血管紧张素Ⅱ及去甲肾上腺素在心肌细胞增殖分化中的生物学意义

目的探讨血管紧张素Ⅱ(angiotensmⅡ,AngⅡ)、去甲肾上腺素(norepinephrine,NE)对心肌细胞增殖、胚胎基因心钠素及转化生长因子β1(trahsforming growth factorβ1,TGF-β1)表达的影响及其生物学意义.方法体外培养的乳鼠心肌细胞,分别以AngⅡ(1×10-6mol/L)、NE(1×10-6mol/L)刺激后,采用流式细胞计数仪检测心肌细胞DNA合成、细胞周期分布;免疫细胞化学检测心肌细胞内TGF-β1表达水平,放射免疫学检测细胞上清心钠素浓度.结果与对照组相比,AngⅡ,NE刺激24 h后,心肌细胞心钠素表达增加,G0/G1,G2/M期DNA合成增强,同时伴有TGF-β1蛋白表达上调(P均＜0.05),但二者不改变心肌细胞的细胞周期分布,细胞仍主要处于G0/G1期.结论AngⅡ,NE能诱导心肌细胞异常增生,但不能刺激细胞增殖.这可能与AngⅡ,NE诱导心肌细胞TGF-β1表达增加有关.     

钙调神经磷酸酶表达与心脏重构的关系

目的：探讨钙调神经磷酸酶(calcineurin,CaN)表达与心脏重构的关系。方法：因瓣膜性心脏病接受外科手术治疗的患者38例,采用彩色多普勒超声心动图测量患者心腔内径和室壁厚度。取患者外周静脉血及少量左心室乳头肌,分别提取外周淋巴细胞和心肌组织总RNA,采用半定量反转录多聚酶链反应技术测定CaN催化亚基(calcineurin subunit A,CnA)α和β异构体的mRNA水平,分析其与心腔内径和室壁厚度的相关性。结果：淋巴细胞CnAα转录水平与主动脉根部内径(r=0.376,P＜0.05)和左心室内径(r=0.383,P＜0.05)呈正相关,淋巴细胞CnAβ与左心室后壁厚度(r=0.324,P＜0.05)和左心房内径(r=0.670,P＜0.01)呈正相关,心肌组织CnAβ与左心房内径呈正相关(r=0.653,P＜0.01)。结论：CaN转录与心脏重构有关,淋巴细胞CnAα mRNA水平可反映心力衰竭和左心室扩大,淋巴细胞和心肌组织CnAβ mRNA水平可反映心肌肥厚和左心房扩大。     

Vascular remodeling and angiotensin II]

Numerous pharmacological approaches have failed to modify the high incidence of restenosis after balloon coronary angioplasty. This inability to alter the restenosis process was caused in part by our incomplete understanding of its pathology. Neointimal formation and geometric remodeling cause the restenosis after angioplasty. The development of restenosis involves vascular smooth muscle cells (VSMCs) migration and proliferation. Angiotensin II has been shown to stimulate the growth of VSMCs via an action on the angiotensin II AT1 receptor subtype. Angiotensin II AT1 receptor antagonists can block the angiotensin II-induced these actions. Angiotensin II AT1 receptor antagonists may block the restenosis after balloon coronary angioplasty.     

小鼠心室肌细胞分离方法的改进

目的：探索更简单实用和重复性好的小鼠心肌细胞分离技术。 方法：实验于2005-05／09在大连大学医学院医学研究中心完成。选择8-16周龄昆明小鼠150只．雌雄不拘，体质量30～40g，由大连大学医学院动物中心提供。采用原位主动脉插管和动脉夹固定法酶解分离小鼠心室肌细胞，并在室温下进行全细胞膜片钳记录。 结果：可将主动脉插管的时间控制在1．5min内，所获心室肌细胞横纹清晰，一次性复钙可获得50％-60％的耐钙细胞，分离所得细胞可在全细胞膜片钳方式下记录到一过性外向钾电流（Ito）。 结论：原位主动脉插管和动脉夹固定酶解分离心室肌细胞的方法简便易行，重复性好，克服了原有方法体外心脏插管时间长，每次插管时间差异较大，分离效果不稳定的弊端。     

Stress-dependent cardiac remodeling occurs in the absence of microRNA-21 in mice

MicroRNAs inhibit mRNA translation or promote mRNA degradation by binding complementary sequences in 3' untranslated regions of target mRNAs. MicroRNA-21 (miR-21) is upregulated in response to cardiac stress, and its inhibition by a cholesterol-modified antagomir has been reported to prevent cardiac hypertrophy and fibrosis in rodents in response to pressure overload. In contrast, we have shown here that miR-21-null mice are normal and, in response to a variety of cardiac stresses, display cardiac hypertrophy, fibrosis, upregulation of stress-responsive cardiac genes, and loss of cardiac contractility comparable to wild-type littermates. Similarly, inhibition of miR-21 through intravenous delivery of a locked nucleic acid-modified (LNA-modified) antimiR oligonucleotide also failed to block the remodeling response of the heart to stress. We therefore conclude that miR-21 is not essential for pathological cardiac remodeling.     

小鼠心室肌细胞分离方法的改进

目的:探索更简单实用和重复性好的小鼠心肌细胞分离技术。方法:实验于2005-05/09在大连大学医学院医学研究中心完成。选择8～16周龄昆明小鼠150只,雌雄不拘,体质量30～40g,由大连大学医学院动物中心提供。采用原位主动脉插管和动脉夹固定法酶解分离小鼠心室肌细胞,并在室温下进行全细胞膜片钳记录。结果:可将主动脉插管的时间控制在1.5min内,所获心室肌细胞横纹清晰,一次性复钙可获得50%～60%的耐钙细胞,分离所得细胞可在全细胞膜片钳方式下记录到一过性外向钾电流(Ito)。结论:原位主动脉插管和动脉夹固定酶解分离心室肌细胞的方法简便易行,重复性好,克服了原有方法体外心脏插管时间长,每次插管时间差异较大,分离效果不稳定的弊端。     

PDGF-D activation by macrophage-derived uPA promotes AngII-induced cardiac remodeling in obese mice

Obesity-induced secretory disorder of adipose tissue–derived factors is important for cardiac damage. However, whether platelet-derived growth factor-D (PDGF-D), a newly identified adipokine, regulates cardiac remodeling in angiotensin II (AngII)–infused obese mice is unclear. Here, we found obesity induced PDGF-D expression in adipose tissue as well as more severe cardiac remodeling compared with control lean mice after AngII infusion. Adipocyte-specific PDGF-D knockout attenuated hypertensive cardiac remodeling in obese mice. Consistently, adipocyte-specific PDGF-D overexpression transgenic mice (PA-Tg) showed exacerbated cardiac remodeling after AngII infusion without high-fat diet treatment. Mechanistic studies indicated that AngII-stimulated macrophages produce urokinase plasminogen activator (uPA) that activates PDGF-D by splicing full-length PDGF-D into the active PDGF-DD. Moreover, bone marrow–specific uPA knockdown decreased active PDGF-DD levels in the heart and improved cardiac remodeling in HFD hypertensive mice. Together, our data provide for the first time a new interaction pattern between macrophage and adipocyte: that macrophage-derived uPA activates adipocyte-secreted PDGF-D, which finally accelerates AngII-induced cardiac remodeling in obese mice.     

Relationship between myocardial remodeling and neurohumoral activation in patients with cardiac failure

50 healthy subjects and 685 patients with heart failure (NYHA functional class I-IV) due to coronary heart disease (CHD) aged 34-74 years (mean age 48.1 +/- 8.22 years) were examined. Cardio-hemodynamics was assessed by M- and B-echocardiography from parasternal, subcostal and apical view on a short and long axis with transmitral Doppler. Plasma levels of epinephrine, norepinephrine, serotonin, aldosterone, STH, hydrocortisone were measured in the patients. Progression of cardiac failure is characterized by evolution of the left ventricular ellipse form into a ball shape primarily due to an increase in cross sectional cavity size. The changes of the left ventricular geometry in heart failure patients are combined with progressive reduction of the relative wall thickness index, intensification of the myocardial stress and impairment of diastolic function. The clearest intercoupling between myocardial remodeling and neurohumoral activation was registered in patients with asymptomatic heart failure. With growing severity of left ventricular dysfunction, plasm activity of serotonin, hydrocortisone and aldosterone increase more than levels of norepinephrine, epinephrine and STH.     

血管紧张素Ⅱ对心肌成纤维细胞蛋白激酶Cε和蛋白激酶Cα表达及转位的影响

目的:探讨血管紧张素Ⅱ(Ang Ⅱ)对大鼠心肌成纤维细胞(MFs)蛋白激酶Cε(PKCε)和蛋白激酶Cα(PKCα)的表达及转位的影响,并探讨这两种PKC亚型在心室重构中的作用.方法:以第2～4代MF8分实验组(加 AngⅡ10-6 mol/L)和对照组(不加 Ang Ⅱ)通过间接免疫荧光法观察PKC亚型ε和α在细胞内的分布和定位,Image-pro-plus 4.0版专业图像处理软件对荧光强度进行半定量统计.结果:荧光显微镜下观察到对照组和实验组PKC亚型ε都存在于膜、胞浆和核-细胞骨架,实验组强度明显增强,尤以核-细胞骨架为甚;对照组PKC亚型α主要存在于核一细胞骨架,而实验组α亚型存在于膜、胞浆和核-细胞骨架,强度都明显增加,尤以核-细胞骨架为甚:且PKC亚型ε和α荧光强度半定量比较实验组均显著高于对照组(P<0.001).结论:Ang Ⅱ对PKC亚型ε和α在MFs的表达和转位有显著影响,提示PKC亚型ε、α可能在MFs信号转导过程中起重要作用.     

血管紧张素Ⅱ及其受体拮抗剂对豚鼠心肌细胞电生理及L-型钙电流的作用

目的 探讨血管紧张素Ⅱ及其受体拮抗剂对豚鼠心肌细胞动作电位间期及L-型钙电流的作用。方法 分离豚鼠乳头肌的单个心室肌细胞，采用内充3mol／LKCl的玻璃微电极记录心肌动作电位。采用膜片钳全细胞技术，钳制电位-40mV，保持时间200ms，指令电位为0，并记录L-型钙电流的最大峰电流。结果 灌注血管紧张素Ⅱ可致多种机制的心律失常。灌注1min，动作电位振幅、动作电位复极90％的间期（APD90）、静息膜电位（RMP）较对照状态显著降低或缩短；灌注3min，动作电位复极30％和50％的间期（APD30和APD50）及有效不应期均较对照状态显著缩短。膜片钳研究示血管紧张素Ⅱ灌注5min，L-型钙电流较对照状态显著增加，氯沙坦灌注1min L-型钙电流显著降低，灌注3min较1min进一步降低，电压-电流关系曲线形状均无显著变化。结论 血管紧张素Ⅱ降低动作电位幅度，缩短动作电位时程及有效不应期，电压依赖性增加L-型钙电流最大峰电流，具有致心律失常作用，氯沙坦电压依赖性地降低L-型钙电流。     

射血分数正常心力衰竭与心脏重构关系的研究

目的 探讨射血分数正常心力衰竭与心脏重构的关系.方法 收集2009年1月至2012年3月龙口市人民医院心内科收治的慢性心力衰竭患者188例,严格依照诊断标准分为射血分数正常心力衰竭109例(HFNEF组)与射血分数下降心力衰竭79例(HFREF组),并按NYHA分级不同又各自分为3个亚组(HFNEF组:心功能Ⅱ级组52例、心功能Ⅲ级组36例、心功能Ⅳ级组21例;HFREF组:心功能Ⅱ级组13例,心功能Ⅲ级组27例,心功能Ⅳ级组39例),测量所有纳入对象的左心室射血分数(LVEF)、左心房内径(LAD)、室间隔厚度(IVST)、左心室后壁厚度(LVPWT)、右心室内径(RVD)等,并对其临床资料进行统计学分析.结果 HFNEF组较HFREF组年龄大[(64.59 ±5.34)岁与(58.89±4.23)岁,t=3.345,P=0.001]、女性患者多[58.7％(64/109)与41.8(33/79),x2=5.265,P=0.022)]、高血压比例高[81.65％(89/109)与63.29％(50/79),x2=8.012,P=0.005].不同心功能分级的HFNEF患者随着心功能下降严重程度的增加,其LVPWT、IVST、LAD、RVD均表现为逐渐增大,但在LVPWT[(9.05±1.89)、(11.30±2.67)、(13.90±2.77) mm,F=3.578,P ＜0.05]、IVST[(9.35±1.75)、(11.51±2.48)、(12.98±3.01) mm,F=3.081,P＜ 0.05]、LAD[(31.23 ±5.98)、(35.55±7.31)、(44.81±10.72)mm,F=6.711,P＜0.001]方面差异有统计学意义,在RVD [(18.95±1.02)、(19.21±1.11)、(19.99±0.98) mm)中差异无统计学意义(F=2.751,P＞0.05].心功能Ⅳ级的HFNEF与HFREF患者在LVPWT[(13.90±2.77)、(7.45±2.01)mm,t=11.439,P＜0.001]、IVST[(12.98±3.01)mm与(7.23±1.94) mm,t=10.318,P＜0.001]、RVD[(19.99±0.98) mm与(23.51±1.10)mm,t=2.838,P＜0.01]方面差异有统计学意义,而LAD [(44.81±10.72)mm与(46.30±11.76) mm]比较差异无统计学意义(=1.451,P＞0.05).结论 HFNEF患者中高龄、女性、高血压患者比例高,并对心脏重构的影响表现为随心功能分级的加重而加大,但对右心室几乎无影响,其心室结构变化与HFREF存在明显不同.因此临床医师须深入认识HFNEF的流行病学、病理生理学特点、诊断标准及治疗原则,从而更好地对该类患者进行诊断与治疗.     

血管紧张素Ⅱ对心肌成纤维细胞增殖及p21,p27表达的影响

目的:探讨血管紧张素Ⅱ(angiotensinⅡ,AngⅡ)对大鼠心肌成纤维细胞(cardiacfibroblasts,CFs)增殖的作用及其分子机制。方法:差速贴壁法体外培养新生Wistar大鼠的CFs,随机分为3组:对照组,AngⅡ处理组,洛沙坦(losartan)+AngⅡ处理组。作用24h后,收集细胞,倒置显微镜下观察活细胞形态;四氮唑盐(MTT)比色法检测细胞的增殖;流式细胞仪分析细胞周期与p21和p27的蛋白表达。结果:AngⅡ可显著提高CFsMTT-OD值,使S期细胞比率增加,G1期细胞比率下降,并降低细胞p27蛋白的表达,而对p21的蛋白表达无影响。AT1受体拮抗剂Losartan可完全阻断AngⅡ的作用。结论:AngⅡ通过AT1受体促进CFs增殖的机制可能与降低细胞p27蛋白的表达有关。     

Dilated cardiomyopathy and impaired cardiac hypertrophic response to angiotensin II in mice lacking FGF-2

FGF-2 has been implicated in the cardiac response to hypertrophic stimuli. Angiotensin II (Ang II) contributes to maintain elevated blood pressure in hypertensive individuals and exerts direct trophic effects on cardiac cells. However, the role of FGF-2 in Ang II-induced cardiac hypertrophy has not been established. Therefore, mice deficient in FGF-2 expression were studied using a model of Ang II-dependent hypertension and cardiac hypertrophy. Echocardiographic measurements show the presence of dilated cardiomyopathy in normotensive mice lacking FGF-2. Moreover, hypertensive mice without FGF-2 developed no compensatory cardiac hypertrophy. In wild-type mice, hypertrophy was associated with a stimulation of the c-Jun N-terminal kinase, the extracellular signal regulated kinase, and the p38 kinase pathways. In contrast, mitogen-activated protein kinase (MAPK) activation was markedly attenuated in FGF-2-deficient mice. In vitro, FGF-2 of fibroblast origin was demonstrated to be essential in the paracrine stimulation of MAPK activation in cardiomyocytes. Indeed, fibroblasts lacking FGF-2 expression have a defective capacity for releasing growth factors to induce hypertrophic responses in cardiomyocytes. Therefore, these results identify the cardiac fibroblast population as a primary integrator of hypertrophic stimuli in the heart, and suggest that FGF-2 is a crucial mediator of cardiac hypertrophy via autocrine/paracrine actions on cardiac cells.     

Myocardial stress and hypertrophy: a complex interface between biophysics and cardiac remodeling

Pressure and volume overload results in concentric and eccentric hypertrophy of cardiac ventricular chambers with, respectively, parallel and series replication of sarcomeres. These divergent patterns of hypertrophy were related 40 years ago to disparate wall stresses in both conditions, with systolic wall stress eliciting parallel replication of sarcomeres and diastolic wall stress, series replication. These observations are relevant to clinical practice, as they relate to the excessive hypertrophy and contractile dysfunction regularly observed in patients with aortic stenosis. Stress-sensing mechanisms in cardiomyocytes and activation of cardiomyocyte death by elevated wall stress continue to intrigue cardiovascular scientists.     

Nitric oxide, atrial natriuretic peptide, and cyclic GMP inhibit the growth-promoting effects of norepinephrine in cardiac myocytes and fibroblasts.

This study tested the hypothesis that nitric oxide (NO) and atrial natriuretic peptide (ANP) can attenuate the effects of adrenergic agonists on the growth of cardiac myocytes and fibroblasts. In ventricular cells cultured from neonatal rat heart, ANP and the NO donor S-nitroso-N-acetyl-D,L-penicillamine (SNAP) caused concentration-dependent decreases in the norepinephrine (NE)-stimulated incorporation of [3H]leucine in myocytes and [3H]thymidine in fibroblasts. In myocytes, the NO synthase inhibitor NG-monomethyl-L-arginine potentiated NE-stimulated [3H]leucine incorporation. In both cell types, ANP and SNAP increased intracellular cGMP levels, and their growth-suppressing effects were mimicked by the cGMP analogue 8-bromo-cGMP. Furthermore, in myocytes, 8-bromo-cGMP attenuated the alpha1-adrenergic receptor-stimulated increases in c-fos. Likewise, ANP and 8-bromo-cGMP attenuated the alpha1-adrenergic receptor- stimulated increase in prepro-ANP mRNA and the alpha1-adrenergic receptor-stimulated decrease in sarcoplasmic reticulum calcium ATPase mRNA. The L-type Ca2+ channel blockers verapamil and nifedipine inhibited NE-stimulated incorporation of [3H]leucine in myocytes and [3H]thymidine in fibroblasts, and these effects were not additive with those of ANP, SNAP, or 8-bromo-cGMP. In myocytes, the Ca2+ channel agonist BAY K8644 caused an increase in [3H]leucine incorporation which was inhibited by ANP. These findings indicate that NO and ANP can attenuate the effects of NE on the growth of cardiac myocytes and fibroblasts, most likely by a cGMP-mediated inhibition of NE-stimulated Ca2+ influx.