尾加压素Ⅱ在糖尿病性心肌病中的意义及其作用机制研究
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摘要
第一部分
尾加压素Ⅱ及其受体在糖尿病性心肌病中表达改变的实验研究
背景
尾加压素Ⅱ(UrotensinⅡ，UⅡ)最早发现于硬骨鱼尾部下垂体，是目前已知最强的缩血管活性物质，其效能可达内皮素-1(ET-1)的10余倍。UⅡ在各种属中普遍存在，目前在包括人类等多个物种中都检测出UⅡ的表达。不同物种UⅡ的结构虽有差异，但都有一环形六肽结构，是UⅡ的活性中心，序列为半胱氨酸-苯丙氨酸-色氨酸-赖氨酸-酪氨酸-半胱氨酸。从鱼类到人类，该环形活性中心相当保守，提示具有重要的生理功能。UⅡ受体是一种孤立的G蛋白偶联受体，即UT。UⅡ及其受体在全身多组织器官中均有表达，其在心血管组织中的广泛分布为UⅡ参与心血管系统调节提供了基础。
除血管张力调节作用外，UⅡ同血管紧张素Ⅱ(AngⅡ)、ET-1等缩血管物质一样，也具有明显的促心脏重构，特别是促心肌纤维化作用。研究发现，血清UⅡ水平在临床心衰患者中明显升高，并在终末期心衰患者心肌组织中呈高表达。动物实验也发现UⅡ及其受体在心梗后心衰及右室肥厚大鼠心肌组织中均呈高表达状态。在体实验发现UⅡ可以增强异丙肾上腺素导致的大鼠心肌纤维化及心肌肥大，而UⅡ受体拮抗剂治疗可以明显减轻心梗后心衰大鼠心肌细胞肥大及间质纤维化，延缓心脏重构，改善心功能，降低死亡率。体外实验也明确证实UⅡ能够增加心脏成纤维细胞胶原合成、促进心肌细胞肥大，而UⅡ受体拮抗剂可以抑制UⅡ诱导的心肌细胞肥大。
近年的研究发现UⅡ还具有代谢调节作用，并与糖尿病及其并发症的发生发展密切相关。糖尿病患者血浆、尿液及肾脏组织UⅡ表达明显高于正常人。动物实验证实脑室内注射UⅡ后，血糖水平明显升高，而大鼠胰腺灌注UⅡ，高糖引起的胰岛素释放被抑制。糖尿病大鼠长期口服选择性UⅡ受体拮抗剂，可以改善存活率，增加胰岛素水平，降低血糖、糖基化血红蛋白及血脂水平，还可以增加肾血流量、延缓蛋白尿及肾功能损害的发生。
随着糖尿病发病率的逐年上升，其并发症糖尿病性心肌病(diabetic cardiomyopathy，DCM)也日益受到人们的重视。DCM的发病机制复杂，涉及代谢紊乱、心肌细胞肥大、心肌纤维化、小血管病变、自主神经功能紊乱和胰岛素抵抗等多个因素，其中心肌细胞肥大及纤维化是DCM时最重要的病理改变之一，但其发生机制尚未完全阐明。
综上所述，鉴于糖代谢紊乱和心脏重构是DCM的特征性病理生理改变，而UⅡ同时参与糖代谢调节及心脏重构过程，我们提出UⅡ／UT系统在DCM的发病中可能具有重要作用的假设。因此，本研究拟在DCM动物模型上，观察UⅡ／UT系统在心脏中的表达情况，以期探讨UⅡ在DCM发病中的病理生理学意义。
目的
1．构建DCM动物模型。
2．探讨DCM动物模型心脏重构及心功能改变情况。
3．明确DCM时UⅡ／UT系统的表达情况。
方法
1．DCM动物模型构建
雄性Wistar大鼠27只，随机分为2组：对照组12只，DCM组15只。标准大鼠饲料喂养1周后，禁食12h，DCM组大鼠给以腹腔内注射链脲佐菌素(STZ)65mg／kg，对照组大鼠注射等量柠檬酸钠／柠檬酸缓冲液。糖尿病大鼠成模标准为：连续2次空腹血糖≥16.7mmol／L。未达成模标准者剔除。DCM组大鼠血糖升高后，继续喂养5个月，处死取材。
2．超声心动图检测
分别于糖尿病建模前、实验末进行常规超声心动图检查，测定如下指标：M型超声心动图测定左室收缩末内径(LVIDs)、左室舒张末内径(LVIDd)、射血分数(EF)、短轴缩短率(FS)；彩色多普勒超声心动图观察瓣膜返流情况；脉冲及连续多普勒超声心动图测定二尖瓣E波最大速度、A波最大速度、E／A比值、E波减速时间(EDT)、等容舒张时间(IVRT)、主动脉血流最大速度(APV)。根据心动周期，计算校正的EDT'(EDT'=EDT／(心动周期)~(1／2))及IVRT'(IVRT'=IVTR／(心动周期)~(1／2))。
3．心肌组织病理学检查及Masson三色染色
动物处死后，进行组织取材、固定、脱水、透明、浸蜡、石蜡包埋、切片，常规HE染色，观察心肌细胞形态并拍片；行Masson三色染色，观察胶原分布形态并定量分析心肌组织胶原含量。
4．实时定量RT-PCR法检测
分别从左室、右室、心房组织提取总RNA，经逆转录反应(RT)得到cDNA，以管家基因β-actin作为参照，通过实时定量RT-PCR技术检测UⅡ和UT的表达。
5．免疫组织化学检测
取组织切片，进行UⅡ及UT免疫组织化学检测，所用一抗包括UⅡ多克隆抗体(SantaCruz公司，1：100稀释)和UT多克隆抗体(SantaCruz公司，1：200稀释)。
6．免疫印迹检测
分别取左室、右室及心房组织，提取总蛋白，经过SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)分离、转膜、蛋白印记、DAB或ECL显色等步骤，检测UT的蛋白表达。
结果
1．实验动物基本情况及血糖检测
实验过程中，对照组大鼠精神状态良好，体重增加明显，反应敏捷，毛色白而光泽。DCM组大鼠出现多食、多饮、多尿和消瘦等症状，体重增加迟缓，精神萎靡，皮毛无光泽，部分出现烂尾、白内障等。整个实验过程中3只大鼠死亡，均为DCM组，死亡原因可能与糖尿病酮症酸中毒、感染或其他相关并发症有关。另有1只大鼠血糖未达成模标准，予以剔除。最终共23只完成实验，其中对照组12只，DCM组11只。
注射STZ之前，对照组与DCM组血糖无明显差异(P＞0.05)。注射STZ 1周后，DCM组血糖明显高于对照组(P＜0.01)，并持续至实验末。
2．超声心动图检测
实验初，对照组及DCM组大鼠超声心动图检测各项指标(包括LVIDs、LVIDd、EF、FS、瓣膜返流、E波最大速度、A波最大速度、E／A比值、EDT’、IVRT’及APV)无显著性差异。实验末，DCM组大鼠LVIDs及LVIDd明显增加，房室瓣瓣膜返流发生率明显增加，E波最大速度下降，A波最大速度增快，E／A比值下降，IVRT’延长，FS降低，APV降低，EDT’及EF无显著性差异。
3．心肌组织病理学检查及Masson三色染色
HE染色：对照组心肌细胞排列整齐，细胞核大小均一，胞浆染色均匀；DCM组心肌细胞排列紊乱，细胞核大小不甚规则。
Masson三色染色：心肌细胞染色呈红色，间质胶原呈蓝绿色，红细胞呈橘黄色。对照组心肌胶原组织分布均匀，DCM组心肌横切面可见心肌内胶原组织明显增多，粗大胶原纤维相互连接成网状，排列紊乱，分布不匀，紧密围绕于心肌细胞周围及小血管周围。定量分析显示，左室心肌组织胶原含量在DCM组明显高于对照组(P＜0.01)。
4．实时定量RT-PCR法检测
与对照组相比，DCM组左室、右室及心房UⅡmRNA表达均明显增高(P＜0.01)，且在同组不同心腔间无显著性差异(P＞0.05)；DCM组左室、右室及心房UT mRNA表达也明显增高(P＜0.01)，且在同组不同心腔间无显著性差异(P＞0.05)。
5．免疫组织化学检测
UⅡ：对照组心肌组织内可见少量、分布均匀、稀疏的浅棕色颗粒，主要定位于心肌细胞及内皮细胞；DCM组心肌细胞胞浆内可见浓密的深棕色颗粒，同时内皮细胞及心脏成纤维细胞亦见明显表达。
UT：对照组心肌组织UT表达很弱，仅见少量分布于心肌细胞；DCM组心肌组织可见浓密的深棕色颗粒，定位于心肌细胞、内皮细胞、心脏成纤维细胞及血管平滑肌细胞。
6．免疫印迹检测
与对照组相比，DCM组左室、右室及心房组织UT蛋白表达均明显增高(P＜0.01)，且在同组不同心腔间无显著性差异(P＞0.05)。
结论
1．通过腹腔注射STZ并喂养5个月，成功建立了糖尿病性心肌病大鼠模型，为DCM发病机制的研究提供了可靠的平台；
2．胶原组织沉积、心肌纤维化是DCM时的主要组织病理改变；
3．心脏舒张功能异常是DCM时的主要功能改变；
4．DCM大鼠心肌组织UⅡ及UT表达增高，提示UⅡ／UT系统可能在DCM的发生发展中发挥重要作用。
第二部分
尾加压素Ⅱ促进心脏成纤维细胞胶原合成信号转导通路的研究
背景
尾加压素Ⅱ(urotensinⅡ，UⅡ)是迄今哺乳动物体内已被证实的最强缩血管活性物质。新近发现UⅡ在心血管系统的病理生理调节中发挥重要作用，UⅡ既具备非内皮依赖性的血管收缩作用，又具备内皮依赖性的血管舒张作用，该作用方式和程度取决于种属及解剖部位。此外，UⅡ在分离的人心房肌小梁表现为正性肌力作用，而负荷量UⅡ注射于短尾猴后，却导致严重的循环衰竭。除了上述血管舒缩、正负性变力等短期心血管调节作用外，UⅡ还参与心脏重构等心血管系统的长期调控过程，具有促进心肌细胞肥大及心肌纤维化作用。
目前有关UⅡ作用的细胞内信号转导机制研究多集中在血管调控方面。研究报道UⅡ可能通过蛋白激酶C、钙／钙调蛋白／肌球蛋白轻链激酶系统及MAPKs等信号转导分子介导血管收缩调节。UⅡ也可以激活大鼠主动脉外膜L-精氨酸／一氧化氮通路，从而引起血管舒张。但是，UⅡ对心脏重构方面信号转导机制的研究较少。有研究发现UⅡ通过G_(αq)、Ras途径引起体外培养的心肌细胞发生肥大表型的改变。以后研究揭示给予UⅡ刺激心肌细胞后，可以激活ERK1／2、P38 MAPK、表皮生长因子受体。上述研究多集中于心肌细胞肥大方面，而关于UⅡ促进心脏重构中胶原合成的信号通路，目前未见相关报道。
心脏重构，特别是心肌纤维化是DCM的重要病理生理机制，而我们的上一部分工作发现UⅡ及其受体在DCM中表达显著升高，结合UⅡ具有促进心脏重构作用，我们推测UⅡ很可能在以心肌纤维化为重要特征的DCM心脏重构中发挥重要作用。胶原累积是心肌间质纤维化的基础，故本工作拟在体外培养的CFBs上观察UⅡ对胶原合成过程的影响及其细胞内信号通路，以期为DCM心脏重构的预防和治疗提供新靶点。
目的
1．观察UⅡ在CFBs胶原合成中的作用。
2．观察UⅡ对CFBs ERK1／2磷酸化的影响。
3．观察UⅡ对CFBs TGF-β1表达的影响。
4．探讨UT、ERK1／2、TGF-β1在UⅡ促CFBs胶原合成过程中的可能作用。
方法
1．CFBs的分离及培养
无菌条件下取出Wistar乳鼠心脏，剪碎、胰酶消化、差速贴壁，获取CFBs。生长至亚融合状态，1：2传代，采用2～4代细胞进行实验。
2．实验设计
1)CFBs给予UⅡ(10~(-7)mol／L、10~(-8)mol／L及10~(-9)mol／L)刺激，部分给予UT受体拮抗剂urantide预处理，测定Ⅰ、Ⅲ型胶原mRNA及胶原蛋白表达；
2)CFBs给予UⅡ(10~(-7)mol／L)刺激5～60min，部分给予urantide或ERK1／2拮抗剂PD98059预处理，测定ERK1／2磷酸化改变；
3)CFBs给予UⅡ(10~(-7)mol／L)刺激4～48h，部分给予urantide或ERK1／2阻断剂PD98059预处理，测定TGF-β1mRNA及蛋白表达；
4)单独给予PD98059或TGF-β1中和抗体预处理、或两者联合预处理后，UⅡ(10~(-7)mol／L)刺激CFBs，测定胶原蛋白表达。
3．实时定量RT-PCR检测Ⅰ、Ⅲ型胶原及TGF-β1 mRNA表达
将所收集的细胞提取总RNA，经逆转录反应得到cDNA，以管家基因β-actin作为参照，通过real-time RT-PCR技术检测Ⅰ、Ⅲ型胶原及TGF-β1 mRNA表达。
4．Western blot检测p-ERK1／2蛋白表达
将所收集的细胞提取总蛋白，经过SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)分离、转膜、蛋白印记、DAB显色等步骤，检测p-ERK1／2的表达。
5．ELISA检测TGF-β1表达
将所收集的细胞上清进行提取、孵育、酶反应、显色等步骤，测定OD450值，并计算出相应浓度。
6．~3H-脯氨酸掺入法检测胶原合成
将CFBs分组给予不同干预的同时，加入~3H-脯氨酸共同孵育48h，经裂解后，用液闪仪测定~3H-脯氨酸掺入量。
结果
1．UⅡ对胶原合成的影响
不同浓度UⅡ(10~(-9)mol／L、10~(-8)mol／L和10~(-7)mol／L)刺激CFBs 24h，结果显示UⅡ呈浓度依赖性促进CFBs的Ⅰ、Ⅲ型胶原mRNA表达，其中以10~(-7)mol／L UⅡ浓度时胶原mRNA表达量最高。不同浓度UⅡ刺激CFBs 48h，结果显示UⅡ呈浓度依赖性促进CFBs ~3H-脯氨酸掺入，同样以10~(-7)mol／L UⅡ作用最明显。
给予UⅡ受体拮抗剂urantide预处理，再给予10~(-7)mol／L UⅡ刺激细胞，与未经urantide预处理组相比，Ⅰ、Ⅲ型胶原mRNA表达及~3H-羟脯氨酸掺入量均明显降低(P＜0.01)；与对照组相比，无显著性差异(P＞0.05)。
2．UⅡ对ERK1／2磷酸化的影响
以10~(-7)mol／L UⅡ孵育CFBs，可以时间依赖性促进ERK1／2磷酸化，5～10min时达高峰，60min时该作用逐渐降至基础水平。给予urantide或PD98059预处理后，再给予10~(-7)mol／L UⅡ刺激CFBs 10min，明显抑制p-ERK1／2表达。
3．UⅡ对TGF-β1表达的影响
用10~(-7)mol／L UⅡ孵育CFBs，时间依赖性增加TGF-β1 mRNA表达，4h时开始增加，8h时达高峰，48h时逐渐回落至基础水平；TGF-β1蛋白表达亦呈时间依赖性增加，该增加于12h时开始，24～48h时达高峰。
使用Urantide或PD98059预处理后，TGF-β1基因及蛋白表达均被明显抑制。
4．ERK1／2及TGF-β1抑制剂对UⅡ促进胶原合成的影响
单独给予TGF-β1中和抗体，UⅡ诱导的胶原合成增加被抑制约为70％(141±7％vs112±5％)；单独给予PD98059，UⅡ诱导的胶原合成增加约50％(141±7％vs120±6％)被抑制；而两者联合应用，抑制程度达90％(141±7％vs105±5％)。
结论
1．UⅡ可以浓度依赖性促进CFBs合成胶原。
2．UⅡ可以时间依赖性激活CFBs ERK1／2及TGF-β1信号通路。
3．在UⅡ诱导的CFBs胶原合成过程中，UT／ERK1／2／TGF-β1信号通路可能发挥重要作用。
PART ONE
Elevated expression of urotensin II and its receptor in
diabetic cardiomyopathy
Background
Diabetes is a major risk factor for cardiovascular disease. Increasing amount of evidence has accumulated for the presence of myocardial dysfunction in diabetic patients in the absence of discernible coronary artery, valvular or hypertensive heart disease. In particular, it has been recently shown that diabetes is independently associated with nonischemic cardiomyopathies. Several mechanisms for the pathogenesis of this diabetic cardiomyopathy (DCM) have been proposed. These include the metabolic derangement, potentially adverse effects of hyperglycemia on endothelial function, autonomic dysfunction, myocardial fibrosis and myocyte hypertrophy. Myocardial fibrosis and myocyte hypertrophy are the most frequently proposed mechanisms to explain cardiac changes in diabetic cardiomyopathy. Several vasoconstrictors have been involved in these processes, such as angiotensin II (Ang II) and endothelin-1 (ET-1).
Recently, the novel vasoconstrictor peptide urotensin II (U II) has emerged as a likely contributor to cardiovascular physiology and pathology. U II is a somatostatin-like cyclic peptide synthesized by proteolytic cleavage from a precursor molecule, prepro-U II, and has been identified as the most potent vasoconstrictor in mammals. In humans, U II binds to a 389 amino acid G-protein coupled receptor termed UT. The G-protein associated with the UT receptor is of the Gq class, which is the same class of G-proteins that bind to angiotensin, endothelin, and α-adrenoceptors. UII induced both endothelium-independent vasoconstriction and endothelium-dependent vasorelaxation, the order and magnitude of which were dependent on the species tested and anatomical location. UII also exerted inotropic effects on the isolated human atrial trabeculea and mitogenic effects on smooth muscle cells. Bolus injection of UII into cynomolgus monkeys resulted in the development of cardiovascular collapse. More recently, a close relationship has been found between U II and congestive heart failure (CHF). Plasma levels of U II are elevated in patients with CHF compared with control subjects. Expression of U II and its receptor is increased in the myocardium of patients with end-stage CHF, in the myocardium of rats with myocardial infarction and in the myocardium of rats with chronic hypoxia induced-right ventricular hypertrophy. Blockage of the UT receptor reduced mortality and improved cardiac function in the rat model of myocardial infarction and CHF. In vitro, U II increases collagen synthesis of cardiac fibroblasts independently and stimulates cellular hypertrophy of cardiac myocytes in conditions of UT upregulation, and the UT receptor antagonist BIM-23127 can inhibit U II-induced hypertrophy in H9c2 cardiomyocytes. All of these suggest an important role for U II/ UT system in the pathogenesis of CHF and in the progression of cardiac remodeling.
Besides its important role in cardiovascular system, U II is also a peptide that has been implicated in metabolic regulation and plays significant roles in diabetes and its complications. The U II gene is localized to 1p36-p32, one of the regions showing potential linkage with type 2 diabetes in Japanese affected sib-pairs. Wenyi showed that S89N polymorphism in the U II gene was associated with development of Type 2 diabetes via insulin sensitivity in the Japanese population. Ong found that haplotypes in the urotensin II gene and urotensin II receptor gene are associated with insulin resistance and impaired glucose tolerance. Following ICV infusion of U II in conscious sheep, plasma glucose increases by 7.0±1.4 mmol/L, compared with vehicle. In the rat pancreas, infusion of U II inhibits insulin's response to glucose through a direct influence of U II on the B-cells. Plasma U II levels are increased in Type 2 diabetic patients and further increased by renal failure. An increased expression of U II and UT was found in diabetic nephropathy. Long-term treatment of STZ-induced diabetic rats with U II receptor antagonist palosuran not only improves survival, increases insulin and slows the increase in glycemia, glycosylated hemoglobin and serum lipids, but also increases renal blood flow and delays the development of proteinuria and renal damage. All of these data strongly suggest an important role of U II in the pathophysiology of diabetes and diabetic nephropathy.
Important role of U II/UT has been found in the pathophysiology of both CHF and diabetes, but whether this system plays a role in diabetic cardiomyopathy still remains unknown. Our aim is to investigate myocardial expression of U II and UT in the hearts of normal and diabetic rats. The data show for the first time that U II and UT are upregulated within diabetic myocardium.
Objective
1. To establish a DCM animal model.
2. To observe the changes of cardiac structure and cardiac function in DCM model.
3. To observe the expression of U II and UT in the myocardium of diabetic rats and controls.
Methods
1. Establishment of DCM animal model
Twenty-seven male Wistar rats were randomly divided into 2 groups: control group (n=12) and DCM group (n=15). Diabetes was induced by a single intraperitoneal injection of STZ (65 mg/kg body wt and dissolved in 0.1 mol/1 citrate buffer, pH 4.2) in rats of DCM group. Control rats received citrate buffer alone. One week after injection of STZ, fasting plasma glucose levels were measured, and rats with plasma glucose at least two times higher than 16.7mmol/L were used. All rats were fed for 5 months after STZ or citrate buffer injections and had free access to standard rat diet and water.
2. Echocardiogram examination
At the beginning and at the end of the study, transthoracic echocardiogram was performed in diabetic and control animals. Rats were placed supine and the anterior chest wall was shaved. Echocardiograms were performed with a Hewlett-Packard Sonos 7500 sector scanner equipped with a 7.5-MHz phased-array transducer. Conventional images included 2-dimensional, M-mode, and continuous wave and pulsed Doppler images.
3. HE staining and Masson staining
HE staining was used to study the pathological changes and Masson staining was used to quantify the collagen content in this study.
4. Real-time RT-PCR
The total RNA was extracted from left ventricles, right ventricles or atriums of control and diabetic rats. The mRNA expression of U II and UT were determined by real-time RT-PCR.
5. Immunohistochemistry
Immunohistochemistry was used to detect the protein expression of U II and UT.
6. Western-blot analysis
Western-blot analysis was used to determine the protein expression of UT.
Results
1. General features of the experimental rats
At the end of the experiment, 11 diabetic rats induced by STZ and 12 control rats survived. Glucose levels were significantly elevated in diabetic rats compared with control rats after STZ injection. Other symptoms, such as lower body weights, polyuria and polyphagia, which are normally associated with diabetic state were also observed in the diabetic rats.
2. Echocardiographic examination
At the beginning of the study, no significant differences of echocardiogram were found between two groups. After 5 months period of diabetes, outstanding echocardiographic changes were detected in diabetic rats, including increased LVIDs and LVIDd; elevated E peak velocity and ration of E/A; decreased A peak velocity, FS and APV; prolongation of IVRT' and increased incidence of valvular regurgition.
3. HE staining
The myocytes from the control group arranged regularly. The size of the nuclear was uniform. The staining cytoplasm was homogeneous. The myocytes from the DCM group arranged irregularly. The nuclear was irregular and the interrupted myofibril arranged irregularly.
4. Masson staining
After five months of diabetes, the cardiac collagen deposition was obviously enhanced in the diabetic groups compared with control groups.
5. Myocardial expression of U II in DCM
Real-time PT-PCR demonstrated a significant increase in U II mRNA transcripts in left ventricles, right ventricles and atriums of diabetic rats compared with controls. There is no difference of mRNA expression among different heart chambers of the same group.
Immunohistochemistry using anti-U II antibody showed little or no U II protein expression in the normal rat heart. Occasionally, certain cell types, such as cardiomyocytes and endothelial cells expressed weak U II immunoreactivity. The myocardium of rats with DCM showed strong U II protein expression, which was mainly concentrated in cardiomyocytes, and to a lesser extent expressed in endothelial cells and cardiac fibroblasts. 6. Myocardial expression of UT in DCM
Similar to U II, real-time RT-PCR also demonstrated a significant increase in UT mRNA transcripts in left ventricles, right ventricles and atriums of diabetic rats compared with controls. There was also no difference of mRNA expression among different heart chambers of the same group.
Immunohistochemistry using anti-UT antibody demonstrated weak UT protein expression in the normal rat heart, which was mainly located in cardiomyocytes. The myocardium of rats with DCM showed strong UT protein expression, which was concentrated in cardiomyocytes, cardiac fibroblasts, endothelial cells and smooth muscle cells.
Western blot analysis demonstrated a significant increase in UT protein expression in left ventricles, right ventricles and atriums of diabetic rats compared with controls.
Conclusions
1. A DCM animal model was established by 5 months period of STZ-induced diabetes in male Wistar rats.
2. The main histopathologic changes of DCM were collagen deposition and myocardial fibrosis.
3. Diastolic dysfunction is characteristic in this DCM model.
4. U II and UT expression were significantly enhanced in the myocardium of DCM group compared with healthy controls.
Part Two
Signal transduction pathways in urotensin II-induced
collagen synthesis in cardiac fibroblasts
Background Urotensin II (U II) is a somatostatin-like cyclic peptide synthesized by proteolytic cleavage from a precursor molecule, prepro-U II, and has been identified as the most potent vasoconstrictor in mammals. In humans, U II binds to a 389 amino acid G-protein coupled receptor termed GPR14, also known as UT. The G-protein associated with the UT receptor belongs to the Gq class, which is the same class of G-proteins that bind to angiotensin, endothelin, and α-adrenoceptors. Recently, U II has emerged as a likely contributor to cardiovascular physiology and pathology. U II induced endothelium-independent vasoconstriction and endothelium-dependent vasorelaxation, the orders and magnitudes of which were dependent on the species tested and anatomical location. U II also exerted inotropic effects on the isolated human atrial trabecular muscles. Bolus injection of U II into cynomolgus monkeys resulted in the development of cardiovascular collapse.
In addition to its short-term roles which include vasoconstriction and chronotropic and inotropic effects on cardiac muscle, recently U II has been implicated in the long-term regulation of growth in the cardiovascular system. A number of studies have suggested an important role of U II in the development of cardiac remodeling. Plasma levels of U II were elevated in patients with congestive heart failure (CHF) compared with control subjects, and this increased plasma level of U II correlates with left ventricular end-diastolic pressure significantly. In vivo studies found that expression of U II and its receptor increased in the myocardium of patients with end-stage CHF, in the myocardium of rats with myocardial infarction and in the myocardium of rats with chronic hypoxia induced-right ventricular hypertrophy. In our previous study, it has been found that the expression of U II/UT system was significantly elevated in diabetic cardiomyopathy on both mRNA and protein levels. Blockade of the UT receptor can result in mortality reduction and cardiac function improvement in the rat model of myocardial infarction and CHF. Moreover, Tzanidis demonstrated that U II promoted collagen synthesis of cardiac fibroblasts independently and stimulated cellular hypertrophy of cardiac myocytes in conditions of UT upregulation. It is also found that the UT receptor antagonist BIM-23127 inhibited U II-induced hypertrophy in H9c2 cardiomyocytes. This U II -induced hypertrophy of cardiac myocytes is promoted by extracelluar signal-regulated kinase1/2 (ERK1/2) and p38 signaling pathways in an epidermal growth factor receptor-dependent manner. Nevertheless, the mechanisms of action of U II in cardiac remodeling, especially in cardiac fibrosis, are still incompletely understood. Further insights into the mechanisms of this effect will have important therapeutic implications.
Objective
1. To identify the function of U II in collagen synthesis in CFBs.
2. To determine whether ERK1/2 pathway and TGF-β1 can be activated by U II in CFBs.
3. To investigate the possible role of UT, ERK1/2 and TGF-β1 in U II-induced collagen synthesis of CFBs.
Methods
1. Cell culture
Neonatal rat cardiac fibroblasts were prepared by the following procedures: three to four hearts from 1- to 3-day-old Wistar rats were finely minced and placed together in 0.25% trypsin. Pooled cell suspensions were centrifuged and resuspended in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/ml penicillin and 100 μg/ml streptomycin. The resuspension was plated onto culture flasks for 90 min, which allowed for preferential attachment of fibroblasts to the bottom of the culture flask. Non-adherent and weakly attached cells were removed and the medium was changed. Cells were grown to confluence and subsequently passaged 1:2 by trypsin. Cell cultures were incubated at 37°C in a humidified atmosphere of 5% CO_2/95% air. Studies were conducted on cardiac fibroblasts (passages two through four) that were grown to subconfluence in serum-containing media and then growth arrested for 24 h in serum-free medium before treatment.
2. Study design
1) To examine the function of U II on collagen synthesis, cells were stimulated with U II (10~(-9)mol/L, 10~(-8)mol/L and 10~(-7)mol/L) for 24 h (for real-time RT-PCR) or for 48 h (for ~3H-proline incorporation) before harvest, part of the cells were pretreated with urantide. Then real-time RT-PCR for type I ,III collagen and ~3H-proline incorporation were performed and the concentration point for maximal effect of U II was used in subsequent experiments.
2) To examine the function of U II on ERK1/2 activation, cells were treated with 10~(-7)mol/L U II for 5 min- 60 min before harvest. Part of the cells were pretreated with urantide or PD98059. Then western blot was performed to evaluate U II -induced phosphorylation of ERK1/2.
3) To examine the function of U II on TGF-β1 production, cells were incubated with 10~(-7)mol/L U II for 4 h to 48 h, and then real-time RT-PCR and sandwish ELISAwere performed to determine the expression of TGF-β1. Pre-treatment of PD98059 was also used to examine the role of ERK1/2 in U II -induced TGF- β1 production.
4) To examine the mechanisms involved in U II-induced collagen synthesis, cells were pretreated with PD98059 or TGF-β1 antibody or both of them, then stimulated with 10~(-7)mol/L U II for 48 h and ~3H-proline incorporation was performed.
3. Real-time RT-PCR
The total RNA was extracted from collected cells. The mRNA expression of collagen I 、 collagen III and TGF-β1 were determined by real-time RT-PCR.
4. Western-blot analysis
The total protein was extracted from collected cells.Western blot analysis was used to determine the protein expression of p-ERK1/2. 4. ELISA analysis ELISA analysis was used to determine the concentration of TGF-β1 in the supernatants collected from cell cultures. 5. ~3H-proline incorporation
~3H-proline incorporation examination was used to determine the collagen synthesis in CFBs.
Results
1. Effects of U II on collagen synthesis by neonatal cardiac fibroblasts
The mRNA expression of both type I and type III collagen mRNA expression was significantly increased by U II in a concentration-dependent manner, with the maximal effect at 10~7mol/L. ~3H-proline incorporation was also increased by U II in a concentration dependent manner with the same maximal effect at 10~(-7) mol/L. Pretreatment of urantide can significantly inhibited these U II-induced collagen synthesis in CFBs.
2. Effects of U II on ERK1/2 activation
U II stimulation resulted in a robust activation of ERK1/2 at 5-10 min and remained above basal up to 60 min after stimulation in CFBs. Pretreatment of urantide or PD98059 can significantly inhibited this U II-induced ERK1/2 activation.
3. Induction of TGF-β1 by U II
Incubation of cardiac fibroblasts with U II (10~(-7)mol/L) induced the expression of both TGF-β1 mRNA and protein in a time-dependent manner. This effect of U II on TGF-β1 mRNA expression began to increase at 4 h, reached a peak at 8 h, and then gradually decreased to the control level at 48 h. Meanwhile, protein expression started to increase at 12 h and reached a plateau at 24 h. Pretreatment of urantide or PD98059 can significantly inhibite this U II-induced TGF-β1 expression.
4. Involvement of ERK1/2 activation and TGF-β1 production in UII-induced collagen synthesis
To determine whether or not ERK1/2 activation and TGF-β1 production are involved in U II-induced collagen synthesis, cardiac fibroblasts were pretreated with TGF-β1 antibody or PD98059 or both of them. Significant inhibition of UII -induced collagen synthesis was observed by using TGF-β1 antibody alone. U II-induced collagen synthesis was reduced about 70%, from 141% to 112% (P<0.01). Similar effect was also observed for ERK1/2 inhibitor PD98059. U II-induced collagen synthesis was reduced by about 50%, from 141% to 120% (P<0.05). Furthermore, the combined inhibition of TGF-β1 and ERK1/2 further reduced U II -induced collagen synthesis by about 90%, from 141% to 105% (P<0.01).
Conclusions
1. U II promoted collagen synthesis in CFBs in a dose-dependent manner.
2. U II promoted ERK1/2 activation and TGF-β1 secrection in CFBs in a time-dependent manner.
3. UT/ERK1/2/TGF-β1 pathway was probably involved in U II -induced collagen synthesis in CFBs.
尾加压素Ⅱ及其受体在糖尿病性心肌病中表达改变的实验研究
背景
尾加压素Ⅱ(UrotensinⅡ，UⅡ)最早发现于硬骨鱼尾部下垂体，是目前已知最强的缩血管活性物质，其效能可达内皮素-1(ET-1)的10余倍。UⅡ在各种属中普遍存在，目前在包括人类等多个物种中都检测出UⅡ的表达。不同物种UⅡ的结构虽有差异，但都有一环形六肽结构，是UⅡ的活性中心，序列为半胱氨酸-苯丙氨酸-色氨酸-赖氨酸-酪氨酸-半胱氨酸。从鱼类到人类，该环形活性中心相当保守，提示具有重要的生理功能。UⅡ受体是一种孤立的G蛋白偶联受体，即UT。UⅡ及其受体在全身多组织器官中均有表达，其在心血管组织中的广泛分布为UⅡ参与心血管系统调节提供了基础。
除血管张力调节作用外，UⅡ同血管紧张素Ⅱ(AngⅡ)、ET-1等缩血管物质一样，也具有明显的促心脏重构，特别是促心肌纤维化作用。研究发现，血清UⅡ水平在临床心衰患者中明显升高，并在终末期心衰患者心肌组织中呈高表达。动物实验也发现UⅡ及其受体在心梗后心衰及右室肥厚大鼠心肌组织中均呈高表达状态。在体实验发现UⅡ可以增强异丙肾上腺素导致的大鼠心肌纤维化及心肌肥大，而UⅡ受体拮抗剂治疗可以明显减轻心梗后心衰大鼠心肌细胞肥大及间质纤维化，延缓心脏重构，改善心功能，降低死亡率。体外实验也明确证实UⅡ能够增加心脏成纤维细胞胶原合成、促进心肌细胞肥大，而UⅡ受体拮抗剂可以抑制UⅡ诱导的心肌细胞肥大。
近年的研究发现UⅡ还具有代谢调节作用，并与糖尿病及其并发症的发生发展密切相关。糖尿病患者血浆、尿液及肾脏组织UⅡ表达明显高于正常人。动物实验证实脑室内注射UⅡ后，血糖水平明显升高，而大鼠胰腺灌注UⅡ，高糖引起的胰岛素释放被抑制。糖尿病大鼠长期口服选择性UⅡ受体拮抗剂，可以改善存活率，增加胰岛素水平，降低血糖、糖基化血红蛋白及血脂水平，还可以增加肾血流量、延缓蛋白尿及肾功能损害的发生。
随着糖尿病发病率的逐年上升，其并发症糖尿病性心肌病(diabetic cardiomyopathy，DCM)也日益受到人们的重视。DCM的发病机制复杂，涉及代谢紊乱、心肌细胞肥大、心肌纤维化、小血管病变、自主神经功能紊乱和胰岛素抵抗等多个因素，其中心肌细胞肥大及纤维化是DCM时最重要的病理改变之一，但其发生机制尚未完全阐明。
综上所述，鉴于糖代谢紊乱和心脏重构是DCM的特征性病理生理改变，而UⅡ同时参与糖代谢调节及心脏重构过程，我们提出UⅡ／UT系统在DCM的发病中可能具有重要作用的假设。因此，本研究拟在DCM动物模型上，观察UⅡ／UT系统在心脏中的表达情况，以期探讨UⅡ在DCM发病中的病理生理学意义。
目的
1．构建DCM动物模型。
2．探讨DCM动物模型心脏重构及心功能改变情况。
3．明确DCM时UⅡ／UT系统的表达情况。
方法
1．DCM动物模型构建
雄性Wistar大鼠27只，随机分为2组：对照组12只，DCM组15只。标准大鼠饲料喂养1周后，禁食12h，DCM组大鼠给以腹腔内注射链脲佐菌素(STZ)65mg／kg，对照组大鼠注射等量柠檬酸钠／柠檬酸缓冲液。糖尿病大鼠成模标准为：连续2次空腹血糖≥16.7mmol／L。未达成模标准者剔除。DCM组大鼠血糖升高后，继续喂养5个月，处死取材。
2．超声心动图检测
分别于糖尿病建模前、实验末进行常规超声心动图检查，测定如下指标：M型超声心动图测定左室收缩末内径(LVIDs)、左室舒张末内径(LVIDd)、射血分数(EF)、短轴缩短率(FS)；彩色多普勒超声心动图观察瓣膜返流情况；脉冲及连续多普勒超声心动图测定二尖瓣E波最大速度、A波最大速度、E／A比值、E波减速时间(EDT)、等容舒张时间(IVRT)、主动脉血流最大速度(APV)。根据心动周期，计算校正的EDT'(EDT'=EDT／(心动周期)~(1／2))及IVRT'(IVRT'=IVTR／(心动周期)~(1／2))。
3．心肌组织病理学检查及Masson三色染色
动物处死后，进行组织取材、固定、脱水、透明、浸蜡、石蜡包埋、切片，常规HE染色，观察心肌细胞形态并拍片；行Masson三色染色，观察胶原分布形态并定量分析心肌组织胶原含量。
4．实时定量RT-PCR法检测
分别从左室、右室、心房组织提取总RNA，经逆转录反应(RT)得到cDNA，以管家基因β-actin作为参照，通过实时定量RT-PCR技术检测UⅡ和UT的表达。
5．免疫组织化学检测
取组织切片，进行UⅡ及UT免疫组织化学检测，所用一抗包括UⅡ多克隆抗体(SantaCruz公司，1：100稀释)和UT多克隆抗体(SantaCruz公司，1：200稀释)。
6．免疫印迹检测
分别取左室、右室及心房组织，提取总蛋白，经过SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)分离、转膜、蛋白印记、DAB或ECL显色等步骤，检测UT的蛋白表达。
结果
1．实验动物基本情况及血糖检测
实验过程中，对照组大鼠精神状态良好，体重增加明显，反应敏捷，毛色白而光泽。DCM组大鼠出现多食、多饮、多尿和消瘦等症状，体重增加迟缓，精神萎靡，皮毛无光泽，部分出现烂尾、白内障等。整个实验过程中3只大鼠死亡，均为DCM组，死亡原因可能与糖尿病酮症酸中毒、感染或其他相关并发症有关。另有1只大鼠血糖未达成模标准，予以剔除。最终共23只完成实验，其中对照组12只，DCM组11只。
注射STZ之前，对照组与DCM组血糖无明显差异(P＞0.05)。注射STZ 1周后，DCM组血糖明显高于对照组(P＜0.01)，并持续至实验末。
2．超声心动图检测
实验初，对照组及DCM组大鼠超声心动图检测各项指标(包括LVIDs、LVIDd、EF、FS、瓣膜返流、E波最大速度、A波最大速度、E／A比值、EDT’、IVRT’及APV)无显著性差异。实验末，DCM组大鼠LVIDs及LVIDd明显增加，房室瓣瓣膜返流发生率明显增加，E波最大速度下降，A波最大速度增快，E／A比值下降，IVRT’延长，FS降低，APV降低，EDT’及EF无显著性差异。
3．心肌组织病理学检查及Masson三色染色
HE染色：对照组心肌细胞排列整齐，细胞核大小均一，胞浆染色均匀；DCM组心肌细胞排列紊乱，细胞核大小不甚规则。
Masson三色染色：心肌细胞染色呈红色，间质胶原呈蓝绿色，红细胞呈橘黄色。对照组心肌胶原组织分布均匀，DCM组心肌横切面可见心肌内胶原组织明显增多，粗大胶原纤维相互连接成网状，排列紊乱，分布不匀，紧密围绕于心肌细胞周围及小血管周围。定量分析显示，左室心肌组织胶原含量在DCM组明显高于对照组(P＜0.01)。
4．实时定量RT-PCR法检测
与对照组相比，DCM组左室、右室及心房UⅡmRNA表达均明显增高(P＜0.01)，且在同组不同心腔间无显著性差异(P＞0.05)；DCM组左室、右室及心房UT mRNA表达也明显增高(P＜0.01)，且在同组不同心腔间无显著性差异(P＞0.05)。
5．免疫组织化学检测
UⅡ：对照组心肌组织内可见少量、分布均匀、稀疏的浅棕色颗粒，主要定位于心肌细胞及内皮细胞；DCM组心肌细胞胞浆内可见浓密的深棕色颗粒，同时内皮细胞及心脏成纤维细胞亦见明显表达。
UT：对照组心肌组织UT表达很弱，仅见少量分布于心肌细胞；DCM组心肌组织可见浓密的深棕色颗粒，定位于心肌细胞、内皮细胞、心脏成纤维细胞及血管平滑肌细胞。
6．免疫印迹检测
与对照组相比，DCM组左室、右室及心房组织UT蛋白表达均明显增高(P＜0.01)，且在同组不同心腔间无显著性差异(P＞0.05)。
结论
1．通过腹腔注射STZ并喂养5个月，成功建立了糖尿病性心肌病大鼠模型，为DCM发病机制的研究提供了可靠的平台；
2．胶原组织沉积、心肌纤维化是DCM时的主要组织病理改变；
3．心脏舒张功能异常是DCM时的主要功能改变；
4．DCM大鼠心肌组织UⅡ及UT表达增高，提示UⅡ／UT系统可能在DCM的发生发展中发挥重要作用。
第二部分
尾加压素Ⅱ促进心脏成纤维细胞胶原合成信号转导通路的研究
背景
尾加压素Ⅱ(urotensinⅡ，UⅡ)是迄今哺乳动物体内已被证实的最强缩血管活性物质。新近发现UⅡ在心血管系统的病理生理调节中发挥重要作用，UⅡ既具备非内皮依赖性的血管收缩作用，又具备内皮依赖性的血管舒张作用，该作用方式和程度取决于种属及解剖部位。此外，UⅡ在分离的人心房肌小梁表现为正性肌力作用，而负荷量UⅡ注射于短尾猴后，却导致严重的循环衰竭。除了上述血管舒缩、正负性变力等短期心血管调节作用外，UⅡ还参与心脏重构等心血管系统的长期调控过程，具有促进心肌细胞肥大及心肌纤维化作用。
目前有关UⅡ作用的细胞内信号转导机制研究多集中在血管调控方面。研究报道UⅡ可能通过蛋白激酶C、钙／钙调蛋白／肌球蛋白轻链激酶系统及MAPKs等信号转导分子介导血管收缩调节。UⅡ也可以激活大鼠主动脉外膜L-精氨酸／一氧化氮通路，从而引起血管舒张。但是，UⅡ对心脏重构方面信号转导机制的研究较少。有研究发现UⅡ通过G_(αq)、Ras途径引起体外培养的心肌细胞发生肥大表型的改变。以后研究揭示给予UⅡ刺激心肌细胞后，可以激活ERK1／2、P38 MAPK、表皮生长因子受体。上述研究多集中于心肌细胞肥大方面，而关于UⅡ促进心脏重构中胶原合成的信号通路，目前未见相关报道。
心脏重构，特别是心肌纤维化是DCM的重要病理生理机制，而我们的上一部分工作发现UⅡ及其受体在DCM中表达显著升高，结合UⅡ具有促进心脏重构作用，我们推测UⅡ很可能在以心肌纤维化为重要特征的DCM心脏重构中发挥重要作用。胶原累积是心肌间质纤维化的基础，故本工作拟在体外培养的CFBs上观察UⅡ对胶原合成过程的影响及其细胞内信号通路，以期为DCM心脏重构的预防和治疗提供新靶点。
目的
1．观察UⅡ在CFBs胶原合成中的作用。
2．观察UⅡ对CFBs ERK1／2磷酸化的影响。
3．观察UⅡ对CFBs TGF-β1表达的影响。
4．探讨UT、ERK1／2、TGF-β1在UⅡ促CFBs胶原合成过程中的可能作用。
方法
1．CFBs的分离及培养
无菌条件下取出Wistar乳鼠心脏，剪碎、胰酶消化、差速贴壁，获取CFBs。生长至亚融合状态，1：2传代，采用2～4代细胞进行实验。
2．实验设计
1)CFBs给予UⅡ(10~(-7)mol／L、10~(-8)mol／L及10~(-9)mol／L)刺激，部分给予UT受体拮抗剂urantide预处理，测定Ⅰ、Ⅲ型胶原mRNA及胶原蛋白表达；
2)CFBs给予UⅡ(10~(-7)mol／L)刺激5～60min，部分给予urantide或ERK1／2拮抗剂PD98059预处理，测定ERK1／2磷酸化改变；
3)CFBs给予UⅡ(10~(-7)mol／L)刺激4～48h，部分给予urantide或ERK1／2阻断剂PD98059预处理，测定TGF-β1mRNA及蛋白表达；
4)单独给予PD98059或TGF-β1中和抗体预处理、或两者联合预处理后，UⅡ(10~(-7)mol／L)刺激CFBs，测定胶原蛋白表达。
3．实时定量RT-PCR检测Ⅰ、Ⅲ型胶原及TGF-β1 mRNA表达
将所收集的细胞提取总RNA，经逆转录反应得到cDNA，以管家基因β-actin作为参照，通过real-time RT-PCR技术检测Ⅰ、Ⅲ型胶原及TGF-β1 mRNA表达。
4．Western blot检测p-ERK1／2蛋白表达
将所收集的细胞提取总蛋白，经过SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)分离、转膜、蛋白印记、DAB显色等步骤，检测p-ERK1／2的表达。
5．ELISA检测TGF-β1表达
将所收集的细胞上清进行提取、孵育、酶反应、显色等步骤，测定OD450值，并计算出相应浓度。
6．~3H-脯氨酸掺入法检测胶原合成
将CFBs分组给予不同干预的同时，加入~3H-脯氨酸共同孵育48h，经裂解后，用液闪仪测定~3H-脯氨酸掺入量。
结果
1．UⅡ对胶原合成的影响
不同浓度UⅡ(10~(-9)mol／L、10~(-8)mol／L和10~(-7)mol／L)刺激CFBs 24h，结果显示UⅡ呈浓度依赖性促进CFBs的Ⅰ、Ⅲ型胶原mRNA表达，其中以10~(-7)mol／L UⅡ浓度时胶原mRNA表达量最高。不同浓度UⅡ刺激CFBs 48h，结果显示UⅡ呈浓度依赖性促进CFBs ~3H-脯氨酸掺入，同样以10~(-7)mol／L UⅡ作用最明显。
给予UⅡ受体拮抗剂urantide预处理，再给予10~(-7)mol／L UⅡ刺激细胞，与未经urantide预处理组相比，Ⅰ、Ⅲ型胶原mRNA表达及~3H-羟脯氨酸掺入量均明显降低(P＜0.01)；与对照组相比，无显著性差异(P＞0.05)。
2．UⅡ对ERK1／2磷酸化的影响
以10~(-7)mol／L UⅡ孵育CFBs，可以时间依赖性促进ERK1／2磷酸化，5～10min时达高峰，60min时该作用逐渐降至基础水平。给予urantide或PD98059预处理后，再给予10~(-7)mol／L UⅡ刺激CFBs 10min，明显抑制p-ERK1／2表达。
3．UⅡ对TGF-β1表达的影响
用10~(-7)mol／L UⅡ孵育CFBs，时间依赖性增加TGF-β1 mRNA表达，4h时开始增加，8h时达高峰，48h时逐渐回落至基础水平；TGF-β1蛋白表达亦呈时间依赖性增加，该增加于12h时开始，24～48h时达高峰。
使用Urantide或PD98059预处理后，TGF-β1基因及蛋白表达均被明显抑制。
4．ERK1／2及TGF-β1抑制剂对UⅡ促进胶原合成的影响
单独给予TGF-β1中和抗体，UⅡ诱导的胶原合成增加被抑制约为70％(141±7％vs112±5％)；单独给予PD98059，UⅡ诱导的胶原合成增加约50％(141±7％vs120±6％)被抑制；而两者联合应用，抑制程度达90％(141±7％vs105±5％)。
结论
1．UⅡ可以浓度依赖性促进CFBs合成胶原。
2．UⅡ可以时间依赖性激活CFBs ERK1／2及TGF-β1信号通路。
3．在UⅡ诱导的CFBs胶原合成过程中，UT／ERK1／2／TGF-β1信号通路可能发挥重要作用。
PART ONE
Elevated expression of urotensin II and its receptor in
diabetic cardiomyopathy
Background
Diabetes is a major risk factor for cardiovascular disease. Increasing amount of evidence has accumulated for the presence of myocardial dysfunction in diabetic patients in the absence of discernible coronary artery, valvular or hypertensive heart disease. In particular, it has been recently shown that diabetes is independently associated with nonischemic cardiomyopathies. Several mechanisms for the pathogenesis of this diabetic cardiomyopathy (DCM) have been proposed. These include the metabolic derangement, potentially adverse effects of hyperglycemia on endothelial function, autonomic dysfunction, myocardial fibrosis and myocyte hypertrophy. Myocardial fibrosis and myocyte hypertrophy are the most frequently proposed mechanisms to explain cardiac changes in diabetic cardiomyopathy. Several vasoconstrictors have been involved in these processes, such as angiotensin II (Ang II) and endothelin-1 (ET-1).
Recently, the novel vasoconstrictor peptide urotensin II (U II) has emerged as a likely contributor to cardiovascular physiology and pathology. U II is a somatostatin-like cyclic peptide synthesized by proteolytic cleavage from a precursor molecule, prepro-U II, and has been identified as the most potent vasoconstrictor in mammals. In humans, U II binds to a 389 amino acid G-protein coupled receptor termed UT. The G-protein associated with the UT receptor is of the Gq class, which is the same class of G-proteins that bind to angiotensin, endothelin, and α-adrenoceptors. UII induced both endothelium-independent vasoconstriction and endothelium-dependent vasorelaxation, the order and magnitude of which were dependent on the species tested and anatomical location. UII also exerted inotropic effects on the isolated human atrial trabeculea and mitogenic effects on smooth muscle cells. Bolus injection of UII into cynomolgus monkeys resulted in the development of cardiovascular collapse. More recently, a close relationship has been found between U II and congestive heart failure (CHF). Plasma levels of U II are elevated in patients with CHF compared with control subjects. Expression of U II and its receptor is increased in the myocardium of patients with end-stage CHF, in the myocardium of rats with myocardial infarction and in the myocardium of rats with chronic hypoxia induced-right ventricular hypertrophy. Blockage of the UT receptor reduced mortality and improved cardiac function in the rat model of myocardial infarction and CHF. In vitro, U II increases collagen synthesis of cardiac fibroblasts independently and stimulates cellular hypertrophy of cardiac myocytes in conditions of UT upregulation, and the UT receptor antagonist BIM-23127 can inhibit U II-induced hypertrophy in H9c2 cardiomyocytes. All of these suggest an important role for U II/ UT system in the pathogenesis of CHF and in the progression of cardiac remodeling.
Besides its important role in cardiovascular system, U II is also a peptide that has been implicated in metabolic regulation and plays significant roles in diabetes and its complications. The U II gene is localized to 1p36-p32, one of the regions showing potential linkage with type 2 diabetes in Japanese affected sib-pairs. Wenyi showed that S89N polymorphism in the U II gene was associated with development of Type 2 diabetes via insulin sensitivity in the Japanese population. Ong found that haplotypes in the urotensin II gene and urotensin II receptor gene are associated with insulin resistance and impaired glucose tolerance. Following ICV infusion of U II in conscious sheep, plasma glucose increases by 7.0±1.4 mmol/L, compared with vehicle. In the rat pancreas, infusion of U II inhibits insulin's response to glucose through a direct influence of U II on the B-cells. Plasma U II levels are increased in Type 2 diabetic patients and further increased by renal failure. An increased expression of U II and UT was found in diabetic nephropathy. Long-term treatment of STZ-induced diabetic rats with U II receptor antagonist palosuran not only improves survival, increases insulin and slows the increase in glycemia, glycosylated hemoglobin and serum lipids, but also increases renal blood flow and delays the development of proteinuria and renal damage. All of these data strongly suggest an important role of U II in the pathophysiology of diabetes and diabetic nephropathy.
Important role of U II/UT has been found in the pathophysiology of both CHF and diabetes, but whether this system plays a role in diabetic cardiomyopathy still remains unknown. Our aim is to investigate myocardial expression of U II and UT in the hearts of normal and diabetic rats. The data show for the first time that U II and UT are upregulated within diabetic myocardium.
Objective
1. To establish a DCM animal model.
2. To observe the changes of cardiac structure and cardiac function in DCM model.
3. To observe the expression of U II and UT in the myocardium of diabetic rats and controls.
Methods
1. Establishment of DCM animal model
Twenty-seven male Wistar rats were randomly divided into 2 groups: control group (n=12) and DCM group (n=15). Diabetes was induced by a single intraperitoneal injection of STZ (65 mg/kg body wt and dissolved in 0.1 mol/1 citrate buffer, pH 4.2) in rats of DCM group. Control rats received citrate buffer alone. One week after injection of STZ, fasting plasma glucose levels were measured, and rats with plasma glucose at least two times higher than 16.7mmol/L were used. All rats were fed for 5 months after STZ or citrate buffer injections and had free access to standard rat diet and water.
2. Echocardiogram examination
At the beginning and at the end of the study, transthoracic echocardiogram was performed in diabetic and control animals. Rats were placed supine and the anterior chest wall was shaved. Echocardiograms were performed with a Hewlett-Packard Sonos 7500 sector scanner equipped with a 7.5-MHz phased-array transducer. Conventional images included 2-dimensional, M-mode, and continuous wave and pulsed Doppler images.
3. HE staining and Masson staining
HE staining was used to study the pathological changes and Masson staining was used to quantify the collagen content in this study.
4. Real-time RT-PCR
The total RNA was extracted from left ventricles, right ventricles or atriums of control and diabetic rats. The mRNA expression of U II and UT were determined by real-time RT-PCR.
5. Immunohistochemistry
Immunohistochemistry was used to detect the protein expression of U II and UT.
6. Western-blot analysis
Western-blot analysis was used to determine the protein expression of UT.
Results
1. General features of the experimental rats
At the end of the experiment, 11 diabetic rats induced by STZ and 12 control rats survived. Glucose levels were significantly elevated in diabetic rats compared with control rats after STZ injection. Other symptoms, such as lower body weights, polyuria and polyphagia, which are normally associated with diabetic state were also observed in the diabetic rats.
2. Echocardiographic examination
At the beginning of the study, no significant differences of echocardiogram were found between two groups. After 5 months period of diabetes, outstanding echocardiographic changes were detected in diabetic rats, including increased LVIDs and LVIDd; elevated E peak velocity and ration of E/A; decreased A peak velocity, FS and APV; prolongation of IVRT' and increased incidence of valvular regurgition.
3. HE staining
The myocytes from the control group arranged regularly. The size of the nuclear was uniform. The staining cytoplasm was homogeneous. The myocytes from the DCM group arranged irregularly. The nuclear was irregular and the interrupted myofibril arranged irregularly.
4. Masson staining
After five months of diabetes, the cardiac collagen deposition was obviously enhanced in the diabetic groups compared with control groups.
5. Myocardial expression of U II in DCM
Real-time PT-PCR demonstrated a significant increase in U II mRNA transcripts in left ventricles, right ventricles and atriums of diabetic rats compared with controls. There is no difference of mRNA expression among different heart chambers of the same group.
Immunohistochemistry using anti-U II antibody showed little or no U II protein expression in the normal rat heart. Occasionally, certain cell types, such as cardiomyocytes and endothelial cells expressed weak U II immunoreactivity. The myocardium of rats with DCM showed strong U II protein expression, which was mainly concentrated in cardiomyocytes, and to a lesser extent expressed in endothelial cells and cardiac fibroblasts. 6. Myocardial expression of UT in DCM
Similar to U II, real-time RT-PCR also demonstrated a significant increase in UT mRNA transcripts in left ventricles, right ventricles and atriums of diabetic rats compared with controls. There was also no difference of mRNA expression among different heart chambers of the same group.
Immunohistochemistry using anti-UT antibody demonstrated weak UT protein expression in the normal rat heart, which was mainly located in cardiomyocytes. The myocardium of rats with DCM showed strong UT protein expression, which was concentrated in cardiomyocytes, cardiac fibroblasts, endothelial cells and smooth muscle cells.
Western blot analysis demonstrated a significant increase in UT protein expression in left ventricles, right ventricles and atriums of diabetic rats compared with controls.
Conclusions
1. A DCM animal model was established by 5 months period of STZ-induced diabetes in male Wistar rats.
2. The main histopathologic changes of DCM were collagen deposition and myocardial fibrosis.
3. Diastolic dysfunction is characteristic in this DCM model.
4. U II and UT expression were significantly enhanced in the myocardium of DCM group compared with healthy controls.
Part Two
Signal transduction pathways in urotensin II-induced
collagen synthesis in cardiac fibroblasts
Background Urotensin II (U II) is a somatostatin-like cyclic peptide synthesized by proteolytic cleavage from a precursor molecule, prepro-U II, and has been identified as the most potent vasoconstrictor in mammals. In humans, U II binds to a 389 amino acid G-protein coupled receptor termed GPR14, also known as UT. The G-protein associated with the UT receptor belongs to the Gq class, which is the same class of G-proteins that bind to angiotensin, endothelin, and α-adrenoceptors. Recently, U II has emerged as a likely contributor to cardiovascular physiology and pathology. U II induced endothelium-independent vasoconstriction and endothelium-dependent vasorelaxation, the orders and magnitudes of which were dependent on the species tested and anatomical location. U II also exerted inotropic effects on the isolated human atrial trabecular muscles. Bolus injection of U II into cynomolgus monkeys resulted in the development of cardiovascular collapse.
In addition to its short-term roles which include vasoconstriction and chronotropic and inotropic effects on cardiac muscle, recently U II has been implicated in the long-term regulation of growth in the cardiovascular system. A number of studies have suggested an important role of U II in the development of cardiac remodeling. Plasma levels of U II were elevated in patients with congestive heart failure (CHF) compared with control subjects, and this increased plasma level of U II correlates with left ventricular end-diastolic pressure significantly. In vivo studies found that expression of U II and its receptor increased in the myocardium of patients with end-stage CHF, in the myocardium of rats with myocardial infarction and in the myocardium of rats with chronic hypoxia induced-right ventricular hypertrophy. In our previous study, it has been found that the expression of U II/UT system was significantly elevated in diabetic cardiomyopathy on both mRNA and protein levels. Blockade of the UT receptor can result in mortality reduction and cardiac function improvement in the rat model of myocardial infarction and CHF. Moreover, Tzanidis demonstrated that U II promoted collagen synthesis of cardiac fibroblasts independently and stimulated cellular hypertrophy of cardiac myocytes in conditions of UT upregulation. It is also found that the UT receptor antagonist BIM-23127 inhibited U II-induced hypertrophy in H9c2 cardiomyocytes. This U II -induced hypertrophy of cardiac myocytes is promoted by extracelluar signal-regulated kinase1/2 (ERK1/2) and p38 signaling pathways in an epidermal growth factor receptor-dependent manner. Nevertheless, the mechanisms of action of U II in cardiac remodeling, especially in cardiac fibrosis, are still incompletely understood. Further insights into the mechanisms of this effect will have important therapeutic implications.
Objective
1. To identify the function of U II in collagen synthesis in CFBs.
2. To determine whether ERK1/2 pathway and TGF-β1 can be activated by U II in CFBs.
3. To investigate the possible role of UT, ERK1/2 and TGF-β1 in U II-induced collagen synthesis of CFBs.
Methods
1. Cell culture
Neonatal rat cardiac fibroblasts were prepared by the following procedures: three to four hearts from 1- to 3-day-old Wistar rats were finely minced and placed together in 0.25% trypsin. Pooled cell suspensions were centrifuged and resuspended in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/ml penicillin and 100 μg/ml streptomycin. The resuspension was plated onto culture flasks for 90 min, which allowed for preferential attachment of fibroblasts to the bottom of the culture flask. Non-adherent and weakly attached cells were removed and the medium was changed. Cells were grown to confluence and subsequently passaged 1:2 by trypsin. Cell cultures were incubated at 37°C in a humidified atmosphere of 5% CO_2/95% air. Studies were conducted on cardiac fibroblasts (passages two through four) that were grown to subconfluence in serum-containing media and then growth arrested for 24 h in serum-free medium before treatment.
2. Study design
1) To examine the function of U II on collagen synthesis, cells were stimulated with U II (10~(-9)mol/L, 10~(-8)mol/L and 10~(-7)mol/L) for 24 h (for real-time RT-PCR) or for 48 h (for ~3H-proline incorporation) before harvest, part of the cells were pretreated with urantide. Then real-time RT-PCR for type I ,III collagen and ~3H-proline incorporation were performed and the concentration point for maximal effect of U II was used in subsequent experiments.
2) To examine the function of U II on ERK1/2 activation, cells were treated with 10~(-7)mol/L U II for 5 min- 60 min before harvest. Part of the cells were pretreated with urantide or PD98059. Then western blot was performed to evaluate U II -induced phosphorylation of ERK1/2.
3) To examine the function of U II on TGF-β1 production, cells were incubated with 10~(-7)mol/L U II for 4 h to 48 h, and then real-time RT-PCR and sandwish ELISAwere performed to determine the expression of TGF-β1. Pre-treatment of PD98059 was also used to examine the role of ERK1/2 in U II -induced TGF- β1 production.
4) To examine the mechanisms involved in U II-induced collagen synthesis, cells were pretreated with PD98059 or TGF-β1 antibody or both of them, then stimulated with 10~(-7)mol/L U II for 48 h and ~3H-proline incorporation was performed.
3. Real-time RT-PCR
The total RNA was extracted from collected cells. The mRNA expression of collagen I 、 collagen III and TGF-β1 were determined by real-time RT-PCR.
4. Western-blot analysis
The total protein was extracted from collected cells.Western blot analysis was used to determine the protein expression of p-ERK1/2. 4. ELISA analysis ELISA analysis was used to determine the concentration of TGF-β1 in the supernatants collected from cell cultures. 5. ~3H-proline incorporation
~3H-proline incorporation examination was used to determine the collagen synthesis in CFBs.
Results
1. Effects of U II on collagen synthesis by neonatal cardiac fibroblasts
The mRNA expression of both type I and type III collagen mRNA expression was significantly increased by U II in a concentration-dependent manner, with the maximal effect at 10~7mol/L. ~3H-proline incorporation was also increased by U II in a concentration dependent manner with the same maximal effect at 10~(-7) mol/L. Pretreatment of urantide can significantly inhibited these U II-induced collagen synthesis in CFBs.
2. Effects of U II on ERK1/2 activation
U II stimulation resulted in a robust activation of ERK1/2 at 5-10 min and remained above basal up to 60 min after stimulation in CFBs. Pretreatment of urantide or PD98059 can significantly inhibited this U II-induced ERK1/2 activation.
3. Induction of TGF-β1 by U II
Incubation of cardiac fibroblasts with U II (10~(-7)mol/L) induced the expression of both TGF-β1 mRNA and protein in a time-dependent manner. This effect of U II on TGF-β1 mRNA expression began to increase at 4 h, reached a peak at 8 h, and then gradually decreased to the control level at 48 h. Meanwhile, protein expression started to increase at 12 h and reached a plateau at 24 h. Pretreatment of urantide or PD98059 can significantly inhibite this U II-induced TGF-β1 expression.
4. Involvement of ERK1/2 activation and TGF-β1 production in UII-induced collagen synthesis
To determine whether or not ERK1/2 activation and TGF-β1 production are involved in U II-induced collagen synthesis, cardiac fibroblasts were pretreated with TGF-β1 antibody or PD98059 or both of them. Significant inhibition of UII -induced collagen synthesis was observed by using TGF-β1 antibody alone. U II-induced collagen synthesis was reduced about 70%, from 141% to 112% (P<0.01). Similar effect was also observed for ERK1/2 inhibitor PD98059. U II-induced collagen synthesis was reduced by about 50%, from 141% to 120% (P<0.05). Furthermore, the combined inhibition of TGF-β1 and ERK1/2 further reduced U II -induced collagen synthesis by about 90%, from 141% to 105% (P<0.01).
Conclusions
1. U II promoted collagen synthesis in CFBs in a dose-dependent manner.
2. U II promoted ERK1/2 activation and TGF-β1 secrection in CFBs in a time-dependent manner.
3. UT/ERK1/2/TGF-β1 pathway was probably involved in U II -induced collagen synthesis in CFBs.
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