【作者】 倪黎；

【导师】 汪道文；

【作者基本信息】 华中科技大学 ， 内科学， 2009， 博士

【摘要】 内质网（endoplasmic reticulum，ER）是真核细胞Ca²⁺储存、脂类合成、蛋白折叠和修饰的重要细胞器，任何影响内质网功能的因素诱发的内质网内非折叠蛋白蓄积（unfolded protein response，UPR）都能导致内质网应激（ER stress）。内质网应激是应激后细胞内的最初反应，是线粒体氧化应激等细胞应激反应的共同初始通路。内质网应激是细胞一种保护性反应，用以减少非折叠蛋白的蓄积，恢复内质网正常功能；但若应激持续，促生存机制向促凋亡途径转变，细胞则发生凋亡。有研究报道，内质网应激反应与众多导致心力衰竭的心血管疾病如动脉粥样硬化、心肌缺血和扩张型心肌病等密切相关，但其具体的作用机制仍了解很少。本研究对心力衰竭中内质网应激反应的具体激活情况、传导途径进行了探索，我们明确了心力衰竭中内质网应激反应被激活，内质网应激诱导的凋亡通路启动，证实并推测了内质网应激在心力衰竭中的激活诱因、传导途径和效应机制。首先，我们用Western免疫印迹检测发现，在晚期心力衰竭行心脏移植术患者的心脏组织中，存在GRP78激活、PERK-eIF2α通路激活和JNKs通路激活的现象，表明在人类慢性心力衰竭中，内质网应激反应激活。然后，我们用两种模拟人类心肌肥大心力衰竭的大鼠模型，压力负荷（腹主动脉缩窄，AAC）与β₁肾上腺素受体持久兴奋（异丙肾上腺素（isoproterenol，Iso）注射）模型，来研究心力衰竭中内质网应激反应的具体途径；并给予选择性β₁受体阻滞剂美托洛尔（metoprolol）干预。通过心脏形态学、病理学、血流动力学检测，发现压力负荷与β₁肾上腺素受体持久兴奋诱导大鼠心肌肥大、左室功能受损，模型制作成功。用Western免疫印迹、免疫组织化学方法和TUNEL凋亡检测发现，心肌肥大心力衰竭大鼠心脏中，内质网分子伴侣GRP78被激活，负责内质网蛋白质控的KDEL受体分子表达大量增加，内质网应激反应IRE1-XBP1途径激活，内质网特异性诱导凋亡的转录因子CHOP表达增加，细胞凋亡增多；用选择性β₁受体阻滞剂美托洛尔干预后，部分抑制了上述途径，心肌凋亡减少，心肌肥大减轻，心功能改善。因此我们可以初步认为压力负荷和β₁肾上腺素受体持久激活诱导了心脏内质网应激，继而引发了心肌细胞凋亡、心肌肥大和心力衰竭。为了验证β₁受体激活是否诱导细胞内质网应激反应，阻断β₁受体能否抑制内质网应激，我们在培养的H9c2（2-1）心肌细胞中给予β₁受体激动剂异丙肾上腺素（Iso）刺激24小时，发现GRP78表达显著增加；预先给予细胞PKA特异性抑制剂PKI、CaMKIIδ特异性抑制剂KN93、以及选择性β₁受体阻滞剂美托洛尔和非选择性β受体阻滞剂普萘洛尔（propranolol）孵育1小时，然后给予Iso刺激，24小时后，我们发现PKI、KN93、美托洛尔干预组GRP78表达与Iso组相比无明显变化，普萘洛尔预先干预组GRP78表达比单纯Iso刺激组明显减少。说明Iso激活β₁受体兴奋诱发的内质网应激可能并不通过传统的PKA或CaMKIIδ途径；非选择性β受体阻滞剂普萘洛尔下调Iso刺激产生的内质网分子伴侣。接着我们使用内质网应激诱导剂毒胡萝卜素（TG）和衣霉素（TM）刺激心肌细胞产生内质网应激，通过Western Blots免疫印迹检测、Hoechst凋亡染色和Anexin V／PI凋亡检测，结果发现与单用TG或TM刺激组相比，美托洛尔组GRP78表达减少，细胞凋亡减轻；普萘洛尔组在24小时的时候，几乎完全阻断了GRP78的表达和抑制了内质网特异的致凋亡效应器caspase-12激活。接着我们用无β受体阻断作用却同样有膜稳定作用的右旋普萘洛尔和仅具有膜稳定作用的钠离子通道阻滞剂美西律干预细胞，发现同样有抑制内质网应激反应及减轻相关凋亡的作用。提示普萘洛尔抑制内质网应激的机制可能独立于β受体阻滞作用之外，与其膜稳定作用有关。为了进一步在体内证实膜稳定作用与内质网应激反应相关，我们选取兼具膜稳定作用的非选择性β受体阻滞剂普萘洛尔，和一种典型的膜稳定剂（钠离子通道阻滞剂）美西律（mexiletine）干预压力负荷大鼠（AAC），发现心肌肥大心力衰竭大鼠心脏GRP78表达减少、PERK-eIF2α途径抑制和CHOP蛋白表达阻断，提示内质网应激反应减轻，细胞凋亡抑制；同时有β受体阻断作用的普萘洛尔减轻了心肌肥大，两者都改善了AAC大鼠左心功能。我们推测在心力衰竭中阻断β受体、抑制钠离子内流增多，可以减轻心肌内质网应激，减少凋亡。上面的研究发现膜稳定剂在体内体外均抑制了内质网应激，并改善了心脏功能，而膜稳定作用实际上是维持细胞钠离子稳态的结果，为了研究内质网应激反应与钠离子活动的关系，我们用一种钠通道激活剂藜芦定（veratridine）刺激H9c2（2-1）细胞，30分钟时GRP78表达增加；24小时时PERK磷酸化和CHOP蛋白表达显著增加，说明钠离子通道过度激活诱发了内质网应激反应。预先给予细胞一种特异的钠离子通道阻断剂河豚毒（tetrodotoxin，TTX）孵育1小时，再分别用TG、TM刺激24小时，结果发现GRP78表达明显减少，磷酸化PERK和CHOP表达被TTX完全阻断，说明阻滞钠通道过度激活能有效阻断内质网应激反应。以上结果提示钠离子通道活动在内质网应激反应中起重要作用。总之我们对心力衰竭中内质网应激反应作用机制和途径进行了系统的研究，初步揭示了心力衰竭中内质网应激反应与β₁受体持久激活、细胞Na⁺内流增多有关。内质网应激反应通过诱导应激分子GRP78，激活PERK-eIF2α和IRE1-XBP1途径，诱导CHOP表达、促使caspase-12活化和激活JNKs通路，引起心肌细胞凋亡而导致心肌肥大心力衰竭。给予β受体阻滞剂和膜稳定剂在体内体外都能有效抑制内质网应激，减少细胞凋亡。内质网应激反应途径的关键分子，可能成为心力衰竭治疗和诊断的靶点，对心力衰竭发生发展的基础研究和临床防治有积极意义。更多还原

【Abstract】 The endoplasmic reticulum （ER） is a central organelle of each eukaryotic cell as theplace of calcium storage,lipid synthesis,protein folding and protein maturation.Disturbances in any of these functions such as accumulation of unfolded protein （unfoldedprotein response,UPR） or excessive protein traffic can lead to so-called ER stress.ERstress is the initial cell response to stress and the common pathway leading to manyintracellular stress responses,such as oxidative stress in mitochondria.ER stress is apro-survival response to reduce the accumulation of unfolded proteins and restore normalER function.However,if stress prolongs,signaling switches from pro-survival topro-apoptotic.It has been reported that ER stress is involved in many heart diseases thatcontribute to heart failure at last,including artherosclerosis,myocardial ischemia,dilatedcardiomyopathy.However,the exact mechanisms of ER stress interfering with heart failurestill remain unclear.This study investigated the inducement and signaling pathway of ERstress in failing heart.We found that heart failure really induced ER stress and theER-initiated apoptosis,and confirm the inducement,pathway and interaction of ER stressin heart failure.We went first to confirm the induction of ER stress in human failing heart from hearttransplant recipients by Western Blots analysis,evaluated by GRP78 increase,PERK-eIF2αpathway and JNKs pathway activation.Then we use two kinds of rattus models with cardiac hypertrophy and heart failure,overload pressure （abdominal aortic constriction,AAC） and sustainedβ₁-adrenergic stimulation （isoproterenol injection,Iso）,to investigate the exact pathway of ER stress inheart failure.And we treated the experiment animals with metoprolol,a selectiveβ₁ blocker.By examination of morphology,pathology,and hemodynamics,we found cardiachypertrophy and left ventricular function impairment induced by overload pressure orchronicβ₁-adrenergic stimulation.And compared with control groups,ER stress wasincreased in the failing hearts of both AAC and Iso rattus,evaluated by increase of GRP78,activation of IRE1-XBP1 and induction of CHOP,by analysis of Western Blots.And wefound KDEL positive cells increased in AAC rattus hearts by histochemical analysis.TUNEL examination showed more apoptosis cells in Iso rattus hearts.Treatment ofmetoprolol attenuated ER stress and apoptosis as well as improving hypertrophy and leftventricular function.The data indicates that overload pressure or sustainedβ₁-adrenergicstimulation induce ER stress leading to cardiomyocytes apoptosis,cardiac hypertrophy andheart failure.To investigate whetherβ₁-adrenergic stimulation induces ER stress,we treatedH9c2（2-1） cells with isoproterenol （Iso） for 24 hours,and found GRP78 expressionincreased.We pretreated H9c2（2-1） cells with PKI,a specific inhibitor of PKA,KN93,aspecific inhibitor of CaMKⅡδ,metoprolol or propranolol for 1 hour before exposing themto Iso.After 24 hours,we found that there was no significant change of GRP78 proteinlevel in PKI,KN93 and metoprolol,but pretreatment of propranolol reduced GRP78expression induced by Iso.It revealed that ER stress induced byβ₁-adrenergic stimulationmay not go through the typical PKA pathway or CaMKⅡδpathway.It may be some wayindependent.Nonselectiveβ-blocker propranolol down-regulation the ER chaperonsinduced by Iso.We pretreated H9c2（2-1） cells with metoprolol or propranolol before exposing them toER stressor TG or TM.By Western Blots analyses,Hoechst staining and Anexin V/PIapoptosis tests,we found pretreatment of metoprolol partially reduced GRP78 andattenuated apoptosis.And propranolol nearly completely cut off the GRP78 and inhibited the activation of pro-caspase-12.We next examined the effect of the propranolol isomerD-propranolol,which compared to L-isomer is approximately 50-fold less potent as aβ-adrenoceptor antagonist,but also plays as a membrane-stabilizer,as well as mexiletine,which is a membrane stabilizer,but not a beta-blocker.We found they also attenuated ERstress and inhibited apoptosis pathway.These data supports that the effect ofpropranolol toattenuate ER stress is dependent of itsβ-blockage.It may be associated with its effects asmembrane stabilizer.To investigate the association of membrane stability and ER stress,we administrateAAC rattus with propranolol or mexiletine.We found that treatment with propranolol andmexiletine both reduced ER stress,assessed by GRP78 decrease,PERK-eIF2αpathwayinhibition,and CHOP reduction.They both improve the left ventricular function of AACrattus,and propranolol attenuated cardiac hypertrophy.We presumed thatβblockade andinhibition of increase Na+ influx in heart failure may attenuate ER stress and decreaseapoptosis.As noted above,membrane-stabilizers unexpectedly improved the heart function andprevented ER stress both in vivo and in cultured cells.We know that the membranestability depends on the sodium homeostasis in fact.To investigate whether ER stress isassociated with sodium channel action,which determins the membrane stability,wepretreated H9c2（2-1） cells with veratridine,a sodium channel activator.GRP78 wasincrease at 30 min,and phosphorylated PERK and CHOP significantly increase after 24hours,suggesting that activation of sodium channel induced ER stress.We then pretreatedcells with TTX （tetrodotoxin）,a specific sodium channel blocker,before adding TG or TM.It showed that GRP78 was remarkably decrease and expression of phosphorylated PERKand CHOP was blocked completely.The data yielded evidence that inhibition ofoveractivation of sodium channel blocked ER stress effectually.Taken together,the datasupposed that sodium channel action played an important role in ER stress.In summary,we have systemically investigated the mechanisms and pathway of ER stress in heart failure.We propose that ER stress is associated with sustainedβ₁-adrenergicstimulation and intracellular Na⁺ increase in heart failure.ER stress induces cardiomyocytesapoptosis by activation of ER chaperons such as GRP78 and CHOP,PERK-eIF2αpathway,IRE1-XBP1 pathway,caspase-12 and JNKs pathway.Blockade of the key molecular of ERstress signaling pathway can inhibit heart failure,suggesting that it is a potential target fordiagnosis and treatment of heart failure.更多还原