血管紧张素-(1-7)在血管紧张素II诱导心肌细胞肥大中的作用

目的：探讨血管紧张素－（１－７）「Ａｎｇ－（１－７）」在血管紧张素Ⅱ（Ａｎｇ Ⅱ）诱导心肌细胞肥大反应中的应用。方法：在Ａｎｇ Ⅱ诱导培养的ＳＤ乳鼠心肌细胞中,应用Ａｎｇ－（１－７）,通过测定心肌细胞蛋白质合成速率,蛋白质含量和细胞表面积等指标,观察心肌细胞肥大情况。结果：Ａｎｇ－（１－７）呈剂量依赖性抑制Ａｎｇ Ⅱ诱导培养的心肌细胞的蛋白质合成速率,Ａｎｇ－（１－７）还能减少Ａｎｇ Ⅱ诱导培养的心肌细胞的细胞蛋白质含量和表面积。基作用受体不是血管紧张素Ⅱ受体１（ＡＴ１）或血管紧张素Ⅱ受体２（ＡＴ２）,而是通过一种特殊受体介导。结论：Ａｎｇ－（１－７）能抑制Ａｎｇ Ⅱ诱导的心肌细胞肥大,其作用是通过一种特殊受体介导。     

阿托伐他汀在1-磷酸鞘氨醇诱导心肌细胞肥大中的作用

目的研究阿托伐他汀对1-磷酸鞘氨醇(S1P)诱导乳鼠心肌细胞肥大反应中的作用。方法原代培养乳鼠心肌细胞,测定心肌细胞体积和[3H]-亮氨酸掺入量作为心肌细胞肥大的指标。乳鼠心肌细胞使用不同浓度的阿托伐他汀(atorvastatin),加入S1P,[3H]-亮氨酸掺入量作为心肌细胞蛋白摄取量;分别用qPCR和Western blot法检测心肌细胞的β-肌球蛋白(β-MHC)的mRNA和蛋白质表达水平;用qPCR检测心肌细胞的心房利钠肽(ANF)的mRNA表达水平。结果与S1P组比较,阿托伐他汀10μmol/L处理组[3H]-亮氨酸掺入率减少[(234.89%±31.23%)比(342.23%±31.60%),P=0.205],β-MHC的mRNA和蛋白表达水平下降[(0.59±0.14)比(0.84±0.20),P=0.318]和[(0.55±0.09)比(0.98±0.15),P=0.223],ANF的mRNA表达水平降低[(0.51±0.13)比(0.76±0.19),P=0.445];与S1P组比较,阿托伐他汀20μmol/L处理组[3H]-亮氨酸掺入率明显减少[(189.07%±17.69%)比(342.23%±31.60%),P<0.01],β-MHC的mRNA和蛋白表达水平显著下降[(0.50±0.12)比(0.84±0.20),P<0.01]和[(0.35±0.08)比(0.98±0.15),P<0.01],ANF的mRNA水平明显降低[(0.47±0.12)比(0.76±0.19),P<0.01]。结论阿托伐他汀可抑制S1P诱导的心肌细胞肥大,并可减少S1P诱导的β-MHC和ANF表达。     

1-磷酸鞘氨醇受体途径诱导的心肌肥大及蛋白激酶C的调节作用

目的研究1-磷酸鞘氨醇受体途径在1-磷酸鞘氨醇(S1P)诱导乳鼠心肌细胞肥大反应中的作用。方法原代培养乳鼠心肌细胞,测定心肌细胞体积和[3H]-亮氨酸掺入量作为心肌细胞肥大的指标。实验分对照组(不加任何干预因素)、S1P组(S1P直接作用于心肌细胞)、VPC23019组(S1P1和S1P3受体抑制剂进行干预30 min后,加入S1P刺激心肌细胞)、JTE组(S1P2受体抑制剂进行干预30 min后,加入S1P刺激心肌细胞)及Staurospo-rine组(蛋白激酶C抑制剂进行干预30 min后,加入S1P刺激心肌细胞),[3H]-亮氨酸掺入法测定心肌细胞蛋白合成速率;用qPCR和Western blot法检测心肌细胞β-肌球蛋白重链的mRNA和蛋白表达水平。结果 10、100和1000 nmol/L的S1P均能增加乳鼠心肌细胞的细胞体积和[3H]-亮氨酸掺入量,尤以1000 nmol/L S1P最为明显,因而选择1000 nmol/L S1P作用心肌细胞48 h为成功的心肌细胞肥大模型。1000 nmol/L S1P处理心肌细胞48 h,可使[3H]-亮氨酸掺入量明显增加,该作用可被S1P2受体抑制剂或蛋白激...     

血管紧张素Ⅱ对培养的心肌细胞中ACE2表达的影响

目的探讨血管紧张素II（Ang II）对培养的心肌细胞血管紧张素转换酶2（ACE2）基因和其蛋白表达的影响。方法体外原代培养新生SD大鼠的心肌细胞,随机分为对照组和Ang II刺激组。测量心肌细胞的直径和表面积。用RT-PCR法检测心肌细胞中ACE2 mRNA的表达,Western blot检测心肌细胞中ACE2蛋白的表达。结果与对照组相比,用10-7mol/L的Ang II处理,可引起心肌细胞的直径和表面积增加（P<0.01）,10-7mol/L Ang II刺激组ACE2 mRNA和其蛋白的表达显著降低（P<0.01）。结论外源性给予10-7mol/L的Ang II,可直接导致心肌细胞肥大,且Ang II刺激引起的心肌细胞中ACE2 mRNA和其蛋白表达的下调,其意义在于使对心肌细胞具有保护作用的Ang（1-7）产生减少,并协同参与了心肌细胞肥大的过程。     

抑制1-磷酸鞘氨醇裂解酶活性加重缺血性心力衰竭

目的:观察1-磷酸鞘氨醇(S1P)裂解酶(SPL)在小鼠缺血性心衰(HF)模型中的作用。方法:将60只成年雄性C57/BL6J小鼠随机分为以下4组:假手术(Sham)组、心肌梗死(MI)组、假手术+THI(Sham+THI)组[THI是SPL的抑制剂]及MI+THI组,每组15只(n=15),将25 mg/L THI溶于饮水中,于手术24 h后连续饲喂2周。MI4周后,采用ELISA试剂盒测定心肌中S1P的含量。根据心脏质量/体质量(HW/BW)评价心肌肥厚。用小动物心脏超声评估小鼠心脏结构和功能,经Masson三色染剂染色法观察心脏纤维化。用Western blot检测转化生长因子-β(TGF-β)蛋白的表达。实时PCR检测Ⅰ、Ⅲ型胶原、心房钠尿肽(ANP)、脑钠尿肽(BNP)和平滑肌肌动蛋白-α(α-SMA)mRNA的水平。结果:与MI组相比,MI+THI组小鼠心肌组织中S1P的含量增加(P0.01);左心室射血分数(LVEF)降低(P0.01),左心室收缩末期内径(LVESD)和舒张末期内径(LVEDD)均增加(均P0.05),HW/BW增加(P0.01),心脏纤维化加重;TGF-β蛋白的表达增加(P0.01);Ⅰ、Ⅲ型胶原、ANP、BNP和α-SMA mRNA的水平均显著增加(均P0.01)。与Sham组相比,Sham+THI组小鼠上述指标无显著差异。结论:抑制SPL的活性可能增加梗死后心肌病理性S1P信号的激活,加重MI后的心脏重构和HF。     

替米沙坦对血管紧张素II诱导的心肌肥大的作用及可能机制

目的探讨替米沙坦对血管紧张素II(AngII)诱发的肥大心肌细胞膜AT1和AT2受体表达变化的影响。方法体外培养心肌细胞,分为三组:对照组,AngII处理组,替米沙坦处理组。构建AngII诱发心肌细胞肥大模型,提取各组心肌细胞膜蛋白,免疫印记(Western Blotting)观察肥大心肌细胞中AT1和AT2表达。结果相对于对照组,AngII处理组心房利钠肽(ANP),脑钠肽(BNP)基因表达明显上升(P<0.05),AT1和AT2受体表达也显著上升(P<0.05);相对于AngII组,替米沙坦处理组ANP,BNP基因和AT1受体表达明显下降(P<0.05)。结论替米沙坦可以明显抑制AngII诱发的心肌细胞肥大,其机制与其降低因AngII诱发AT1受体表达升高和维持AngII引起的AT2受体高表达有关。     

心肌营养素-1在血管紧张素Ⅱ致心肌细胞肥大中的表达

目的利用血管紧张素Ⅱ(AngⅡ)刺激心肌细胞造成肥大,观察心肌细胞肥大过程中心肌营养素-1(cardiotrophin-1,CT-1)的表达情况。方法培养原代心肌细胞,给予AngⅡ刺激心肌细胞造成肥大,在不同时间取细胞进行反转录聚合酶链反应(RT-PCR)观察CT-1 mRNA表达,免疫组化法检测CT-1蛋白表达,相差显微镜下观察细胞形态变化。结果经AngⅡ刺激后在相差显微镜下可见心肌细胞面积变大。CT-1 mRNA水平随着AngⅡ刺激时间延长而有增高的趋势,CT-1蛋白表达亦增加。结论AngⅡ致心肌细胞肥大过程中伴有CT-1表达增加,CT-1有可能参与心肌细胞的肥大机制。     

血管紧张素Ⅱ及其受体拮抗剂对心肌细胞肥大的影响

目的 观察血管紧张素Ⅱ（AngⅡ）、AT1受体拮抗剂氯沙坦和AT2受体拮抗剂PD123177对心肌细胞蛋白质合成速率和AT1受体mRNA表达的影响。方法 采用^3H－亮氨酸掺入法测定培养的心肌细胞蛋白质合成速率,RT－PCR方法检测心肌细胞AT1受体mRNA表达。结果 在培养的心肌细胞中加入AngⅡ可明显增加心肌细胞^3H－亮氨酸的掺入量,并呈剂量依赖性,氯沙旦可显著抑制AngⅡ引起的蛋白质合成增加,而PD123177对其无影响;AngⅡ上调AT1受体基因表达,氯沙坦抑制其上调,PD123177无影响。结论 AngⅡ可通过上调AT1受体引起心肌细胞肥大,氯沙坦下调AT1受体,抑制心肌细胞肥大。     

鞘氨醇-1-磷酸在支气管哮喘中作用的研究进展

鞘类脂质是一类广泛存在于各种真核细胞膜上的分子类物质.鞘脂类最初被认为是细胞膜的结构组成部分,随着研究的深入发现此类物质同时也是重要的信号分子.鞘氨醇-1-磷酸(sphingosine-1-phosphate,S1P)是鞘脂类的代谢产物,在细胞的变异、增殖、凋亡以及血管生成的过程中是重要的生物活性信号.S1P在支气管哮喘的发病机制中扮演了重要角色.近期研究表明在肥大细胞中鞘氨醇激酶的激活通过FcεRI信号通路使鞘氨醇转变为S1P,参与诱导了气道平滑肌的收缩和气道重塑.这些新发现的信号途径为支气管哮喘患者提供了潜在的治疗靶点.     

鞘氨醇-1-磷酸在支气管哮喘中作用的研究进展

鞘类脂质是一类广泛存在于各种真核细胞膜上的分子类物质。鞘脂类最初被认为是细胞膜的结构组成部分,随着研究的深入发现此类物质同时也是重要的信号分子。鞘氨醇-1-磷酸（sphingosine-1-phosphate,S1P）是鞘脂类的代谢产物,在细胞的变异、增殖、凋亡以及血管生成的过程中是重要的生物活性信号。S1P在支气管哮喘的发病机制中扮演了重要角色。近期研究表明在肥大细胞中鞘氨醇激酶的激活通过FceRI信号通路使鞘氨醇转变为SIP,参与诱导了气道平滑肌的收缩和气道重塑。这些新发现的信号途径为支气管哮喘患者提供了潜在的治疗靶点。     

Inpp5f抑制血管紧张素Ⅱ诱导的心肌细胞肥大反应

目的 探讨磷酸酶Inpp5f在血管紧张素Ⅱ(AngⅡ)诱导的心肌细胞肥大反应中的作用。方法 用AngⅡ(1×10-7 mol/L)诱导大鼠心肌细胞H9c2发生肥大反应,qRT-PCR 和Western blot分别检测Myh7、Myh6和Inpp5f的表达变化;构建Inpp5f 过表达载体,给予细胞过表达外源性Inpp5f后,在AngⅡ诱导的心肌肥大反应中通过qRT-PCR 和Western blot查看Myh7、Myh6的表达变化。结果 在心肌细胞肥大反应中,Inpp5f表达降低。与对照相比,外源性过表达Inpp5f能显著减少AngⅡ引起的Myh7表达升高和Myh6表达降低。结论 在心肌肥大反应中,Inpp5f表达降低,而增加Inpp5f的表达能抑制心肌肥大反应,提示其作为心肌肥大保护因子的可能。     

Involvement of calcineurin in angiotensin II-induced cardiomyocyte hypertrophy and cardiac fibroblast hyperplasia of rats

A rapidly emerging body of literature implicates a pivotal role for the Ca2+-calmodulin-dependent phosphatase, calcineurin, as a cellular target for a variety of Ca2+-dependent signaling pathways culminating in cardiac hypertrophy. The aim of the present study was to test whether calcineurin is involved in the signal transduction of angiotensin II (AngII)-induced cardiac myocyte hypertrophy and fibroblast hyperplasia. Firstly, we observed that calcineurin activity was significantly increased in AngII-stimulated cardiac myocytes as well as fibroblasts, but was markedly inhibited by Losartan (50 micromol/l), H7 (50 micromol/l), and Fura-2/AM (5 micromol/l). It is indicated that AngII-induced activation of calcineurin is through an ATI receptor, may be dependent on the sustained increases of [Ca2+]i, and be regulated by protein kinase C. In a second experiment, we found that cyclosporin (0.1-10micromol/l), a specific inhibitor of calcineurin, decreased the protein synthesis rate in AngII-stimulated cardiomyocytes and the DNA synthesis rate in AngII-treated fibroblasts in a dose-dependent manner. In the latter experiment, calcineurin inhibition reduced the mRNA level of the atrial natriuretic factor gene. These results indicate that calcineurin is involved in the signal transduction of AngII-induced cardiomyocyte hypertrophy and fibroblast hyperplasia.     

Sulforaphane protects H9c2 cardiomyocytes from angiotensin II-induced hypertrophy

Background

Cardiac hypertrophy is an adaptive process of the heart in response to various stimuli, but sustained cardiac hypertrophy will finally lead to heart failure. Sulforaphane—extracted from cruciferous vegetables of the genus Brassica such as broccoli, brussels sprouts, and cabbage—has been evaluated for its anticarcinogenic and antioxidant effects.

Aims

To investigate the effect of sulforaphane on angiotensin II (Ang II)-induced cardiac hypertrophy in vitro.

Methods

Embryonic rat heart-derived H9c2 cells were co-incubated with sulforaphane and Ang II. The cell surface area and mRNA levels of hypertrophic markers were measured to clarify the effect of sulforaphane on cardiac hypertrophy. The underlying mechanism was further investigated by detecting the activation of Akt and NF-κB signaling pathways.

Results

We found that H9c2 cells pretreated with sulforaphane were protected from Ang II-induced hypertrophy. The increasing mRNA levels of ANP, BNP, and β-MHC in Ang II-stimulated cells were also down-regulated after sulforaphane treatment. Moreover, sulforaphane repressed the Ang II-induced phosphorylation of Akt, GSK3β, mTOR, eIF4e, as well as of IκBα and NF-κB.

Conclusion

Based on our results, sulforaphane attenuates Ang II-induced hypertrophy of H9c2 cardiomyocytes mediated by the inhibition of intracellular signaling pathways including Akt and NF-κB.     

FHL2/SLIM3 decreases cardiomyocyte survival by inhibitory interaction with sphingosine kinase-1

Sphingosine kinase-1 (SK1) is a key enzyme catalyzing the phosphorylation of sphingosine to sphingosine-1-phosphate (S1P). Recent studies suggest that SK1, and its product S1P, regulate diverse biological functions, including cell growth, differentiation, proliferation, and apoptosis. S1P may also play an important role in cardiac development and ischemic preconditioning, but the mechanism underlying these effects is not known. Using a yeast 2-hybrid screen with SK1 as bait and a cardiac cDNA library to identify novel proteins involved in regulating SK1 activity in cardiomyocytes, we identified the LIM-only factor FHL2 (SLIM3) as a SK1-interacting protein in both yeast and mammalian cells. FHL2, but not FHL1 or FHL3, interacted with SK1, and FHL2 colocalized with SK1 in the cytoplasm. The interaction sites with SK1 consisted of at least 4 LIM domains in FHL2, whereas the C-terminal portion of SK1 mediates the binding of FHL2 in SK1. Overexpression of FHL2 attenuated the activity and antiapoptotic effects of SK1. Indeed, endothelin-1, which is a potent survival factor in cardiomyocytes, inhibited FHL2-SK1 association and increased SK1 activity. These findings indicate that FHL2 is a novel inhibitor of SK1 activity in cardiomyocytes and suggest that targeting FHL2 for inhibition may prevent myocardial apoptosis through activation of SK1.     

NADPH oxidase-derived superoxide anion mediates angiotensin II-induced cardiac hypertrophy

Cardiac hypertrophy is an adaptive response to increases in blood pressure. Recent studies indicate that the hypertrophic process is associated with increases in intracellular oxidative stress in cardiomyocytes. We hypothesize that superoxide anion mediates the hypertrophic response and that antioxidant therapy may be effective in attenuating cardiac hypertrophy. Neonatal rat cardiac myocytes were stimulated with angiotensin II (AngII, 1 microM) with and without various antioxidants. N-acetylcysteine (NAC, 10 mM) and probucol (50 microM), and to a lesser extent, vitamin C (500 microM) and reduced glutathione (1 mM), inhibited AngII-induced [(3)H]-leucine uptake and atrial natriuretic factor (ANF) promoter activity. The hypertrophic response is mediated by superoxide anion (O(2)(-).) since cell-permeable polyethylene glycol (PEG)-conjugated superoxide dismutase (50 U/ml), but not PEG-catalase (500 U/ml), attenuated AngII-induced [(3)H]-leucine uptake and ANF promoter activity. Furthermore, NAC blocked AngII-induced increase in myocardial oxidative stress, decreased the expression of ANF and myosin light chain-2v, and inhibited the re-organization of cytoskeletal proteins, desmin and alpha-actinin. These effects of AngII were abolished by angiotensin type 1 receptor blocker, losartan, but not type 2 receptor blocker, PD123319. Indeed, co-administration of losartan (10 mg/kg/d, 14 d) or NAC (200 mg/kg/d, 14 d) inhibited AngII-induced O(2)(-). production and cardiac hypertrophy in rats without affecting blood pressure. These findings indicate that the generation of O(2)(-). contributes to oxidant-induced hypertrophic response and suggest that antioxidant therapy may have beneficial effects in cardiac hypertrophy.     

Endothelin-converting enzyme inhibition ameliorates angiotensin II-induced cardiac damage

We tested the hypothesis that endothelin-converting enzyme (ECE) inhibition ameliorates end-organ damage in rats harboring both human renin and human angiotensinogen genes (dTGR). Hypertension develops in the animals, and they die by age 7 weeks of heart and kidney failure. Three groups were studied: dTGR (n=12) receiving vehicle, dTGR receiving ECE inhibitor (RO0687629; 30 mg/kg by gavage; n=10), and Sprague-Dawley control rats (SD; n=10) receiving vehicle, all after week 4, with euthanasia at week 7. Systolic blood pressure was not reduced by ECE inhibitor compared with dTGR (205+/-6 versus 206+/-6 mm Hg at week 7, respectively). In contrast, ECE inhibitor treatment significantly reduced mortality rate to 20% (2 of 10), whereas untreated dTGR had a 52% mortality rate (7 of 12). ECE inhibitor treatment ameliorated cardiac damage and reduced left ventricular ECE activity below SD levels. Echocardiography at week 7 showed reduced cardiac hypertrophy (4.8+/-0.2 versus 5.7+/-0.2 mg/g, P<0.01) and increased left ventricular cavity diameter (5.5+/-0.3 versus 3.1+/-0.1 mm, P<0.001) and filling volume (0.42+/-0.04 versus 0.16+/-0.06 mL, P<0.05) after ECE inhibitor compared with untreated dTGR. ECE inhibitor treatment also reduced cardiac fibrosis, tissue factor expression, left ventricular basic fibroblast growth factor mRNA levels, and immunostaining in the vessel wall, independent of high blood pressure. In contrast, the ECE inhibitor treatment showed no renoprotective effect. These data are the first to show that ECE inhibition reduces angiotensin II-induced cardiac damage.     

Caloric restriction ameliorates angiotensin II-induced mitochondrial remodeling and cardiac hypertrophy

Angiotensin II-induced cardiac damage is associated with oxidative stress-dependent mitochondrial dysfunction. Caloric restriction (CR), a dietary regimen that increases mitochondrial activity and cellular stress resistance, could provide protection. We tested that hypothesis in double transgenic rats harboring human renin and angiotensinogen genes (dTGRs). CR (60% of energy intake for 4 weeks) decreased mortality in dTGRs. CR ameliorated angiotensin II-induced cardiomyocyte hypertrophy, vascular inflammation, cardiac damage and fibrosis, cardiomyocyte apoptosis, and cardiac atrial natriuretic peptide mRNA overexpression. The effects were blood pressure independent and were linked to increased endoplasmic reticulum stress, autophagy, serum adiponectin level, and 5' AMP-activated protein kinase phosphorylation. CR decreased cardiac p38 phosphorylation, nitrotyrosine expression, and serum insulin-like growth factor 1 levels. Mitochondria from dTGR hearts showed clustered mitochondrial patterns, decreased numbers, and volume fractions but increased trans-sectional areas. All of these effects were reduced in CR dTGRs. Mitochondrial proteomic profiling identified 43 dTGR proteins and 42 Sprague-Dawley proteins, of which 29 proteins were in common in response to CR. We identified 7 proteins in CR dTGRs that were not found in control dTGRs. In contrast, 6 mitochondrial proteins were identified from dTGRs that were not detected in any other group. Gene ontology annotations with the Panther protein classification system revealed downregulation of cytoskeletal proteins and enzyme modulators and upregulation of oxidoreductase activity in dTGRs. CR provides powerful, blood pressure-independent, protection against angiotensin II-induced mitochondrial remodeling and cardiac hypertrophy. The findings support the notion of modulating cardiac bioenergetics to ameliorate angiotensin II-induced cardiovascular complications.     

丙戊酸对AngⅡ诱导的心肌细胞肥大与HDAC2表达的抑制作用

目的: 观察组蛋白脱乙酰基酶抑制剂—丙戊酸（valproic acid,VPA）抑制心肌细胞肥大和组蛋白脱乙酰基酶2（histone deacetylase 2，HDAC2）的表达。方法: 常规方法培养原代心肌细胞，分为5组:对照组、肥大组、低浓度VPA组(5×10-6 mol/L)、中浓度VPA组(10-5 mol/L)和高浓度VPA组(2×10-5 mol/L)。给予血管紧张素Ⅱ(AngⅡ)刺激心肌细胞造成肥大后，给予不同浓度的VPA进行干预。AngⅡ作用24 h后,于相差显微镜下观察心肌细胞面积的变化。用RT-PCR检测HDAC2 mRNA的表达；免疫组化染色法检测HDAC2蛋白的表达。结果: 经AngⅡ刺激后，在相差显微镜下可见心肌细胞面积变大，HDAC2 mRNA的水平增高，HDAC2蛋白表达亦增加。给予不同浓度的VPA后，上述指标随着VPA浓度的增加而逐渐下降（P<0.05）。结论: AngⅡ致心肌细胞肥大的过程中伴有 HDAC2表达增加，给予HDAC抑制制后，可使心肌细胞面积减少，HDAC2表达减少，提示VPA可抑制心肌细胞的肥大，HDAC2有可能参与心肌细胞肥大的机制。