P<0.01) in the vehicle group. At the 36th week of immunization, QTc was 0.51±0.015 in anti-β1-AR-ECⅡgroup (n=8) which was longer than 0.33±0.018 (P<0.01) in the vehicle group (n=6, Fig.18C).
The above results suggested that different kinds and degrees of arrhythmia could be induced and prolongation of Q-T interval could be observed in the process of passive immunization which was coincident with the findings in active immunization model. The phenomenon indicated that anti-β1-AR-ECⅡwas the main cause of arrhythmia. But how it induced arrhythmia or the exact mechanisms of arrhythmia induced by anti-β1-AR-ECⅡneed further study.
2. The possible mechanisms of anti-β1-AR-ECⅡinducing cardiac remodeling of electricity
2.1 Long-term effects of anti-β1-AR-ECⅡon AP waveforms and RMP in rat ventricular myocytes at the 36th week after passive immunization
Anti-β1-AR-ECⅡexisting for a long time in the sera could induce the attenuation of RMP and prolongation of APD in rats’ventricular myocytes. Anti-β1-AR-ECⅡgroup: immunize rats with antibody againstβ1-AR-ECⅡ(n=6); negative sera group: immunize rats with IgG from negative sera (n=6). Representative APs and RMP were recorded in the negative sera group (black traces) and anti-β1-AR-ECⅡgroup (dash line) in Fig.19. RMP was attenuated to -62.37±0.69 mv in anti-β1-AR-ECⅡgroup which was lower than -81.27±0.91 mv in the negative sera group (P<0.05). Moreover APD was prolonged to 51.09±2.66 ms in anti-β1-AR-ECⅡgroup which was longer than 33.26±2.58 ms in the negative sera group (P<0.05, Table 2).
2.2 Anti-β1-AR-ECⅡexisting for a long time in th
引文
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5. Chiale P.A., Rosenbaum M. B.,Elizari M. V., Hjalmarson A, Magnusson Y, Wallukat G, Hoebeke J. High Prevalence of Antibodies Against Beta 1- and Betaz-Adrenoceptors in Patients With Primary Electrical Cardiac Abnormalities. J Am Coll Cardiol. 1995; 26:864-9.
6. Christ T, Schindelhauer S, Wettwer E, Wallukat G, Ravens U. Interaction between autoantibodies against the beta1-adrenoceptor and isoprenaline in enhancing L-type Ca2+ current in rat ventricular myocytes. J Mol Cell Cardiol.2006; 41:716-23.
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