【作者】 王丽；

【导师】 刘慧荣；

【作者基本信息】 山西医科大学 ， 生理学， 2014， 博士

【摘要】 研究背景心血管疾病,特别是心力衰竭已经成为影响人类健康和威胁生命的主要因素。尽管针对目前已知原因所采取的治疗有很大的进展,但心力衰竭的死亡率仍居高不下,提示心力衰竭的发生发展过程中还有一些未知因素参与。因此,进一步探讨其致病因素是有效降低心力衰竭死亡率的关键所在。由于心肌细胞的反复丢失是心力衰竭发生发展的重要因素,所以,对心肌细胞丢失机制的研究具有十分重要的意义。目前认为,造成心肌细胞丢失的方式中,程序性细胞死亡,即凋亡和自噬(I型和II型程序性细胞死亡)由于具有可调控性,因而成为研究心肌损伤机制的主要目标。但是,导致心肌细胞程序性死亡的机制中,还有许多不清楚之处。抗p1-肾上腺素受体自身抗体(β1-AA)是新近发现的一种存在于许多类型心血管疾病患者血清中的自身抗体,能够与β1-肾上腺素受体(β1-AR)细胞外第二环抗原肽段结合,发挥类受体激动剂样的作用。临床研究发现,采用免疫吸附技术去除心衰患者血液中的β1-AA可以大大改善心衰患者的心功能,提示β1一AA与心衰的发生发展密切相关。我们的前期研究发现,心衰可以诱导β1-AA的产生,后者反过来可以导致大鼠心功能衰竭。即,β1-AA与心力衰竭互为因果关系。对β1-AA致病机制的研究发现,β1-AA的存在可以引起成鼠心肌细胞死亡。我们和他人在近几年的研究中均发现,β1-AA可以诱导心肌细胞凋亡,但是单纯抑制细胞凋亡并不能完全逆转β1-AA所引起的心肌细胞的死亡,提示在β1-AA诱导的心肌细胞死亡的过程中还有其他因素的参与。自噬作为一种广泛存在的正常生理过程,又是细胞对不良环境的一种防御机制,对于维持细胞稳态发挥着重要的作用。在心脏中,心肌细胞自噬对于维持心脏功能和活力具有重要的作用。自噬的异常可以导致各种心脏疾病,但是导致心肌细胞自噬异常的机制还不十分清楚。有研究表明,β肾上腺素受体激动剂异丙肾上腺素可以引起自噬水平的下降。但是,β1-AA这种类β肾上腺素受体激动剂样的自身抗体,是否也会对心肌细胞的自噬产生影响并不清楚。如果有影响,那么与细胞凋亡相比,自噬一这种维持稳态的重要机制在β1-AA引起的心功能下降及心肌细胞死亡过程中的意义又有多大,值得我们深入探讨。因此,本研究的研究目的是：(1)观察β1-AA对于心肌组织自噬水平的影响,并进一步探讨这种影响在β1-AA诱导的心功能下降中的作用；(2)观察β1-AA诱导心肌细胞自噬水平变化的时间规律并进一步与细胞凋亡对比,明确自噬在β1-AA引起的心肌细胞死亡中的意义；(3) β1-AA引起心肌细胞自噬水平下降的受体机制。第一部分心肌组织自噬水平下降在β1-AA引起心功能降低中的作用研究目的本部分实验旨在通过建立β1-AA主动免疫大鼠模型,观察β1-AA对于心肌自噬水平的影响以及自噬的变化在β1-AA引起的心功能下降过程中的作用。材料和方法1.实验动物选用由山西医科大学实验动物中心提供的8周龄,体重180-200g的健康雄性Wistar大鼠。2.实验方法2.1抗原肽段的合成委托吉尔生化上海有限公司,根据人β1肾上腺素受体细胞外第二环的氨基酸序列(β1-AR-ECⅡ,197aa-223aa,人鼠同源性为100%)合成人β1-AR-ECⅡ肽段作为免疫用抗原肽段,纯度>95%。2.2建立主动免疫大鼠模型健康雄性Wistar大鼠(8周龄)作为免疫对象,随机分组：(1)免疫组：β1-AR-ECⅡ抗原肽段主动免疫大鼠(2)溶剂对照组即伪免疫组各组均于每次免疫前一天鼠尾静脉采血,分离血清,用链酶亲合素一酶联免疫吸附实验(SA-ELISA法)检测p1-AA的产生情况,在免疫后不同时间点(4周,8周,12周)处理动物,留取血清备用,并取心室肌组织。2.3检测指标2.3.1大鼠在体心功能检测(颈总动脉插管)2.3.2心肌组织中LC3II和Beclin1的蛋白含量测定(Western blot)2.3.3心肌组织中LC3和Beclin1的mRNA含量测定(Real-time PCR)2.3.4大鼠心肌组织LC3和Beclin1蛋白的原位表达(冰冻切片免疫荧光染色)2.3.5线粒体膜电位测定(99mTc-MIBI心肌显像和JC-1染色)2.3.6成年大鼠心肌细胞的分离培养及线粒体膜电位检测(JC-1染色)结果1.p1-AA的长期存在可以引起大鼠在体心功能下降1.1主动免疫大鼠模型建立成功在主动免疫2周时,与伪免疫组相比,免疫组大鼠血清中β1-AA的OD值已明显升高,并且随着免疫时间的延长逐渐升高,到第6周时免疫组大鼠血清中β1-AA的OD值达到2.18+0.27,并且一直保持较高水平,直至实验结束。1.2β1-AA的长期存在可以引起大鼠在体心功能下在主动免疫12周时,与同期伪免疫组相比,免疫组大鼠的左心室收缩压(LVSP)、左心室压力上升的最大速率(+dp/dtmax)和左心室压力下降的最大速率(-dp/dtmax)明显下降,左心室舒张末期压(LVEDP)明显升高。以上结果提示：在主动免疫12周时,免疫组大鼠在体心功能出现了明显下降。1.3β1-AR-ECⅡ主动免疫大鼠可导致心肌线粒体膜电位选用核素99mTc-MIBI心肌显像和JC-1染色两种方法来反映心肌线粒体膜电位的变化。核素99mTc-MIBI心肌显像方法选用心／纵膈比值(H/M ratio)来表示99mTc-MIBI摄取率,以此来反映心肌线粒体膜电位的变化。结果发现,与伪免疫组相比,在主动免疫大鼠4周时,免疫组大鼠99mTc-MIBI’心/纵膈比值明显降低。同时选用JC-1荧光染色来反映心肌细胞线粒体膜电位变化。结果显示,与伪免疫组相比,免疫组大鼠心肌组织红/绿荧光比值明显降低。为了进一步验证这一现象,我们分离伪免疫组和免疫组大鼠的成鼠心肌细胞,再给予JC-1染色,选用SpectraMax(?) M2e多功能酶标仪进行荧光定量。结果显示,与伪免疫组相比,免疫组大鼠心肌细胞的红/绿荧光比值明显降低,以上结果均提示,β1-AA的长期存在可以引起心肌线粒体膜电位下降。2.β1-AA的长期存在可以引起心肌组织自噬水平降低LC3II是自噬形成的标志蛋白,是目前较为公认的自噬标记物。而Beclin1是参与自噬调控的重要基因,其水平可反映机体自噬水平的变化。我们的研究结果显示,β1-AR-ECⅡ主动免疫大鼠4周时心肌组织LC3和Beclin1蛋白水平和mRNA水平均下降。3. β1-AA引起的心肌自噬水平下降参与了心功能下降的过程3.1给予雷帕霉素可以使β1-AA免疫组大鼠心肌组织自噬水平增加雷帕霉素(RAPA)可以上调心肌组织LC3II和Beclin1的蛋白水平以及mRNA水平。3.2雷帕霉素上调自噬可以逆转免疫组大鼠心功能的下降本实验选用雷帕霉素上调心肌组织自噬水平,再观察免疫组大鼠心功能的变化情况。结果显示,在主动免疫12周时,与同期免疫组相比,免疫+RAPA组大鼠的心功能明显好转,表现为LVSP、+dp/dtmax和-dp/dtmax的下降均明显恢复、LVEDP的升高出现明显下降。以上结果提示：自噬水平的变化可以对免疫组大鼠的心功能产生影响。4.β1-AA引起的心肌自噬水平下降参与了心肌线粒体膜电位的下降使用RAPA上调心肌组织自噬水平,采用核素99mTc-MIBI心肌显像方法和JC-1染色来分析主动免疫大鼠心肌线粒体膜电位水平。结果显示,与免疫组相比,免疫+RAPA组大鼠的线粒体膜电位明显恢复。以上结果提示,上调自噬可以恢复β1-AA长期存在引起的心肌线粒体膜电位的下降。小结一1.β1-AA长期存在可以引起心肌组织自噬水平的降低；2.β1-AA诱导的心肌自噬水平的降低可以通过影响线粒体膜电位进而参与心功能下降的过程。第二部分β1-AA诱导心肌细胞自噬水平变化的时间规律及其在心肌细胞死亡中的意义研究目的本部分实验选用H9c2细胞来模拟心肌细胞,旨在观察β1-AA导致自噬水平变化的时间规律；通过与β1-AA导致心肌细胞凋亡的时间规律及凋亡在心肌细胞死亡中的作用进行对比分析,进一步阐明自噬在β1-AA诱导的心肌细胞死亡中的意义。材料与方法1.细胞株H9c2(2-1)细胞株：购于中国科学院上海细胞库。2.实验方法2.1亲和层析法提纯β1-AA阳性或阴性血清IgGs利用IgGs亲和纯化试剂盒对主动免疫后β1-AA阳性组以及伪免疫组的大鼠血清分别进行提纯,并用SDS-PAGE凝胶电泳检测提纯的抗体纯度,然后进行BCA蛋白定量。2.2乳鼠心肌细胞分离和培养选取1-3天龄Wistar大鼠的乳鼠心脏,剪成组织块,用II型胶原酶加胰蛋白酶联合消化法分离细胞,再通过差速贴壁法去除成纤维细胞,获取乳鼠心肌细胞,48h后换液,待细胞生长到90%融合时给予不同处理,观察乳鼠心肌细胞跳动频率。2.3H9c2(2-1)心肌细胞培养选用DMEM高糖培养基+10%胎牛血清+青链霉素混合液并置于37℃,5%C02,95%空气的培养箱中进行培养,待细胞密度达70-80%即可进行传代。2.4构建Beclin1过表达载体以及RNA干扰载体,转染H9c2心肌细胞构建Beclin1过表达质粒pcDNA3.1-Beclin1,并将其转染H9c2细胞,以上调细胞自噬水平；构建Beclin1RNA干扰质粒Beclin1-shRNA转染H9c2细胞,以抑制细胞自噬水平。2.5检测指标2.5.1检测细胞存活率(细胞计数Kit-8法)2.5.2乳酸脱氢酶(LDH)活性测定(ELISA)2.5.3H9c2心肌细胞凋亡情况检测(Annexin V-FITC/PI双染流式细胞术分析和Caspase-3活性测定)2.5.4H9c2心肌细胞线粒体膜电位的测定(JC-1染色流式细胞仪分析)2.5.5H9c2心肌细胞LC311和Beclin1的蛋白表达及mRNA含量测定(Western blot和Real-time PCR)结果1.从主动免疫大鼠血清中成功提纯β1-AA1.1β1-AA蛋自定性及定量检测亲和柱纯化后的p1-AA经SDS-PAGE凝胶电泳检测纯度,结果有两条带出现,分别是IgG重链(55KD)和轻链(25KD),这两条条带与IgG标准品相一致,表明纯化效果理想。纯化后的抗体经BCA试剂盒测定：蛋白浓度为8mg/mL。1.2提纯的β1-AA可以使乳鼠心肌细胞跳动频率增加将提纯的p1-AA加入培养的乳鼠心肌细胞,结果显示：p1-AA刺激30min使乳鼠心肌细胞跳动频率明显增加,这一结果提示从大鼠血清中提纯的p1-AA具有生物活性。2.β1-AA可以引起心肌细胞死亡2.1β1-AA使H9c2心肌细胞的存活率降低在本实验中通过CCK8法检测细胞的存活率。结果显示,与对照组相比,细胞存活率在β1-AA刺激12小时出现了明显的下降,36小时下降最明显,48小时细胞存活率恢复。2.2β1-AA使H9c2心肌细胞培养上清中乳酸脱氢酶活力增加用提纯的p1-AA干预H9c2心肌细胞,结果显示,与对照组相比,漏出的乳酸脱氢酶(LDH)活力在6小时即明显升高并且这种活力的增加延续到24h,在36h降低。以上结果提示p1-AA可以直接引起H9c2心肌细胞损伤。3.抑制细胞凋亡可以部分逆转β1-AA引起的心肌细胞死亡3.1β1-AA可以引起H9c2心肌细胞凋亡增加用β1-AA在不同的时间点刺激H9c2心肌细胞,通过Annexin V/PI双染流式细胞术以及检测Caspase-3活性来反映心肌细胞凋亡情况。流式细胞分析结果显示：β1-AA刺激6h时细胞凋亡明显增加,12h仍高于对照组,24h恢复正常。Caspase-3活性检测显示出类似的结果。由此可见,β1-AA可以引起H9c2心肌细胞凋亡增加,并且这种凋亡的增加出现在β1-AA刺激6h。在细胞凋亡早期,其线粒体跨膜电位的降低是一种不可逆事件。用JC-1染色流式细胞分析发现β1-AA的刺激可以引起线粒体膜电位下降的细胞数增多,进一步印证在p1-AA刺激6h时可以引起心肌细胞凋亡。3：2细胞凋亡的抑制可以部分逆转β1-AA透导的心肌细胞存活率的下用Caspase的广谱抑制剂Z-VAD-FEK预处理H9c2心肌细胞,可使β1-AA诱导的Caspase-3活性增加明显降低；并可部分逆转p1-AA诱导的细胞存活率下降,这一结果提示细胞凋亡的抑制仅可以部分减少β1-AA诱导的心肌细胞的死亡。4.自噬水平的下降是β1-AA引起心肌细胞死亡的重要原因4.1H9c2心肌细胞株受到β1-AA刺激以后自噬水平将β1-AA作用于H9c2细胞不同时间,结果显示：(1) β1-AA干预12h后心肌细胞的LC3Ⅱ蛋白表达和mRNA水平明显降低,24h继续下降至干预后36h降至最低,干预后48h恢复至正常水平；(2) β1-AA干预H9c2细胞24h后心肌细胞的Beclin1蛋白表达下降,36h降至最低,48h恢复至正常水平。而Beclin1的mRNA水平在p1-AA干预12h即明显下降,36小时降至最低,之后逐渐恢复。4.2构Beclin1过表达质粒cDNA3.1-Beclin1染H9c2细胞后自噬水平上调Beclinl过表达质粒pcDNA3.1-Beclin1构建成功以后,将pcDNA3.1-Beclin1转染H9c2细胞,与未转染细胞相比,LC3和Beclin1mRNA含量及蛋白水平均升高；而空质粒pcDNA3.1(-)对细胞LC3和Beclin1的mRNA水平和蛋白水平基本无影响。4.3构建Beclin1RNA王扰质粒Beclin1-shRNA转染H9c2细胞后自噬水平下调将构建成功的Beclin1-shRNA转染H9c2细胞后胞浆呈现绿色荧光,并且转染细胞的LC3和Beclin1mRNA含量及蛋白水平均降低,而空质粒Control-shRNA对LC3和Beclin1的mRNA水平和蛋白水平无明显影响。4.4自噬水平的降低是β1-AA引起心肌细胞死亡的重要原因用p1-AA刺激H9c2心肌细胞,在36小时可以使心肌细胞的存活率降至最低,因此本研究选用36小时作为p1-AA处理的时间点。用Beclin1过表达质粒上调自噬水平,再给予p1-AA刺激36小时,结果显示,自噬水平上调的H9c2细胞存活率有所恢复,并且明显高于凋亡抑制对于p1-AA引起的心肌细胞存活的恢复。而自噬水平被抑制的H9c2细胞存活率进一步降低。以上结果提示p1-AA引起的心肌细胞自噬水平的降低参与了心肌细胞死亡的过程。4.5β1-AA作用王心肌细胞后,其自噬水平呈现先升高后降低的变化趋势为了进一步验证p1-AA究竟是直接抑制自噬还是由于自噬的耗竭而导致的自噬水平的降低,我们将p1-AA的刺激时间提前,在刺激30min,1h,3h,6h以及后续的12h分别收集H9c2细胞。结果显示,细胞自噬水平在早期有一个增高的趋势,其蛋白水平在β1-AA处理细胞30min时即出现明显升高,这种增高持续到3h,至6h恢复正常,刺激12h开始出现下降。LC3的mRNA水平也呈现类似的变化趋势。4.6β1-AA刺激早期引起的细胞自噬水平的升高有利于心肌细胞存活为了证明p1-AA刺激早期细胞自噬水平升高对于心肌细胞死亡的影响,我们选用RNA干扰技术干扰H9c2心肌细胞的Beclin1基因从而抑制自噬水平。另外,由于自噬水平的增高现象在6小时基本恢复,为了排除p1-AA刺激后期自噬水平本身的降低对于细胞存活率的影响,我们选择6小时作为观察时间点。结果发现,在β1-AA刺激早期,抑制细胞自噬可以明显降低心肌细胞的存活率。小结二在p1-AA引起心肌细胞死亡的过程中,自噬水平升高是先于细胞凋亡出现的应激性保护反应,而后期自噬水平的降低则有可能是p1-AA引起心肌细胞死亡的重要原因。第三部分β1-AA引起自噬水平下降的p-肾上腺素受体机制探讨研究目的本部分实验着重从β-肾上腺素受体的角度探讨p1-AA引起心肌细胞自噬变化的受体机制。材料和方法1.细胞株H9c2(2-1)细胞株和HEK293细胞株：购于中国科学院上海细胞库。2.实验方法2.1HEK293细胞的培养此种细胞选用含10%胎牛血清的DMEM高糖培养基培养,细胞冻存复苏以及传代培养同H9c2心肌细胞。2.2构建β1-AR过表达质粒转染HEK293细胞构建β1-AR(ADRB1)过表达质粒pEGFP-Nl-ADRB1转染HEK293细胞,使HEK293细胞表面表达β1-AR。2.3检测指标2.3.1H9c2心肌细胞和HEK293细胞的LC3Ⅱ、Beclin1和β1-AR的蛋白含量检测(Western blot)2.3.2H9c2心肌细胞和HEK293细胞的LC3Ⅱ、Beclinl和p1-AR的mRNA含量测定(Real-time PCR)2.3.3β1-AR和β2-AR的原位表达(免疫荧光染色)2.3.4检测细胞的cAMP水平(ELISA)2.3.5β1-AR的内吞检测(激光共聚焦显微镜)结果1.β1-AA可以通过β1-AR信号通路引起心肌细胞自噬水平下降1.1β1-AA阻断割阿替洛尔可以部分阻断β1-引起的自噬水平下将p1-AA作用于H9c2细胞36h以后,LC3II和Beclin1的蛋白表达下降；如果给予p1-AR阻断剂阿替洛尔(Atenolol)预处理,可以部分逆转p1-AA引起的LC3II和Beclin1的蛋白表达下降。此外,LC3和Beclin1的mRNA水平也表现出类似的趋势。以上结果提示,β1-AR可能在p1-AA引起的自噬水平降低过程中发挥着作用。1.2PK抑制剂H-89可以部分逆转β1-AA引起的自噬水平蛋白激酶A(PKA)是β1-AR活化后的主要信号蛋白。我们选用PKA抑制剂H-89预处理H9c2心肌细胞,再给予β1-AA干预,结果发现H-89可以部分逆转p1-AA引起的自噬水平的降低。以上结果提示,β1-AA可以通过βi-AR/PKA通路引起心肌细胞自噬水平下降。2.β2-AR可能参与了β1-AA引起的心肌细胞自噬水平下降的过程2.1β2-AA阻断剂ICI118551可以部分阻断β1-AA引起的心肌细胞自噬水平将β2-AR阻断剂IC1118551预处理H9c2心肌细胞,结果发现,与p1-AA组相比,LC3II和Beclin1的蛋白表达水平及mRNA水平均部分恢复。这一结果提示,β2-AR可能也参与了β1-AA诱导的心肌细胞自噬水平的降低。2.2以HEK293细胞为模型,进一步证实β1-AR和β2-AR在β1-AA引起的自噬水平降低过程中发挥的作用选择胞膜没有p1-AR表达,有β2-AR表达的HEK293细胞作为细胞模型,并构建p1-AR过表达质粒pEGFP-N1-ADRB1转染HEK293细胞。选用cAMP作为p1-AR功能鉴定的指标。结果显示,β1-AA干预后,转染细胞的cAMP水平明显增高。以上结果提示转染的β1-AR有生物活性。将细胞进一步分组：HEK293细胞对照组(Vehicle), HEK293细胞+p1-AA组(AA),转染细胞对照组(A+Vehicle),转染细胞+p1-AA组(A+-AA),转染细胞+β1-AA+ICI118551(A+-AA-ICI),转染细胞+单纯ICI118551(A+-ICI)。结果显示,与对照组相比,单纯给予未转染的HEK293细胞p1-AA的刺激并没有引起LC3mRNA水平的变化；与A+Vehicle相比,p1-AA刺激β1-AR过表达的细胞,可以引起自噬水平的明显下降,提示p1-AR在p1-AA诱导的自噬水平降低过程中发挥作用。同时,给转染细胞加用β2-AR阻断剂ICI118551以及β1-AA处理,结果显示,与A+-AA组相比,加入阻断剂可以使自噬水平部分恢复,提示p2-AR在p1-AA诱导的自噬水平降低过程中同样发挥着作用。3.β1-AR的内吞特性参与p1-AA引起的自噬水平下降3.1异西肾上腺素可以引起H9c2心肌细胞自噬水平下降,并且很快恢：将β-AR激动剂异丙肾上腺素(ISO)加入培养的H9c2心肌细胞,结果显示,ISO引起的心肌细胞LC3mRNA水平的下降在刺激6h即发生,12h恢复正常。而β1-AA则可以长时间引起H9c2心肌细胞自噬水平的降低,即存在不脱敏的特性。3.2与异丙肾上腺素相比,β1-AA的刺激使β1-AR内吞减弱将p1-AA加入培养的H9c2心肌细胞,结果发现,作用0mmin时,细胞上的p1-AR大部分位于细胞膜上；作用10min时,细胞膜上的β1-AR有很少一部分内吞入细胞膜；作用30min时,进入细胞内的p1-AR增多,提示p1-AA作为p1-AR的类激动剂,可以导致p1-AR的内吞。同ISO作用相比,β1-AA引起的内吞明显减少。以上结果提示p1-AR内吞程度的减弱可能参与了β1-AA致β1-AR不脱敏的机制。小结三β1-AA可能通过使p1-AR内吞减弱,持续激活后续经典的cAMP/PKA通路,进而引起心肌细胞自噬水平下降。同时,β2-AR可能也参与了p1-AA诱导的心肌细胞自噬水平下降的过程。更多还原

【Abstract】 BackgroundCardiovascular diseases, especially heart failure have become a major factor affecting human health. Despite advances in cardiac dysfunction treatment, mortality remains high, which suggest that there are still some unknown factors involved in the development of heart failure. Therefore, further exploration of its etiology is the key to reducing the mortality of cardiac dysfunction effectively. In recent years, it has emerged that loss of myocardial cells is an important factor in the development of heart failure and therefore the study of the mechanism of myocardial cell loss has a very important significance.Loss of cardiomyocytes through programmed cell death (apoptosis and autophagy) has become the major mechanism of myocardial injury. However, there is much uncertainty concerning the mechanisms of programmed cardiomyocytes death. In recent years, anti-β1-adrenoceptor autoantibodies (β1-AAs) have been shown to be prevalently distributed in the sera of patients with kinds of cardiovascular diseases. Binding of the specific autoantibody with the second extracellular loop of β1-adrenergic receptor (β1-AR-ECⅡ) resulted in agonist-like effects. Complete elimination of cardiodepressant autoantibodies by immunoadsorption in patients could greatly improve cardiac function of patients with heart failure. Our research found that cardiac function deterioration could induce the generation of β1-AA which could lead to rat myocardial dysfunction conversely. In other words, there is a causal relationship between β1-AA and heart failure.Studies have shown that β1-AA could lead to cell death in adult rat ventricular myocytes. Our and others research have discovered that β1-AA could induce cardiomyocyte apoptosis. Caspase inhibitor Z-VAD-FEK was used to inhibit apoptosis and discovered that inhibition of apoptosis could not completely reverse cardiomyocyte death that induced by β1-AA, which suggested that there were still other factors involved in the process of cell death. As another way of programmed cell death, autophagy plays an important role in maintaining cell homeostasis. Some reports have shown that autophagy plays an important role in maintaining cardiac function and cardiac activity. Another research showed that P-adrenergic agonist isoproterenol could induce the decline of autophagy. However, whether β1-AA which resembles β-adrenergic receptor agonists has effects on autophagy in cardiomyocytes or not? If it does, compared with apoptosis, what is the contribution of autophagy to the changes in the decreased cardiac function and the death of cardiomyocytes?Therefore, the aims of the present study:(1) to explore the influence of β1-AA on the level of cardiomyocyte autophagy, and further investigate the role of this effect in cardiac function deterioration induced by β1-AA.(2) To observe the time course of autophagy induced by β1-AA and demonstrate the significance of autophagy in cardiomyocyte death caused by β1-AA compares with apoptosis.(3) The receptor mechanism for the decreased autophagy in cardiomyocytes induced by β1-AA.Section1The role of decreased autophagy in the decline of cardiac function induced by anti-β1-adrenoceptor autoantibodiesObjectiveThe active immunization rat models were established by injection of Pi-AR-ECⅡ to identify the effects of β1-AA on cardiac autophagy and to determine whether autophagy was involved in the decreased heart function.Materials and methods1. Experimental animalsHealthy male Wistar rats (weighing180g-200g, the Experimental Animal Center of Shanxi Medical University).2. Methods2.1PeptideApeptide corresponding to amino acids197-223of human β1-AR-ECⅡ (197aa-223aa) was synthesised by GL Biochem Ltd (Shanghai, China)(purity was greater than95%).2.2The establishment of active immunization rat modelsThe rats in the β1-AA group were injected with this antigen emulsified in complete Freund’s adjuvant (CFA,0.4μg/g, subcutaneous injection) posteriorly along the back at multiple sites for the first time. The rats in the Vehicle group were injected with1ml of normal saline (1mg/ml) mixed with1ml of Freund’s adjuvant in the same manner as described above. Blood samples were collected1day before booster injection to test the generation of βi-AA after immunization. The experiment lasts12weeks.2.3Detection of indicators2.3.1The detection of rat cardiac function in vivo2.3.2Detection of the myocardial tissue LC3Ⅱ, Beclin1protein level by Western blot2.3.3Determination of the myocardial tissue LC3Ⅱ, Beclin1mRNA level by Real-time PCR2.3.4Immunofluorescence staining of frozen tissue sections from the myocardial tissue2.3.5The detection of mitochondrial membrane potential by99mTc-MIBI myocardial imaging and JC-1staining2.3.6The detection of mitochondrial membrane potential by JC-1staining in cultured adult rat cardiomyocytesResults1. Cardiac function was decreased with the existence of β1-AA1.1Pi-AA-positive rat models were establishedIn both experimental groups, the OD values of β1-AA in the sera before treatment were at a very low level. However, these were markedly increased in the β1-AA group after two weeks of immunization. Moreover, the serum levels remained relatively high until the end of the experiment.1.2Cardiac function was decreased with the existence of β1-AAAt the12th week after immunization, the left ventricular systolic and diastolic functions, expressed by LVSP,+dP/dtmax and LVEDP,-dP/dtmax, decreased significantly in the β1-AA group compared with the Vehicle group. These results indicated that cardiac function was declined as a result of immunization.1.3The△ψm in rat cardiac myocytes declined with the existence of Pi-AAMyocardial radionuclide imaging technology and JC-1staining were executed to evaluate the alteration in△ψm.Myocardium uptake of99m Tc-MIBI, expressed by the H/M ratio [regions of interest positioned over the heart (H) and upper mediastinum (M)], can be used to reflect the△ψm in cardiac myocytes. The H/M ratio declined at the4th week after immunization in βi-AA group versus the Vehicle group. Meanwhile, JC-1staining was used to detect the△ψm. Frozen tissue sections from the myocardial tissue were stained by JC-1and showed that the red/green ratio decreased in the β1-AA group. To further confirm the result, at the4th week after immunization, ventricular cardiomyocytes were isolated and stained with JC-1. After adding JC-1, the intensity of the red fluorescence of JC-1aggregates was significantly weaker and the green fluorescence of JC-1monomers was stronger in the Pi-AA group compared with the Vehicle group. These results demonstrated that the△ψm was declined with the existence of β1-AA.2. Cardiac autophagy was declined with the existence of β1-AALC3and Beclin1, which have been used as molecular markers of autophagic activity, were measured in the present study. Both protein and mRNA levels of LC3and Beclin1were significantly decreased at the4th week in the β1-AA group compared with the Vehicle group.3. Decreased myocardial autophagy contributes to the decline in cardiac function with the existence of β1-AA3.1Rapamycin (RAPA) could increase the level of autophagy in cardiac tissue during long-term presence of β1-AARAPA could increase both protein and mRNA levels of LC3and Beclin1.3.2Autophagy induced by RAPA could improve cardiac function in active immunization of rats with Pi-AAThe results showed that pretreatment with RAPA significantly increased LVSP、+dp/dtmax和-dp/dtmax and decreased LVEDP at the12th week in the β1-AA+RAPA group compared with the β1-AA group and demonstrated that β1-AA-induced autophagic changes may have an effect on heart function of the immune rats.4. Decreased myocardial autophagy contributes to the decline in△ψm with the existence of Pi-AAPretreatment with RAPA significantly increased the myocardium uptake of99mTc-MIBI and the red/green ratio of JC-1staining. The results demonstrated that the induction of autophagy could improve△ψm in cardiac myocytes due to the existence of β1-AA.Summary1. Cardiac autophagy was decreased during long-term presence of β1-AA;2. The decreased cardiomyocyte autophagy induced by β1-AA is a critical contributor to cardiac mitochondrial damage and the followed cardiac dysfunction.Section2The time course of β1-AA-induced autophagy and its significance in myocardial cell deathObjectiveIn this experiment, the time course of autophagy induced by Pi-AA was detected in H9c2cells. And the study was to clarify the significance of autophagy that was induced by Pi-AA in cardiac cell death.Materials and methods1. Cell linesH9c2(2-1) cell lines were purchased from the Shanghai Cell Bank, Chinese Academy of Sciences.2. Methods2.1Acquirement of β1-AA positive or negative IgGsExtracting and purifying IgGs by MAb Trap Kit. The purity of the antibody solution was detected by SDS-polyacrylamide gel electrophoresis.2.2Culture of neonatal cardiomyocytesThe hearts were removed aseptically from1-to3-day-old Wistar rats. Single cells were dissociated from the minced ventricles with a solution of collagenase type II and trypsin. Then, fibroblasts were removed by differential adhesion method and get neonatal rat cardiomyocytes. When cells tend to grow to fuse with each other, and the changes in beating frequency were measured after the addition of the tested agents.2.3Culture of H9c2cells H9c2cells were cultured with high glucose DMEM medium+10%FBS+penicillin-streptomycin mixture and placed in37℃,5%CO2,95%air incubator until the density of cells reached70to80%can be passaged.2.4The Beclin1overexpression vector and RNA interference vector were structured, and H9c2cells were transfected to upregulate or inhibit the level of autophagy2.5Determination of indicators2.5.1Cell survival was detected by Cell Counting Kit-8(CCK-8)2.5.2LDH activity was measured2.5.3Apoptosis of H9c2cells was detected by Annexin V-FITC/PI double staining flow cytometry analysis and Caspase-3Activity Assay2.5.4Mitochondrial membrane potential of H9c2cells was measured by JC-1staining followed by flow cytometry analysis2.5.5Determination of the expression of LC3and Beclin1in H9c2cells by western blot and real-time PCRResults1. Preparation of β1-AA from the sera of active immunized rats1.1Quantitative and qualitative of β1-AAThe results showed that two straps of55KD and25KD appeared which represented one heavy chain and one light chain respectively and the pattern of purified IgG was virtually identical to commercial IgG The concentration of purified IgG was8mg/mL.1.2Purified β1-AA had a positive chronotropic effect on cultured neonatal rat cardiomyocytesThe result showed that β1-AA markedly increased cardiomyocyte contraction frequency after30min of exposure, which suggested that purified β1-AA had biological activity.2. β1-AA could cause myocardial cell death2.1β1-AA could reduce cell viability in H9c2cellsCompared with the vehicle group, cell viability decreased significantly after cultured12hours with β1-AA and the viability decreased to the lowest after cultured36h. After48h with Pi-AA, cell viability was recovery.2.2Pi-AA could increase the activity of LDH in the supernatant of cultured H9c2cellsCompared with the vehicle group, the activity of LDH was significantly increased which was cultured6hours with β1-AA until24h, and the level of LDH was recovery after36h with Pi-AA. The results suggested that β1-AA could directly injury the H9c2cells.3. Inhibition of apoptosis only partially improved β1-AA-induced myocardial cell death3.1Pi-AA could increase apoptosis in H9c2cellsH9c2cells were stimulated with β1-AA at different points, and Annexin V/PI double staining and Caspase-3activity were measured to reflect apoptosis. Flow cytometry analysis showed that apoptosis significantly increased at6h. The level of apoptosis still increased at12h with β1-AA and the level of apoptosis was recovered at24h. Caspase-3activity assay showed similar results. Thus,β1-AA could cause apoptosis in H9c2cells after6hours of culture with β1-AA.It was an irreversible event that mitochondrial transmembrane potential decreased at the early stage of apoptosis. JC-1staining followed flow cytometry analysis showed that β1-AA could lead to decreased mitochondrial membrane potential. And the result further confirmed that β1-AA could cause apoptosis in H9c2cells after6hours of culture with β1-AA.3.2Inhibition of apoptosis could partially restore the cell viability which was reduced by Pi-AAThe Caspase-3activity was decreased and the cell viability was recovered partially when the H9c2cells were pretreated with Z-VAD-FEK, which suggested that inhibition of apoptosis may only partially reduce cell death induced by β1-AA.4. Decreased autophagy was an important cause of β1-AA-induced myocardial cell death4.1The levels of autophagy reduced after stimulation by β1-AA in H9c2myocardial cellsThe result of autophagy levels at different time courses in H9c2cells stimulated by β1-AA shown:the protein expression and mRNA levels of LC3II obviously decreased at12h after Pi-AA intervention in H9c2cells, and at36h after intervention reached its minimum which restored to normal at48h.Beclin1protein expression decreased at24h after β1-AA intervention in H9c2cells, and at36h after intervention reached its minimum which restored to normal at48h. Nevertheless, the mRNA levels of Beclin1significantly reduced at12h after β1-AA intervention, and at36h after intervention reached the minimum which recovered gradually.4.2After pcDNA3.1-Beclin1construction and transfection successfully, the level of autophagy in H9c2cells was increasedBeclin1over-expression plasmid (pcDNA3.1-Beclin1) was constructed successfully and then transfected to H9c2cells. After pcDNA3.1-Beclin1transfection in H9c2cells, the mRNA content and protein levels of LC3and Beclin1obviously increased. But the mRNA content and protein levels of LC3and Beclin1had no significant change after control-pcDNA3.1treatment.4.3After Beclin1siRNA construction and transfection successfully, the levels of autophagy reduced in H9c2cellsBeclin1RNA interference plasmid (Beclin1-siRNA) was constructed successfully and the cytoplasm of transfected cells showed green fluorescence indicated that the transfection was successful. After Beclin1siRNA transfection in H9c2cells, the mRNA content and protein levels of LC3and Beclin1obviously decreased. However, the mRNA content and protein levels of LC3and Beclin1had no significant change after control-siRNA treatment.4.4Decreased autophagy was an important cause of β1-AA-induced myocardial cell deathIn the study,36hours was chosen as the time point of Pi-AA treatment, due to H9c2cell viability reached to the minimum after36h β1-AA stimulation.Firstly autophagy was up-regulated by Beclin1over-expression plasmid, and then H9c2cell viability could be restored after36h β1-AA stimulation. Moreover, the effect of Beclin1over-expression plasmid was significantly stronger than apoptosis inhibitor. H9c2cell viability further decreased when autophagy was inhibited. The above results suggested that decreased cardiomyocyte autophagy induced by β1-AA was involved in the process of myocardial cell death.4.5Autophagy increased in the early stage of β1-AA stimulation and then decreasedTo further verify whether reduced autophagy caused directly by β1-AA or depletion of autophagy, we collected cells after30min,1h,3h,6h and12h β1-AA treatment. The results found that the level of autophagy had an increasing trend in the early. The autophagic protein levels were significantly increased after30min Pi-AA treatment, and it was still increased at3h after stimulation. At6h after treatment the autophagic protein levels returned to normal, while at12h after stimulation it began to decline. LC3mRNA levels also showed a similar trend. 4.6Elevated autophagy was good for the survival of cardiomyocyte in the early stage of Pi-AA stimulationTo prove the effect of elevated autophagy in the early stage of Pi-AA stimulation on myocardial cell death, autophagy was inhibited by interfering with Beclin1gene expression. In addition, increased autophagy recovered6h after β1-AA stimulation. In order to exclude the impact of reduced autophagy in the late stage of β1-AA stimulation on cell viability, we chose6h after β1-AA stimulation as the observation point. The results showed that inhibiting autophagy could obviously decrease myocardial cell viability in the early stage of β1-AA stimulation.SummaryIn the process of β1-AA-induced myocardial cell death, increased autophagy was protective stress response prior to apoptosis in the early stage, while decreased autophagy may be an important cause of β1-AA-induced myocardial cell death in the late stage.Section3β-adrenoceptor mechanism of decreased autophagy induced by β1-AAObjectiveThis part of the experiment focuses on the mechanism of changes in autophagy induced by β1-AA from the perspective of receptorMaterials and Methods1. Cell linesH9c2(2-1) cell lines and HEK293cell lines:purchased from the Shanghai Cell Bank, Chinese Academy of Sciences.2. Methods2.1Culture of HEK293cells2.2Establishment of HEK293cells transfected with plasmid pEGFP-Nl-ADRBl that overexpressed Pi-AR2.3Determination of indicators2.3.1Detection of the protein levels of LC3Ⅱ、Beclin1and β1-AR in H9c2myocardial cells and HEK293cells by Western blot2.3.2Determination of mRNA expressions for LC3, Beclin1and β1-AR in H9c2myocardial cells and HEK293cells by Real-time PCR2.3.3Immunofluorescence staining of β1-AR and P2-AR2.3.4Detection of cellular cAMP level by ELISA2.3.5Detection of β1adrenergic receptor endocytosisResults1. β1-AA could decrease the levels of autophagy through Pi-AR signaling pathway1.1Declined autophagy induced by β1-AA can be reversed partially.by the β1-AR antagonist atenololThe result showed that the protein expression of LC3II and Beclin1reduced at36h after β1-AA intervention in H9c2cells, and pretreated with Pi-AR antagonist atenolol recovered the protein expression of LC3II and Beclin1partially. mRNA levels for LC3II and Beclin1displayed a similar trend. These results suggested that Pi-AR may play a role in decreased autophagy induced by β1-AA.1.2Decreased autophagy induced by Pi-AA can be recovered partially by PKA inhibitor H-89Cardiomyocytes was pretreated with PKA inhibitor H-89, and the result showed that decreased autophagy induced by β1-AA can be reversed by H-89partially. These result revealed that β1-AA could reduce autophagy of cardiomyocytes through β1-AR/PKA signaling pathway.2. β1-AR contributed to the decreased autophagy induced by β1-AA2.β2-AR antagonist ICI118551could reverse the level of autophagy induced by β1-AA partiallyThe results displayed that after pretreatment of H9c2cardiomyocytes with β2-AR antagonist ICI118551, protein expression and mRNA levels of LC3Ⅱ and Beclin1recovered partially compared with β1-AA group. These results demonstrated that β2-AR may also be involved in declined autophagy induced by β1-AA.2.2To clarify the role of β1-AR and β2-AR during the process of decreased autophagy induced by Pi-AA in HEK293cell model Pi-AR transfected HEK293cells as positive control, Real time PCR and immunofluorescence staining were used to determine that β1-AR are not concentrated on HEK293cell surface membranes but β2-AR are expressed on HEK293cell surface. β1-AR over-expression plasmid (pEGFP-Nl-ADRB1) was constructed successfully and then transfected to HEK293cells. cAMP was chosen as an indicator. When β1-AR-transfected cells were interfered with β1-AA, cAMP level in transfected cells increased significantly and the results suggested that the transfected Pi-AR is bioactive.Then, the cells were further grouped:HEK293cell control group (Vehicle), HEK293cell+Pi-AA group (AA), transfected cell group (A+Vehicle), transfected cell+β1-AA group (A+-AA), transfected cell+β1-AA+ICⅡ18551group (A+-AA-ICI), transfected cell+ICⅡ18551group (A+-ICI). The result showed that LC3mRNA level was not changed by giving β1-AA to non-transfected HEK293cells compared with Vehicle group. Compared with A+Vehicle group, the levels of autophagy was obviously declined by giving Pi-AA to overexpression of β1-AR, which suggested that Pi-AR took effects in process of decreasing autophagy levels induced by β1-AA. Simultaneously, transfected cells were pretreated with β2-AR antagonist ICI118551and β1-AA and the result showed that, the level of autophagy restored compared with A+-AA group, which suggested that P2-AR play a role in the process of declined autophagy induced by Pi-AA.3. Endocytosis characteristics of Pi-AR contributes to the change in autophagy induced by Pi-AA3.1Isoproterenol (ISO) could decrease the level of autophagy in H9c2cells but restored soonThe result showed that LC3mRNA levels of cardiomyocytes declined with the existence of ISO at6h and restored to normal at12h.3.2Compared with ISO, the endocytosis of β1-AR is weakened by stimulation of β1-AAThe result showed that0minute after the exposure to β1-AA, most of β1-AR located at the cell membrane and a few β1-AR at the cell membrane got inside after10minutes. After30minutes, more β1-AR got into cytoplasm which suggested that β1-AA as β1-AR agonist could lead to the endocytosis of β1-AR. The endocytosis of Pi-AR induced by β1-AA was obviously decreased compared with ISO, which suggested that declined endocytosis of β1-AR might participate in the mechanism of β1-AR non-desensitization induced by Pi-AA.Summary β1-AA could lead to the decline in the level of autophagy by weakening endocytosis of β1-AR and sustained activation of classic cAMP/PKA signaling pathway. Simultaneously, β2-AR also may takes part in the process of decreased autophagy in cardiomyocytes induced by β1-AA.更多还原