【作者】 周宁；

【导师】 邹云增；

【作者基本信息】 复旦大学 ， 内科学， 2010， 博士

【摘要】 心肌细胞膜血管紧张素Ⅱ-1型(ATl)受体的过度活化是心肌肥厚发生、发展的重要原因。AT1受体敲除或活性受抑均可以有效的缓解压力超负荷引起的心肌肥厚和损伤的进展。近年来研究表明,AT1受体不但可以被其配体血管紧张素Ⅱ(AngⅡ)所激活,而且还可以直接被机械刺激所活化,继而引起心肌肥厚。机械刺激直接激活AT1受体是一个全新的发现,目前对其发生机制、调控因素以及引发心肌肥厚的主要细胞内信号转导途径均不明确。本研究在内源性AngⅡ缺失的细胞株(COS7细胞和HEK293细胞)中分别转染野生型AT1受体和钙调蛋白激酶Ⅱ(CaMKⅡ)质粒,然后给予体外机械牵张,证实了CaMKⅡ对于机械牵张激活AT1受体具有重要作用。另外我们在不表达AngⅡ的血管紧张素原基因敲除(ATG-/-)小鼠中实施主动脉缩窄(transverse aorta constriction, TAC)手术构建了压力超负荷心肌肥厚模型,并给予AT1受体阻断剂Losartan和特异性CaMKⅡ抑制剂KN93干预。结果表明持续2周的TAC在ATG-/-小鼠中引起了显著的代偿性心肌肥厚,Losartan和KN93在没有明显影响血流动力学的情况下均有效的缓解了心肌肥厚的发生发展。在机械刺激诱导肥厚的心肌细胞中,CaMKⅡ向心肌细胞膜聚集定位并与AT1受体结合,Losartan可以抑制二者结合,而KN93对二者之间的结合无显著抑制作用。在构建不同部位的CaMKⅡ突变体并将其和野生型AT1受体共转染进COS7细胞之后,牵张并检测肥厚反应,发现将CaMKⅡ中间自我抑制结构敲除之后,对AT1受体的感受机械刺激能力的抑制最为显著,因此推测CaMKⅡ与AT1受体蛋白的结合位点可能出于其中间结构域。鉴于钙调神经磷酸酶(CaN)在AT1受体介导的AngⅡ致心肌肥厚的细胞内信号通路中的核心地位,我们推测CaN可能在AT1受体介导的细胞外机械信号的胞内信号通路中也扮演者重要的角色。因此我们在压力超负荷构建的的ATG-/-小鼠心肌肥厚模型中以Losartan和特异性的CaN抑制剂FK506干预,证实Losartan和FK506均有效抑制了压力超负荷引起的心肌肥厚,抑制AT1受体活性也显著性降低了心肌细胞内的CaN表达,证实CaN是AT1受体介导的细胞外机械信号的不可或缺的胞内信号蛋白。综合以上研究结果,我们认为机械刺激引起心肌细胞内CaMKⅡ向膜聚集并与AT1受体结合和相互作用,使得AT1受体可以直接被机械刺激活化并介导心肌肥厚。某些AT1受体阻断剂如Losartan可以抑制CaMKⅡ与ATl受体之间的结合,而CaMKⅡ与AT1受体的结合部位可能位于其中间自我抑制结构。在AT1受体介导的心肌细胞外机械刺激的信号通路中,CaN是其中重要的信号分子。第一部分CaMKⅡ参与了机械刺激直接诱导的AT1受体激活目的：验证CaMKⅡ是否参与了机械刺激直接诱导的AT1受体活化过程。方法：在硅胶皿培养的COS7细胞中单转染或共转染野生型CaMKⅡ与AT1受体质粒,机械牵张8分钟后收集细胞总蛋白以免疫印迹法(Western-blot)检测磷酸化细胞外信号调节蛋白激酶(extracellular signal-regulated protein kinases, p-ERKs)的表达水平,提取细胞总RNA,以逆转录PCR检测胎儿型基因ANP、BNP和骨骼肌α-肌动蛋白(skeletal-a actin, SAA)基因表达水平。结果：在没有转染AT1受体质粒的COS7细胞中,无论是AngⅡ还是牵张刺激,均不能引起p-ERKs和ANP、BNP以及SAA的表达上调。在单独转染AT1受体但是没有转染CaMKⅡ质粒的COS7细胞中,机械牵张引起了p-ERKs和ANP表达的轻度上调。在共转染CaMKⅡ与ATl受体质粒的COS7细胞中,牵张刺激引起了明显的p-ERKs和ANP、BNP以及SAA的表达上调。结论：AT1受体是介导机械刺激引起的增殖反应的最主要膜受体,CaMKⅡ参与了机械刺激活化AT1受体的过程。第二部分机械刺激诱导CaMKⅡ向心肌细胞膜聚集并与AT1受体结合目的：研究在机械刺激引起的心肌肥厚中,CaMKⅡ与心肌细胞膜AT1受体的关系。方法：32只8-10周龄的血管紧张素原基因敲除(ATG-/-)小鼠被均分为4组：假手术+生理盐水干预组,TAC+生理盐水干预组,TAC+Losartan (3mg/Kg/d)干预组,TAC+KN93 (3mg/Kg/d)干预组。生理盐水和干预药物溶液均通过术前3天埋植在小鼠背部皮下的渗透压泵24小时持续给药。TAC术后2周,所有小鼠在行经胸心脏超声测定心脏形态和心功能,侵入性心导管检测主动脉以及左室压力后处死,取心脏测量心重并计算心重体重比值,部分心脏石蜡包埋后HE染色测定心肌细胞横截面积,部分心脏做冰冻切片以备后期免疫荧光观察,部分心脏液氮冻存或者直接用于抽提总蛋白、膜蛋白或者总RNA。Western-blot检测抽提总蛋白中p-ERKs表达,检测膜蛋白中CaMKⅡ和AT1受体蛋白表达。免疫共沉淀(co-immunoprecipitation, Co-IP)检测CaMKⅡ和AT1受体蛋白的结合,用免疫荧光激光共聚焦成像方法观察CaMKⅡ和AT1受体分布和共定位状态。体外培养COS7细胞并转染野生型CaMKⅡ和AT1受体质粒,牵张刺激24小时后,提取细胞膜蛋白行Western-blot检测膜CaMKⅡ含量,Co-IP检测CaMKⅡ和AT1受体的结合。结果：TAC手术在ATG-/-小鼠中显著升高了主动脉血压和左心室收缩末期压力,Losartan和KN93干预并没有明显影响TAC引起的血流动力学异常。2周的TAC在ATG-/-小鼠中引起了代偿性心肌肥厚,心超表现为左心室前壁和后壁增厚,左心室内径减小,反应性左心室射血分数增加而左室短轴缩短率无明显变化。TAC后2周小鼠心重体重比和心肌细胞横截面积均显著增加,肥厚相关标志物包括p-ERKs和ANP、BNP和SAA表达均显著升高,SERCA2表达下降。Losartan和KN93干预显著抑制了左心室前、后壁增厚,左室内径的减小和左心室射血分数异常,对FS无明显改变,同时也显著降低了心重体重比、心肌细胞横截面积和肥厚相关标志物的表达异常。TAC引起心肌细胞膜CaMKⅡ表达明显增加。Losartan显著抑制了CaMKⅡ的在心肌细胞膜的表达上调,KN93对心肌细胞膜的CaMKⅡ聚集抑制作用更为明显。心脏切片免疫荧光证实TAC之后CaMKⅡ在心肌细胞膜区域高浓度聚集,KN93比Losartan更为显著的抑制这种趋势。Co-IP和免疫荧光发现,TAC引起了CaMKⅡ和AT1受体在心肌细胞膜结合,Losartan可以明显抑制二者的结合,而KN93对二者结合的抑制作用不明显。在体外培养的COS7细胞转染野生型CaMKⅡ和AT1质粒,牵张刺激24小时后的受体的膜蛋白检测提示机械牵张促使CaMKⅡ向细胞膜聚集并和AT1受体结合。结论：机械刺激引起心肌细胞内CaMKⅡ向膜聚集并与AT1受体结合,可能是AT1受体直接被机械刺激活化的重要机制。虽然KN93更为明显的抑制了CaMKⅡ向心肌细胞膜的聚集,但却不能像Losartan那样抑制CaMKⅡ与AT1受体之间的结合。第三部分机械刺激后CaMKⅡ与AT1受体结合的部位目的：研究在机械刺激下CaMKⅡ与AT1受体的可能结合部位。方法：在硅胶皿培养的COS7细胞中单转染或共转染野生型AT1受体质粒与CaMKⅡ突变体质粒,牵张刺激8分钟后收集细胞总蛋白以Western-blot法检测转染目的基因的蛋白表达效率和p-ERKs的表达水平。结果：Western-blot检测提示转染的目的基因AT1受体质粒与CaMKⅡ突变体质粒的蛋白表达显著,转染效率可靠稳定。牵张刺激之后,共转染野生型AT1受体质粒与CaMKⅡ的野生型、N端敲除突变体、C端敲除突变体质粒的COS7细胞中p-ERKs表达均有显著上调,而转染M段敲除突变体的COS7细胞牵张后的p-ERKs表达变化并不明显,提示N、C端敲除对CaMKⅡ的功能无显著影响,而M段对其促进AT1受体结合的作用显著。结论：CaMKⅡ的中间自我抑制区域可能是其与AT1受体的结合部位。第四部分AT1受体受机械刺激活化后引起心肌肥厚的细胞内信号转导途径目的：研究在AT1受体介导的细胞外机械刺激的心肌细胞内信号通路中钙调神经磷酸酶(calcineurin, CaN)的作用。方法：8-10周龄的ATG-/-小鼠行如下处理：假手术+生理盐水干预(n=8),TAC+生理盐水干预(n=8), TAC+Losartan (3mg/Kg/d, n=8)干预,TAC+FK506 (1mg/Kg/d, n=8)干预。生理盐水和干预药物溶液均通过术前3天埋植在小鼠背部皮下的渗透压泵24小时持续给药。TAC术后2周,所有小鼠在行经胸心脏超声测定心脏形态和心功能,侵入性心导管检测主动脉以及左室压力后处死,取心脏测量心重并计算心重体重比值,部分心脏石蜡包埋后HE染色测定心肌细胞横截面积,部分心脏直接用于抽提总蛋白和总RNA。Western-blot法检测心肌细胞CaN表达水平。Realtime-PCR检测肥厚相关基因ANP、SAA表达。结果：TAC手术在ATG-/-小鼠中成功构建代偿性心肌肥厚模型。FK506干预作用同Losartan干预作用相似,在并没有明显影响小鼠血流动力学的情况下,明显抑制了左心室前、后壁增厚,左室内径的减小和左心室射血分数异常,对FS无明显改变。同时也显著降低了心重体重比、心肌细胞横截面积和肥厚相关标志物的表达上调。Losartan干预也有效抑制了心肌细胞CaN的表达。结论：AT1受体介导了压力超负荷诱发的心肌肥厚,并上调了细胞内CaN表达,后者是AT1受体介导的细胞外机械信号在细胞内的极为重要的信号转导蛋白。结论1、机械刺激直接活化AT1受体需要CaMKⅡ的参与。2、机械刺激引起心肌细胞内CaMKⅡ向膜聚集并与AT1受体结合和相互作用,使得AT1受体可以直接被机械刺激活化,介导心肌肥厚作用。3、CaMKⅡ的中间自我抑制区域可能是其与AT1受体的结合部位。4、Losartan可以抑制CaMKⅡ与AT1受体之间的结合。5、钙调神经磷酸酶是AT1受体介导的机械刺激信号在细胞内的重要信号转导蛋白。创新点和研究意义1、本课题是针对机械刺激可以不依赖于AngⅡ直接激活AT1受体从而诱发心肌肥厚、损伤这一全新发现所做的深入机制研究。2、揭示了机械刺激诱导CaMKⅡ和AT1受体的相互作用是导致AT1受体活化的重要机制。3、发现了CaMKⅡ在机械刺激下向细胞膜的迁移定位现象,证实其并非只是单纯的执行细胞内信号分子作用。4、证实某些ARBs抗心肌肥厚的机制在于通过抑制CaMKⅡ与AT1受体的结合从而抑制AT1受体的活化,为寻找更为有效的ARBs提供理论基础。更多还原

【Abstract】 The excessive activation of angiotensin II type 1 receptor (AT1-R) was critically involved in the initiation and development of cardiac hypertrophy evidenced by marked attenuation of pressure overload induced-cardiac hypertrophy attributing to the blockade or knockout of AT1-R. Recently published data revealed a new finding that AT1-R can be activated by mechanical stress independent of AngⅡand consequently induced cardiac hypertrophy, although the mechanism, upstream regulating factors and intracellular signaling transduction pathways remained unclear. Here we demonstrated that Ca2+/calmodulin-dependent protein kinaseⅡ(CaMKⅡ) plays a crucial role in the activation of AT1-R induced by mechanical stress independent of AngⅡin the development of cardiac hypertrophy.We firstly performed in vitro studies by using mechanical stretched-COS7 cells or HEK293 cells which both in lack of endogenous AngⅡ. Mechanical stress fail to induce hypertrophic responses in COS7 cells artificially expressed with AT1-R without CaMKII, indicating the critical role of CaMKII in the mechanical stress induced-activation of AT1-R.Next cardiac hypertrophy model was established in angiotensinogen gene knockout (ATG-/-) mice subjected to transverse aorta constriction (TAC) for 2 weeks. AT1-R and CaMKII were inhibited by treating the TAC ATG-/- mice with Losartan or KN93, respectively. At 2 weeks after TAC, the ATG-/- mice with KN93 treatment showed a less cardiac hypertrophy and unchanged hemodynamics, which were similar to that in the TAC mice treated with Losartan, than the TAC mice with vehicle treatment characterized by adaptive cardiac hypertrophy. Accumulated CaMKII and marked binding of CaMKII with AT-R were detected in mechanical stress induced-hypertrophied cardiomyocytes, which were significantly inhibited by Losartan. Mutation in the middle autoinhibitory sequence of CaMKII failed to induce the hypertrophy response elicited by mechanical stress, indicating a possible binding site with AT1-R in the middle autoinhibitory sequence.Calcineurin functioned as a key molecule in the intracellular signal transduction pathway. We therefore hypothesized that it may also play a critical role in the cardiac hypertrophy resulting from the directly activated AT1-R by extracellular mechanical stimuli. FK506, a specific inhibitor of calcineurin, significantly reversed the cardiac hypertrophy in ATG-/-mice imposed with TAC for 2 weeks, suggesting an essential role of calcineurin in the intracellular signal transducing the extracellular mechanical stimuli mediated by AT1-R.Part1CaMKⅡwas involved in the activation of AT1-R induce by mechanical stress independent of AngⅡObjective: To elucidate the role CaMKⅡplays in the activation of AT1-R induced by mechanical stress without the involvement of AngⅡ.Methods:COS7 cells transfected with the plasmids of wild type of CaMKⅡand AT1-R plasmids were cultured in silicon-based plates pre-coated with collagen and incubated in serum and antibiotic-free conditions for 24 hours before mechanical stretch to 120% for 8 minutes. Losartan (10-6mol/L) or KN-93 (10-9mol/L) pre-administered in the medium for 30 minutes. After mechanical stretch, the cells were collected for the extraction of total protein and RNA for the following experiments.Results:Mechanical stretch did not induce the upregulation of the phosphorylation of ERKs and reprogramming of ANP, BNP and SAA in COS7 cells without any transfection or only transfected with CaMKⅡalone because of lacking AT1-R. In COS7 cells enforced expressing AT1-R but without CaMKⅡ, mechanical stretch slightly upregulated the phosphorylation of ERKs and the reprogramming of ANP, BNP and SAA, which were largely upregulated in those doubly transfected by CaMKⅡand AT1-R.Conclusion:These evidences suggested that CaMKⅡmay play an important role in the activation of AT1-R induced by mechanical stress independent of AngⅡ.Part 2Mechanical Stress Elicited the Translocation of CaMKⅡfrom Cytoplasm to Membrane and Induced the Binding of CaMKⅡand AT1-RObjective:Although evidences showed that CaMKⅡis involved in the activation of AT1-R induced by mechanical stress directly, CaMKⅡis mainly located in cytoplasm which is disconnected with AT1-R locating in the membrane of cardiomyocytes. So we hypothesized that CaMKII may translocate to the membrane and interacted with the AT1-R in the condition of mechanical stress.Methods:ATG-/- mice imposed with TAC for 2 weeks were continuously administered with Losartan (3 mg/kg/day) or KN93 (3 mg/kg/day) by osmotic minipumps subcutaneously implanted in the back of ATG-/- mice. Transthoracic echocardiographic analysis and invasive hemodynamics measurement were performed at the end of 2ed week. Excised hearts were weighed, perfused with PBS followed by 4% polyformaldehyde for global morphometry and fixed in 10% formalin for histological analysis. Paraffin embedded hearts were sectioned at 4-μm thickness and stained with hematoxylin and eosin (H-E). Cardiomyocytes were chosen from each section at a high magnification, and cross sectional area (CSA) of cardiomyocytes was measured. Total RNA was isolated from the left ventricular (LV) tissues and reverse transcription and polymerase chain reaction was performed. Total proteins extracted from LV tissues were subjected to Western blot analysis for phosphorylation of ERKs. The amounts of CaMKII were examined after the membrane fraction was divided from the cytoplasm fraction. Extracted membrane proteins were used for Co-immunoprecipitation with an anti-AT1-R or anti-CaMKII antibody. Frozen slices of heart tissue of mice were incubated with anti-CaMKII or anti-AT1-R antibodies served as the primary antibody, and then with secondary antibodies. The fluorescence was observed under confocal microscopy.Results:Two weeks after TAC, although compensated cardiac hypertrophy characterized by increases in left ventricular (LV) wall thickness, global heart size, heart weight/body weight ratio (HW/BW) and cross-sectional area (CSA) of cardiomyocytes in LV section and decreases of LV cavity, was obviously formed and fractional shortening (FS) was preserved in all TAC mice, it was attenuated by KN93 as effectively as Losartan without impacting the BP and LVESP of TAC mice, consisting with the upregulation of phosphorylation of ERKs and reprogramming of ANP, BNP, SAA and SERCA2. Although data from Max dP/dT and contractibility index indicated that Losartan may be more effective on improving the contractibility of hearts of TAC mice, there were no statistical differences on the inhibitory effects of cardiac hypertrophy between KN-93 and Losartan, suggesting inhibition of CaMKII blocked the activation of AT1-R resultant in the cardiac hypertrophy. We employed the method of co-immuneprecipitation using both anti-CaMKII and anti-AT1-R antibodies. Western blot analyses of immunoprecipitate with anti-AT1-R antibody from membrane fraction of heart tissue of mice for CaMKII expression showed a combination of CaMKII with AT1-R, which was much more significant in the TAC mice than in sham-operated mice. Consisting with the results from co-immuneprecipitation, immunofluorescent staining with CaMKII and AT1-R revealed that much more CaMKII was located in cell-surface area and co-existed with AT1-R in cardiomyocytes of TAC mice than in sham-operated mice. Taken the data from co-immuneprecipitation and immunofluorescent staining into consideration, we found it is Losartan, but not KN93, that inhibited the combination of CaMKII and AT1-R.Conclusion:These findings suggested that mechanical stress elicited the translocation of CaMKII from cytoplasm to membrane and induced the binding of CaMKII and AT1-R. Although KN93 was more effectively inhibited the translocation of CAMKII from cytoplasm to membrane, it can hardly inhibit the combination of CaMKII and AT1-R as Losartan did.Part 3The binding sites of CaMKII with AT1-R in the mechanical stress stimulated-myocardiocytesObjective: To determine the binding sites of CaMKII with AT1-R in the mechanical stress stimulated-myocardiocytes.Methods:Three CaMKII mutants were constructed, which were depleted in N terminal (8N), in middle autoinhibitory sequences (δM), in C terminal (δC).We detected the phosphorylation of ERKs in the COS7 cells doubly transfected by AT1-R and each mutants of CaMKII before and after mechanical stretch for 8 minutes.Results:Significant differences were observed in the phosphorylation of ERKs in the COS7 cells doubly transfected by AT1-R and wild type,δC or 8N mutants before and after mechanical stretch, suggesting that CaMKII is essential in the activation of AT1-R and depletion of the C terminal and N terminal of CaMKII has no obvious effect on the function conducing activation of AT1-R. However, no significant differences in phosphorylation of ERKs were found in COS7 cells co-transfected with AT1-R andδM mutant of CaMKII before and after mechanical stretch which indicated that middle sequence of CaMKII is a critical site for the binding of CaMKII to AT1-R.Conclusion:The middle sequence of CaMKII is essential in mediating the activation of AT1-R and binding with AT1-R.Part 4Activation of AngiotensinⅡreceptor 1 by mechanical stress independent of AngiotensinⅡinduces cardiac hypertrophy through calcineurin pathwayObjective:Mechanical stress activates angiotensinⅡ(AngⅡ)type 1(AT1)receptor without the involvement of angiotensinⅡand therefore induces cardiac hypertrophy. However, the intracellular signal transduction pathway of AT1 receptor-mediated excellular mechanical signaling remains indeterminate.Methods:ATG-/- mice subjected to TAC for 2 weeks were continuously administered with Losartan (3 mg/kg/day) or FK506 (1mg/kg/day) by osmotic minipumps subcutaneously implanted in the back of the ATG-/- mice. Transthoracic echocardiographic analysis and invasive hemodynamics measurement were performed at the end of 2ed week. Excised hearts were weighed, perfused with PBS followed by 4% polyformaldehyde for global morphometry and fixed in 10% formalin for histological analysis. Paraffin embedded hearts were sectioned at 4-μm thickness and stained with hematoxylin and eosin (H-E). Cardiomyocytes were chosen from each section at a high magnification, and CSA of cardiomyocytes was measured. Total RNA was isolated from the LV tissues and realtime PCR was performed to detect the expression of ANP and SAA. Total proteins extracted from LV tissues were subjected to Western blot analysis for phosphorylation of ERKs.Results:We demonstrated that Losartan attenuated the cardiac hypertrophic responses and the upregulation of myocardial Ca2+ -dependent phosphatase calcineurin (CaN) in ATG-/- mice subjected to TAC for 2 weeks without an antihypertensive effect, indicating CaN is a downstream effector of AT1 receptor-mediated mechanical signaling inducing hypertrophic responses. Compared with Losartan, FK506, a specific inhibitor of CaN, similarly reversed the myocardial hypertrophy manifested with a preserved LVEF, less thicker ventricular wall thickness and enlargement of left ventricular cavity, decreased heart weight/body weight ratio and cross sectional area as well as the inhibition of up-regulated expression of fetal type genes including ANP and SAA, implicating an essential role of CaN in the mechanical stress induced-cardiac hypertrophy independent of AngⅡ.Conclusion:These findings collectively suggested that activation of AT1 receptor by mechanical stress independent of AngⅡinduces cardiac hypertrophy through CaN pathway.Conclusion1, CaMKII is involved in the activation of AT1-R induced by mechanical stress independent of AngⅡ.2, Mechanical stress elicited the translocation of CaMKII from cytoplasm to membrane and induced the binding of CaMKII and AT1-R, which enable AT1-R responding to the mechanical stress directly and consequently resulting in cardiac hypertrophy.3, The middle sequence of CaMKII is essential in mediating the activation of AT1-R and binding with AT1-R.4, Losartan, but not KN93, inhibited the combination of CaMKII and AT1-R induced by mechanical stress.5, Activation of AT1-R by mechanical stress independent of AngⅡinduces cardiac hypertrophy through calcineurin pathway.The potential application and novelty of this project1. We explored the mechanism of a new finding that mechanical stress activates AT1-R without the involvement of AngⅡ.2. The bindings and interaction between CaMKII and AT1-R is critically involved in the direct activation of AT-R induced by mechanical stress.3. Our findings indicated that mechanical stress promoted the translocation of CaMKII from cytoplasm to membrane, indicating a new function of CaMKII besides of its signaling transduction in cardiomyocytes.4. We found the anti-hypertrophied effect of Losartan partly attributed to its inhibition of the binding of CaMKII and AT1-R, which may provide new evidences for the development of ARBs in the cardioprotection.更多还原