【作者】 梁艳艳；

【导师】 邹云增；

【作者基本信息】 复旦大学 ， 心血管内科学， 2011， 博士

【摘要】 兰尼碱2型受体(Ryanodine receptor type 2 RyR-2)主要存在于心肌细胞内质网上,介导心肌细胞内钙离子(Calciumion Ca2+)释放,Ca2+在心肌细胞的兴奋-收缩耦联中起关键作用,心肌细胞的每一次舒缩都伴有胞浆内Ca2+浓度的增高和恢复,除此之外,越来越多研究表明Ca2+也参与病理条件下心室重构及心力衰竭的发生过程。然而,在心室重构及心力衰竭的发病机制中,RyR-2受体的作用机制依然不甚明了。应用野生型小鼠(WT)及RyR-2基因敲除小鼠(RyR-2+/-)通过升主动脉缩窄的方法构建压力超负荷引起的心室重构模型,进行RyR-2的分子机制研究。结果表明,在基础生理条件下,与RyR-2+/+小鼠相比,RyR-2+/-小鼠尽管已经出现明显Ca2+释放异常,但在心肌细胞大小、心脏结构形态和心肌收缩功能等方面却无明显差异；主动脉缩窄后,小鼠心脏压力负荷增加,3周后,各组小鼠均发生了明显的心室重构--心肌肥厚,而相对wT小鼠,Ryy-2+/-小鼠的肥厚程度明显减弱、心肌纤维化程度以及收缩功能的减弱程度明显减轻,这些改变与心肌细胞凋亡增加而心肌细胞自噬减少有关,与心脏中微血管生成无关。进一步研究发现,一系列Ca2+调节蛋白包括L型钙通道(L-type Ca2+ channel, LCC)、钠钙交换体(Na1+/Ca2+ exchanger, NCX)、钙离子ATP酶2(SR Ca2+-ATPase 2, SERCA2)的基因表达紊乱；压力超负荷后RyR-2+/-小鼠心脏中与RyR-2受体结合的结合蛋白FKBP量减少而另一种与RyR-2受体结合的蛋白PKA量却增加。除此之外,我们还进一步分析了Ca2+依赖的蛋白激酶途径发现RyR-2是钙神经素(calcineurin)、细胞外信号调节激酶(extracellular signal-regulated protein kinases, ERK)和蛋白激酶B (protein kinase B, Akt)的调节因子,而在Ca2+/钙调蛋白依赖蛋白激酶Ⅱ(Ca2+/calmodulin-dependent protein kinaseⅡ, CaMKⅡ)途径中影响不大。由此可见,RyR-2受体的缺失破坏了细胞内钙稳态、增加心肌细胞凋亡而减少自噬、影响Ca2+相关蛋白激酶的活性从而减弱了心肌细胞对压力负荷增加所产生的重构改变,影响心脏功能,参与心力衰竭的发生。第一部分：RyR-2基因敲除对压力超负荷后心肌肥厚发生和心功能的影响目的：通过敲除RyR-2基因探讨该基因对心肌细胞内Ca2+稳态的影响并观察RyR-2基因敲除小鼠在压力超负荷情况下心肌肥厚的发生情况。方法：应用Langendorff装置体外灌流的方法获得并培养成年RyR-2+/+和RyR-2+/-小鼠心肌细胞,用AngⅡ作为刺激因素之一,3-4μmol/L fluo-3 AM孵育标记心肌细胞内Ca2+,在波长485nm时,分别激发心肌细胞荧光。应用荧光显微镜,经过530 nm带通滤波,观察激发出的荧光,并收集数据用来反应细胞内钙离子的浓度。,用0.25HZ电场刺激2min诱发心肌细胞收缩,随后将10 mmol/L咖啡因加入细胞中以诱导细胞内钙贮存的释放,分析细胞内钙瞬变。实验分RyR-2+/+小鼠组和RyR-2+/-组。分别将RyR-2+/+小鼠和RyR-2+/-小鼠随机分为TAC(Transverse aorta constriction)组和Sham组,分别给予缩窄升主动脉或假手术,三周后行心脏超声和新导管检查评价小鼠心脏结构和功能改变。HE染色和V-G染色分别观察心室重构和纤维化情况。结果：1)RyR-2基因敲除后,心肌细胞内钙瞬变幅度、收缩期胞内Ca2+浓度升高幅度、缩短分数百分率(FS%)等反应心脏收缩功能指标均明显降低。2)咖啡因诱导细胞收缩后细胞半舒张期时间在两组之间无明显变化。3)生理条件下,wT小鼠和RyR-2+/-小鼠的心脏无论在结构和收缩功能上均没有明显差别。4)TAC三周后,无论是wT小鼠还是RyR-2+/-小鼠均发生心肌肥厚,但二者之间肥厚程度有显著差别。与WT小鼠i相比,RyR-2+/-小鼠肥厚程度明显减轻。5)TAC三周后,与WT小鼠相比,RyR-2+/-小鼠的室间隔厚度(IVSTd)、左室射血分数(LVEF)、dP/dtmax著减少而,左室舒张末压力增高。6)组织化学结果显示TAC术后,与WT小鼠相比,RyR-2+/-小鼠心肌细胞增大程度、肌纤维增粗和纤维化程度均减弱。7) AngⅡ刺激并没有引起收缩期或舒张期细胞内Ca2+水平的改变。结论：1)RyR-2基因敲除后,小鼠心肌细胞内Ca2+稳态发生明显改变,提示RyR-2基因可能参与Ca2+介导的心肌肥厚的发生。2)尽管在生理条件下RyR-2之基因敲除无明显心脏异常,但在RyR-2敲除动物中压力负荷增引起的心肌肥厚明显减弱。第二部分：RyR-2基因对心肌肥厚相关基因和Ca2+调节蛋白影响目的：观察RyR-2基因敲除后,压力负荷增加时心肌肥大标志基因和钙调控蛋白变化情况。方法：RyR-2+/+小鼠和RyR-2+/-小鼠行TAC术,2天和三周后分别获取标本,应用northern blotting方法检测肥大基因ANP、BNP、钙离子通道蛋白LCC、SERCA2、NCX的基因表达情况；应用免疫共沉淀方法观察RyR-2结合蛋白FKBP、PKA等量的改变。同时假手术组作为对照。结果：1)TAC引起ANP和BNP基因表达增高,TAC引起ANP和BNP基因表达增高在RyR-2+/-小鼠组中程度有所减轻2)TAC2天和三周后RyR-2+/+小鼠α-SKA表达增高水平一致,而RyR-2+/-小鼠中TAC3周后α-SKA出现更明显的增高。3)TAC诱导RyR-2+/+小鼠RyR-2和SERCA2基因表达下调而LCC和NCX基因表达上调。TAC诱导RyR-2+/-小鼠NCX因表达下调而SERCA2和LCC基因表达不变4)基础条件F,与RyR-2结合的FKBP的量在RyR-2+/-小鼠有所减少,而与RyR-2结合的PKA的量却呈现出增加。TAC 3周后,与RyR-2结合的FKBP的量在WT和RyR-2+/-小鼠中均减少,与RyR-2结合的PKA的量均增加。结论：RyR-2基因敲除后影响了压力负荷增加所致肥厚基因的表达并引起钙通道蛋白表达异常。第三部分：RyR-2基因敲除减弱肥大信号传导途径的激活目的：观察TAC后WT和RyR-2+/-小鼠心脏中不同肥大信号途径中激酶如CaMKⅡ、钙神经素(calcineurin)、ERK和AKT的表达变化。方法：手术和分组同前,获取小鼠心脏组织标本,应用western blotting的方法检测CaMKⅡ、calcineurin、ERK和AKT蛋白表达；体外培养心肌细胞以不同浓度AngⅡ刺激,检测CaMKⅡ和calcineurin活性改变。结果：1)AngⅡ以剂量依赖方式迅速激活CaMKⅡ, CaMKⅡ活性显著增加；而对calcineurin, AngⅡ并没有显著激活作用。2)WT小鼠中压力超负荷增加CaMKⅡ和calcineurin表达；RyR-2+/-小鼠中压力超负荷引起CaMKⅡ表达增加而calcineurin途径无明显激活。3)WT小鼠中TAC 3周后显著激活ERK、AKT通路而RyR-2+/-小鼠两条通路无明显激活改变。结论：敲除RyR-2基因减弱压力超负荷引起肥大反应是通过影响calcineurin信号途径实现的。第四部分：RyR-2基因敲除后压力超负荷引起心功能减退的可能机制研究目的：从凋亡、自噬及血管发生情况方面分析RyR-2基因可能的影响心功能的机制。方法：临床收集5例心脏组织标本,分为心衰组和正常对照组,分析RyR-2基因表达,动物分组及手术方法同前,获取心脏标本,组织切片染色,TUNEL方法检测心肌细胞凋亡情况,免疫染色和Western Blotting方法检测细胞自噬和微血管计数情况。结果：1)衰竭心脏中RyR-2基因表达降低,降低程度与心功能状态正相关。2)RyR-2+/-小鼠中TAC后,心肌细胞凋亡较RyR-2+/+明显增加。3)RyR-2+/-小鼠中TAC后,心肌细胞自噬较RyR-2+/+明显增加。4)RyR-2+/-小鼠中TAC后,血管生成较RyR-2+/+无明显改变。结论：RyR-2基因敲除后压力负荷增加引起心功能减退可能与心肌细胞凋亡增多、自噬减少有关,而与血管生成无明显关系。更多还原

【Abstract】 Ryanodine receptor type 2 (RyR-2) mediates Ca2+ release from sarcoplasmic reticulum (SR) and plays a key role in the pathogenesis of contractile dysfunction that is associated with altered intracellular Ca2+ handling. However, the role of RyR-2 in the development of cardiac hypertrophy remains unclear. Here, mice with reduction of RyR-2gene (RyR-2+/-) and their littermate wild type (WT) mice were analyzed. At baseline, there was no difference in cardiomyocyte size, heart morphology and cardiac contractile function between WT and RyR-2+/- mice, although Ca2+ release from SR was impaired in isolated RyR-2+/- cardiomyocytes. When these animals were subjected to the pressure overload stimulation, comparing to WT ones, RyR-2+/- mice exhibited attenuated cardiac hypertrophy, myocardial fibrosis and contractile functions associated with increased apoptosis and decreased autophagy of cardiomyocyte, but unrelated to the impaired cardiac angiogenesis. Pressure overload-induced increases in activation of calcineurin, extracellular signal-regulated protein kinases (ERKs) and protein kinase B/Akt in WT heart was disappeared in RyR-2+/- mice, but that of Ca2+/calmodulin-dependent protein kinase II (CaMKII) was similar between RyR-2+/- and WT mice, suggesting that RyR-2 is a regulator for calcineurin, ERK and Akt but not for CaMKII during pressure overload. Although the detailed mechanism is unclear, our data indicate that RyR-2 contributes to the development of cardiac hypertrophy and adaptation of cardiac function during pressure overload through regulation of Ca2+ handing, activation of calcineurin, ERKs and Akt and cardiomyocyte survival. PART 1:Reduction of RyR-2 gene results in altered Ca2+ handling in adult cardiac myocytes and attenuated cardiac hypertrophy and contractility in response to pressure overloadObjective:To examined the impact of heterozygous RyR-2 gene disruption on Ca2+ handling and contractility in isolated adult cardiac myocytes and to evaluate cardiac morphology and contractile function of WT and RyR-2+/- mice after TAC by echocardiography and catheterization.Method:Adult cardiomyocytes were isolated from mice by Langendorff perfusion method. The intracellular Ca2+ transients were electrically ctivatedat0.25Hz,stabilized within 2 min, and then 10 mmol/L caffeine was rapidly applied to the cardiomyocytes just afte the pacing was turned off. The cells were excited by UV light of 485 nm, and the emission of 530 nm was collected. TAC was performed on WT and RyR-2+/- mice for 2 days and 3 weeks respectively. The right carotid blood pressure was measure and the thickness of left ventricular wall and heart function were measured by echocardiography. H-E and V-G staining analysis were used to reveal the myofiber size and the extent of fibrosis.Results:1)The amplitude of Ca2+ tntracellular Ca2+ concentration during systole, and percentile fractional shortening were significantly reduced in myocytes isolated from RyR-2+/- mice compared to those isolated from WT mice.2) The half time of relaxation from caffeine-induced contracture was comparable between WT and RyR-2+/- myocytes.3)There was no significant difference in dimension and contractility between WT and RyR-2+/(?) mice.4) 3 weeks after TAC, thickness of interventricular septum in diastole (IVSTd) and LV ejection fraction (LVEF) in RyR-2+/- mice were significantly reduced compared to those of WT mice and hemodynamic studies revealed increased LVEDP、decreased dP/dtmax in RyR-2+/- mice. 5) Morphological studies showed heart weight/body weight ratio of RyR-2+/- mice 3 weeks after TAC was significantly smaller than that of WT mice.6) Histological analysis revealed that myofiber size and the extent of fibrosis were significantly reduced in RyR-2+/- mice compared to WT mice.The size of isolated cardiomocytes was comparable between WT and RyR-2+/- cells at baseline but was significantly reduced in RyR-2+/- cells compared to WT cells after TAC.7) AngⅡ(10 nmol/L) could not induce increases in cytosolic free Ca2+ levels at both the systolic and diastolic phases in cultured cardiomyocytes of neonatal rats.Conclusion:1) The heterozygous deletion of RyR-2 gene resulted in altered Ca2+ handling in isolated cardiac myocytes.2) These observations suggest that, although there is no apparent cardiac phenotype at baseline, heterozygous deletion of RyR-2 gene resulted in attenuated cardiac hypertrophy and impaired contractility in response to pressure overload.PART 2:Reduction of RyR-2 gene results in the alteration of hemodynamic load-responsive gene expression programObjective:To examine induction of hypertrophic responses of the heart to hemodynamic overload in the heart at 2 days and 3 weeks after TAC.Method:Mice were divided in to WT and RyR-2+/- groups. TAC and sham operation was performed respectively. The strategy of northern blotting was used to test the fetal-type cardiac genes such as ANP, BNP and a-SKA and also the expression of genes encoding Ca2+ handling proteins such as SERCA2, LCC, NCX.Results:1) TAC-mediated induction of ANP and BNP genes was downregulated whereas induction of a-SKA gene was not altered at day 2 and enhanced 3 weeks after TAC in the heart of RyR-2+/- mice.2) In WT hearts,3 weeks of pressure overload induced downregulation of RyR-2 and SERCA2 genes and upregulation of LCC and NCX genes.3) In RyR-2+/- hearts, the expression levels of SERCA2 and LCC genes were not altered whereas that of NCX gene was downregulated.4) Association of FKBP with RyR-2 was further decreased and phosphorylation of RyR-2 by PKA was further increased in the WT heart compared to WT hearts after TAC.Conclusion:1) Pressure overload-responsive gene expression program is impaired or altered by heterozygous deletion of RyR-2 gene.2) Hypertrophic responses of the gene program regulating the expression of Ca2+ handling proteins are also altered by heterozygous deletion of RyR-2 gene.PART 3:Reduction of RyR-2 gene results in the attenuated activation of hypertrophic signaling pathways in response to pressure overloadObjective:To examine whether the load-induced activation of hypertrophic signaling pathways are impaired in RyR-2+/- hearts.Method:Since pressure overload-induced cardiac hypertrophy is attenuated in RyR-2+/- mice and CaMKII and calcineurin are critical regulators of Ca2+ -mediated cardiac hypertrophy, histochemical staining. The expression of CaMKⅡ、calcineurin、ERK and AKT in myocardium were detected by Western blotting.Results:1) Although AngⅡrapidly (at 1 min) increased activities of CaMKII in a dose-dependent manner, it could not induce an increase in calcineurin activation.2) Pressure overload activated CaMKII and calcineurin in WT hearts.3) Activation of calcineurin in response to pressure overload was impaired in RyR-2+/- hearts whereas the activation of CaMKII was comparable between WT and RyR-2+/- hearts. 4) TAC for 3 weeks induced robust activation of ERK1/2 and Akt in WT hearts but not in RyR-2+/- hearts.Conclusion:These observations indicate that heterozygous deletion of RyR-2 gene led to the attenuated activation of hypertrophic signaling pathways in response to pressure overload, which may account for the reduced growth of the heart in RyR-2+/ mice after TAC.PART 4:Reduction of RyR-2 gene results in increased apoptosis and decreased autophagy but unchanged angiogenesis in pressure overloaded heartObjective:To find out the possible additional mechanisms that contribute to impaired cardiac function in RyR-2+/- mice independently of the alteration in Ca2+ homeostasisMethod:Human failing hearts were obtained from 3 end-stage heart failure patients with dilated cardiomyopathy (DCM). Two donor hearts that could not be transplanted for technical reasons were used for controls. The expression of RyR-2 at the mRNA level was evaluated using RT-PCR. Mice were divided in to RyR-2+/+ and RyR-2+ groups. TAC and sham operation was performed respectively.The density of capillaries in the myocardium was examined by CD31 immunostaining, and the number of CD31-positive microvessels per 100 cardiomyocytes was calculated. Apoptotic death of cardiomyocytes was detected in situ by TUNEL kit in paraffin-embedded heart tissue sections. Autophagy of cardiomyocytes was evaluated by LC3b immunostaining on paraffin sections. Immunostaining was performed using rabbit anti-LC3b antibody after minimal antigen retrieval.Results:1) Comparing with controls, the DCM hearts showed a decrease of RyR-2 gene expression, and the decrease seemed to be more significant in patients with worse contractile functions.2) At baseline condition, the number of apoptotic cardiomyocytes in the heart as evidenced by TUNEL positivity was comparable between WT and RyR-2+/- mice, however, after chronic pressure overload, cardiomyocyte apoptosis in RyR-2+/- heart was significantly increased compared to that in WT hearts.3) The extent of autophagy in the myocardium after pressure overload as evidenced by LC3b positive dots or increased expression of beclinl was attenuated in RyR-2+/-hearts compared to that inWT hearts.4) The number of capillaries and the expression of vascular endothelial growth factor (VEGF) were not altered between WT and RyR-2+/- hearts after chronic pressure overload.Conclusion:1) The heterozygous deletion of RyR-2 gene led to increased myocyte apoptosis after chronic pressure overload.2) The heterozygous deletion of RyR-2 gene led to decreased autophagy in the heart after chronic pressure overload.3) The myocardial ischemia does not contribute to contractile dysfunction of RyR-2+/- hearts after pressure overload.更多还原