【作者】 张弋；

【导师】 陈敏生；

【作者基本信息】 广州医学院 ， 内科学， 2008， 博士

【摘要】 【背景】左心室肥厚(LVH)是高血压最常见的并发症,它是以心肌细胞肥大为主要特征的一系列心脏结构重建,LVH既是导致心力衰竭的危险因素,也是心血管疾病预后的独立预测因子。因此,心肌肥厚机制的研究一直是心血管病领域的热门研究课题。胞内游离钙是体内最广泛、最重要的信使物质之一,在细胞信号网络中处于枢纽地位,通过其在浓度、时相及空间位置上的变化,编码和传递外来刺激信息,参与生命活动的信息传递与调控,包括细胞收缩功能、节律变化、生长死亡等。钙信号参与心肌细胞肥大的发生、发展及功能改变。神经肽Y(NPY)是脑和心脏组织内表达最丰富的肽类物质,作为神经递质与去甲肾上腺素(NE)共存于交感神经末稍。NPY对心脏的作用分为短效及长效两部分。短效作用即作为神经递质对心脏节律、收缩、传导的急性作用,一般发生在数秒或数分钟之内;长效作用是指NPY具有生长因子样作用,在刺激数小时或数天后,可诱导心肌细胞肥大、血管平滑肌增殖等。两个效应均与钙信号相关,但两者之间是否存在相互影响或因果关系,目前还不清楚。以往有关NPY对细胞内钙信号的研究大多是急性试验,即观察NPY刺激即刻的效应。研究发现,NPY使钙瞬变幅度增加的作用在4～5min后消失,由此推断NPY对心肌钙活动以及收缩力的影响只是暂时的。而我们的前期研究提示NPY对钙信号的作用是持久的,并与心肌细胞肥大效应相关。因此,有必要探讨较长时间NPY刺激对钙活动及稳态的作用,及其与心肌细胞肥大的关系。肌浆网钙调控是心肌细胞钙动态活动的核心环节,其中最重要的调控蛋白是RyR2, SERCA2a,分别控制钙的释放和摄取;CASQ2,PLB是两个重要辅助蛋白,对SR的钙调控也有重要意义。NPY是否通过影响上述蛋白而改变细胞钙活动,目前还未见报道。近年来,钙/钙调素依赖蛋白激酶Ⅱ(CaMKⅡ)在心血管系统中的作用广受关注,它可以通过磷酸化作用调节SR钙调控蛋白的活性,影响SR钙的释放和摄取。NPY对心肌细胞钙活动的作用是否与CaMKⅡ有关,目前还不清楚。因此,本课题用NPY诱导心肌细胞肥大,观察细胞内钙瞬变、钙分布、SR内钙调控系统的改变以及CaMKⅡ在其中的作用,进一步探讨心肌细胞肥大发病机理。第一部分NPY诱导心肌细胞肥大中钙信号的改变1研究目的用NPY诱导心肌细胞肥大,观察肥大心肌细胞胞浆钙瞬变(反映胞内钙的动态变化)及钙分布(反映胞内钙的静态变化)的变化,以探讨钙信号的变化在NPY诱导心肌细胞肥大效应中的作用。2研究方法2.1新生大鼠原代心肌细胞培养,将细胞分为2组(n=6):ⅰ对照组:培养液中不加任何刺激药品。ⅱNPY组:培养液中加入终浓度为100 nmol/L的NPY。2.2用NPY刺激心肌细胞24h后,应用3氚-亮氨酸(3H-Leu)掺入量法测定心肌细胞蛋白质合成速率、荧光定量聚合酶链法(FQ-PCR)测心肌细胞胚胎期基因ANF以及β-MHC mRNA的表达。2.3用NPY刺激心肌细胞24h后,以Fluo-4 AM和Fluo-5N AM分别标记胞浆游离钙和肌浆网游离钙,激光共聚焦显微镜记录心肌细胞钙影像:静息状态下胞浆以及肌浆网内游离钙离子浓度、场刺激诱发的胞浆钙瞬变(Ca2+ transient)以及咖啡因诱导的胞浆钙瞬变(Caffeine-induced Ca2+ transient,CCT)。3结果3.1 NPY组心肌细胞3H-Leu掺入量高于对照组( P< 0.05)。3.2 NPY组心肌细胞ANF以及β-MHC mRNA的表达较对照组上调( P< 0.05)。3.3场刺激诱导下NPY组心肌细胞胞浆钙瞬变幅度高于对照组(P<0.05),NPY组钙瞬变恢复时间短于对照组(P< 0.01),但钙瞬变上升时间两无显著差别(P>0.05)。3.4与对照组相比,NPY组心肌细胞胞浆游离钙浓度增高( P< 0.05);3.5与对照组相比,NPY组心肌细胞肌浆网内游离钙含量降低(P < 0.05);肌浆网内总钙容量有低于对照组的趋势,但其差异无统计学意义(P>0.05)。4小结4.1 NPY刺激可诱导心肌细胞肥大效应,表现为心肌细胞蛋白合成速率增加、胚胎期基因ANF以及β-MHC mRNA表达上调。4.2 NPY刺激可显著影响兴奋-收缩耦联过程中钙的动态活动,即胞浆钙瞬变幅度增加、恢复时间明显缩短。提示NPY刺激可使心肌细胞收缩力增强,并可能与心肌细胞肥大时心脏泵功能增强有关。4.3 NPY刺激可导致心肌细胞内游离钙出现空间重分布的变化,即胞浆钙浓度增高而肌浆网内钙负荷减少。胞浆钙浓度增高与钙依赖的肥大信号途径活化有关;肌浆网内钙负荷减少则可能导致细胞从肥大向衰竭方向转变。第二部分NPY诱导心肌细胞肥大中肌浆网钙调控系统的作用1研究目的观察NPY对肥大心肌细胞肌浆网(SR)内钙调控蛋白(Ca2+ -ATP酶、ryanodine受体、CASQ和PLB)的作用。2研究方法2.1新生大鼠心肌细胞培养,分组同第一部分。2.2用NPY刺激心肌细胞24h后,应用FQ-PCR法测定心肌细胞SR内钙调控相关蛋白:Ca2+ -ATP酶(SERCA2a)、ryanodine受体(RyR2)、集钙蛋白(CASQ)以及受磷蛋白(PLB)mRNA的表达。2.3用NPY刺激心肌细胞24h后,应用免疫印迹分析法(Western-blot)测定心肌细胞SR内钙调控相关蛋白:Ca2+ -ATP酶(SERCA2a)、ryanodine受体(RyR2)蛋白含量的变化。2.4用NPY刺激心肌细胞24h后,应用免疫荧光法同步检测SERCA2a和RyR2的分布及量的变化。3结果3.1与对照组相比,NPY组心肌细胞SERCA2a、RyR2、CASQ以及PLB mRNA的表达上调( P< 0.05)。3.2免疫印迹结果显示,NPY组心肌细胞SERCA2a、RyR2蛋白表达量较对照组上调( P< 0.05)。3.3免疫荧光法结果显示, SERCA2a、RyR2在心肌细胞内分布均匀,NPY刺激后两者表达同步上调,高于对照组( P< 0.05)。4小结NPY刺激后肌浆网主要的钙调控蛋白SERCA2a、RyR2水平同步升高,辅助调节蛋白CASQ、PLB mRNA表达也上调。这种改变对肌浆网钙释放和摄取均有刺激作用,进而导致钙瞬变特征改变。而从心肌细胞内钙分布的变化推断,NPY对两者的作用可能存在不平衡。第三部分钙调素依赖的蛋白激酶II参与NPY诱导的钙调控异常1研究目的观察NPY对钙调素(Ca2+ /CaM)依赖蛋白激酶II(CaMKⅡ)的活化效应,以及CaMKⅡ在NPY诱导钙调控变化中的作用。2研究方法2.1新生大鼠心肌细胞培养,将细胞分为3组(n=6):ⅰ对照组:培养液中不加任何刺激药品。ⅱNPY组:培养液中加入终浓度为100 nmol/L的NPY。ⅲKN- 93(CaMKⅡ特异性抑制剂)组:培养液中加入NPY(100 nmol/L)+KN-93(10μmol/L)。2.2心肌细胞CaMKⅡ酶活性的测定。2.3激光共聚焦显微镜记录静息状态下心肌细胞胞浆以及肌浆网内游离钙离子浓度。2.4应用FQ-PCR法测定心肌细胞SR内钙调控相关蛋白:Ca2+ -ATP酶(SERCA2a)、ryanodine受体(RyR2)、集钙蛋白(CASQ)以及受磷蛋白(PLB)mRNA的表达。2.5应用蛋白印迹法(Western-blot)测定心肌细胞SR内钙调控相关蛋白:Ca2+ -ATP酶(SERCA2a)、Ryanodine受体(RyR2)蛋白含量的变化。2.6应用免疫荧光法检测SERCA2a和RyR2的分布及量的变化。3结果3.1 NPY组心肌细胞CaMKⅡ酶活性较对照组增高( P< 0.05),而KN-93组的CaMKⅡ酶活性与对照组则无差异( P>0.05)。3.2 NPY组心肌细胞胞浆游离钙浓度高于对照组,给予KN-93干预后,心肌细胞胞浆游离钙浓度则下降( P< 0.05);而NPY组心肌细胞肌浆网内游离钙含量低于对照组(P < 0.05);给予KN-93干预后,心肌细胞肌浆网游离钙浓度则出现上升,但差异无统计学意义( P>0.05)。3.3与对照组相比,NPY组心肌细胞SERCA2a、RyR2、CASQ以及PLB mRNA的表达上调( P< 0.05);而KN-93可减轻上述作用( P< 0.05)。3.4与对照组相比,应用Western-blot检测显示NPY组心肌细胞SERCA2a、RyR2蛋白表达量上调( P< 0.05);而KN-93可减轻上述作用( P< 0.05)。3.5与对照组相比,应用免疫荧光法检测显示NPY组心肌细胞SERCA2a、RyR2蛋白表达量上调( P< 0.05);而KN-93可减轻上述作用( P< 0.05)。4小结4.1 NPY刺激可以活化心肌细胞内CaMKⅡ。4.2活化CaMKⅡ通过调整SR钙调蛋白SERCA2a、RyR2、CASQ以及PLB的表达,进而调控SR的钙转运。4.3 CaMKⅡ介导NPY刺激下的心肌细胞内钙重分布,最终启动心肌细胞肥大相关的信号转导途径。全文结论NPY刺激24h后,心肌细胞发生肥大反应,并伴随显著的钙信号改变,具体如下:1.NPY刺激导致心肌细胞兴奋-收缩偶联中钙瞬变特征改变,这与心肌细胞肥大时心肌收缩力增强有关。2.NPY刺激导致心肌细胞内游离钙出现空间重分布的变化。其中,胞浆钙浓度增高与钙依赖的肥大信号途径活化有关;肌浆网内钙负荷减少则可能导致细胞从肥大向衰竭方向转变。3.NPY刺激后肌浆网主要的钙调控蛋白SERCA2a、RyR2水平同步升高,辅助调节蛋白CASQ、PLB mRNA表达也上调。4. NPY刺激可以活化心肌细胞内CaMKⅡ;CaMKⅡ是上述钙信号变化的一个关键调控位点。更多还原

【Abstract】 BackgroundLeft ventricular hypertrophy(LVH) is the most complication in patients with hypertension. Cardiomyocyte hypertrophy is the main feature of a series of cardiac structure remodeling. Cardiomyocyte hypertrophy is not only the risk factor of heart failure but also a marker for determining the prognosis of cardiovascular diseases. Mechanism of cardiomyocyte hypertrophy has been one of the most popular research topicses in cardiovascular disease field .Intracellular calcium is one of the most extensive and important messengers in vivo. It plays a central role in network of cell signal to regulate information of activities of life, including systolic function, rhythm changes, cell growth and death.Calcium signal codes and transmits external stimulation information through the changes of calcium concentration, spatial distribution and phase. Calcium signal involved in the occurrence and development of cardiomyocyte hypertrophy.Neuropeptide Y(NPY) is the most abundant peptide in the mammalian heart and brain. NPY is released from sympathetic neurons with norepinephrine. NPY exerts an acute action as a neurotransmitter on cardiac function that occurs over a period of seconds or minutes. On the other hand, NPY plays long-term effect (defined as several hours or days) as a trophic factor responsible for cardiac hypertrophy and angiogenesis. Both of them are closely related to calcium signal. However, the relationship and mutual influence of them are poorly understood.The most of researchs about NPY’s effect on calcium signal were acute test.It was suggested that the effects of NPY on calcium signal and cardiac contractility were temporary,as the enhance of calcium transcient was disappeared 4~5min after NPY stimulation. However, in our previous study, NPY had sustained effect on calcium signals and correlated with cardiomyocyte hypertrophy. Therefore, it is necessary to explore the alterations of calcium activity and homeostasis indued by sustained NPY stimulation and the relationships with cardiac hypertrophy.Sarcoplasmic reticulum (SR) Ca2+ handling is central in intracellular Ca2+ homeostasis. The Ca2+ handling protein RyR2 and SERCA2a,control the release and uptake of calcium respectively. The coupling of auxiliary proteins PLN and CASQ2 seem to mediate the functions of calcium handling. However, the alteration of SR Ca2+ handling in hypertrophic myocytes induced by NPY remains unclear. CaMKⅡplays a important role in cardiovascular system, which regulats SR calcium handling protein activity through phosphorylation and influences on SR calcium release and uptake. It is unclear that the role of CaMKⅡplays in the alteration of SR Ca2+ handling in hypertrophic myocytes induced by NPY.This study was the first investigation about above questions. We explored Ca2+ mobilizations, distribution, SR calcium handling and CaMKⅡin hypertrophic cardiomyocytes induced by 24 hr stimulation of NPY. All of them were to explore the relating mechanism for cardiac hypertrophy and find effective drug prevention.Part one The alteration of calcium signals in hypertrophic cardiomyocytes induced by NPY1 ObjectivesTo investigate the changes of distribution of intracellular calcium and calcium transient in hypertrophic cardiomyocytes induced by NPY.2 Methods2.1 Cardiomyocytes of neonatal Sprague-Dawley rats were incubated with NPY 100 nmol/L for 24 h. The cardiomyocytes were randomly divided into two groups(n=6) :ⅰcontrol group :Without any treatment ;ⅱNPY group:treated with 100 nmol/L NPY.2.2 24 hours after incubation with NPY, the protein synthesis rate was determinded by 3H-Leu incorporation, the ANF andβ-MHC mRNA expression were determinded by FQ-PCR..2.3 Fluorescent indicator Fluo-4 AM was used to detect [Ca2+] in plasma and calcium transient. Fluo-5N AM was used to detect [Ca2+] in sarcoplasmic reticulum .Calcium images were recorded by laser scanning confocal microscope. The SR Ca2+ load was estimated by Caffeine-induced Ca2+ transient(CCT).3 Results3.1 Compared with control group, 3H-Leu incorporation was significantly elevated in NPY group (P<0.05).3.2 Compared with control group, the ANF andβ-MHC mRNA expression were significantly elevated in NPY group (P<0.05).3.3 Compared with control group, the concentration of free Ca2+ in plasma([Ca2+]i) was significantly elevated in NPY group (P<0.05); and the concentration of free Ca2+ in SR([Ca2+]SR) was significantly decreased in NPY group (P<0.05).3.4 Under the field stimulation ,the evoked Ca2+ transient was of higher amplitude and of faster decay in the presence of NPY(P< 0.01).The time-to-peak-Ca2+ was no significant change.The peak of CCT was slightly attenuated by NPY(P).4 Brief summary4.1 NPY(100 nmol/L) can induce hypertrophy of cardiomyocytes, which appeared upregulation of ANF andβ-MHC mRNA expression and the increasing of myocardial protein synthesis rate.4.2 NPY stimulation can significantly impact excitation-contraction coupling process of dynamic activity of calcium, namely the enhancement of amplitude and the decrease of decay time in Ca2+ transient, which can contributed to promote cardiomyocytes contractility and was related with enhancement pumping function in cardiomyocyte hypertrophy.4.3 NPY stimulation caused redistribution of free calcium in cardiomyocytes, namely the elevation in [Ca2+]i and decline in [Ca2+]SR. The elevation in [Ca2+]i may activate intracellular signaling pathways responsible for cardiac hypertrophy. Reduce SR calcium content can occur during the development of cardiac failure.Part two Effects of sarcoplasmic reticulum Ca2+ handling in hypertrophic cardiomyocytes induced by NPY1 ObjectivesTo investigate the effects of NPY on sarcoplasmic reticulum Ca2+ handling in hypertrophic cardiomyocytes..2 Methods2.1 Cardiomyocytes of neonatal Sprague-Dawley rats were incubated with NPY 100 nmol/L for 24 h. The Cardiomyocytes were randomly divided into two groups(n=6) :ⅰcontrol group :Without any treatment ;ⅱNPY group:treated with 100 nmol/L NPY.2.2 24 hours after incubation with NPY, SERCA2a, RyR2, PLB and CASQ2 mRNA expression were determinded by FQ-PCR..2.3 The changes of the expression of SERCA2a and RyR2 were detected with Western blot and double immunofluorescence method.3 Results3.1 Compared with control group, SERCA2a, RyR2, PLB and CASQ2 mRNA expression were significantly elevated in NPY group (P<0.05).3.2 According to the analyze of Western blot, SERCA2a and RyR2 protein expression in NPY group were significantly elevated compared to control group (P<0.05).3.3 By the detection of double immunofluorescence, SERCA2a and RyR2 protein expressions in NPY group were elevated sychronously compared to control group (P<0.05). 4 Brief summaryBy sustained stimulation of NPY, the levels of Serca2, RyR2 in myocytes were elevated synchronically, accompanied with enhances of CASQ2 and PLN mRNA. These alterations were consistent with the changes of calcium transcient. However, the promotions of Ca2+ release and Ca2+ uptake in SR by NPY could not be imbalanced.Part three CaMKⅡparticipates in alteration of Ca2+ handling induced by NPY1 ObjectivesTo investigate the activation of CaMKⅡin hypertrophic cardiomyocytes induced by NPY and its roles in sarcoplasmic reticulum Ca2+ handling.2 Methods2.1 Cardiomyocytes of neonatal Sprague-Dawley rats were incubated with NPY 100 nmol/L for 24 h. The Cardiomyocytes were randomly divided into two groups(n=6) :ⅰcontrol group :Without any treatment ;ⅱNPY group:treated with 100 nmol/L NPY;ⅲKN- 93group:Co-treated with NPY+KN-93(10μmol/L)。2.2 The activity of CaMKⅡwas determined.2.3 Fluorescent indicator Fluo-4 AM was used to detect [Ca2+] in plasma and calcium transient. Fluo-5N AM was used to detect [Ca2+] in sarcoplasmic reticulum .Calcium images were recorded by laser scanning confocal microscope.2.4 SERCA2a, RyR2, PLB and CASQ2 mRNA expression were determinded by FQ-PCR..2.5 The changes of the expression of SERCA2a and RyR2 were detected with Western blot and immunofluorescence method.3 Results3.1 Compared with control group, the activity of CaMKⅡwas elevated in NPY group (P<0.05) and the effect was blunted by KN-93,a inhibitor of CaMKⅡ.3.2 Compared with control group, the concentration of free Ca2+ in plasma([Ca2+]i) was significantly elevated in npy group(P<0.05), the concentration of free Ca2+ in SR([Ca2+]SR) was significantly decreased in npy group (P<0.05) and the effects were attenuated by KN-93.3.3 Compared with control group, SERCA2a, RyR2, PLB and CASQ2 mRNA expression were significantly elevated in NPY group (P<0.05) and the effects were blunted by KN-93.3.4 SERCA2a and RyR2 protein expression were the same result of mRNA expression.4 Brief summary Conclusions4.1 NPY activated CaMKⅡin cardiomyocytes.4.2 The activated CaMKⅡcan influence calcium mobilizations in SR by regulating calcium handling proteins in SR, including SERCA2a,RyR2 , PLB and CASQ2.4.3 CaMKⅡis responsible critically for Ca2+ redistribution induced by NPY, which can activate intracellular signaling pathways of cardiac hypertrophy.ConclusionsCalcium signals were altered remarkably in hypertrophic cardiomyocytes induced by 24 hr stimulation,including:1.NPY stimulation can significantly impact excitation-contraction coupling process of dynamic activity of calcium, which can contributed to the enhancing pump function in cardiomyocyte hypertrophy.2.NPY stimulation caused redistribution of free calcium in cardiomyocytes. The elevation in [Ca2+]i may activate intracellular signaling pathways responsible for cardiac hypertrophy. Reduce SR calcium content can occur during the development of cardiac failure.3.By sustained stimulation of NPY, the levels of Serca2, RyR2 in myocytes were elevated synchronically, accompanied with enhances of CASQ2 and PLN mRNA. 4. NPY activated CaMKⅡin cardiomyocytes, which takes critical roles in alterations of Ca2+ signal induced by NPY.更多还原