微小RNA451a对心肌肥厚的影响

目的探讨微小RNA(miRNA)-451a在心肌肥厚的作用。方法腹主动脉缩窄术(abodminal aortic constriction,AAC)建立大鼠心肌肥厚模型,超声检测、心肌肥厚指数及组织病理学观察评估模型建立效果,蛋白印迹检测LC3II/I的比值,实时定量聚合酶链反应(quantiative real-time polymerase chain reaction,qRT-PCR)检测心肌miRNA-451a及肥厚标志基因的表达。乳鼠心肌细胞分空白对照组及异丙肾上腺素(isoproterenol,ISO)组,qRT-PCR检测miRNA-451a及肥厚标志分子基因的表达,免疫荧光测定细胞表面积及测定LC3II/I的比值。乳鼠心肌细胞分4组,即miR-451a mimic组及其阴性对照组、miR-451a inhibitor组及其阴性对照组,分别转染乳鼠心肌细胞24 h再予ISO处理48 h,qRT-PCR检测肥厚标志基因的表达,测定细胞表面积及LC3II/I比值。结果 AAC组大鼠心肌肥厚模型建立成功,其肥厚标志基因表达上调,miRNA-451a表达下调,LC3II/I比值增大。ISO组心肌细胞比空白对照组肥厚标志基因表达上调,miRNA-451a表达下调,LC3II/I比值及细胞表面积增大。miRNA-451a mimic+ISO组心肌细胞比阴性对照组肥厚标志基因表达下调,细胞表面积及LC3II/I比值减小;而miRNA-451a inhibitor+ISO组心肌细胞则表现相反。结论 miRNA-451a可能在心肌肥厚过程中具有调控作用。     

miR-350在腹主动脉部分缩窄大鼠心肌组织中的表达及意义

目的观察腹主动脉部分缩窄大鼠心肌组织miR-350在心肌肥厚形成过程中的表达变化并探讨其临床意义。方法将20只雄性SD大鼠随机分成正常组5只、假手术组5只和模型组10只,采用腹主动脉部分缩窄术制备大鼠心肌肥厚模型。采用Trizol法提取心肌组织RNA,芯片技术检测miRNA表达。构建过表达miR-350质粒载体,转染大鼠的胚胎心肌细胞H9c2;采用Real-time PCR方法检测心肌细胞中ANP、BNP、β-myosin及α-actin的表达。结果 miR-350在模型组的表达量明显高于对照组;过表达miR-350的细胞中ANP mRNA及BNP mRNA升高(P<0.05或<0.01)。结论在腹主动脉部分缩窄大鼠的心肌组织中miR-350表达升高,且介导心肌细胞肥厚。     

DJ-1蛋白在去氧肾上腺素诱导的心肌细胞肥厚中的作用

目的 探讨DJ-1对去氧肾上腺素（phenylephrine,PE）诱导的心肌细胞肥厚的调控作用.方法 应用PE诱导乳鼠导致心肌细胞肥厚;通过Western blot观察心肌细胞DJ-1在PE刺激下的表达量改变;通过腺病毒载体过表达DJ-1,检测过表达效率;荧光定量聚合酶链反应（polymerase chain reaction,PCR）检测心肌肥厚标志物心房利钠肽（atrial natriuretic peptide,ANP）和B型利钠肽（B type-natriuretic peptide,BNP） mRNA表达水平改变;显微镜观察心肌细胞面积改变.结果 在PE诱导的心肌细胞肥厚过程中,DJ-1表达水平下调;PE可诱导ANP和BNP表达水平升高,心肌细胞面积增加;过表达DJ-1则显著抑制ANP和BNP的上调,减少心肌细胞面积的增大.结论 DJ-1可抑制PE诱导的心肌细胞肥厚.     

生理性和病理性心肌肥厚组织中心钠素表达的差异研究

［摘要］　目的　研究心钠素（ANP）在生理性和病理性心肌肥厚组织中表达的差异及其在心肌肥厚发生、发展中的作用。方法　选择无特定病原体（SPF）级8周龄雄性Sprague-Dawley(SD)大鼠15只,随机分为对照组（CON组）、异丙肾上腺素组(ISO组)和运动组(EXE组),每组5只。ISO组通过颈部多点皮下注射异丙肾上腺素构建病理性心肌肥厚模型。EXE组采用有氧运动（游泳）诱导生理性心肌肥厚。CON组不予特殊干预。在第8周末采用过量麻醉法处死大鼠,取心脏组织。采用HE染色法观察心脏组织病理、生理改变情况。采用Western-blot法和免疫组化染色法检测三组心肌组织中ANP的表达情况。结果　与CON组相比,EXE组和ISO组的心肌纤维明显增粗,且ISO组还出现了心肌纤维间隙增宽、排列紊乱等结构受损的特征,而EXE组的心肌纤维则排列整齐,结构完整。ANP主要表达于心肌细胞的胞浆中,ISO组ANP表达水平高于EXE组和CON组,差异有统计学意义（P<0.05）。结论　ANP在病理性心肌肥厚组织中表达升高,可作为鉴别生理性和病理性心肌肥厚的辅助性指标。     

环状RNA _005647通过结合miR-99b-5p抑制心肌细胞中肥厚相关基因的表达

目的研究环状RNA circRNA_005647抑制心肌肥厚相关基因表达的作用机制。方法建立血管紧张素Ⅱ(AngⅡ)灌注诱导的心肌肥厚小鼠模型。原代分离获得C57BL/6乳小鼠心肌细胞(NMVC),AngⅡ处理NMVC建立心肌细胞肥大模型。利用NMVC,分别感染circRNA_005647重组腺病毒(rAd-circRNA_005647)和转染miR-99b-5p模拟物来研究对NMVC肥大的影响。通过双荧光素酶报告基因实验和RNA Pull-down实验验证circRNA_005647与miR-99b-5p的结合作用。实时荧光定量PCR和Western blot检测NMVC中心肌肥厚相关基因的mRNA和蛋白表达水平。结果在AngⅡ诱导的小鼠心肌肥厚模型和心肌细胞肥大模型中,circRNA_005647及其宿主基因肌球蛋白IXA(Myo9a)表达升高。过表达circRNA_005647可抑制AngⅡ诱导的NMVC中肥厚相关基因心房钠尿肽(Anp)和肌球蛋白重链7(Myh7)基因表达升高。circRNA_005647与miR-99b-5p间存在结合作用。过表达circRNA_005647可抑制miR-99b-5p促心肌细胞肥大的作用。结论circRNA_005647通过结合miR-99b-5p发挥抑制心肌细胞肥大的作用。     

RNA干扰下调可溶性环氧化物水解酶表达对异丙肾上腺素诱导心肌细胞凋亡的影响

目的:构建含有可溶性环氧化物水解酶(sEH)基因特异性的RNA干扰序列质粒,选择性下调小鼠心肌细胞sEH的表达,筛选出抑制sEH效果最明显的表达质粒;观察其对异丙基肾上腺素(ISO)诱导的心肌细胞的凋亡及相关基因的影响.方法:构建2条靶向sEH基因的特异性小干扰RNA(siRNA)质粒 EH-1和 EH-2,以含非特异性siRNA编码序列的质粒(PCN)为阴性对照,用FuGENE HD转染原代培养的心肌细胞,通过半定量RT-PCR和Western blot法检测sEH的mRNA和蛋白的表达情况,筛选出抑制sEH效果最明显的表达质粒,命名为EH-R.采用浓度为10μmol*L-1ISO诱导心肌凋亡.分为正常对照组、ISO组、PCN+ISO组和EH-R+ISO组.利用EH-R下调心肌细胞sEH基因的表达,观察其对ISO诱导的心肌细胞凋亡的影响,流式细胞仪检测各组细胞凋亡的发生率,Western blot法检测各组Bcl-2和Bax蛋白的表达变化.结果:质粒EH-R组心肌细胞sEH的mRNA和蛋白质表达较其余组明显降低,差异有统计学意义(P<0.01).与正常对照组相比,应用ISO各组,心肌细胞凋亡率明显升高,心肌细胞的Bax表达升高,而Bcl-2表达下降(P<0.01).然而EH-R+ISO组与ISO组和PCN+ISO组相比心肌细胞凋亡率明显降低;心肌Bax表达降低,Bcl-2表达升高(P<0.01).结论:构建了特异性小干扰RNA质粒,利用RNAi技术成功下调了原代培养的心肌细胞中sEH的表达,从而增加了抑凋亡基因Bcl-2的表达,减轻了ISO诱导心肌细胞的凋亡.为进一步进行心肌细胞的RNA干扰研究奠定了基础.     

潜阳合剂对血管紧张素Ⅱ诱导的心肌细胞MYH7、ACTA1、BNP mRNA及蛋白表达的影响

目的观察潜阳合剂对血管紧张素Ⅱ(AngⅡ)诱导的心肌细胞β-肌球蛋白重链(MYH7)、骨骼肌肌动蛋白α1(ACTA1)、脑钠肽(BNP)mRNA及蛋白表达的影响,探讨潜阳合剂改善心肌纤维化的机制。方法建立AngⅡ诱导的心肌细胞异常增殖的细胞模型,并采用潜阳合剂干预。用CCK-8法测定潜阳合剂对心肌细胞的细胞毒性和拮抗AngⅡ作用的最佳浓度,并分成空白组、AngⅡ组(800 nmol/L)、AngⅡ+潜阳合剂组(稀释4000倍),应用蛋白质免疫印迹法(Western Blot)检测MYH7、ACTA1及BNP蛋白表达,实时荧光定量聚合酶链式反应(q-PCR)检测加入潜阳合剂后MYH7、ACTA1、BNP mRNA表达。结果与空白组比较,AngⅡ组MYH7、ACTA1及BNP mRNA及蛋白表达明显上升(P<0.05或P<0.01);与AngⅡ组比较,AngⅡ+潜阳合剂组MYH7、ACTA1、BNP mRNA及蛋白表达明显下降(P<0.05或P<0.01)。结论潜阳合剂可以通过降低MYH7、ACTA1、BNP mRNA及蛋白的表达起到改善心肌纤维化的作用。     

酸性富含亮氨酸的核磷蛋白32家族成员A对心肌细胞肥大的影响及机制研究

目的探讨酸性富含亮氨酸的核磷蛋白32家族成员A(Anp32a)对心肌细胞肥大的作用及机制。方法通过挖掘数据库小鼠心脏假手术组及主动脉缩窄手术组芯片数据,进行差异基因分析。选取8~10周龄小鼠,随机分为模型组和假手术组(n=7或8),终末取材提取心脏组织RNA检测Anp32amRNA表达水平。构建Anp32a敲低(AdshAnp32a),Anp32a过表达(AdAnp32a)及其相应的对照组AdshRNA,AdGFP腺病毒并转染H9C2心肌细胞,以血管紧张素Ⅱ(AngⅡ)刺激H9C2心肌细胞诱导心肌细胞肥大,提取细胞RNA通过实时荧光定量聚合酶链式反应(qPCR)检测心肌肥厚指标[心房利钠肽(AnP),脑利钠肽(Bnp),β-肌球蛋白重链(β-Mhc)]表达变化,并通过心肌细胞骨架蛋白(α-actinin)的免疫荧光染色观察Anp32a对心肌细胞大小的影响。机制研究方面,通过蛋白免疫印迹(Western blot)检测Anp32a在调节心肌肥厚过程中涉及的信号通路的变化。结果 Anp32a在心肌肥厚模型中表达下调。AngⅡ刺激细胞,免疫荧光染色检测AdAnp32a组细胞明显较其对照组(AdGFP)减小,而AdshAnp32a组心肌细胞与其对照组(AdshRNA)相比显著增大。qPCR检测显示AdAnp32a组心肌肥厚基因(Anp、Bnp、β-Mhc)的mRNA表达降低,而AdshAnp32a组表达则相反。Western blot检测发现Anp32a能够抑制AngII诱导的Akt蛋白磷酸化水平的升高。结论 Anp32a通过AKT信号通路抑制心肌细胞肥大,可作为防治心肌肥厚及心力衰竭的潜在治疗靶点。     

耐力训练对异丙肾上腺素致大鼠心肌肥厚中核因子κB信号通路的影响

目的探讨耐力训练对异丙肾上腺素诱导的大鼠心肌肥厚的保护作用及其机制。方法 SD大鼠32只,随机分成4组:正常对照组、异丙肾上腺素组、异丙肾上腺素+耐力训练组、耐力训练组。心肌肥厚模型建立采用腹腔注射异丙肾上腺素,耐力训练采用每天1 h无负重游泳训练,6天/周,共4周。采用称重法计算全心质量指数(HMI)、左心室质量指数(LVMI);常规HE染色观察心肌细胞横截面积;real-time PCR测定心房钠尿肽(ANP)、脑钠尿肽(BNP)、转化生长因子β1(TGF-β1)、肿瘤坏死因子α(TNF-α)和白细胞介素1β(IL-1β)mRNA表达水平;Western blot检测心肌组织中p65、IκB蛋白表达水平。结果与正常对照组相比,异丙肾上腺素组大鼠的HMI、LVMI和心肌细胞表面积显著增加;ANP和BNP mRNA表达上调,TGF-β1、TNF-α和IL-1βmRNA表达水平增加;心肌细胞核内p65表达上调,而IκB表达下调。与异丙肾上腺素组相比,异丙肾上腺素+耐力训练组能显著降低HMI、LVMI和心肌细胞表面积,同时能下调ANP和BNP mRNA表达水平;降低炎症因子TGF-β1、TNF-α以及IL-1βmRNA的表达水平,同时减少心肌细胞核内p65的表达增加IκB的表达。结论耐力训练可以有效改善异丙肾上腺素诱导的实验性心肌肥厚,其机制可能与抑制核因子κB炎症信号通路有关。     

Peroxisome proliferator-activated receptor beta/delta activation improves angiotensin II-induced cardiac hypertrophy in vitro

Agonists of the peroxisome proliferator-activated receptor alpha (PPARalpha) and gamma (gamma) exert anti-proliferative and anti-inflammatory effects that led to the testing of these drugs in experimental cardiac hypertrophy. However, the effect of PPAR beta/delta (beta/delta) agonists in hypertrophy is not yet known. In this paper, an experiment was conducted to explore whether PPARbeta/delta activation has an effect on cardiac hypertrophy. An in vitro cardiomyocyte hypertrophy from neonatal rats was induced with Angiotensin II (Ang II1micromol x L(-1)) stimulation. For the examination of PPAR beta/delta effect, the cultured rat cardiac myocytes were pretreated with GW0742 (10 micromol.L(-1)), an agonist of PPARbeta/delta, for 48h before Ang II stimulation. The following parameters in the cultured cells were determined: surface areas of myocytes were measured by the NIH Image Software; (3)H-leucine incorporation into myocytes was counted by liquid scintillometer; mRNA expression of PPARbeta/delta, ANP, BNP, MMP9, MMP2, and IL-1beta was detected by RT-PCR; PPARbeta/delta protein expression was evaluated with immunofluorescence staining; GW0742 could ameliorate Ang II-induced cardiomyocyte hypertrophy, as indicated by its inhibitory effects on the surface area of myocytes, and ANP and BNP mRNA expressions in myocytes and (3)H-leucine incorporation into myocytes. Meanwhile, GW0742 pretreatment exerted inhibition on mRNA expression augmentation of such cytokines as MMP9, MMP2, and IL-1beta in hypertrophic myocytes. In addition, the down-regulated expression of PPARbeta/delta mRNA and protein in hypertrophic myocytes was also significantly reversed by GW0742. We demonstrate for the first time that GW0742 exerts a beneficial effect on Ang II-induced cardiac hypertrophy and the relation to inflammation response.     

过氧化物酶体增殖物活化型受体β/δ改善心肌中炎性因子的表达

目的探讨过氧化物酶体增殖物活化型受体(PPAR)β/δ的特异性激动剂GW0742体外抑制心肌肥厚作用的可能机制。方法取体外用血管紧张素Ⅱ(AngⅡ)诱导新生大鼠的心室肌细胞,建立心肌细胞肥厚模型,以GW0742作用于肥大的心肌细胞,设立正常组、AngⅡ组、AngⅡ+GW0742组,并测量心肌细胞表面积、3H-亮氨酸掺入法检测心肌细胞蛋白合成速率及使用RT-PCR半定量测定心钠素、脑钠素及炎性因子的表达变化。结果与正常组心肌细胞比较,AngⅡ组诱导的肥大心肌细胞的表面积、3H-亮氨酸掺入、心钠素、脑钠素表达显著增加(P<0.01);AngⅡ+GW0742组在终浓度为10μmol/L时可明显逆转此改变(P<0.01);同时GW0742抑制肥大心肌细胞的基质金属蛋白酶-2和基质金属蛋白酶-9、白细胞介素-1β的mRNA过度表达。结论GW0742抑制AngⅡ介导的体外心肌细胞肥大,其机制可能为通过调控炎性因子所致。     

阿托伐他汀上调过氧化物酶体增殖物活化型受体α、γ表达及抑制心肌细胞肥大的作用

目的探讨阿托伐他汀在体外对血管紧张素Ⅱ（Ang Ⅱ）介导的肥大心肌细胞的作用,分析过氧化物酶体增殖物活化型受体（PPAR）α、γ在其中的可能作用.方法体外培养新生大鼠的心室肌细胞,用Ang Ⅱ诱导建立心肌肥厚模型,在模型中加入不同浓度阿托伐他汀,用软件分析观察心肌细胞表面积,应用3H-亮氨酸掺入实验检测心肌细胞蛋白合成速率及使用RT-PCR半定量测定心钠素、脑钠素、基质金属蛋白酶9、基质金属蛋白酶2、白介素1β和PPARα、γ mRNA的表达变化.结果 Ang Ⅱ可使体外培养的心肌细胞表面积和3H-亮氨酸的掺入增加,升高心钠素、脑钠素、基质金属蛋白酶9、基质金属蛋白酶2和白介素1β的表达,PPARα、γ表达下降;阿托伐他汀可逆转上述变化并呈剂量依赖性,而作为溶剂的DMSO对心肌肥厚无影响.结论阿托伐他汀具有抑制Ang Ⅱ介导的体外心肌细胞肥大的作用,PPARα及PPARγ很可能参与该过程.     

微小RNA-199a对大鼠心肌肥厚的影响

目的 探讨微小RNA(miRNA)-199a在心肌肥厚中的作用.方法 (1)Sprague-Dawley (SD)大鼠12只,分为腹主动脉缩窄(abdominal aortic constriction,AAC)组(AAC组,n=6)和假手术组(n=6).AAC组通过腹主动脉缩窄术建立大鼠心肌肥厚模型.实时定量聚合酶链反应(qRT-PCR)检测心肌中部分miRNA表达的变化.(2)SD乳鼠心肌细胞分为两组,即miRNA-199a重组腺病毒(Ad-miRNA-199a)组(n=8)和腺病毒载体(Ad-vector)组(n=8),分别转染SD乳鼠心肌细胞48 h后qRT-PCR检测心肌细胞中miRNA-199a以及心肌肥厚标志分子α肌球蛋白重链(α-myosin heavy chain,αMHC)、β肌球蛋白重链(β-myosin heavy chain,βMHC)、心房钠尿肽(atrial natriuretic peptide,ANP)编码基因myh6、myh7、Nppa的表达变化,并利用免疫荧光分析检测细胞表面积的变化.(3)SD乳鼠心肌细胞分为两组,即miRNA-199a的反义寡核苷酸(As-miRNA-199a)组(n=8)和混杂寡核苷酸(As-ctl)组(n=8),分别转染SD乳鼠心肌细胞48 h后qRT-PCR检测心肌细胞中miRNA-199a的表达变化.(4)SD乳鼠心肌细胞分为4组,即空白对照组(n=8)、苯肾上腺素组(phenylephrine,PE)(n=8)、PE+As-cd组(n=8)和PE+As-miRNA-199a绀(n=8),分别转染SD乳鼠心肌细胞48 h,qRT-PCR检测心肌肥厚标志分子编码基因的表达变化,免疫荧光检测细胞表面积的变化.结果 (1)qRT-PCR结果显示,AAC组大鼠造模后1周miRNA-1、miRNA-133、miRNA-181a及miRNA-499的表达均显著低于假手术组,而miRNA-199a表达显著高于假手术组.(2)qRT-PCR结果显示,AdmiRNA-199a组SD乳鼠心肌细胞中miRNA-199a的表达显著高于Ad-vector组,myh7的表达亦显著高于Ad-vector组,而myh6的表达低于Ad-vector组.免疫荧光显示Ad-miRNA-199a组SD乳鼠心肌细胞的表面积大于Ad-vector组,P<0.05.(3)qRT-PCR结果显示,As-miRNA-199a组SD乳鼠心肌细胞中miRNA-199a的表达显著低于As-ctl组,P<0.05.(4)qRT-PCR结果显示,PE组Nppa及myh7的表达量显著高于空白对照组,而myh6的表达量低于空白对照组,P<0.05.免疫荧光检测显示,PE+As-miRNA-199a组SD乳鼠心肌细胞的表面积显著小于PE+As-ctl组,P<0.05.结论 miRNA-199a在心肌肥厚过程中可能发挥着调节作用.

Abstract：

Objective To investigate the role of miRNA-199a on cardiac hypertrophy.Methods (1)Male Sprague-Dawley rats were subjected to pressure overload induced by abdominal aortic constriction (AAC,n=6)and quantitative real-time polymerase chain reaction(qRT-PCR)was used to detect the change of microRNAs(miRNAs).(2)Neonatal rat ventricular myocytes were isolated from 2-day old Sprague-Dawley rats.The myocytes were divided into two groups:adenovirus miRNA-199a(Ad-miRNA199a) or adenovirus vector(Ad-vector).They were transfected in cardiomyocytes for 48 h using Lipofectamine 2000.qRT-PCR was used to detect the change of myocardial hypertrophy markers α-myosin heavy chain(αMHC,myh6),β-myosin heavy chain(βMHC,myh7)and atrial natriuretic peptide(ANP,Nppa).Software Axio Vision was used to detect the change of cardiomyocytes surface areas.(3)Neonatal rat ventricular myocytes were divided into two groups:antisense oligonucleotide-miRNA-199a(As-miRNA199a) and scramble oligonucleotides (As-ctl). They were transfected to cardiomyocytes respectively for 48 h. qRT-PCR was used to detect the change of miRNA-199a. (4) Neonatal rat ventricular myocytes were divided into four groups : A : control ( ctl), B : phenylephrine ( PE), C : PE + As-ctl, D : PE + As-miRNA199a. qRT-PCR was used to detect the change of myh6 ,myh7 and Nppa. Software Axio Vision was used to detect the change of cardiomyocytes surface areas. Results ( 1 ) qRT-PCR results showed that miRNA-1,miRNA-133, miRNA-181a and miRNA-499 were significantly decreased, while the miRNA-199a was significantly increased at 1 week post AAC hearts compared with the sham group. (2) qRT-PCR results showed that miRNA-199a and myh7 were increased and myh6 was decreased significantly in Ad-miRNA199a group compared with Ad-vector group. The cardiomyocytes surface area was increased in Ad-miRNA199a group detected by immunofluorescence. (3) qRT-PCR results showed that miRNA-199a was significantly decreased in As-miRNA-199a group compared with Ad-vector group. (4) The Nppa and myh7were significantly increased and myh6 was decreased in cardiomyocytes stimulated by PE for 48 h. The cardiomyocytes surface area determined by immunofluorescence was increased in PE + As-miRNA-199a groups compared with PE + As-ctl groups. Conclusion miRNA-199a may play a regulatory role in cardiac hypertrophy.     

Autonomous and growth factor-induced hypertrophy in cultured neonatal mouse cardiac myocytes. Comparison with rat

Cultured neonatal rat cardiac myocytes have been used extensively to study cellular and molecular mechanisms of cardiac hypertrophy. However, there are only a few studies in cultured mouse myocytes despite the increasing use of genetically engineered mouse models of cardiac hypertrophy. Therefore, we characterized hypertrophic responses in low-density, serum-free cultures of neonatal mouse cardiac myocytes and compared them with rat myocytes. In mouse myocyte cultures, triiodothyronine (T3), norepinephrine (NE) through a beta-adrenergic receptor, and leukemia inhibitory factor induced hypertrophy by a 20% to 30% increase in [(3)H]phenylalanine-labeled protein content. T3 and NE also increased alpha-myosin heavy chain (MyHC) mRNA and reduced beta-MyHC. In contrast, hypertrophic stimuli in rat myocytes, including alpha(1)-adrenergic agonists, endothelin-1, prostaglandin F(2alpha), interleukin 1beta, and phorbol 12-myristate 13-acetate (PMA), had no effect on mouse myocyte protein content. In further contrast with the rat, none of these agents increased atrial natriuretic factor or beta-MyHC mRNAs. Acute PMA signaling was intact by extracellular signal-regulated kinase (ERK1/2) and immediate-early gene (fos/jun) activation. Remarkably, mouse but not rat myocytes had hypertrophy in the absence of added growth factors, with increases in cell area, protein content, and the mRNAs for atrial natriuretic factor and beta-MyHC. We conclude that mouse myocytes have a unique autonomous hypertrophy. On this background, T3, NE, and leukemia inhibitory factor activate hypertrophy with different mRNA phenotypes, but certain Gq- and protein kinase C-coupled agonists do not.     

Calcium-calmodulin kinase II is the common factor in calcium-dependent cardiac expression and secretion of A- and B-type natriuretic peptides

Peptides derived from the precursor of A- and B-type natriuretic peptides (ANP and BNP) are powerful clinical markers of cardiac hypertrophy and dysfunction. It is known that many stimuli affecting the intracellular calcium concentration also induce ANP and BNP secretion. It was our intention to study the mechanisms by which calcium regulates the secretion of ANP and BNP. The effects of pacing and calcium-calmodulin kinase II activity on natriuretic peptide secretion were studied in isolated perfused rat atria and cultured rat neonatal cardiomyocytes. In isolated rat atrium pacing induced an increase in diastolic, systolic, and averaged intracellular free calcium concentration and a frequency-dependent increase in the secretion of both ANP and BNP. The molar ratio of the secreted natriuretic peptides (ANP to BNP) remained nearly constant ( approximately 1000) at all the pacing frequencies tested (1, 3, 6, and 8 Hz). Calmodulin kinase II inhibitor KN-93 (3 mum) did not affect intracellular free calcium concentration but showed a frequency-dependent inhibitory effect on ANP and BNP secretion without a change in ANP to BNP ratio. In the neonatal cardiomyocytes, KN-93 (3 mum) suppressed the secretion and gene expression of both ANP and BNP. Overexpression of constitutively active (T286D) or nuclear (delta(B)) calcium-calmodulin kinase II induced an increase in ANP and BNP gene expression. The results indicate that the calcium-dependent secretion and gene expression of A- and B-type natriuretic peptides are similarly regulated by calmodulin kinase II-dependent mechanisms. This is a plausible mechanism contributing to exercise-induced natriuretic peptide secretion and the augmented secretion in heart dysfunction due to impaired calcium handling.     

Increased connective tissue growth factor relative to brain natriuretic peptide as a determinant of myocardial fibrosis

Excessive fibrosis contributes to an increase in left ventricular stiffness. The goal of the present study was to investigate the role of connective tissue growth factor (CCN2/CTGF), a profibrotic cytokine of the CCN (Cyr61, CTGF, and Nov) family, and its functional interactions with brain natriuretic peptide (BNP), an antifibrotic peptide, in the development of myocardial fibrosis and diastolic heart failure. Histological examination on endomyocardial biopsy samples from patients without systolic dysfunction revealed that the abundance of CTGF-immunopositive cardiac myocytes was correlated with the excessive interstitial fibrosis and a clinical history of acute pulmonary congestion. In a rat pressure overload cardiac hypertrophy model, CTGF mRNA levels and BNP mRNA were increased in proportion to one another in the myocardium. Interestingly, relative abundance of mRNA for CTGF compared with BNP was positively correlated with diastolic dysfunction, myocardial fibrosis area, and procollagen type 1 mRNA expression. Investigation with conditioned medium and subsequent neutralization experiments using primary cultured cells demonstrated that CTGF secreted by cardiac myocytes induced collagen production in cardiac fibroblasts. Further, G protein-coupled receptor ligands induced expression of the CTGF and BNP genes in cardiac myocytes, whereas aldosterone and transforming growth factor-beta preferentially induced expression of the CTGF gene. Finally, exogenous BNP prevented the production of CTGF in cardiac myocytes. These data suggest that a disproportionate increase in CTGF relative to BNP in cardiac myocytes plays a central role in the induction of excessive myocardial fibrosis and diastolic heart failure.     

促红细胞生成素促心肌细胞肥大的实验研究

目的观察促红细胞生成素（Erythropoietin,Epo）对离体乳鼠心肌细胞肥大形态学及分子生物学表现的影响。方法（1）新生大鼠原代心肌细胞分为空白对照、AngⅡ1×10^-6M和Epo10U／ml干预组,取24h、48h和72h三个时间点固定细胞,cTnI免疫组化染色,比较各组细胞面积;（2）RT．PCR法检测比较空白对照和Epo10U／ml干预30min c-los mRNA和2hANP和BNPmRNA表达;（3）Western blot检测Epo10U／ml刺激p-ERK表达的时间变化。4）Western blot检测不同浓度Epo刺激p-ERK表达的差异。结果（1）AngⅡ和Epo刺激均使心肌细胞面积增大（P〈0．05,24h、48h和72h,AngII组VSCon组;P〈0．05,48h和72h,Epo组VSCon组）;（2）Epol0U／ml干预组心肌细胞30minc．fosmRNA表达量与对照组相比差异不明显,2hANP mRNA和BNP mRNA表达量和对照组相比,明显升高（P〈0．05）;（3）Western blot结果显示,Epo干预心肌细胞后P—ERK2表达随时间出现先升高后下降的变化,30min时达到高峰,与干预前和干预后120min差别具有统计学意义（P〈0．05,30minVS0,120min）;（4）随Epo干预浓度增大,P-ERK2表达水平升高（P〈0．05,Epo10U／ml和Epo100U／ml组VS Con组）。Epo刺激p-ERKl表达也出现相同的时间浓度趋势,但各组差别无统计学意义。结论Epo刺激可使心肌细胞表面积增大,促进肥大基因ANP-BNP mRNA表达的升高,并刺激p-ERK出现时间相关性和浓度依赖性表达。