【作者】 王晨；

【导师】 王海昌；

【作者基本信息】 第四军医大学 ， 内科学， 2008， 博士

【摘要】 APJ是1993年发现的7次跨膜的G蛋白偶联受体,其内源性配体由Tatemoto等于1998年发现并命名为Apelin （APJ endogenous ligand）。人APJ受体由380个氨基酸组成,与血管紧张素受体AT1高度相似,跨膜区有54%的氨基酸序列相同。Apelin由77个氨基酸的前体蛋白水解生成不同长度的活性肽段如Apelin-36,Apelin-31,Apelin-17,Apelin-13等。APJ受体及其内源性配体apelin在体内分布广泛,中枢与外周组织均有不同水平的表达,尤其心、脑、肺、血管、脾、肠道、乳腺等器官与组织有高水平表达。有报道称apelin具有强的正性肌力作用,但国内尚无相关研究,其机制也尚不清楚。因此本实验从体外灌流整体心脏水平和单心肌细胞水平研究Apelin对大鼠心脏收缩功能的影响及其作用机制。实验目的:1.证实Apelin对大鼠心肌在整体心脏水平和单细胞水平均具有正性肌力作用。2.证实Apelin对大鼠正性肌力作用的剂量效应关系。3.比较Apelin与其他几种常见正性肌力作用药物之间的异同。4.证明Apelin正性肌力作用的特异性。5.寻找Apelin正性肌力作用的细胞内信号通路。6.证明不同前负荷对Apelin正性肌力作用的影响。7.发现PLC,PKC在Apelin正性肌力作用中的参与机制。8.研究SERCA,RyR,NCX,NHE等蛋白在Apelin作用的信号通路中的位置和作用。实验方法:心脏灌流。断头法处死大鼠,迅速取下心脏行Langendorff逆行灌注Krebs-Henseleit碳酸氢盐缓冲液。心脏收缩力测量。连接一个力位移换能器至心尖部,初始前负荷拉力为2g。60分钟稳定平衡后,记录5分钟的对照期,之后往灌流液中加入各种试剂记录30分钟（0.5ml/min）。首先测量Apelin-16的浓度效应曲线（0.01-1nmol/L）。之后比较Apelin与内皮素-1,肾上腺髓质素,以及β-肾上腺素能受体激动剂异丙肾上腺素作用效果之间的差异。研究Apelin作用的特异性,灌流液中加入了多种受体的拮抗剂。所选浓度为所报道的离体心脏有效阻断浓度。研究内源性NO在大鼠心脏正性肌力作用中的角色,灌流液中添加了L-NAME,300μmol/L的L-NAME能够有效地阻断心肌中的一氧化氮合酶。信号通路研究。灌流液中加入PLC抑制剂PKC抑制剂NHE抑制剂和NHE-1抑制剂,反向NCX抑制剂。等容心脏灌流研究。为了研究Apelin对不同前负荷下的心脏的收缩力的影响,进行了离体等容心脏灌流实验。等容左室压力的测量是通过使用充液球囊置于左心室内并连接压力传感器从而完成。20分钟的平衡稳定后加入Apelin （1 nmol/L）或者对照液灌流30分钟。前10分钟内球囊是松的,之后球囊以10μL为单位逐渐膨胀使LVEDP达到1,5,10,15mmHg。当收缩数据稳定后记录。心肌细胞的分离。以常规酶解法分离成年大鼠心肌细胞。细胞内钙的测量。将Fura-2/AM （0.5 mmol/L）与心肌细胞避光孵育30 min （25°C）,采用双激发荧光光电倍增系统检测荧光信号。将indo-1/AM（5μmol/L）与心肌细胞避光孵育30 min （25°C）,用新鲜的Hepes溶液漂洗两次,静置15分钟。负载好的细胞置于灌注腔内,灌注腔内液可在100ms内更换。Indo-1荧光应用340nm激光激发,在410nm和516nm波长处测量发射光。用可视化动缘探测系统检测心肌细胞收缩功能。该系统由倒置显微镜、Ion Optix细胞影像适配器、Ion Optix Myc Cam摄像系统及其控制器、Ion Optix光电倍增系统（PMT）、荧光系统控制器（FS1）、Ion Optix光源及其电源、监视器、电子刺激器、程控灌流泵和计算机等部分组成。咖啡因挛缩的测量。咖啡因挛缩引起的钙瞬变C[Ca²⁺]_i不同于电刺激引起的细胞内钙瞬变E[Ca²⁺]_i,C[Ca²⁺]_i的幅度常被用来作为SR Ca²⁺含量的指标。冷挛缩的测量。冷挛缩（Rapid cooling contracture,RCC）可导致心肌细胞内质网（SR）钙的完全释放,而释放出来的大量钙被限制在细胞质内。利用可控灌流器可以迅速更换冰的Tyrode’s溶液并在200ms内将温度降至0–1°C。SERCA活性检测。心肌细胞肌浆网的制备参照经Kodavanti改良Jones等的差速离心方法。采用南京建成生物公司微量ATP酶试剂盒,以比色定磷法测定无机磷含量,以含Ca²⁺与不含Ca²⁺管之差计算SERCA活性。实验结果:1. Apelin（0.01-10nmol/L）可引起离体大鼠心脏剂量依赖性的心肌张力（developed tension,DT）的增高。最大效应剂量点出现在1nmol/L的浓度上,半效浓度（EC50）为33.9±1.8pmol/L。2.与其他几种常见的正性肌力药物相比,Apelin的作用时程为逐渐起效并且保持较长的作用时间≥30min。3. Apelin产生的正性肌力作用并不伴随最大张力产生时间（Time-to-peak tension）的明显变化。4.当LVEDP升高到10及15mmHg后,Apelin能够明显增加dP/dtmax值。5. AT1受体拮抗剂CV-11974对心肌活动张力（DT）无明显影响,并且对1nmol/L的Apelin的正性肌力作用亦无明显影响。灌流液中加入L-NAME对Apelin的正性肌力作用无明显影响。6. PLC的抑制剂U-73122 100nmol/L单独使用对心肌收缩力无明显影响,但与Apelin（1nmol/L）合用时却可以显著降低Apelin的正性肌力作用效果,最多可以减弱其68%的正性肌力作用。7.广谱的蛋白激酶抑制剂Staurosporine,以及特异性PKC抑制剂GF-109203X均可减弱Apelin的正性肌力作用。8. NHE抑制剂MIA（1μmol/L）单独使用对心肌收缩力无影响（P=NS）。但MIA（1μmol/L）能够显著减弱Apelin（1nmol/L）的正性肌力作用最多可达55%（F=12.7,P<0.001）。9.反向NCX的选择性抑制剂KB-R7934（250nmol/L）本身对心肌收缩的基线值无明显影响（P=NS）,但却能够显著减弱Apelin（1nmol/L）作用的60%（F=18.5,P<0.001）。10.在电刺激的情况下Apelin能够升高E[Ca²⁺]_i transients的幅度。11. Apelin能够增强细胞的收缩功能（FS）并且作用持续至少30分钟。12.钙瞬变峰值时间(time to peak,TTP of E[Ca²⁺]_i)被Apelin显著的降低了12.53±3.8%。13.半效衰减时间(T50 of E[Ca²⁺]_i)被Apelin降低了30.82±1.17%。14. Apelin组咖啡因挛缩中测得的SR Ca²⁺ content轻微但却显著的下降了8.41±0.92%。15. Apelin能够显著减少16.22±1.36%的咖啡因挛缩的半效衰减时间(T50 of C[Ca²⁺]_i)。16. Apelin降低了冷挛缩法测得的SR Ca²⁺ content值（n=5, P<0.05）。17. Apelin增强了SR Ca²⁺-ATPase的活性（n=7, P<0.05）。18. PKC抑制剂CHE能够显著减弱Apelin对NCX、SERCA活性的提高作用。PKC抑制剂CHE能够显著减弱Apelin的正性肌力作用。实验结论:1.在大鼠整体心脏灌流水平,Apelin具有剂量依赖的正性肌力作用。2.在大鼠单心肌细胞灌流水平,1nmol/L的Apelin具有正性肌力作用。3.钙瞬变幅度的升高是Apelin正性肌力作用的原因之一。但仍然存在着不依赖钙瞬变幅度升高的可能机制,该机制与PKC的激活有关。4.Apelin能够降低SR钙容量,该作用与PKC的激活有关,其中NCX和SERCA是两个关键的作用蛋白。更多还原

【Abstract】 Regulation of myocardial contractility by endogenous peptides is important in physiological and pathophysiological conditions and may be a crucial therapeutic target.An autocrine or paracrine system which potentially regulates heart function is the recently discovered angiotensin receptor like-1 （APJ） with its endogenous ligand apelin.Apelin and APJ are widely expressed throughout the cardiovascular system. Apelin is actively synthesized by cardiac myocytes, vascular endothelial and smooth muscle cells, and has specific receptors in the heart. Ex vivo studies using isolated perfused rat hearts have identified apelin as one of the most potent inotropic substances yet recognised. Moreover, apelin has been reported to increase left ventricular contractility in vivo following acute as well as chronic infusion in rodents.The APJ receptor and apelin have been implicated in the pathophysiology of human heart failure. Plasma concentration of apelin has been shown to decrease in patients with congestive heart failure and long-term cardiac resynchronization therapy could restore plasma levels of the peptide. Moreover, mechanical ventricular support increases APJ mRNA levels in patients with heart failure. Apelin has also been implicated in the pathophysiology of arrhythmias. Ellinor et al. demonstrated that plasma apelin levels decrease in patients with lone atrial fibrillation. Despite recent advances in our understanding of the cardiovascular effects of the apelin-APJ system in vivo, the direct effects of apelin on cardiomyocyte contractility remain unknown.Aim:1. To find Apelin’s effect on whole heart and isolated myocyte of rat.2. To find the Dose-Effect relationship of Apelin’s positive inotropic action.3. To find the differences between Apelin and other medicines.4. To find the specificity of Apelin’s positive inotropic action.5. To find the signal pathway of intracellular transduction of Apelin.6. To find different pre-load’s effect on Apelin’s positive inotropic action.7. To find the role of PLC and PKC in the mechanism of Apelin’s positive inotropic action.8. To find the role of SERCA, RyR, NCX and NHE in the mechanism of Apelin’s positive inotropinc action.Material and Methods:Rats were decapitated and hearts were quickly removed and arranged for retrograde perfusion by the Langendorff technique as described previously.Heart rate was maintained constant （304±1 beats/min） by atrial pacing using a Grass stimulator （model S88, 11 V, 0.5 ms）. Contractile force （apicobasal displacement） was obtained by connecting a force displacement transducer （Grass Instruments, FT03） to the apex of the heart at an initial preload stretch of 2 g. A 60-minute equilibration period and a 5-minute control period was followed by addition of various drugs to the perfusate by an infusion pump at a rate of 0.5 mL/min for 30 minutes. Initially, we determined the concentration-dependent effect of apelin-16 （0.01 to 1 nmol/L） on cardiac contractility. Next, we compared the effect of apelin to the responses of endothelin-1, adrenomedullin, and theβ-adrenergic receptor agonist isoproterenol. To test the specificity of the effect of apelin, it was infused in the presence of various receptor antagonists. To define the role of endogenous nitric oxide in apelin-induced inotropic response, the peptide was infused in the presence of L-NAME. L-NAME at a concentration of 300μmol/L effectively inhibited nitric oxide synthase in the myocardium.For signal transduction studies, the concentration of U-73122 （100 nmol/L）, staurosporine （10 nmol/L） and GF-109203X （90 nmol/L）, MIA （1μmol/L） and zoniporide （1μmol/L）, and KB-R7943 （250 nmol/L） were selected because these concentrations have been demonstrated to suppress phospholipase C and protein kinase C activity and inhibit Na+/H+ exchange and the reverse mode Ca²⁺/Na+ exchange, respectively.The perfusion technique and the composition of the Krebs-Henseleit bicarbonate buffer was similar to that described above, however, left ventricular contractility was assessed by measuring isovolumic left ventricular pressure. Isovolumic left ventricular pressure was measured using a fluid-filled balloon, which was placed in the left ventricular chamber and connected to a pressure transducer. The balloon was large enough so that negligible pressure resulted, when the balloon alone was filled up to the maximum volume used. The following parameters were obtained: peak systolic left ventricular pressure, left ventricular end-diastolic pressure （LVEDP）, left ventricular developed pressure （DP）, maximum and minimum values of the first derivative of isovolumic pressure （dP/dtmax, dP/dtmin）, time from peak systolic pressure to 60% relaxation （RT 60%）, time from peak systolic pressure to 90% relaxation （RT 90%）. A 20-minute equilibration was followed by addition of apelin （1 nmol/L） or vehicle to the perfusate for 30 minute. During the first 10 minutes of infusion the left ventricular balloon was flaccid, thereafter the balloon was inflated in 10μL steps to achieve a LVEDP of 1, 5, 10 and 15 mmHg. Parameters of left ventricular contractility were obtained when a new steady-state was reached. Myocytes were isolated using standard procedures.We used two different Ca²⁺ indicators and protocols to measure intracellular Ca²⁺. The first: Freshly isolated myocytes were placed on laminin-coated glass coverslips and allowed to attach for 30 minutes before they were loaded with fura 2-AM. The free [Ca²⁺]_i of loaded cardiac myocytes were measured as the fluorescence ratio （360/380nm）. The second: Cells were loaded during 30 minutes with 5μmol/l indo-1/AM and 0.01% pluronic befor each experiment. The field stimulation （0.5Hz） was elicited and indo-1 fluorescence was measured in dual emission mode, excited at 340nm with xenon lamp flashes （100 W）. Dual wavelength emission was measured at 410 and 516 nm, respectively. Shortening of myocytes was simultaneously measured with E[Ca²⁺]_i. Myocytes were perfused with Tyrode’s solution at 22°C and 1 ml/min flow rate. Myocytes were equilibrated at 22°C for about 20 minutes before use. All experimental protocols were carried out at room temperature. The contractileshortening of ventricular myocyte was measured by a video-based motion edge-detection system and an inverted microscope.Caffeine-induced Ca²⁺ (C[Ca²⁺]_i) transient amplitude was used as a measurement of SR Ca²⁺ content.SR Ca²⁺ content was measured by another way since this was the most important observation in the current study. RC causes complete depletion of calcium from SR and calcium released remains confined to the cytoplasm.The activity of Ca²⁺-ATPase was determined with a kit （Jiancheng, Nanjing, China） by measuring the inorganic phosphate （Pi） liberated from ATP hydrolysis.Results:1. In isolated perfused rat hearts, infusion of apelin （0.01 to 10 nmol/L） induced a dose-dependent positive inotropic effect （EC50: 33.1±1.5 pmol/L）.2. Moreover, preload-induced increase in dP/dtmax was significantly augmented （P<0.05） in the presence of apelin.3. Inhibition of phospholipase C （PLC） with U-73122 and suppression of protein kinase C （PKC） with staurosporine and GF-109203X markedly attenuated the apelin-induced inotropic effect （P<0.001）.4. In addition, zoniporide, a selective inhibitor of NHE isoform-1, and KB-R7943, a potent inhibitor of the reverse mode NCX, significantly suppressed the response to apelin （P<0.001）.5. Compared with control, treatment with apelin caused a 55.7±13.9% increase in sarcomere fraction shortening （FS） and a 43.6±4.56% increase in amplitude of E[Ca²⁺]_i transients （n=14,P<0.05）.6. SR Ca²⁺ content measured by caffeine-induced Ca²⁺ (C[Ca²⁺]_i) transient was decreased 8.41±0.92% in response to apelin （n=14,P<0.05）.7. NCX function was increased since half-decay time （T50） of C[Ca²⁺]_i was decreased 16.22±1.36% in response to apelin.8. Sarcoplasmic reticulum Ca²⁺-ATPase （SERCA） activity was also increased by apelin.9. These responses can be partially or completely blocked by chelerythrine chloride （CHE）, a PKC inhibitor.10. In addition, to confirm our data, we used indo-1 as another Ca²⁺ indicator and rapid cooling（RC） as another way to measure SR Ca²⁺ content, and we observed the similar results. Conclusions:1. In the whole rat heart level, Apelin has dose-dependent positive inotropic action. PLC, PKC and NHE, NCX are involved.2. In the isolated myocyte level, 1nmol/l Apelin has positive inotropic action.3. The increase of amplitude of E[Ca²⁺]_i is one of reasons of Apelin’s positive inotropic action. Some other reason is remained, and is PKC dependent.4. Apelin decreases SR Ca²⁺ content, with a PKC dependent way, and NCX, SERCA are two key proteins.更多还原