【作者】 赵卓；

【导师】 康维强； 葛志明；

【作者基本信息】 山东大学 ， 内科学， 2009， 博士

【摘要】 论文Ⅰ自发性高血压大鼠心肌组织蛋白激酶D1的表达与左室重塑的关系及阿托伐他汀的干预研究背景高血压左心室重塑是心脏事件重要的独立危险因素,与心律失常、猝死、心力衰竭等密切相关。高血压病时心脏的后负荷增加,血流动力学改变伴随神经-内分泌异常,左心室肥厚、心肌纤维化以及心腔扩大,此过程即为左心室重塑。早期左室重塑是对血流动力学负荷增加的一种适应性代偿反应,能降低室壁张力,维持甚至增加心排血量。但到后期,进展到左室肥厚时则成为危险因素作为疾病进程重要机制之一,左心室重塑导致了临床症状的恶化,是心力衰竭发生、发展的基础。近年来,随着分子细胞生物学的发展,对心衰发生发展机制的研究已有了新的认识,目前认为导致心衰不断进展的基本机制是心肌重塑。心肌重塑是一个极为复杂的病理过程,其确切的机制仍不十分明确,研究发现,包括神经内分泌激活、氧化应激、细胞因子的活化等多种因素参与。其中,细胞因子的活化对心肌重塑的调控作用正日益受到重视。高血压发展过程中可出现心肌肥厚、心肌纤维化等左室重塑的变化,其发病机制以及所涉及的信号传导途径是心血管研究领域的热点问题之一。蛋白激酶D（protein kinase D,PKD）作为一类特殊蛋白激酶家族,从蛋白激酶C家族中独立出来,其在细胞间信息传递过程中发挥的重要作用近几年才逐渐为我们所认识。PKD家族主要包括PKD1、PKD2,PKD3等三个成员。作为Ca⁺/钙调蛋白依赖性蛋白激酶,PKD1与PKD3又分别称作PKC_μ和PKC_ν。有研究发现,受生长因子、GPCR拮抗剂、佛波酯等的刺激,PKD活化,PKD1可能参与信号调节激酶ERK和JNK途径。Friederike等人对研究表明,腺病毒转染增强成年大鼠心室肌细胞PKD1表达后发现,PKD1可以使肌钙蛋白I磷酸化,同时降低肌丝Ca⁺的敏感性,提示PKD1调节心室肌细胞的功能。PKD1的活化诱导PKD1的质膜转位,通过RIN1磷酸化和封锁与Ras的结合激活Ras-Raf-MEK.ERK途径,可能通过磷酸化C-Jun下调MEKK-MKK-JNK途径,进而调节细胞的增殖和分化。Fielitz等人发现PKD1敲除小鼠模型中,PKD1基因缺失使心肌肥厚明显减轻,心功能有改善。PKD1在自发性高血压大鼠心肌组织中的活性表达会是什么样呢?它是否在左室重塑过程中起到关键作用呢?对于PKD1在左室重塑中的作用机制还不是很明确。截止目前,在国内外文献中尚未发现对上述相关问题的研究。近年来,对HMG-CoA还原酶抑制剂——他汀类药物的研究表明:该类药物除了本身的降脂效应外,还具有独立于调脂以外的多种效应。本研究应用阿托伐他汀观察其在自发性高血压大鼠（SHR）动物模型中对SHR左室重塑的影响,评价阿托伐他汀在高血压心肌重塑发生发展中的作用,探讨他汀类药物与PKD1通路间的相关关系。目的1.验证自发性高血压大鼠左室肥厚、心肌纤维化的发生发展;2.探讨蛋白激酶D1在自发性高血压大鼠模型心肌组织中的表达;3.探讨自发性高血压大鼠心肌组织的蛋白激酶D1活性表达与左室肥厚、心肌纤维化的相关性。4.评价阿托伐他汀在高血压心肌重塑发生发展中的作用及与PKD1的关系。方法8周龄WKY大鼠28只、自发性高血压（SHR）大鼠41只。WKY大鼠随机分为四组,A组:8周龄（n=7只）:B组:16周龄（n=7只）;C组:24周龄（n=7只）,G组:WKY安慰剂（n=7只）。SHR大鼠随机分为五组:D组:8周龄（n=7只）;E组:16周龄（n=7只）;F组:24周龄（n=7只）;H组:SHR安慰剂（n=10只）,I组:SHR阿托伐他汀（n=10只）。以标准大鼠饲料喂养及普通饮水。另外,WKY安慰剂组、SHR安慰剂组及SHR阿托伐他汀治疗组,每天以蒸馏水或阿托伐他汀(50 mg·kg^-1·d^-1)分别灌胃,喂养16周。于8、16、24周处死动物,留取心肌组织标本备用。实验过程中,进行以下检测:（1）各组大鼠每隔2周测量体重及尾动脉血压一次:（2）分别于喂养前、喂养至16周、24周抽血测定空腹血脂;（3）测定各组大鼠LVM及LVMI值;（4）分别于实验开始前、实验末进行常规超声心动图检查,测定左室舒张末期内径（LVEDD）、左室收缩末期内径（LVESD）、室间隔舒张末期厚度（IVSTd）、左室后壁舒张末期厚度（LVPWTd）,同时计算相对室壁厚度（RWT）=（IVSTd+LVPWTd）/LVEDD、左室射血分数（LVEF）;（5）于实验末做心肌组织超微结构和病理学检查。（6）Westen blot法检测PKD1、p-PKD1744/748、p-PKD1916蛋白的表达及与心肌肥厚各项指标相关性。（7）心肌胶原定性和定量分析及与PKD1、磷酸化PKD1的相关性。结果:1.实验动物基本情况:实验过程中共有3只大鼠死亡,WKY大鼠16周龄组一只,SHR大鼠组16及24周龄组各一只。在普通喂养10～24周内死亡。SHR阿托伐他汀治疗组无死亡。共66只大鼠完成实验,其中WKY组27只,SHR组39只。2.各组大鼠基本指标比较喂养前,各组大鼠体重、尾动脉收缩压、血脂均无差异（P＞0.05）。16周、24周后,与WKY对照组及8周龄SHR组比较16周SHR组、24周SHR大鼠组尾动脉收缩压升高明显,差异有显著性意义（P＜0.01）;SHR阿托伐他汀治疗组尾动脉收缩压较WKY对照组及8周龄SHR组明显升高,有显著性差异（P＜0.01）,而与16周SHR组水平无明显差异（P＞0.05）。虽然SHR组与WKY组比较,未干预各组大鼠体重、血甘油三酯、总胆固醇及高密度脂蛋白分别有降低趋势,但各组间差异无显著性统计学意义（P＞0.05）。干预各组大鼠中SHR阿托伐他汀组血清TC,TG及HDL均较WKY安慰剂、SHR安慰剂组明显降低,差异有统计学意义（P＜0.05～0.01）。3.测定各组大鼠LVM及LVMI值随着周龄的增加,与同龄WKY大鼠比较,SHR大鼠LVM及LVMI值逐渐升高,表明高血压组大鼠左心室发生明显肥厚。SHR阿托伐他汀组与WKY安慰剂组相比,LVM及LVMI值无明显差异（P＞0.05）,而与SHR安慰剂组相比具有显著性差异（P＜0.01）,表明阿托伐伐他汀对SHR高血压的左室肥厚有逆转作用。4.超声心动图检查分别于喂养前和喂养至16、24周后行超声心动图检查。由专人采用美国CATEMAY超声诊断仪,测取连续3个心动周期的舒张期室间隔厚度（IVSTd）、左室后壁厚度（LVPWT）和左心室舒张未期内径（LVEDD）,取其均值。测定左室射血分数（LVEF）。SHR大鼠16周龄组与24周龄组IVSd、PWT、LVM、LVMI均较WKY大鼠各周龄组及SHR大鼠8周龄组明显升高,有显著性差异（P＜0.01）。LVEF各组没有显著性差异。阿托伐他汀组IVSd、PWT、LVM、LVMI值与SHR16周组比较无明显差异,而较SHR24周组明显减低（P＜0.05）。5.心肌组织病理观察HE染色显示:WKY大鼠组心肌细胞体积较小,大小均一,饱满,排列较整齐;SHR大鼠组心肌细胞体积变大,大小不均一,形态不规则,排列紊乱;细胞壁不完全清楚,细胞相交处可见融合。随着周龄的增加,上述心肌细胞表现愈加明显。6.Westen blot检测各组心肌组织中PKD1及磷酸化PKD1的表达与同周龄WKY对照组相比,SHR8、16、24周龄组大鼠心肌组织中p-PKD1744/748、p-PKD1916表达明显升高（P＜0.01）;随着周龄的增加SHR组p-PKD1744/748、p-PKD1916表达水平逐渐升高（P＜0.01）。同期WKY各组大鼠的p-PKD1744/748、p-PKD1916没有显著性变化。SHR阿托伐他汀组的磷酸化PKD1较SHR24周组的表达减弱,有统计学意义（P＜0.01）。SHR各组PKD1表达无明显变化。相关性分析显示,SHR组p-PKD1744/748、p-PKD1916表达与IVST、LVM、LVMI呈明显的正相关（r=0.74,P＜0.01）。7.心肌纤维化定性及定量分析Masson染色胶原含量的检测:WKY组心肌纤维排列整齐、紧致,小动脉周围少量胶原沉积,但很少向周围间质延伸,心肌间质内胶原多呈散在或长条索状分布;SHR组与WKY组比较,心肌细胞肿大,肌纤维排列疏松紊乱,血管周围及心肌间质胶原显著增多,且交联成网格状。阿托伐他汀组心肌细胞肿大的现象明显减少。定量分析心肌组织羟脯氨酸含量、心肌胶原容积分数（CVF）和心肌内血管周围胶原面积（PVCA）,并对所得指标进行相关分析。检测结果为SHR大鼠D、E、F组心肌羟脯氨酸含量随着周龄的增加。与同龄WKY对照组相比,SHR16、24周龄组左室心肌组织胶原含量明显升高（P＜0.01）。阿托伐他汀用药组左室心甲橹涸拷蟂HR24周龄组明显减低（P＜0.05）。相关性分析显示:SHR组大鼠的左室心肌组织胶原含量与周龄及p-PKD1744/748表达、p-PKD1916表达、IVST、PWT、LVM、LVMI呈明显的正相关（r为0.746,P＜0.01,）,而与PKD1表达无明显相关性。结论1.SHR大鼠随着喂养时间延长呈增龄性心肌肥厚和心肌纤维化,为高血压左室重塑发病机制的研究提供了可靠动物模型平台。2.SHR大鼠心肌组织中磷酸化PKD1表达升高,且与心肌肥厚相关指标呈明显正相关,提示磷酸化PKD1参与了自发性高血压大鼠心肌肥厚的发生发展过程。3.自发性高血压大鼠心肌组织纤维化与p-PKD1有相关性。4.阿托伐他汀可以抑制自发性高血压大鼠左室重塑的发展,其作用靶点可能与PKD1有关。论文Ⅱ蛋白激酶D1在血管紧张素Ⅱ诱导心肌细胞肥大、心肌成纤维细胞增殖和胶原合成中的作用及其信号转导机制背景心肌重构是一系列复杂的分子及细胞学的改变引起的心肌结构及功能的重要变化,这些变化主要包括:心肌重构的结构改变,表现为心室腔的扩大,室壁增厚及心室腔几何构型的改变。当后负荷增加时,心肌在收缩期压力超负荷,心室壁张力增加,心肌细胞中肌小节数量增加成横向排列,结果细胞的直径增大导致心室壁增厚,心室腔相对变小。而前负荷增加时,心腔容量超负荷,舒张期室壁张力升高,肌小节数量增多并呈纵向排列,细胞变长,心腔呈离心性重构。Ganau等对未经治疗的高血压病患者按超声测定的左室心肌重量指数（LVMI）与相对室壁厚度（RLVT）将心室重构分为正常构型、向心性重构、向心性肥厚、离心性肥厚四种构型。后负荷增重所致左室重构主要表现为左室肥厚和心肌纤维化,在细胞水平表现为心肌细胞肥大、心肌成纤维细胞增殖和胶原合成的增加。AngⅡ是RAS系统的主要血管活性肽,实验证实,AngⅡ可以通过调节各种细胞因子最终导致细胞的增殖、肥大及炎症反应。在高血压的病理状态下,神经体液因子的激活比压力负荷的升高更为重要。业已证实,全身的RAS,尤其是血管和心肌组织的RAS在高血压的发生、发展中起着重要作用。而RAS的中心环节是AngⅡ识别其特异性受体,并通过一定的信号途径而发挥作用。不同的细胞类型及不同的细胞因子环境下,AngⅡ可引起不同的生长反应,增殖或肥大,这取决于细胞周期蛋白依赖激酶（CDK）表达的改变,研究表明,AngⅡ刺激心肌细胞时,CDK2活性受到抑制,使细胞周期停滞在G1期,最终导致细胞肥大。MAPKs通路在心肌重塑中的激活是一个三级级联反应,MAP3K激活MAP2K（MEK）,而MEK激活MAPK。激活后的MAPKs转到细胞核周围靶向作用于下游的转录因子及一些肥厚基因如ANP,BNP,β—MHC和α—SKA。最后心肌细胞体积增大（直径增宽或长度增加）,肌节数量增多、心肌胶原纤维含量增多,心肌发生肥厚的表型改变。本研究的第一部分结果显示,PKD1在心脏组织的表达升高与左室重塑相关,提示PKD1可能通过心脏组织参与了心肌肥厚的病理生理过程。最新研究表明,PKD1能通过多种途径参与肥大反应,增加多种细胞因子的表达。而MAPK后的信号通路与心肌肥厚的信号传导存在交叉作用。因此,我们设想PKD1可能通过调节心脏组织ERK5/MEF2C信号通路促进心肌肥厚,从而导致高血压左室重塑的发生。目的1.验证AngⅡ诱导心肌细胞肥大、促成纤维细胞增殖和胶原合成作用;2.明确AT1/PKC/PKD1/ERK5/MEF2C信号转导通路在AngⅡ诱导心肌细胞肥大中的作用机制;3.探讨PKD1参与AngⅡ诱导心肌成纤维细胞增殖、胶原合成的信号转导通路。方法取1-3天SD新生乳鼠,进行原代培养。体外培养心肌细胞加入不同的刺激因素,研究心肌细胞在干预后的变化,分别进行以下处理:无刺激因素孵育细胞;心肌细胞用一定浓度的AngⅡ刺激;AngⅡ刺激+AT₁拮抗剂Losartan孵育细胞;AngⅡ刺激+AT₂拮抗剂PD123319孵育细胞;AngⅡ刺激+PKC抑制剂G（o|¨）6983孵育细胞;AngⅡ刺激+PKεsiRNA孵育细胞;AngⅡ刺激+PKD1 siRNA孵育细胞;AngⅡ刺激+ERK5 siRNA孵育细胞。对不同时间段的心肌细胞进行如下检测:（1）电镜观察心肌细胞形态学改变;（2） ³H-亮氨酸摄入率测定,评估细胞肥大的程度;（3） Western blot测定p-PKCa/β、δ、ζ、ε、PKC以及PKD1、p-PKD1、ERK1/2、p-ERK1/2、p38、p-p38、ERK5、p-ERK5、MEF2C、p-MEF2C的表达;（4）加入特异性siRNA观察通路分子的关系;（5）免疫荧光测定各分子的表达及ERK5胞浆胞核之间的转位。体外培养的新生大鼠心肌成纤维细胞,通过观察血管紧张素Ⅱ刺激后大鼠心肌成纤维细胞PKD1、p-PKD1的表达变化,观察PKDsiRNA对血管紧张素Ⅱ诱导的成纤维细胞增殖、胶原合成的影响,旨在探讨PKD1信号通路在心肌细胞肥大及心肌纤维化中的发病机制中的作用。结果1.AT1/PKC/PKD/ERK5/MEF2C信号通路参与AngⅡ诱导的心肌细胞肥大（1） AngⅡ刺激心肌细胞的形态学变化AngⅡ刺激组加血管紧张素Ⅱ（以无血清培养液溶解）使终浓度为100nM;正常对照组（control）:培养液中加等量无血清培养液。处理不同时间（0min、5min、15min、30min、60min、120min）后在显微镜下观察细胞形态学变化。镜下观察可见在加用血管紧张素Ⅱ刺激后,心肌细胞较正常对照组表现明显肥大。随着时间的延长,细胞肥大明显。AngⅡ刺激心肌细胞15分钟,表面积呈剂量依赖性增加,其中10nM、100nM、1000 nM的AngⅡ组心肌细胞的表面积分别为1422.31±139.26μm²、1931.79±142.66μm²、2032.71±195.73μm²,均明显高于对照组心肌细胞表面积（816.39±92.11μm²）,差异非常显著（P＜0.01）。（2） AngⅡ刺激的心肌细胞³H—亮氨酸掺入的测定将3×10⁵ cells/ml的心肌细胞接种于24孔板上,24h后换成无血清培养液,48h后加处理因素。AngⅡ浓度分为5组:空白组、1nM、10nM、100nM、1000nM。用LKB液体闪烁仪测定³H放射性强度。结果发现:AngⅡ100nM 15分钟、30分钟刺激的大鼠心肌细胞蛋白质合成速率明显增加,³H-亮氨酸掺入较空白组、1nM组明显增高（P＜0.01）。随着AngⅡ浓度的增加,心肌细胞蛋白合成速率呈剂量依赖性增加,其中10nM、100nM、1000 nM的AngⅡ组心肌细胞[3H]-亮氨酸掺入率分别为1588.71±144.52、2008.14±151.25、2177.38±165.76cpm/well,均明显高于对照组心肌细胞[3H]-亮氨酸掺入率（1141.27±134.95cpm/well）,差异非常显著（P＜0.01）。（3） Western blot测定AT₁/PKC/PKD1/ERK5/MEF2C信号通路①AngⅡ通过AT1激活PKD1AngⅡ受体两个亚型AT1、AT2。应用AT1特异性拮抗剂缬沙坦（0.3、1.0、3.0μmol/L）及AT2特异性拮抗剂PD123319（20μmol/L）预处理心肌细胞,然后AngⅡ100nM刺激半小时,结果显示缬沙坦3μmol/L完全阻断p-PKD17441748、p-PKD1916的表达,而PD123319对p-PKD的表达没有影响。②AngⅡ激活PKD1是依赖于PKC通路应用PKC的抑制剂G（o|¨）6983不同浓度（0.3、1、3μmol/L）预处理心肌细胞1小时,AngⅡ100nM刺激心肌细胞,结果显示随着浓度的增加,G（o|¨）6983抑制磷酸化PKD1的表达,由此证明PKC参与AngⅡ激活PKD1的过程。③PKCε特异性介导AngⅡ激活PKD1选择PKC的多个亚型PKCα,β,δ,εandζ,观察AngⅡ刺激心肌细胞各亚型的表达。结果显示:只有PKCε的激活随着时间的变化而变化,表达呈时间依赖性变化,而PKCα/β、PKCδ、PKCζ磷酸化表达没有变化。p-PKCε与p-PKD1的表达有显著相关性（p＜0.05）。④AngⅡ对心肌细胞中促分裂原活化蛋白激酶（MAPKs）的激活AngⅡ100nM不同时间（0、5、15、30、60min）刺激心肌细胞,观察MAPKs中ERK1/2、p-38、ERK5总蛋白及磷酸化蛋白的表达,结果发现磷酸化ERK5表达呈时间依赖性变化,在5分钟时开始表达,随后表达水平开始逐渐上升,15min到30min达到表达高峰,以后则呈逐渐下降,趋势1小时表达恢复基线水平。与0、5、60min组相比均有显著性差异（p＜0.01）。磷酸化ERK1/2、磷酸化p-38表达较早,5min表达达高峰,较0、15、30、60min有显著性差异（p＜0.01）。ERK1/2、p-38、ERK5总蛋白表达在各时间段没有变化。⑤PKCε及PKD1参与了AngⅡ介导的MEF2C的激活AngⅡ100nM不同时间（0、5、15、30、60min）刺激心肌细胞,观察MEF2C的激活。结果显示:磷酸化MEF2C在5分钟时开始表达,随后表达水平开始逐渐上升,15min到30min达到表达高峰,1小时表达恢复基线水平。与0、5、60min组相比均有显著性差异（p＜0.01）。磷酸化MEF2C与磷酸化PKCε、磷酸化PKD1及磷酸化ERK5表达有显著相关性（p＜0.01）。（4）加入特异性siRNA观察通路分子的关系①PKCεsiRNA应用PKCεsiRNA转染心肌细胞,观察下游分子PKD1、ERK5、MEF2C的表达。结果显示:PKCεsiRNA抑制p-PKD1、p-ERK5、p-MEF2C的表达,与对照组有显著性差异（P＜0.01）.②PKD1 siRNA应用PKD1 siRNA转染心肌细胞,观察下游分子ERK5、MEF2C的表达。结果显示:PKD1 siRNA抑制p-ERK5、p-MEF2C的表达,与对照组有显著性差异（P＜0.01）。③ERK5 siRNA应用PKD1 siRNA转染心肌细胞,观察下游分子MEF2C的表达。结果显示:ERK5 siRNA抑制p-MEF2C的表达,与对照组有显著性差异（P＜0.01）。（5）免疫荧光测定各分子的表达及ERK5胞浆胞核之间的转位用免疫荧光细胞化学染色观察AngⅡ处理心肌细胞15min的p-PKD1744/748、p-PKD1916、p-ERK5、p-MEF2C表达。刺激细胞15分钟,结果显示:p-PKD1744/748、p-PKD1916胞质表达,p-ERK5、p-MEF2C胞核表达。随着时间的变化,p-ERK5由胞质转移到核内,在由核内转移到胞质。2.PKD1参与心肌成纤维细胞增殖、胶原合成建立原代新生SD大鼠心肌成纤维细胞（CFs）培养模型,探讨AngⅡ促成纤维细胞增殖和胶原合成中的细胞信号转导过程中PKD1的参与机制。（1） Angll促CFs增殖和胶原合成的浓度和时间效应①AngⅡ不同浓度对心肌成纤维细胞增殖和胶原合成的影响AngⅡ10、100、1000nM刺激CFs24小时,A490值均高于无AngⅡ刺激组及FBS组,差异有统计学意义（P＜0.05～0.01）;羟脯氨酸量亦均高于无AngⅡ刺激组及FBS组。随着浓度的增加,CfsA490值和羟脯氨酸量的增加更为明显,不同的AngⅡ刺激浓度组间差异有统计学意义（p＜0.05）。②AngⅡ对心肌成纤维细胞增殖和胶原合成影响的时间效应予100nM AngⅡ刺激CFs,在24h内,随着刺激时间的延长,平均吸光度值及胶原合成量逐渐增高。（2） AngⅡ刺激新生SD大鼠CFs PKD1表达和活性情况Western Blot分析显示,AngⅡ（100nM）刺激心肌成纤维细胞12小时后,即出现p-PKD1744/748、p-PKD1916表达的增高,12、24、48h蛋白质表达比对照组分别增加52%、153%、110%,高峰时间为24小时。AngⅡ刺激各实验时间点p-PKD1744/748、p-PKD1916表达与对照组比较差异有统计学意义（P＜0.05～0.01）。（3）抑制PKD1的表达对AngⅡ刺激CFs增殖和胶原合成的影响应用PKD1 siRNA预处理CFs后,再用AngⅡ刺激CFs。PKD1 siRNA+AngⅡ组p-PKD1水平显著低于AngⅡ刺激组（P＜0.01）,而与对照组差异无统计学意义（P＞0.05）。PKD1 siRNA+AngⅡ组平均吸光度值和胶原合成量均低于AngⅡ刺激组,差异有统计学意义（P＜0.01）,单用PKD1 siRNA预处理而未加AngⅡ刺激组平均吸光度值和胶原合成量与对照组间差异无统计学意义。结论1.进一步验证了AngⅡ具有诱导心肌细胞肥大,促成纤维细胞增殖和促胶原合成作用,这些作用均呈剂量和时间依赖性。2.首次证明AT1/PKC/PKD1/ERK5/MEF2C信号通路参与AngⅡ诱导的心肌细胞肥大的发生发展过程。3.AngⅡ可增加新生SD大鼠心肌成纤维细胞的p-PKD1蛋白表达,PKD1siRNA可显著抑制AngⅡ刺激的CFs增殖和胶原合成。PKD1在Ang Il刺激CFs增殖和胶原合成中可能发挥重要作用。更多还原

【Abstract】 DissertationⅠ:The Relationship between Expression of Protein Kinase D1 and Left Ventricular Remodeling in Spontaneously Hypertensive Rats as well as Atorvastatin Interventive StudyBackgroundHypertension is the most important risk factor for cardiovascular disease.The adult heart undergoes myocardial remodeling when subjected to longstanding hypertension. Hypertensive myocardial remodeling is an independent risk factor for lethal cardiovascular events,and is the key pathological manifestations during the transition of heart function from compensation to decompensation.Hearts respond to such stress stimuli by increasing cell size and extracellular matrix,reorganizing sarcomeres and activating a fetal cardiac gene program.Although these responses may initially normalize wall stress,the prolonged hypertrophy increases the risk for chamber dilation,heart failure and sudden death.Recent years,with the development of molecular biology,cardiac remodeling has been shown to be the basic mechanism of heart failure.Cardiac remodeling is a complex course,and the pathogenesis mechanism still remains unclear.It is well-known that abnormal activation of neurohumoral factors,oxidative stress and cytokines are involved in cardiac remodeling.A complex set of signal-transduction pathways and downstream transcription factors underlie hypertensive myocardial remodeling.Therefore,the researches on the pathogenesis and control of cardiac remodeling have become a worldwide hot topic. Protein Kinase D（PKD） is a recent addition to the calcium/calmodulin-dependent serine/threonine protein kinase.PKD family consists of 3 isoforms,these are the original PKD1 which now is also referred to as PKD, PKD2 and PKD3.Increasing evidence now points toward important roles for PKD-mediated signaling pathways in the cardiovascular system,particularly in the regulation of myocardial contraction,hypertrophy and remodeling.Lots of studies have reported that cardiac specific expression of a constitutively active PKD mutant in transgenic mice leads to cardiac hypertrophy,and a chronic increase in PKD activity is sufficient to induce adverse myocardial remodeling.Studies also show that mice lacking cardiac PKD display an impaired response to stress signals that normally lead to cardiac hypertrophy.As hypertension is often accompanied by dyslipidemia,the treatment frequently involves 3-hydroxy-3-methylglutaryl coenzymeA（HMG-CoA） reductase inhibitors （statins）.More and moer evidences have been established that statins not only efectively reduced serum cholesterol level,but also exerted pleiotropic beneficial efects on cardiovascular disease,including improvement of endothelial function, reduction of plaque thrombogenicity,prevention of cardiac hypertrophy or remodeling. However,the exact mechanism remains unclear.This study was therefore designed to observe the temporal profile of the expression of PKD and analyze its relationship with hypertensive cardiac remodeling; to study the role of PKD related signal-transduction pathways and downstream factors; to investigate the effects and the mechanism of atorvastatin in the prevention and treatment of cardiac remodeling.The purpose of the study is to elucidate the cellular and molecular mechanisms of hypertensive cardiac remodeling and the interventional effects of atorvastatin on it,and to provide novel theoretical evidences and strategy for prevention and treatment of hypertensive cardiac remodeling.Objectives1.To investigate the expression of PKD and related molecular pathway at both the mRNA and protein levels in myocardium of spontaneously hypertensive rats （SHR）. 2.To investigate the involvement and the signal-transduction pathway of PKD in left ventricular remodeling in SHR.3.To evaluate the effects and the mechanism of atorvastatin in the prevention and treatment of cardiac remodeling.MethodsTwenty-eight 8-week-old WKY、fourty-one SHRs were obtained from company. WKY rats were randomly divided into the following four groups:A Group:8 weeks （n=7）;B Group:16 weeks（n=7）;C Group:24 weeks（n=7）;G Group:WKY placbo （n=7）.SHR rats were randomly divided into the following five groups:D Group:8 weeks（n=7）;E Group:16 weeks（n=7）;F Group:24 weeks（n=7）;H Group:SHR placbo（n=10）;I Group:SHR atorvastatin(（n=10）.All animals were feeded by normo-fortage and water.WKY placbo、SHR placbo、SHR atorvastatin receiving distilled water or atorvastatin at 50 mg/kg/day for 16 weeks by intragastric administration.Animals were killed when they were 8 weeks,16 weeks and 24 weeks old by decapitation.The hearts were immediately harvested and weighed.The follwing parameters were measured during the study:（1） All the rats have their body weight,heart rat and tail blood pressure measured once per 2 week;（2） Blood was collected from jugular vein at 8 weeks,16 weeks and 24 weeks respectively.Plasm lipid was determined using routine method;（3） Echocardiography was used to evaluate the function of heart, LVEDd,IVSd and LVPWd were measured,RWT and LVEF were calculated;（4） Histopathological study of heart tissue,for the detection of remodeling,the ventdcular tissue was stained with H-E and Masson’s trichrome staining;（5） The left ventricular mass index（LVMI） was used to estimate the degree of cardiac hypertrophy.（6） Hydroxyproline content assay,CVF and PVCA were used to estimate the cardiac fibrosis;（7） Western-blot,RT-PCR for the expression of PKD, ERK5 and MEF2C.Results1.The experimental animals Two rats of SHR group died in the entire experiment,one of WKY group died. A total of 66 rats finished the study,27 rats in WKY group,39 rats in SHR group.2.Comparisons of SBP,BW,HR and Lipids level between WKY and SHR groups.There were no significant differences in terms of body weight,heart rat,tail systolic blood pressure and lipids at the beginning of the experiment.SBPs in SHRs at 16 and 24 weeks are higher than those in WKY rats（P＜0.01）.There were no significant differences in the BW,HR and total cholesterol level among the SHR and WKY groups..In SHR group,SBP increased during the course,while the one in WKY-V group remained unchanged.After treatment with atorvastatin,SBP decreased significantly（P＜0.01）.3.Eehoeardiographie evaluationEchocardiography was taken at 8,16 and 24 weeks respectly.Interventricular septum （IVS）,left ventricular posterior wall（LVPW） and left ventricular diastolic diameter （LVDd） of SHR in 16W and 24W group increased significantly（P＜0.01） compared with the SHR 8W and those in WKY group,and these changes were attenuated by atorvastatin（P＜0.01）.4.HE stainingHE staining slides under optical microscopy showed that in WKY group,cell size of cardiomyocytes was smaller,uniform,and array was regular;In SHR 16W and 24W groups,cardiomyocytes size was greater,not uniform,cell form was irregular,and cell arrange was disorder;Cell wall was not clear and cell fusion was observed at intersection of cell.Cardiomyocytes in atorvastatin group lied between WKY and SHR group.5.Left Ventricular Mass Index analysisThe left ventricular mass index（LVMI） is the ratio of left ventricular weight（in milligrams） to body weight（in grams）（LVW/BW）,LVMI of SHR in 16W and 24W group increased significantly（P＜0.01） compared with the SHR 8W and those in WKY group,and these changes were attenuated by atorvastatin（P＜0.01）.6.Cardiac fibrosis of SHRs As depicted in Masson,marked deposition of collagen was detected in the cardiac interstitial and perivascular areas of the SHRs in 16W and 24W compared with the WKY rats,which was ameliorated by atorvastatin.Consistent with this, hydroxyproline content,CVF and PVCA were elevated in the left ventricle of SHRs in 16W and 24W compared with WKY rats（P＜0.01）,and atorvastatin significantly attenuated this elevation（P＜0.05）7.Expression of PKD,p-PKD1 by Western-blotCompared to WKY group,the groups of SHRs in 16W and 24W showed a significant rise in p-PKD protein content in heart tissue（P＜0.01）.In SHR group,expression of p-PKD increased during the course（P＜0.01）,while the one in WKY-V group remained unchanged.Consistent with this,p-ERK5 and p-MEF2C in SHRs of 16W and 24W increased significantly（P＜0.05）,and these changes were attenuated by atorvastatin （P＜0.01）.8.Relationship among blood pressure,cardiac hypertrophy,fibrosis and expression of p-PKD1744/748、p-PKD1916The LVMI,IVS,LVPW,LVDd,hydroxyproline content,CVF and PVCA were all positively related to the systolic blood pressure,and reduction of blood pressure by atorvastatin contributes to the attenuation of cardiac hypertrophy and fibrosis.The LVMI was also significantly correlated with the hydroxyproline content.Cardiac hypertrophy was positively correlated with the expression of p-PKD1744/748 and p-PKD1916,p-PKD1744/748 and p-PKD1916 was positively related to the collagen content.ConclusionsThe aging-related myocardial hypertrophy and fibrosis occured with the development and process of hypertension in SHR model which offers reliable animal mode for the researches on the mechanism of hypertensive LV myocardial remodeling.2.The over-expression of PKD1 at both the mRNA and protein levels has been confirmed in the heart tissue of SHR,and which associated significantly with the myocardial hypertrophic-related parameters.The activation of PKD1 involved in the development and process of myocardial hypertrophy in SHR.3.There was relationship between p-PKD1 and myocardial fibrosis in SHR.4.Atorvastatin could partially reverse the hypertension-induced myocardial remodeling through the down-regulation of PKD activation. DissertationⅡ:The Role of Protein kinase D in Cardiomyocyte Hypertrophy And Cardiac Fibroblasts Proliferation Induced by AngiotensinⅡStimulation:Involvement of Signalling PathwayBackgroundThe reason of cardiac remodeling are a series of complex molecular and celluar mechanisms,which contribute to the changes of cardiac structure,function and phenotye.These changes including:myocyte hypertrophy,apoptosis,fibroblast proliferation、reexpression of fetal gene and protein.Among these changes,myocyte hypertrophy and fibroblast proliferation are the key factors of cardiac remoedling. AngiotensinⅡ（AngⅡ） is an octapeptide that exerts inotropic,hypertrophic and apoptotic effects on cardiomyocytes[Fabris et al.,2007;Mollmann et al.,2007].Thus, AngⅡis central for any process involved in control of hypertrophy and heart failure. The corresponding signal transduction pathways have been demonstrated in fetal, neonatal and adult cardiomyocytes.The renin-Ang system is an important component of the physiological and pathological responses of the cardiovascular system.Through AngⅡreceptor-1（AT₁）.AngⅡcarries out its functions,including hypertrophic remodeling of cardiomyocytes,which involves various downstream signal transduction mechanisms However,the regulatory molecular mechanisms, specifically the signaling cascades,involved in AngⅡ-induced cardiomyocyte hypertrophy are not fully understood.Mitogen-activated protein kinases（MAPKs） are a family of serine/threonine kinases that play a central role in transducing extracellular cues into a variety of intracellular responses.Activated MAPKs phosphorylate multiple intracellular targets,including numerous transcription factors that induce the reprogramming of gene expression. More recently,some reports have showed that PKD1 is implicated in AngⅡ-induced proliferation of vascular smooth muscle cells.However,little is known about how extracellular hypertrophic stimulation angtensinⅡis perceived and converted into intracellular signals and how these signals change the transcriptional program that eventually leads to cardiac hypertrophy and fibros in vivo.Protein Kinase D（PKD） is a recent addition to the calcium/calmodulin-dependent serine/threonine protein kinase,increasing evidence now points toward important roles for PKD-mediated signaling pathways in the cardiovascular system,particularly in the regulation of myocardial contraction,hypertrophy and remodeling.The laboratories of Olson and McKinsey have reported that cardiac specific expression of a constitutively active PKD mutant in transgenic mice leads to cardiac hypertrophy, and a chronic increase in PKD activity is sufficient to induce adverse myocardial remodeling.Studies also show that mice lacking cardiac PKD display an impaired response to stress signals that normally lead to cardiac hypertrophy.These exciting data,together with the preliminary evidence that PKD expression may be increased in human heart failure,necessitate further investigation of the role of PKD in the development of cardiac remodeling and failure in vivo in response to clinically relevant stresses such as pressure overload and myocardial infarction.Objective1 To validate further the effect of cardiomycyte hypertrophy,fibroblast proliferation and syncesis of collagen induced by AngⅡ.2 To investigate the mechanism of the signal-transduction pahtway bout AT1/PKC/PKD1/ERK5/MEF2C in hypertrophic cardiomyocytes induced by AngⅡ.3 To probe the investigate the involvement and the signal-transduction pathway of PKD1 in fibroblast proliferation and syncesis of collagen.MethodsTake 1-3days SD neonatal rats,primary culture in vitro.Study changes of cadiomyocyte after stmulation.Cardiomyocyts were divided into different groups: Control Group:no stmulation factor;AngⅡstmulation Group:different dosges AngⅡ; AngⅡstmulation +Losartan Group;AngⅡstmulation + PD123319 Group;AngⅡstmulation+ PKC inhibitor G（o|¨）6983 Group;AngⅡstmulation + PKCεsiRNA Group; AngⅡstmulation+ PKD1 siRNA Group;AngⅡstmulation+ ERK5 siRNA Group. Dtecting different time group myocyte below:（1） Observing changes of cardiomyocyte form by electron microscope;（2） ³H-leu incorporation rate,evaluated the level of cell hypertrophy;（3） Western blot dectected expression of p-PKCα/β、δ、ζ、εand PKD、p-PKD、ERK1/2、p-ERK1/2、p38、p-p38、ERK5、p-ERK5、MEF2C、p-MEF2C;（4） Added special siRNA to study the relationship among pathway molecule;（5） Determine expression of pathway rnolecul by Immunofluorescence method and EKR5 translocation between cytoplasm and nucelus.The neonate rat CFs as a experimental model were cultured primarily by the different time.To detcet effect of AngⅡon CFs hyperplasy and collogen synthesis and expression of PKD1、p-PKD1.Results1.AT1/PKC/PKD/ERK5/MEF2C signal-transduction pathway was involved in hypertrophic cardiomyocytes induced by AngⅡ.（1） Morphological changes of cardiomyocytes stimulated by AngⅡAngⅡgroup:the final concentration is 100nM:Control group:cardiomyocytes were incubated in serum-free medium.Morphological changes were observed by electron microscope after treatment at different times（0min、5min、15min、30min、60min、120min）.Hypertrophy phenomenon of cardiomyocytes was significantly greater in AngⅡtreated cells than in controls,which was in a time-dependent way.Moreover, AngⅡtreatment increased the cell surface area in a dose-dependent way.The cell surface area of 10nM,100nM and 1000 nM groups were 1422.31±139.26μm²、1931.79±142.661μm²、2032.71+195.73μm² repectively,which were significantly higher than those of the control group（816.39±92.11μm²）（P＜0.01）.（2） Measurement of[3H]-Leu incorporation Cardiomyocytes（3×10⁵ cells/ml） which were incubated in 24 orifice plates were made quiescent by incubation in serum-free DMEM medium for 24 h.Cells were stimulated by different concentrations of AngⅡ（0,1nM,10nM,100 nM and 1000nM）.[3H]-Leu incorporation were determined by using a scintillation counter. 100nM AngⅡrapidly increased[3H]-Leu incorporation with peak incorporation at 15 and 30 min.[3H]-Leu incorporation was concentration dependent,beginning at 10 nmol/1 AngⅡand with maximum effect at 1000 nmol/1 AngⅡ.[3H]-Leu incorporation of 10nM,100nM and 1000 nM groups were 1588.71±144.52、2008.14±151.25、2177.38±165.76cpm/well repectively,which were significantly higher than those of the control group（1141.27±134.95cpm/well）（P＜0.01）.（3） Western blot for AT₁/PKC/PKD1/ERK5/MEF2C signal-transduction pathway●AngⅡstimulates PKD activation through a AT1-dependent pathway AngⅡreceptor has two subtypes:AT1 and AT2,cells were pretreated for 1 hr with with losartan(0.3,1.0,3.0μmol/L,a specific antagonist for AT1,or PD123319（20μmol/L）,an antagonist for AT2,then stimulated with AngⅡ（100 nmol/L） for 0.5 hr. Losartan at 3.0μmol/L completely blocking PKD phosphorylation at Ser744/748 and Ser916,whereas PD123319 had no effect on AngⅡactivation of PKD.●Activation of PKD by AngⅡis PKC-dependent Cells were pretreated with the general PKC inhibitor G（o|¨）6983（0.3,1,3μmol/L） for 1 hr before exposure to AngⅡ（100 nmol/L） for 1 hr.G（o|¨）6983 blocked PKD phosphorylation in a dose-dependent manner,which suggests that PKC is involved in AngⅡ-stimulated PKD phosphorylation in cardiomyocytes.●PKCεspecifically mediated AngⅡ-induced PKD phosphorylation Several members of PKC isoforms,includingα,β,δ,εandζ,are expressed in cardiomyocytes,AngⅡtreatment induced marked phosphorylation of PKCεwithin 15 min but did not induce phosphorylation of PKCα/β,PKCδ,or PKCζ.Expression of p-PKCεis significantly correlated with phosphorylation of PKD（P＜0.05）.●Activation of MAPKs by AngⅡin neonatal rat cardiomyocytes Cardiomyocytes were stimulated by AngⅡat 100nM for different times（0,5,15,30, 60min）,we examined the potential role of ERK1/2,P38,ERK5.AngⅡ（100 nmol/L） induced phosphorylation of ERK5 after 5 min,with peak phosphorylation between 15 and 30 min（P＜0.01）,which returned to base line after 1 hr.However,the phosphorylation of ERK1/2 and p38 were earlier than that of ERK5,with peak phosphorylation at 5 min（P＜0.01）. Total protein level of ERK5,ERK1/2 and P38 did not change.●PKCεand ERK5 are involved in AngⅡ-induced activation of MEF2C Cardiomyocytes were stimulated by AngⅡat 100nM for different times（0,5,15,30, 60min）,then examined the activation of MEF2C.AngⅡsignificantly stimulated the phosphorylation of MEF2C by 5min treatment,with peak phosphorylation at 15min （P＜0.01）,which returned to basal levels by 60 min.Activation of MEF2C is positively related to phosphorylation of PKCε,PKD and ERK5.●Further research in the signal-transduction pathway by specific siRNA PKCεsiRNACardiomyocytes were infected with PKCεsiRNA before stimulated by AngⅡat 100nM,then examined the activation of PKD,ERK5 and MEF2C.PKCεsiRNA inhibited the phosphorylation ofPKD,ERK5 and MEF2C（P＜0.01）.PKD siRNACardiomyocytes were infected with PKD siRNA before stimulated by AngⅡat 100nM,then examined the activation of ERK5 and MEF2C.PKD siRNA inhibited the phosphorylation of ERK5 and MEF2C（P＜0.01）.ERK5 siRNACardiomyocytes were infected with ERK5 siRNA before stimulated by AngⅡat 100nM,then examined the activation of MEF2C.ERK5 siRNA significantly inhibited the phosphorylation of MEF2C（P＜0.01）.（4） Immunofluorescence staining for expression of all moleculars and translocation of ERK5 The expression of p-PKD 1744/748、p-PKD 1916、p-ERK5、p-MEF2C was observed by immunofluorescence staining.After stimulation by AngⅡfor 15 min, p-PKD744/748 and p-PKD1916 were expressed in cytoplasm while p-ERK5 and p-MEF2C were in the nucleus.Before stimulation with AngⅡ,ERK5 was located primarily in the cytoplasm of cardiomyocytes;ERK5 nuclear entry was seen at 15 min after AngⅡstimulation,with a striking translocation from the cytoplasm to the nucleus.At 60 min of AngⅡtreatment,ERK5 was gradually shuttled back to the cytoplasm.2.PKD was involved in the proliferation of cardiac fibroblasts（CFs） and collagen synthesis（1） AngⅡinduced the proliferation of CFs and collagen synthesis in concentration and time dependent manner●AngⅡconcentration-dependently stimulate the proliferation of CFs and collagen synthesisCompared with the control and FBS groups,A490 of AngⅡ10,1O0,1000nM groups were significant higher（P＜0.05～0.01）,Consistent with this,hydroxyproline content was also elevated（P＜0.05）.A490 and hydroxyproline content were concentration dependent,beginning at 10 nmol/1 AngⅡand with maximum effect at 1000 nmol/1 AngⅡ.●AngⅡtime-dependently stimulate the proliferation of CFs and collagen synthesisCFs were stimulated by AngⅡat 100nM,absorbance value and hydroxyproline content were time-dependently elevated within 24 hr.（2） Activation of PKD in CFs stimulated by AngⅡCFs were stimulated by AngⅡat 100nM for different times（12,24,48 hr）,we examined the phosphorylation of PKD.AngⅡ（100 nmol/L） induced phosphorylation of PKD at Ser744/748 and Ser916 after 12 hr with peak phosphorylation at 24 hr. Compared with the control group,the p-PKD content in 12,24 and 48hr increased 52%,153%and 110%respectively（P＜0.05～0.01）.（3） PKD specific siRNA inhibited the proliferation of CFs and collagen synthesis CFs were infected with PKD siRNA before stimulated by AngⅡ,then measured the absorbance value and hydroxyproline content.Compared with the AngⅡgroup, p-PKD expression in the PKD siRNA+ AngⅡgroup was significantly inhibited （P＜0.01）,and was similar with the control group（P＞0.05）.PKD siRNA decreased both the absorbance value and the hydroxyproline content.（P＜0.01）. Conclusions1.AngⅡinduced hypertrophy of cardiomyocytes,proliferation of cardiac fibroblasts and synthesis of collagen.2.AT1/PKC/PKD1/ERK5/MEF2C signal pathway was involved in hypertrophy of cardiomyocytes induced by AngⅡ.3.PKD1 plays an important role in AngⅡinduced proliferation of cardiac fibroblasts and synthesis of collagen.更多还原