PTEN对心衰患者心肌MAPK/PKC信号通路的作用

摘要：目的 观察不同程度充血性心力衰竭（CHF）患者心肌重构病理过程中PTEN及丝裂原活化蛋白激酶（MAPK）/蛋白激酶C（PKC）的作用。方法 通过手术取材，选择因瓣膜性心脏病接受二尖瓣置换术的CHF病人39例，正常对照38例(其中8例来自意外伤亡的器官捐献者)。竞争蛋白结合法检测心肌组织PKC及MAPK活性，免疫沉淀法检测PTEN蛋白表达。结果 CHF患者心肌组织呈典型的重构心肌的病理改变。心肌组织PTEN蛋白表达光密度值（A）（PTEN/β-actin）比值随心功能恶化而降低, 各CHF组与正常组相比差异有统计学意义（P < 0.01）; 相反，CHF患者心肌组织PKC和MAPK活性明显高于对照组(P <0.01)，并随心功能恶化其表达逐渐增加，各CHF组与正常组相比差异有统计学意义（P < 0.01）。 结论 PTEN及MAPK/PKC信号通路共同参与调节CHF患者心肌重构的病理过程， PTEN在心肌重构病理过程中起负性调节作用。     

肾素血管紧张素系统诱导心力衰竭患者心肌重构的细胞内信号传导

目的 探讨丝裂素活化蛋白激酶(mitogen-activated protein kinase,MAPK)系统、心肌肥大相关基因参与心力衰竭(CHF)患者心肌重塑的机制.方法 选择2004年1月至2004年12月于成都军区总医院心内科因瓣膜性心脏病接受二尖瓣置换术的CHF患者39例,正常对照组38例(其中8例来自意外伤亡的器官捐献者).彩色多普勒超声心动图仪检测心脏扩大和心功能参数.放免法检测血浆及心肌组织AngⅡ质量浓度.免疫沉淀法测心肌组织c-jun NH2末端激酶(JNK)、细胞外信号调节酶(ERK)、P38MAPK蛋白表达及磷酸化及原癌基因c-myc、c-myb和c-jun蛋白表达.结果 CHF心功能Ⅱ级组患者心肌组织ERK磷酸化明显强于正常对照组(P＜0.01),明显强于CHF心功能Ⅲ、Ⅳ级组(P＜0.01),CHF心功能Ⅲ、Ⅳ级组间差异无统计学意义(P＞0.05);CHF心功能Ⅱ、Ⅲ、Ⅳ级组JNK磷酸化明显强于正常对照组(P＜0.01),P38MAPK磷酸化明显强于对照组(P＜0.05),随心功能恶化,其表达逐渐增加;正常心肌组织中无c-myc、c-myb和c-jun蛋白表达,随心功能恶化CHF组其蛋白表达明显增高(P＜0.01).结论 肾素血管紧张素系统(RAS)激活的MAPK系统导致原癌基因蛋白表达在CHF患者心肌重构机制中可能起重要作用.     

心衰患者重构心肌组织细胞外基质的变化

目的 探讨心衰患者重构心肌组织细胞外基质（ECM）纤连蛋白（FN）和肌腱蛋白（TN-C）与心功能损害的关系。 方法 选择因二尖瓣关闭不全心脏病接受二尖瓣置换术的CHF病人39例，正常对照38例（其中8例来自意外伤亡的器官捐献者）。光镜检查心肌组织病理变化，免疫沉淀法检测心肌组织FN、TN-C蛋白表达，免疫荧光法检查心肌组织细胞外基质FN、TN-C的分布。 结果 瓣膜病所致心力衰竭病人心肌组织呈典型的心肌重构病理改变。心肌组织TN-C蛋白表达吸光度值（A）（A/β-actin）比值正常对照组分别为0，心功能Ⅱ级组1.69±0.05，心功能Ⅲ级组1.89±0.06，心功能Ⅳ级组3.36±0.40, 各CHF组与正常对照组相比差异有统计学意义（P<0.05 或0.01）; 相反，心肌组织FN蛋白表达（A/β-actin）比值，正常组分别为2.01±0.02，心功能Ⅱ级组1.13±0.01，心功能Ⅲ级组0.70±0.02，心功能Ⅳ级组0.42±0.05，各CHF组与正常对照组相比差异有统计学意义（P<0.05 或0.01）。 结论 心衰患者TN-C表达量的增高及FN的降低参与心肌重构的病理过程，重构心肌组织通过改变心肌细胞外基质进一步恶化心功能。     

PTEN/AKT在子宫内膜癌中的表达及临床意义

目的研究抑癌基因PTEN在子宫内膜癌中的表达及其与蛋白激酶B（PKB/AKT）表达。方法采用RT-PCR及Western印迹杂交法检测30例子宫内膜癌、20例非典型增生、20例正常子宫内膜组织中PTEN、AKTmRNA及蛋白的表达。结果子宫内膜癌组织中PTEN的表达低于非典型增生和正常子宫内膜组织（P〈0.01）;子宫内膜癌组织中AKT表达明显高于非典型增生和正常子宫内膜组织（P〈0.01）。结论子宫内膜癌组织中AKT的表达水平的明显增高,是由于PTEN表达下调或缺失而引起,AKT在子宫内膜癌的发生中起着重要的作用。     

醛固酮诱导心肌成纤维细胞增殖的信号转导

目的探讨钙调神经磷酸酶（CaN）、丝裂素活化蛋白激酶（MAPK）、蛋白激酶C（PKC）信号通路在醛固酮（aldosterone,ALD）诱导的乳鼠心肌成纤维细胞（fibroblasts,FBs）增殖中的作用。方法以培养的FBs为模型,以3H-亮氨酸及3H-胸腺嘧啶掺入量作为反映FBs增殖的指标,用ALD诱导FBs增殖,并检测FBsCaN、MAPK、PKC活性。结果ALD呈浓度依赖性增高FBs蛋白及核酸合成速率;螺旋内酯（spironolactone,SPI）能明显阻断ALD诱导的FBs蛋白及核酸合成。ALD诱导组FBsCaN、MAPK、PKC活性明显升高,与对照组相比差异显著（P<0.05或P<0.01）。SPI能阻断ALD诱导的FBsCaN、MAPK、FBsPKC活性增高。结论CaN、MAPK、PKC信号通路均参与了ALD诱导的FBs增殖。     

PTEN和VEGF在肺癌中的表达及意义

目的研究PTEN和血管内皮生长因子（VEGF）在肺癌中的表达、临床意义及其相关性.方法应用组织微阵列仪制作肺癌组织芯片.用免疫组织化学S-P法检测PTEN、VEGF在76例肺癌和30例正常肺组织中的表达.结果肺癌组织中PTEN蛋白阳性表达率显著低于正常肺组织（P＜0.01）,VEGF蛋白阳性表达率显著高于正常肺组织（P＜0.01）;PTEN、VEGF的表达与组织分化程度、淋巴结转移和临床病理分期有关;两者表达呈负相关（P＜0.01）.结论联合检测PTEN、VEGF对肺癌的恶性程度及预后判断具有重要的临床意义,应用组织芯片大规模高效检测临床组织样本具有快速、准确、方便经济的特点.     

脂联素在慢性压力负荷性心力衰竭大鼠心肌中的表达

目的：探讨脂联素（adiponectin）蛋白在慢性心力衰竭尤其是伴有2型糖尿病的大鼠心肌组织中的分布及表达,以及糖尿病对心衰大鼠心肌中脂联素表达的影响.方法：选取Sprague-Dawley雄性大鼠,将其随机分为假手术组（SHAM组）、单纯2型糖尿病组（DM组）、单纯心力衰竭组（HFNDM组）和心力衰竭伴2型糖尿病组（HFDM组）.建立慢性压力负荷性心力衰竭的大鼠模型及高脂高糖饮食后小剂量链脲佐菌素（STZ）诱导的2型糖尿病大鼠模型.运用苏木素-伊红（HE）染色观察大鼠的心脏组织学形态.通过免疫组化、Western Blot定位、定量研究脂联素在心肌中的表达. 结果：大鼠模型建立8周后,DM组及HFDM组大鼠体质量明显低于SHAM组（均P＜0.01）.HFNDM组、DM组及HFDM组的心脏重量指数均显著增加（P＜0.01）.HE染色显示心力衰竭大鼠心肌细胞肥大、排列不整齐、细胞间连接松散,心肌纤维紊乱.免疫组化结果表明各组大鼠心肌中均有脂联素蛋白的表达,定位于细胞浆.Western Blot显示脂联素在糖尿病心肌中低表达（P＜0.05）,心力衰竭心肌中高表达（P＜0.01）. 结论：尽管糖尿病会影响脂联素在大鼠心肌组织中的表达,但在心衰伴糖尿病时心肌中脂联素的表达量仍高于正常大鼠.     

柚皮苷对糖尿病心肌病大鼠心肌的保护作用

目的:观察柚皮苷对糖尿病心肌病大鼠心肌蛋白激酶C(PKC)表达及超微结构的影响. 方法:将60只大鼠随机分为空白对照组、模型对照组和柚皮苷低、中、高剂量组.各组处理后检测血糖,用免疫组织化学方法检测心肌组织PKC表达情况,电子显微镜观察心肌细胞超微结构. 结果:经实验处理6周后,柚皮苷高、中、低剂量组大鼠的血糖及心肌PKC表达较模型对照组显著下降(P＜0.05),心肌超微结构柚皮苷各组均较模型对照组更为接近正常,以柚皮苷高、中剂量组保护作用较为显著. 结论:柚皮苷能有效地改善糖尿病心肌病大鼠心肌组织病理形态学,下调PKC表达,具有确切的心肌保护作用.     

蛋白激酶C活性对缺血再灌注心肌环氧化酶表达的影响

目的探讨心肌缺血再灌注过程中蛋白激酶C（PKC）活性变化对缺血再灌注损伤心肌的影响及机制。方法将40只大鼠随机分为模型组、观察1组、观察2组及对照组各10只,前三组均采用冠状动脉分支结扎术制备心肌缺血再灌注模型,再灌注1 h后结束实验;其中观察1组和观察2组分别于冠状动脉结扎术前20 min经股静脉注射佛波酯及多黏菌素B;对照组为假手术组。实验结束后各组均取静脉血,采用化学比色法测定肌酸激酶（CK）和丙二醛（MDA）水平;处死动物,取心肌组织,采用免疫组化法检测PKC和环氧化酶-2（COX-2）表达。结果与对照组比较,模型组血清CK、MDA水平明显升高（P〈0.01）,心肌PKC表达无明显差异、COX-2表达明显升高（P〈0.05）;与观察2组相比,观察1组血清CK、MDA水平明显降低（P〈0.05）,心肌PKC表达明显升高、COX-2表达明显降低（P均〈0.01）。结论 PKC激活有利于减轻心肌氧化应激损伤,可能机制为下调COX-2表达;此为心肌缺血再灌注损伤的防治提供了依据。     

p38MAPK抑制剂SB203580对脓毒症大鼠心功能的影响及机制探讨

目的 探讨丝裂原活化蛋白激酶p38MAPK抑制剂SB203580对脓毒症大鼠心功能的影响及其机制.方法 30只雄性SD大鼠随机分为对照组、模型组、干预组,各10只.模型组和干预组采用盲肠结扎穿刺术（CLP）制备脓毒症大鼠模型.干预组给予p38MAPK抑制剂SB203580干预.干预后1、3、6、12、24h检测各组血清肿瘤坏死因子α（TNF-α）、IL-6水平,24h后行心脏彩超检测各组大鼠的左室射血分数（EF）及短轴缩短率（FS）;并分离左室心肌采用Western blot方法检测p38MAPK蛋白表达.结果 模型组、干预组术后血清TNF-α、IL-6水平迅速升高,于24h到达高峰,但干预组升高幅度小于模型组（P均＜0.05）.术后24h模型组、干预组左心室EF和FS较对照组显著降低（P均＜0.05）.干预组左心室EF和FS下降程度小于模型组（P均＜0.05）.与对照组比较,术后24h模型组、干预组心肌组织中p38MAPK蛋白相对表达量升高,干预组低于模型组（P均＜0.05）.结论 p38MAPK抑制剂SB203580对脓毒症大鼠心功能有保护作用,降低TNF-α、IL-6水平可能为其主要机制.     

细胞外信号调节激酶的变化在心力衰竭患者心肌重构中的意义

目的了解慢性心力衰竭（CHF）患者心肌组织细胞外信号调节激酶（ERK）的变化与心肌重构和心功能的关系。方法采用Westernblot技术测定31例瓣膜病所致CHF患者不同心功能组心肌细胞ERK蛋白基础表达和激活情况。结果瓣膜病所致CHF患者心肌组织呈心肌重构的病理改变,轻度CHF患者ERK基础表达受抑,激活的磷酸化ERK显著增高。重度CHF患者ERK蛋白大多以无活性的形式存在,激活降低。结论ERK表达与CHF患者心肌重构和心功能关联密切,重度CHF患者较轻度患者心肌组织的磷酸化ERK活性显著降低。     

PARP inhibition prevents postinfarction myocardial remodeling and heart failure via the protein kinase C/glycogen synthase kinase-3beta pathway

The inhibition of glycogen synthase kinase-3beta (GSK-3beta) via phosphorylation by Akt or protein kinase C (PKC), or the activation of mitogen-activated protein kinase (MAPK) cascades can play a pivotal role in left ventricular remodeling following myocardial infarction. Our previous data showed that MAPK and phosphatidylinositol-3-kinase/Akt pathways could be modulated by poly(ADP-ribose)polymerase (PARP) inhibition raising the possibility that cardiac hypertrophic signaling responses may be favorably influenced by PARP inhibitors. A novel PARP inhibitor (L-2286) was tested in a rat model of chronic heart failure following isoproterenol-induced myocardial infarction. Subsequently, cardiac hypertrophy and interstitial collagen deposition were assessed; additionally, mitochondrial enzyme activity and the phosphorylation state of GSK-3beta, Akt, PKC and MAPK cascades were monitored. PARP inhibitor (L-2286) treatment significantly reduced the progression of postinfarction heart failure attenuating cardiac hypertrophy and interstitial fibrosis, and preserving the integrity of respiratory complexes. More importantly, L-2286 repressed the hypertrophy-associated increased phosphorylation of panPKC, PKC alpha/betaII, PKC delta and PKC epsilon, which could be responsible for the activation of the antihypertrophic GSK-3beta. This work provides the first evidence that PARP inhibition beneficially modulates the PKC/GSK-3beta intracellular signaling pathway in a rat model of chronic heart failure identifying a novel drug target to treat heart failure.     

心力衰竭病人心肌组织血管紧张素Ⅱ受体基因表达与心肌重塑

目的：了解不同程序充血性心力衰竭（CHF）病人心肌细胞血管紧张素Ⅱ型受体（AT1）、AT2的mRNA表达与心肌重塑和心功能的关系。方法：通过手术取材,采用RT－PCR技术测定31例瓣膜病所致CHF病人不同心功能组与5例正常人心肌细胞AT1和AT2的mRNA表达。结果：瓣膜病所致心力衰竭（心衰）病人心肌组织呈心肌重塑的病理改变。轻度心衰病人心肌细胞AT1mRNA表达较正常人增高,中重度心衰组AT1mRNA表达明显降低,AT2mRNA表达差异无显著性,但AT1／AT2比值明显降低。结论：心肌细胞AT1在轻度心衰病人增高可能与心肌肥厚的发生有关,重度心衰病人心肌细胞占优势的受体亚型从AT1转变为AT2,其功能意义可能是对心衰的局部保护机制,也可能通过AT2诱导心肌细胞凋亡,致心功能进一步恶化。     

Alterations in protein kinase A and protein kinase C levels in heart failure due to genetic cardiomyopathy.

BACKGROUND: It is becoming evident that both cardiac and skeletal muscles are affected in congestive heart failure. Although protein kinases are known to regulate cardiac function, very little is known about their status in cardiac and skeletal muscles during the development of congestive heart failure. OBJECTIVE: To determine changes in the activities and protein levels of protein kinase A (PKA) and protein kinase C (PKC) in cardiac and skeletal muscles in congestive heart failure due to genetic cardiomyopathy on the basis that PKA and PKC are crucial for protein phosphorylation. ANIMALS AND METHODS: Genetically cardiomyopathic UM-X7.1 hamsters (250 to 300 days old) and age-matched Syrian hamsters were used in this study. PKA and PKC activities were assayed by measuring 32P from [gamma-32P]ATP incorporated into synthetic substrates. Relative protein contents of these protein kinases were obtained by using immunoblot analysis in control and failing hamster hearts and skeletal muscles. RESULTS: PKC activity was significantly increased in the failing hearts compared with control preparations. The relative protein contents of cytosolic PKC-alpha and -epsilon , and of particulate PKC-epsilon isozymes were significantly increased in failing hearts. PKC activity was also markedly increased in cardiomyopathic skeletal muscle. Furthermore, PKA activity and protein level in both cardiac and skeletal muscles were significantly increased in the failing heart group compared with control values. CONCLUSIONS: Increased PKC activity in heart failure may be due to changes in PKC-alpha and -epsilon isozymes in cardiomyopathic hearts. Alterations of PKA and PKC in congestive heart failure were not limited to the heart because similar changes in enzyme activities were evident in skeletal muscle.     

钙蛋白酶参与心力衰竭患者心肌重构的信号传导

目的 探讨钙敏感的信号物质钙蛋白酶(calpain)对心力衰竭(心衰)患心肌重构信号传导的调控。方法选择因二尖瓣狭窄伴关闭不全心脏病接受二尖瓣置换术的心衰患39例,正常对照38例(其中8例来自意外伤亡的器官捐献)。彩色多普勒超声心动图检测心功能参数,放免法检测心衰患血浆及心肌组织血管紧张素Ⅱ含量,免疫印迹法检测心肌组织calpain、钙调神经磷酸酶(CaN)的抑制蛋白(cain/cabin 1),cain／cabin 1降解产物(cain／cabin 1△)蛋白表达、CaN磷酸化。结果心衰患血浆及心肌组织血管紧张素Ⅱ浓度、心肌组织u-calpain、m-calpain、cain／cabin 1降解产物cain／cabin 1△蛋白表达及CaN磷酸化明显高于对照组,且随心功能恶化逐渐增加,cain／cabin 1蛋白表达随心功能恶化逐渐降低。结论心衰患通过calpain降解cain／cabin 1进而激活CaN信号通路,提示其在肾素一血管紧张素系统等介导的心肌重构中起一定作用。     

冠心病患者血小板PKC、PKCI及红细胞PKC活性变化

目的 研究蛋白激酶C(PKC)在冠心病的发生发展中的作用。方法 测定87例心绞痛(AP)患者、91例急性心肌梗死(AMl)患者、90例健康对照(HC)者的血小板胞膜、胞浆PKC、胞浆蛋白激酶C抑制剂(PKCl)活性和红细胞胞膜、胞浆PKC活性。结果 AP组和AMI组血小板胞膜中PKC活性明显高于HC组,而胞浆中PKC活性明显低于HC组;AP组和AMI组血小板胞浆中PKCI活性明显低于HC组;AP组和AMI组红细胞胞膜中PKC活性明显高于HC组,而胞浆中PKC活性低于HC组。结论 PKC可能参与冠心病的发病。     

肿瘤坏死因子α和基质金属蛋白酶家族与左室重构的关系

目的　探讨肿瘤坏死因子 (TNF)α与基质金属蛋白酶家族 (MMPs)MMP 2、3、9与心肌重构、心功能损害的关系。方法　采用双抗体夹心ELISA法测定心力衰竭 (心衰 )患者TNFα血浓度 ,Westernblot法对 31例风湿性心脏病 (RHD)心衰患者及 8例健康人心室肌组织中MMP 2、3、9蛋白表达含量进行半定量分析 ;RHD患者术前心功能指标采用超声心动图检查。结果　心衰患者TNFα血浓度随心衰加重而增高 (P <0 0 5或 <0 0 1) ,心肌组织MMP 2、3、9蛋白表达随心衰程度加重而增加 (P <0 0 5或程 <0 0 1) ,TNFα血浓度与心肌组织MMP 2、3、9蛋白表达存在明显的正相关关系 (P <0 0 1或 <0 0 0 1)。结论　TNFα可能通过刺激MMPs的表达 ,引起细胞外基质结构发生改变 ,加重心衰时的左室重构 ,引起心功能恶化     

Matrix metalloproteinase-9 is a marker of heart failure after acute myocardial infarction

BackgroundMatrix metalloproteinases (MMPs) have been associated with the development of left ventricular remodeling after myocardial infarction (MI). We sought to determine whether peripheral levels of MMPs can be used as a risk marker for the development of congestive heart failure (CHF) after acute MI.Methods and ResultsPlasma levels of MMP-2, MMP-9, C-reactive protein (hs-CRP), tumor necrosis factor-α (TNF-α), and pro-brain natriuretic peptide (pro-BNP) were measured in 109 consecutive patients with acute MI treated with primary mechanical reperfusion. Echocardiographic assessment of left ventricular wall motion index was performed during admission. Patients were followed for the development of CHF. Left ventricular function and volumes were determined after 2 years with radionuclide ventriculography. During 2-year follow-up, 15 patients developed congestive heart failure (CHF). Using multivariate analysis, MMP-9 levels were the only circulating factor predictive of late onset CHF. Patients who had high MMP-9 levels had a significant risk of late onset CHF (OR of 6.5, P ≤ .006) and left ventricular remodeling (ΔEF = −9%, P = .03, and Δend-diastolic volume = +13 mL, P = .03). MMP-2, TNF-α, hs-CRP, creatine kinase, and pro-BNP were not predictive of late onset CHF.ConclusionMMP-9 levels may hold prognostic significance in MI patients.     

Differential induction of protein kinase C isoforms at the cardiac hypertrophy stage and congestive heart failure stage in Dahl salt-sensitive rats.

Several protein kinase C (PKC) isoforms may play important roles in cellular signaling pathways. Recent reports have suggested that PKC plays critical isoform-specific roles in the development of cardiac hypertrophy and heart failure. The purpose of the present study was to examine the expression profiles of PKC isoforms in models of cardiac hypertrophy and heart failure. We examined the cardiac expression of individual PKC isoforms at the cardiac hypertrophy stage and the heart failure stage in Dahl salt-sensitive rats by Western blot analysis. The levels of all PKC isoforms increased at the cardiac hypertrophy stage and the heart failure stage, but the pattern of increase differed among PKC isoforms at the heart failure stage. The expressions of PKCalpha, beta, and delta increased at the cardiac hypertrophy stage and remained elevated at the heart failure stage. On the other hand, the expression of PKCepsilon and atypical PKCs (aPKCs) increased at the cardiac hypertrophy stage, but this increase tended to decline at the congestive heart failure stage. These results suggest that there are two groups of PKC isoforms. Several reports have shown that PKCalpha, beta, and delta are involved in the development of cardiac hypertrophy and heart failure, and that PKCepsilon plays a role in the physiological hypertrophic responses and cardioprotective actions. These facts suggest that all PKC isoforms (PKCalpha, beta, delta, epsilon, and aPKCs) expressed in the heart may have similar functions at the cardiac hypertrophy stage, but that two groups of PKC isoforms (PKCalpha, beta, delta, and PKCepsilon, aPKCs) have different functions at the congestive heart failure stage.     

Alterations in G protein and MAP kinase signaling pathways during cardiac remodeling in hypertension and heart failure

The present study was undertaken to elucidate the G-protein and mitogen-activated kinase (MAP kinase) coupled signaling profile in a genetic model of hypertension and congestive heart failure (CHF) that mimics similar disease in humans. At the receptor level, Ang II type 1 receptor (AT1R) increased in left ventricular hypertrophy (LVH) and reverted to normal in CHF, whereas there was a downregulation of the Ang II type 2 receptor (AT2R) in CHF. At the transducer level, Galphaq and Galpha12 protein levels were unchanged during LVH but decreased significantly in CHF. In contrast, Gbeta and Galpha13 protein content were markedly upregulated in CHF. Furthermore, using phospho-specific antibodies in Western blots and in vitro kinase assays, we found at the effector level an upregulation of the small G-protein Rac1 activity during LVH but a decrease during CHF. In parallel, small G-protein Rho activity was significantly increased during LVH but was unchanged in failure. We found at the downstream level that MAP kinase isoforms extracellular signal regulated-kinase (ERK1/2), big mitogen-activated kinase (BMK1/ERK5), C-jun N-terminal-activated kinase (JNKs/SAPKs), and stress-activated kinase (p38) bioactivities were increased during LVH. During CHF, ERK1/2 and JNK1/2 kinase activities were decreased, whereas BMK1/ERK5 kinase activity reverted to normal values. In conclusion, this study demonstrates, for the first time, multistep alterations of G-protein and MAP kinase signaling pathways in LVH and progression to failure in a genetic model of hypertension and failure.