【作者】 李磊；

【导师】 邹云增；

【作者基本信息】 复旦大学 ， 生物化学与分子生物学， 2010， 博士

【摘要】 多年来的基础和临床研究显示,心脏肥大不仅是心脏对各种刺激的代偿反应,而且是许多心血管疾病发病及死亡的独立危险因素。在许多内在和外在的诱发心脏肥大的因素中,压力超负荷是最重要的激发因素,因此确定压力超负荷所导致的心脏肥大的分子机制非常重要,其中血管紧张素Ⅱ受体(AT1)的活化在心脏肥大中的作用在近年来备受瞩目。越来越多的证据表明血管紧张素Ⅱ型受体拮抗剂(ARB)在降低病人血压的同时,还能够保护靶器官并降低心血管事件的发病率和死亡率。当前许多ARB在临床实践中得到广泛运用,如氯沙坦,厄贝沙坦,替米沙坦,坎地沙坦,奥米沙坦和缬沙坦等。虽然有研究认为-些ARB在降低血压方面要优于一些其他的抗高血压药物,如β受体阻滞剂和钙离子通道拮抗剂,也有一些报道认为ARB和其他的抗高血压药物在靶器官保护中有不同的作用。由此我们假设不同的ARB除降压之外,它们在机械牵张导致心肌肥大中的作用和机制也有可能不同。AT1受体属于G蛋白偶联受体家族,ARB高度选择性的结合于AT1受体并因此阻断AT1受体介导的各种效应。从结构上看,各种ARB除了有一个共同的母核-联苯四唑环外,还有一个在该环上特异的侧链基团,这可能是导致它们之间药效和药物代谢不同的最主要的原因。包括我们在内许多离体研究提示不同ARB抑制AT1受体活化的作用和机制都有所不同,但目前为止还没有报道比较不同ARB在压力超负荷下导致的心脏肥厚中的作用。我们最近报道坎地沙坦能够不依赖AngⅡ直接抑制机械牵张引起的心肌肥大,但其他的ARB是不是有同样的作用以及它们抑制机械牵张触发的心肌肥大的分子机制还少见报道。我们的结果提示不同的ARB在压力超负荷下对心肌的保护是不一样的,这与他们的化学结构差异密切相关。因此,确定机械牵张条件下的ARB的化学结构和AT1受体的相互作用模型以及AT1受体活化的分子机制有助于我们进一步理解机械牵张触发的心肌细胞信号传导通路,基于抑制AT1受体活化的方式的不同来区别ARB以及开发出既能降压又能很好的靶器官保护的新型ARB。此外,心脏肥厚是心力衰竭的独立危险因素,确定心脏肥大的分子机制有利于我们进一步预防和治疗心衰。因此,结合先前的研究结果,我们就机械牵张导致的心肌肥大的分子机制以及不同ARB在该过程中的不同作用做些探讨。第一部分不同ARB逆转压力超负荷诱导的小鼠心脏肥厚的差异比较目的：比较五种ARB(氯沙坦,替米沙坦,坎地沙坦,奥米沙坦和缬沙坦)对压力超负荷导致的心脏肥厚的作用强度和作用方式的差异。方法：缩窄小鼠主动脉弓(TAC)作为小鼠压力超负荷心脏肥大模型。10周龄野生型和血管紧张素原基因敲除小鼠随机各分为7个组：sham组(sham, n=5和n=4),生理盐水组加TAC组(saline,n=9和n=8),奥美沙坦组加TAC组(olmesartan, n=10和n=9),坎地沙坦加TAC组(condesartan,n=10和n=8),氯沙坦加TAC组(losartan,n=8和n=8),替米沙坦加TAC组(telmisartan,n=10和n=8)和缬沙坦加TAC组(valsartan,n=9和n=8)。在TAC2周后,米勒导管经右颈总动脉行至左心室测得左心室压力。五个指标用于评价TAC2周后心脏肥大和重构程度：1、行小动物高频超声检查心脏心态学和评价心脏功能2、心重体重比用于比较心脏大小3、通过RT-PCR检测肥大基因心房利钠肽(ANP),a-骨骼肌肌动蛋白(SAA)和肌浆网ATP钙泵(SERCA2),并通过Real-time PCR进一步验证4、病理切片染色用于显示心脏横切面大小(ESA),心肌细胞横切面积大小(CSA)和心肌纤维化程度。结果：仅仅左心室收缩末压(LVSP)大于155mmHg且小于180mmHg的小鼠用于统计分析,在各组行TAC手术中的小鼠的LVSP无统计学差异。对于野生型小鼠,五种ARB均能逆转压力超负荷引发的心脏肥厚。但对于ATGKO的小鼠缬沙坦和替米沙坦不能抑制TAC导致的心脏肥大的发展而氯沙坦,坎地沙坦和奥米沙坦仍然可以。结论：我们的数据提示氯沙坦,坎地沙坦和奥米沙坦不依赖AngⅡ而缬沙坦和替米沙坦依赖AngⅡ逆转压力超负荷导致的心肌肥大。第二部分不同ARB在机械牵张触发的Jak2信号通路中的作用目的：通过比较奥米沙坦和替米沙坦对机械牵张触发的心肌细胞肥大的抑制效应来探讨机械牵张触发的Jak2信号通路方法：(1)从四个化学合成的siRNA中筛选能有效干扰血管紧张素原(ATG)siRNA片段,将差速贴壁法分离得到的新生大鼠乳鼠心肌细胞分为五组：分别为阳性对照组(转染阴性siRNA)和转染四种针对血管紧张素原siRNA片段；用Real-time PCR方法检测ATG来确定五个siRNA片段干扰效率,从中筛选出一个干扰效果最好的片段用于后继实验。(2)新生大鼠乳鼠心肌细胞分为两大组：野生型组(不转染siRNA)和siRNA干扰组；野生型组再分成四小组分别为空白对照组(不牵张),牵张组,Olmesartan组(Olmesartan加牵张),Temisartan组(Temisartan加牵张)；siRNA干扰组同样分为空白对照组(转染siRNA不牵张),牵张组(转染siRNA并牵张),Olmesartan组转染(Olmesartan加siRNA并牵张),Temisartan组(Temisartan加siRNA并牵张)。通过Real-time PCR检测即刻早期反应基因c-fos,c-jun和心室利钠肽(BNP)在各组中的表达量。Western bloting检测AT1,Gαq11,p-ERKs, Jak2, p-Jak2的各组蛋白的表达量。结果：(1)相比阴性对照siRNA片段,四个化学合成siRNA片段都能不同程度的降低ATG的表达,其中第三个片段干扰效率最高。(2)对于野生型组,Olmesartan和Telmisartan都能够显著抑制因牵张引起c-fos,c-jun和BNP的表达；牵张后Gαq11在心肌细胞胞浆中含量明显升高,Olmesartan和Telmisartan都能够显著抑制Gαq11在胞浆中的升高；牵张后,与AT1受体结合的JaK2量显著增加,Olmesartan和Telmisartan能显著抑制AT1和JaK2的结合；Olmesartan和Telmisartan都能够显著抑制因牵张引起p-ERKs的表达。对于siRNA干扰组,相对Telmisartan, Olmesartan能够显著抑制因牵张引起c-fos和c-jun的表达量的增加,Gαq11在心肌细胞胞浆中含量的升高以及与AT1受体结合的JaK2量和p-ERKs表达量的增加。结论：机械牵张能够不依赖AngⅡ通过AT1-JaK2-ERKs信号途径直接引起心肌细胞肥大,Olmesartan而不是Telmisartan能有效阻断该信号通路。第三部分CaMKII参与机械牵张直接激活AT1受体下游的信号通路目的：初步探讨CaMKII在机械牵张导致的AT1受体活化的信号通路中的作用并进一步明确CaMKII的那个结构域参与这一重要信号通路。方法：(1)对培养在硅胶皿中的心肌细胞转染siRNA后,分别用AT1受体的拮抗剂奥米沙坦和CaMKII的特异性抑制剂KN-93处理并牵张,用WB检测CaMKII和p-ERK的表达。(2)将接种于硅胶皿的HEK293AT1细胞分为四个大组,分别为转染CaMKIδB组；转染CaMKIIδC组；转染CaMKIIδN组；转染CaMKIIδM组。这四个大组再亚分为不牵张和牵张两小组。牵张10分钟后检测的p-ERKs的表达。结果：(1)在心肌细胞中,在没有AngⅡ的情况下,相对于不牵张组,机械牵张可明显升高CaMKII的表达；奥米沙坦能明显抑制机械牵张导致CaMKII的表达升高；KN-93和奥米沙坦通过分别抑制CaMKII和AT1受体明显降低机械牵张导致的ERK的磷酸化。(2)相对于各自的未牵张组,转染CaMKIIδC, CaMKIIδN, CaMKIIB质粒的HEK293AT1细胞牵张后p-ERKs明显上升,但转染CaMKII8M质粒的HEK293AT1细胞牵张后p-ERKs不见升高。结论：机械牵张能够不依赖AngⅡ直接触发AT1-CaMKII-ERKs信号途径,位于CaMKII中部的自我调节域可能是参与这一通路的关键结构域。结论1.建立了升主动脉缩窄的压力超负荷小鼠心肌肥大模型。2.氯沙坦,坎地沙坦和奥米沙坦不依赖AngⅡ而缬沙坦和替米沙坦依赖AngⅡ逆转压力超负荷导致的小鼠心脏肥大。3.机械牵张能够不依赖AngⅡ通过AT1-JaK2-ERKs信号途径直接引起心肌细胞肥大,Olmesartan而不是Telmisartan能有效阻断该信号通路。4.机械牵张能够不依赖AngⅡ直接触发AT1-CaMKII-ERKs信号途径,位于CaMKII中部的自我调节域可能是参与这一通路的关键结构域。潜在价值和创新点1.能够为以后进行药物心脏保护实验提供稳定可靠的小鼠心脏肥厚模型。2.发现并初步证实了在心肌细胞中机械牵张能导致JaK2和CaMKII的磷酸化,并提示这两个蛋白在压力超负荷导致的信号通路中起非常重要的作用。从而可以针对这两个蛋白的关键结构域开发相应的药物来治疗心脏肥厚。3.通过我们的小鼠和细胞实验,从心脏保护的观点发现临床常用的五种ARB在逆转压力超负荷导致的心脏肥大的作用方式和强度均有所不同,从而为临床用药提供理论参考。更多还原

【Abstract】 Cardiac hypertrophy is not only a physiological adaptively state before heart failure, but also an independent risk factor of major cardiac events. Although there are many factors that can induce cardiac hypertrophy, hypertensive stimulation is a major hypertrophy-inducing factor. AngiotensinⅡ(AngⅡ) and its type 1-receptor (AT1-R) have been known to be greatly involved in the pressure overload-induced cardiac hypertrophy. AT1-R blockers (ARBs) are clinically available as a highly effective and well-tolerated class of drugs for the treatment of hypertension. In addition, a variety of clinical evidences have shown that ARBs provide cardiovascular protection beyond blood pressure (BP)-lowering. Especially, ARBs administration effectively prevents cardiac hypertrophy and improves the cardiovascular outcomes in patients with hypertension. At present, there are several ARBs widely used in clinics such as Losartan, Irbesartan, Telmisartan, Candesartan, Valsartan, Olmesartan, etc. Although some of them have been shown to have more beneficial effects than other types of antihypertensive agents such asβ-adrenergic receptor blockers and calcium channel antagonists, there are also reports indicating that cardiovascular outcomes by ARBs-based treatment do not differ from those by other agents-based treatment. We therefore supposed whether there were differences in cardiac protection among ARBs besides antihypertensive potency.AT1-R is a member of the G protein-coupled receptor (GPCR) superfamily [10] and ARBs are highly selective in binding to the AT1 receptor and blocking diverse effects mediated by AT1 receptor. Structurally, ARBs not only have the common biphenyl-tetrazole ring but also the unique side chains which contribute to drug-specific differences in pharmacokinetic and pharmacodynamic properties and antihypertensive potency. Many in vitro studies including ours have suggested that effects of ARBs on suppression of AT1-R activation differ. However, a rigorous in vivo comparison of different ARBs to regress pressure overload-induced cardiac hypertrophy has not been performed. Moreover, we have recently reported that Candesartan inhibited activation of AT1-R and attenuated cardiac hypertrophy induced by pressure overload in the absence of AngⅡ, however, it is unknown whether this holds true for other ARBs. The efficacies of ARBs on cardiac protection during hypertension are different, which associate with the differences in chemical structures of the drugs. Elucidation of the molecular interaction model for the chemical structure of ARB and AT1-R and the molecular mechanisms of ATl-R activation induced by pressure overload will be of help to discriminate the ARBs according to the efficacies in AT1-R inactivation, to understand the Signaling pathway induced by pressure overload, and to develop a novel ARB with superb efficacy not only in blood pressure-lowering but also in organ protection.PartIDifferent efficacies and mechanisms of ARBs in reversal of pressure-overload cardiac hypertrophy in miceObjectives:This study is designated to compare efficacies and mechanisms of different angiotensin II type 1 (AT1) receptor receptor blockers (ARBs) (including olmesartan, candesartan, losartan, telmisartan and valsartan) in reversal of cardiac hypertrophy induced by pressure overload.Methods:Constricting transverse aorta (TAC) mice were used as a model for pressure-overload cardiac hypertrophy. Wild and angiotensinogen-deficient C57BL/6J mice of 8-10 weeks were each divided into seven groups: sham-operated group (n=5 and n=4), drinking water plus TAC group (n=9 and n=8), olmesartan plus TAC group (n=10 and n=9), candesartan plus TAC group (n=10 and n=8), losartan plus TAC group (n=8 and n=8), telmisartan plus TAC group (n=10 and n=8), and valsartan plus TAC group (n=9 and n=8). After 2 weeks of TAC, mice were subjected to catheterization via the right carotid artery into left ventricle to measure the left ventricular end-systolic pressure (LVESP). Including examination of myocardial fibrosis, four endpoint measurements were made to assess the phenotype of cardiac hypertrophy after 2 weeks of TAC: 1).Two-dimensional guided M-mode echocardiographic assessments were performed with 30MHz high frequency scanhead for the geometry and functions of hearts,2). Heart weight to body weight of ratio (HW/BW) was used to compare the weights of hearts,3). The expression of the genes which are associated with cardiac hypertrophy such as atrial natriuretic peptide (ANP), skeletal a-actin (SAA) and the sarcoplasmic reticulum Ca2+-ATPase (SERCA2) were determined by RT-PCR and confirmed further by qRT-PCR,4).Cardiac hypertrophy was confirmed further by histological analysis on the cross-sectional area (CSA) of cardiac myocytes and mid-transverse sectional area (MSA) in the left ventricle.Results:Only the mice with more than 155mmHg and less than 180mmHg were considered to be successfully TAC-operated and were used for statistic analysis, and the haemodynamics have no significant differences among all the TAC-operated groups. For wild-type mice, all the five ARBs can reverse cardiac hypertrophy and have the same efficacies. However, when angiotensinogen of the mice were knock-out, valsartan and telmisartan can’t reverse cardiac hypertrophy, although olmesartan, candesartan and losartan can still.Conclusions:Unlike valsartan and telmisartan that depend on angiotension II, omlesartan, losartan and candesartan do not depend on angiotensin II to inhibit the pressure-overload-induced AT1 receptor activation and therefore act as inverse agonists.Part IIComparison of Olmesartan and Telmisartan to inhibit stress-induced Jak2 pathway in cardiomyocytes of neonatal ratsObjective:To compare the effects of olmesartan and telmisartan on Jak2 pathway induced by stress in cardiomyocytes of neonatal rats(RCM).Methods:To select the better small interfering RNA siRNA segment down-regulating the expression of ATG,4 pairs of chemically synthetical siRNA(siRNA-1, siRNA-2, siRNA-3, siRNA-4) were transfected into RCM isolated by different adherent time method by use of NeoFX, respectively. Negative group and positive group were designed as control. The effects of silence for ATG were determined by Real-time PCR. (2) RCMs were divided into two big groups:wild type group (no siRNA was transfectd), siRNA group (siRNA was transfected). The two big groups were divided into four sub-groups, respectively:control group (no stress), stress group (stress), group treated by olmisartan and group treated by telmisartan. The mRNA level of immediate-early-response genes of c-fos, c-jun and BNP were determined by Real-time PCR and the protein level of ATI,Gaq11, p-ERKs, Jak2, p-Jak2 were detected by western blotting.Results:(1) compared to positive group,4 pairs of chemically synthetical siRNA could down-regulating the expression of ATG, but siRNA-3 segment has the best effect of silence for ATG. (2) As for wild-type group, olmesartan and telmisartan can effectively inhibit stress-induced the expression of c-fos, c-jun and BNP. After mechanical stress, there was a significantly increase for level of Gaq11 in Cytosol of RCM, treatment with olmesartan and telmisartan both significantly inhibited the increment; Mechanical stretch induced association of Jak2 with the AT1 receptor in RCM.Pretreatment of the cells with olmesartan and telmisartan significantly suppressed association of Jak2 with the AT1 receptor; In addition, olmesartan and telmisartan can inhibit the activation of ERKs induced by mechanical stress. For siRNA group in which ATG was down-regulating, compared with telmisartan, olmesartan can significantly inhibited the increment of c-fos and c-jun, level of Gaq11 in cytosol, association of Jak2 with the AT1 receptor, andp-ERKs after stress.Conclusions:mechanical stress activated the AT1-JaK2-ERKs pathway independently of AngII and unlike telmisatan, olmesartan can block the pathway even in the absence of AngII.PartIIIActivation of CaMKII is crucially involved in the stretch-induced activation of AT1 receptorObjective:To explore the effects of CaMKII on the pathway of the activation of AT1 receptor induced by mechanical stress in vitro.Methods:(1) RCM cultured in silica gel dish were each treated with olmesartan and KN-93, the specifical inhibitor of CaMKII. After mechnical stress, p-CaMKII and p-ERKs were detected by WB. (1) HEK293AT1 cultured in silica gel dish randomly divided into four groups accordingly to the plasmids transfected:CaMKIIδB group, CaMKIIδC group, CaMKIIδN group and CaMKIIδM group. The four groups were divided into two groups:not-stress and stress, respectively. After stretching the cells by 20%for 10 min,p-ERKs of all the groups were detected by WB. Results:(1) mechanical stress can signicicantly increase the expression of p-CaMKII without AngII in RCM; Olmesartan inhibited the acivation of CaMKII induced by stress; KN-93 and olmesartan inhibited the acivation of ERKs through suppressing CaMKII and ATI receptor, respetively. (2) compared to corresponding to no-stress group,p-ERKs of the stress groups transfected into the plasmids of CaMKII8C, CaMKII8N, CaMKIIB were increased significantly, butp-ERKs of CaMKIIδM group have no change after stress.Conclusions:Mechanical stress activated the ATl-CaMKII-ERKs pathway without the involvement of AngII and the autoregulatory domain lacated at the middle of CaMKII may play an important role in the pathway. Conclusions1. Unlike valsartan and telmisartan that depend on angiotension II, omlesartan, losartan and candesartan do not depend on angiotensin II to inhibit the pressure-overload-induced AT1 receptor activation and therefore act as inverse agonists.2. Mechanical stress activated the AT1-JaK2-ERK pathway independently of AngII and unlike telmisartan, olmesartan can block the pathway even in the absence of AngII.3. Mechanical stress activated the AT1-CaMKII-ERKs pathway without the involvement of AngII and the autoregulatory domain located at the middle of CaMKII may play an important role in the pathway.Novelty and Value1. We can supply stable and repetitive TAC mice model for the drug screen that regressed cardiac hypertrophy.2. We found JaK2 and CaMKII play an important roles in signalling pathway of cardiac hypertrophy induced by pressure overload. So some active domains of JaK2 and CaMKII may be potential drug targeting for treatment of cardiac hypertrophy.3. We found efficacies and mechanisms of ARBs available in clinical use is different in reversal of pressure-overload cardiac hypertrophy in vivo and in vitro, which will be helpful to discriminate the ARBs according to the efficacies in AT1-R inactivation for the protection of heart.更多还原