地高辛对慢性心力衰竭大鼠心肌细胞作用的蛋白质组学研究
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摘要
背景
心力衰竭是由于心脏器质性或者功能性疾病损害心脏充盈和射血能力而引起的一组临床综合征。心力衰竭的主要临床表现有引起运动耐量受限的呼吸困难和疲乏,以及导致肺脏充血和肢体水肿的液体潴留。心力衰竭这一临床综合征可由于心包、心肌、心内膜或者大血管疾病所致,但是大多数患者有左心室功能受损的症状。冠状动脉疾病仍是心力衰竭的主要原因。心力衰竭是一个严重的公共卫生问题,中国心力衰竭患病率达0.9%,住院率占同期心血管疾病的20%。美国有超过500万患者,每年有近50万新诊断的心力衰竭患者。心力衰竭主要是老年人的疾病,中国65岁老年人群中大约13%患有心力衰竭。美国每年花费在心力衰竭上的费用约为37亿美元。
洋地黄药物作为治疗心力衰竭的正性肌力药物,应用于临床已有200多年历史,对其作用机制的现代研究证实该类药物通过抑制Na~+-K~+-ATP酶活性而发挥正性肌力作用。近年来研究发现洋地黄类药物在细胞水平表现了多靶位的作用机制。在不影响Na~+-K~+-ATP酶活性的情况下,可以直接作用于肌浆网上的兰尼碱受体,促进Ca~(2+)释放,产生正性肌力作用。洋地黄类药物和Na~+-K~+-ATP酶结合的同时,还可以发挥信号传导功能,低剂量时激活了T型Ca~(2+)通道,提高了心肌收缩力,促进了细胞增殖。而高剂量则可以通过抑止核转录因子促进细胞凋亡,反映了该类药物有双重作用。洋地黄可抑制心衰中神经内分泌的过度激活,增强副交感神经活性,是其用于心衰治疗的重要机制之一。近期的DIG实验证实地高辛在低浓度下能够降低心力衰竭患者的住院率和死亡率,可以节省大量的医疗费用。但是治疗量与中毒量30%的重叠这一严重制约了其在临床广泛应用的重大难题,但迄今尚无可以替代该类药物的新型高效低毒的强心药物。
2000年6月人类基因组框架图的完成,标志着生命科学的研究进入一个新纪元—后基因组时代,蛋白质组学即成为这一前沿研究领域的核心技术。蛋白质组的研究不仅能为生命活动规律提供物质基础,也能为多种疾病机理的阐明及攻克提供理论根据和解决途径。通过对正常个体及病理个体间的蛋白质组比较分析,可以找到某些疾病特异性的蛋白质分子,它们可成为新药物设计的分子靶点,或者也会为疾病的早期诊断提供分子标志。人类疾病的蛋白质组研究通常采用比较蛋白质组学分析,同位素标记相对和绝对定量技术(isobaric tags for relative and absolute quantitation,iTRAQ)是其中重要的研究方法之一。iTRAQ是一种新的、功能强大的可同时对四种及以上样品进行绝对和相对定量研究的方法。iTRAQ试剂为可与氨基酸N端及赖氨酸侧链连接的胺标记同重元素。在质谱图中,任何一种iTRAQ试剂标记的不同样本中的同一蛋白质表现为相同的质荷比。而在串联质谱中,信号离子表现为不同质荷比的峰值,因此根据波峰的高度及面积,可以得到蛋白质的定量信息。
目前国际国内心血管疾病等重大疾病已进行了富有成效的研究。蛋白质组学技术日趋完善与成熟,因而,借助于先进的分子生物学手段,研究地高辛对心肌细胞的作用,深入探讨地高辛在细胞水平生物学作用机制,寻找其作用靶蛋白和靶位相关蛋白,对于拓宽临床对洋地黄药物的作用机制的认识,进一步明确慢性心力衰竭等重要心血管疾病的病理生理机制,寻找药物治疗的新靶位,开辟新的心血管疾病治疗途径具有重要的理论和实际意义。
目的
采用蛋白质组学方法寻找地高辛对心力衰竭大鼠心肌细胞作用的差异蛋白,阐述地高辛治疗慢性心力衰竭的作用机制,进一步确定其正性肌力作用的靶位蛋白或相关蛋白,为开辟心血管疾病治疗的新途径奠定理论基础。
方法
1心力衰竭大鼠动物模型的建立
采用Johns方法结扎大鼠冠状动脉前降支,造成急性心肌梗死后形成慢性心力衰竭动物模型。健康雄性Wistar大鼠50只,取35只行手术造成急性心肌梗死,15只作为假手术组。建立心肌梗死后慢性心力衰竭大鼠动物模型,分为心力衰竭对照组、地高辛治疗组。超声心动图动态检测心肌梗死后大鼠心脏结构和心功能的改变。测定左心室舒张末期及收缩末期内径(LVIDd,LVIDs)、左心室舒张末期及收缩末期后壁厚度(LVPWd,LVPWs)。计算左心室射血分数(LVEF)及左心室短轴缩短分数(FS),记录二尖瓣瓣口舒张早期最大血流峰值速度(E),舒张晚期最大血流峰值速度(A)和收缩期应变率峰值(SRs)。分别记录冠状动脉手术前、手术8周和手术16周后的体重、收缩压(SBP)心电图的变化。观察病理结构和电镜超微结构的改变。
2地高辛对心肌细胞的差异蛋白质筛选
分别选取假手术组、对照组和治疗组大鼠的心肌细胞,提取心肌细胞总蛋白。每组选取6个标本研磨成粉末,裂解、离心和bradford法定量分析,进行蛋白质分离,将蛋白质裂解为肽段,然后用iTRAQ试剂进行差异标记,将标记的样本相混合。选取3种iTKAQ 8plex kit试剂115、117和119进行标记,假手术组样品用115、对照组用117、治疗组用119标记。与样本结合后,用MALDI-TOF/TOF检测每个组分中的肽段组成情况,对于肽段含量较少的组分合并。
3蛋白质谱鉴定
电喷雾四极杆飞行时间串联质谱仪micro-QTOF对肽段进行鉴定分析。分别用DATA ANALYSIS 4.0和WARP-LC软件分析质谱数据。根据搜索结果中每个蛋白匹配肽段出现次数为1次,Rank为1的肽段用于该蛋白的定量。DATAANALYSIS 4.0提取选定肽段的对应iTRAQ标记物的峰面积。115、119的峰面积分别与117的峰面积比较,求取比值。
结果
1大鼠心肌梗死后8周,心室结构发生变化,表现为心腔显著扩大,室壁变薄。心脏运动幅度明显减弱,收缩和舒张功能下降。心功能检测结果:治疗前对照组、治疗组大鼠较假手术组LVEF、FS和SRs明显减低;E和E/A明显增高,A值减低。治疗后治疗组大鼠LVEF、FS高于对照组,SRs升高明显;E减低,A值升高,E/A减低。治疗后对照组和治疗组心律失常发生率无差异,未观察到药物中毒心电图改变。相关性分析:三组治疗前后的LVEF与SRs成正相关,FS与SRs成正相关,所有动物治疗前后的LVEF和FS与SRs均成明显正相关。
治疗8周后光镜下观察假手术组大鼠肌纤维大小近似,排列规整。对照组心肌纤维排列不规则,组细胞肿胀明显,部分心肌纤维断裂,细胞间纤维组织增生明显。治疗组心肌纤维排列不规整,纤维组织增生,心肌细胞微肿胀。电镜超微观察假手术组心肌细胞膜外基板完整,肌原纤维排列规则,肌节长短一致,线粒体结构和分布基本正常,少量肌浆网肿胀。对照组肌节长短基本一致,Z线结构和肌原纤维破坏,线粒体大小不一并灶性聚集,可见线粒体空化,肌浆网肿胀。治疗组心组心肌细胞肌节长短基本一致,部分Z线结构和肌原纤维破坏,大多数线粒体和肌浆网结构基本正常,但线粒体增生明显,数量多,肌浆网肿胀。
2电喷雾四极杆飞行时间串联质谱仪micro-QTOF鉴定分析,发现主要有32个差异蛋白点,其中二个或以上肽段鉴定蛋白13个,单个肽段鉴定蛋白19个。分别用DATA ANALYSIS 4.0和WARP-LC软件分析质谱数据,MASCCOT搜索质谱匹配蛋白标本差异表达蛋白点,鉴定的32个蛋白,其中治疗组较对照组18个蛋白上调,14个下调;对照组较假手术组有13个蛋白上调,17个蛋白下调,2个变化不明显。
质谱鉴定得出32个差异蛋白包括线粒体蛋白、肌原纤维相关蛋白、胞浆蛋白、核蛋白和肌浆网蛋白等,其中以线粒体蛋白为主占53%,其次是肌原纤维相关蛋白占25%。鉴定的32个差异蛋白进行功能分析。在鉴定的32个蛋白质中,涉及能量代谢、细胞收缩、信号转导等,所占比例分别为50%、25%、13%。
结论
1心肌梗死后大鼠模型是研究慢性心力衰竭机制的有效手段。地高辛可以改善慢性心力衰竭大鼠左心室收缩和舒张功能。超声应变率技术可以用来评价心力衰竭的收缩功能。地高辛作用的差异蛋白分布于线粒体和肌原纤维等,涉及能量代谢、细胞收缩、信号转导等:以调节Ca~(2+)浓度变化的正性肌力作用依旧是改善心功能的基础,其中annexinⅥ可能在心肌细胞收缩和舒张功能中发挥重要的作用。另外,优化心肌能量代谢也可能是地高辛是改善心功能的主要因素之一。
3本实验提供了地高辛对心力衰竭心肌细胞作用蛋白质表达的重要信息,为全面阐述该药物作用分子机理提供了重要方法和新线索,为寻找新的心血管疾病治疗途径提供了有意义的靶位蛋白,为探讨心血管疾病新的防治措施奠定了初步依据。
Background
Heart failure(HF) is a complex clinical syndrome that can result from any structural or functional cardiac disorder that impairs the ability of the ventricle to fill with or eject blood.The cardinal manifestations of HF are dyspnea and fatigue,which may limit exercise tolerance,and fluid retention,which may lead to pulmonary congestion and peripheral edema.The clinical syndrome of HF may result from disorders of the pericardium,myocardium,endocardium,or great vessels,but the majority of patients with HF have symptoms due to an impairment of LV myocardial function.Coronary artery disease,is the primary cause of HF.HF is a major and growing public health problem.Approximately 5 million patients in the United States have HF,and over 500 000 patients are diagnosed with HF for the first time each year. The incidence of HF approaches 0.9%of the population in china.Heart failure is primarily a condition of the elderly,and the incidence of HF approaches 10%of the population after age 65 in china.It has been estimated that the total direct and indirect cost of HF in the US will be equal to＄37 billion.
Digoxin has been used in the treatment of chronic heart failure for more than 200 years.Its ability to bind to and inhibit the Na-K-ATPase(NKA) has been well established,as has the resulting increase[Ca~(2+)]i in cellular responsible for its positive inotropic action and its toxicity as well.However,recent reports have challenged this view,suggesting that Na+-independent mechanisms contribute significantly to the cardioactive glycoside-induced inotropy.These include glycosides:directly activating SR Ca~(2+) release(via ryanodine receptors;RyRs);increasing Ca~(2+) selectivity and Ca~(2+) influx via TTX-sensitive Na~+ channels;and othersignalling mechanisms,activatiing protein tyrosine kinases and mitogen-activated protein kinases(MAPKs),which causes increased Ca~(2+) transients and also regulatesthe transcription of growth-related factors.DIG showed Digoxin at low SDC significantly reduced mortality and hospitalizations in ambulatory chronic systolic and diastolic HF patients.
With the discovery of most human genes in 2000,it is now apparent that a factory approach' to address biological problems is desirable if we are to gain a comprehensive understanding of complex biological processes.Proteomics is the large scale study of proteins,usually by biochemical methods.Many studies of cardiovascular disease and cancer have used proteomics techniques.Nonetheless, proteomics has already revolutionized the discovery process for antimicrobial drugs by accelerating target identification and evaluation,assay development,mechanism of action studies,and follow-up support for medicinal chemistry programs.It is important to note that proteomics techniques not only discovery of novel biomarkers, but also of proteins that may actively participate in cardiovascular process.An important starting point for drug discovery is to supply a validated therapeutic target, and the proteomics study ofdigoxin on heart failure may yet provide insights into the mechanism of cardiovascular disease,and provide new starting points for therapeutic intervention.
Objective
The aim of this work was to characterize the effect of digoxin on myocardial cell in the rats wih chronic heart failure,and to determinate differently expressed proteins, further to supply a validated therapeutic target and provide new avenues for the treatment of cardiovascular diseases.
Methods
1 The model of chronic heart failure
The anterior descending coronary branch was ligated in the 35 rats.This procedure is followed by evelopment of a large anterior myocardial infarction.Within 8 weeks,the animals reproducibly underwent CHF.A concurrent group of 15 animals was subjected to sham coronary ligature.Echocardiographic images were acquired. Conventional measurements LV end-diastolic diameter(LVEDD),LV end-systolic diameter(LVESD),systolic and diastolic posterior(LVPWs,LVPWd) were obtained from grayscale.LV ejection fraction(LVEF) and fractional shortening(FS) were measured.Myocardial mean radial peak systolic strain rate(SRs) were computed from a region of interest that was positioned the posterior wall.
2 Preparation for iTRAQ analysis
The tissues were disrupted using intermittent sonication on ice in 20 vol of homogenization buffer.Add 70uL of ethanol to each iTRAQ Reagent vial.Transfer the iTRAQ Reagent vial to one sample tube.Incubate the tubes at room temperature for 1hr,and then add 100uL of Milli-Q water to each tube to quench the iTRAQ reaction.Incubate at room temperature for 30 minutes.Combine the contents of all iTRAQ Reagent-labeled sample tubes into one tube,and then dry the tube.Three samples(sham,NS,digoxin group) were labelled with iTRAQ reagents having molecular weights 115,117 and 119 Da,respectively.
3 Mass spectrometry identify the differently expressed proteins
The diferential protein spots were cut from the gels using proteomework spot cutter and subjected to in-gel digestion with trypsin.The digested peptides' separation was conducted by micro-QTOF coupled with a Surveyor HPLC system.
Results
1 Model of heart failure
The 25 rats that survived 8 weeks after MI were randomly allocated to either NS (n=13) or di goxin(n=12).The 13 sham-operated rats was excellent and displayed normal echocardiographic parameters,whereas MI rats showed clear signs of LV dysfunction.Substantial LV dilation was increased when compared with shamoperated rats.Systolic and diatolic performance was worsened in MI rats,as indicated by the EF,FS,SRs,E,A and E/A.In the 12 rats treated with a low dose of digoxin and followed serially by Echocardiography,when compared with both NS-treated contemporaneous controls and baseline,changed significantly.A good correlation was present between mean LVEF,FS and SRs,including three groups,and all time points.Cardiocyte morphology and ultrastructures of rats changed significantly in NS group.
2 Mass spectrometry identify the differently expressed proteins
32 differentially expressed protein spots were selected and identified with micro-QTOF.The data was analyzed by micro-QTOF,searched from DATA ANALYSIS 4.0 and WARP'-LC software,and compared to MASCCOT.Among these 32 protein spots,the upregulated preoteins were 18,while down regulated were 14 between NS and digoxin group;the upregulated preoteins were 13,while down regulated were 17 between sham and NS group.The subcellar localization of 32 proteins consists of mitochondria,muscle fiber,endochylema,nucleus and sarcoplasmic reticulum.The function of all 32 proteins consists of metabolish, construction,signal transport and proliferation and stress.
Conclusion
1 The identified proteins involved in various aspect of myocardial cell,including metabolism,cell construction,signal transduction,cell proliferation as well.This indictes that digoxin plays its role in cardiovascular disease not only through its inhibiting NKA,but also have another potential target sites.AnnexinⅥprotein may play an important role for the protein in cardiac performance.This effect on cardiac performance is mediated via interaction of the annexinⅥwith cellular proteins responsible for maintaining Ca~(2+) homeostasis in the cell.
2 This study provides an important knowledge of protein expression in vivo and should be helpful for the further elucidatation of the molecular mechanisms involved in digoxin-involved cardiovascular disease.
心力衰竭是由于心脏器质性或者功能性疾病损害心脏充盈和射血能力而引起的一组临床综合征。心力衰竭的主要临床表现有引起运动耐量受限的呼吸困难和疲乏,以及导致肺脏充血和肢体水肿的液体潴留。心力衰竭这一临床综合征可由于心包、心肌、心内膜或者大血管疾病所致,但是大多数患者有左心室功能受损的症状。冠状动脉疾病仍是心力衰竭的主要原因。心力衰竭是一个严重的公共卫生问题,中国心力衰竭患病率达0.9%,住院率占同期心血管疾病的20%。美国有超过500万患者,每年有近50万新诊断的心力衰竭患者。心力衰竭主要是老年人的疾病,中国65岁老年人群中大约13%患有心力衰竭。美国每年花费在心力衰竭上的费用约为37亿美元。
洋地黄药物作为治疗心力衰竭的正性肌力药物,应用于临床已有200多年历史,对其作用机制的现代研究证实该类药物通过抑制Na~+-K~+-ATP酶活性而发挥正性肌力作用。近年来研究发现洋地黄类药物在细胞水平表现了多靶位的作用机制。在不影响Na~+-K~+-ATP酶活性的情况下,可以直接作用于肌浆网上的兰尼碱受体,促进Ca~(2+)释放,产生正性肌力作用。洋地黄类药物和Na~+-K~+-ATP酶结合的同时,还可以发挥信号传导功能,低剂量时激活了T型Ca~(2+)通道,提高了心肌收缩力,促进了细胞增殖。而高剂量则可以通过抑止核转录因子促进细胞凋亡,反映了该类药物有双重作用。洋地黄可抑制心衰中神经内分泌的过度激活,增强副交感神经活性,是其用于心衰治疗的重要机制之一。近期的DIG实验证实地高辛在低浓度下能够降低心力衰竭患者的住院率和死亡率,可以节省大量的医疗费用。但是治疗量与中毒量30%的重叠这一严重制约了其在临床广泛应用的重大难题,但迄今尚无可以替代该类药物的新型高效低毒的强心药物。
2000年6月人类基因组框架图的完成,标志着生命科学的研究进入一个新纪元—后基因组时代,蛋白质组学即成为这一前沿研究领域的核心技术。蛋白质组的研究不仅能为生命活动规律提供物质基础,也能为多种疾病机理的阐明及攻克提供理论根据和解决途径。通过对正常个体及病理个体间的蛋白质组比较分析,可以找到某些疾病特异性的蛋白质分子,它们可成为新药物设计的分子靶点,或者也会为疾病的早期诊断提供分子标志。人类疾病的蛋白质组研究通常采用比较蛋白质组学分析,同位素标记相对和绝对定量技术(isobaric tags for relative and absolute quantitation,iTRAQ)是其中重要的研究方法之一。iTRAQ是一种新的、功能强大的可同时对四种及以上样品进行绝对和相对定量研究的方法。iTRAQ试剂为可与氨基酸N端及赖氨酸侧链连接的胺标记同重元素。在质谱图中,任何一种iTRAQ试剂标记的不同样本中的同一蛋白质表现为相同的质荷比。而在串联质谱中,信号离子表现为不同质荷比的峰值,因此根据波峰的高度及面积,可以得到蛋白质的定量信息。
目前国际国内心血管疾病等重大疾病已进行了富有成效的研究。蛋白质组学技术日趋完善与成熟,因而,借助于先进的分子生物学手段,研究地高辛对心肌细胞的作用,深入探讨地高辛在细胞水平生物学作用机制,寻找其作用靶蛋白和靶位相关蛋白,对于拓宽临床对洋地黄药物的作用机制的认识,进一步明确慢性心力衰竭等重要心血管疾病的病理生理机制,寻找药物治疗的新靶位,开辟新的心血管疾病治疗途径具有重要的理论和实际意义。
目的
采用蛋白质组学方法寻找地高辛对心力衰竭大鼠心肌细胞作用的差异蛋白,阐述地高辛治疗慢性心力衰竭的作用机制,进一步确定其正性肌力作用的靶位蛋白或相关蛋白,为开辟心血管疾病治疗的新途径奠定理论基础。
方法
1心力衰竭大鼠动物模型的建立
采用Johns方法结扎大鼠冠状动脉前降支,造成急性心肌梗死后形成慢性心力衰竭动物模型。健康雄性Wistar大鼠50只,取35只行手术造成急性心肌梗死,15只作为假手术组。建立心肌梗死后慢性心力衰竭大鼠动物模型,分为心力衰竭对照组、地高辛治疗组。超声心动图动态检测心肌梗死后大鼠心脏结构和心功能的改变。测定左心室舒张末期及收缩末期内径(LVIDd,LVIDs)、左心室舒张末期及收缩末期后壁厚度(LVPWd,LVPWs)。计算左心室射血分数(LVEF)及左心室短轴缩短分数(FS),记录二尖瓣瓣口舒张早期最大血流峰值速度(E),舒张晚期最大血流峰值速度(A)和收缩期应变率峰值(SRs)。分别记录冠状动脉手术前、手术8周和手术16周后的体重、收缩压(SBP)心电图的变化。观察病理结构和电镜超微结构的改变。
2地高辛对心肌细胞的差异蛋白质筛选
分别选取假手术组、对照组和治疗组大鼠的心肌细胞,提取心肌细胞总蛋白。每组选取6个标本研磨成粉末,裂解、离心和bradford法定量分析,进行蛋白质分离,将蛋白质裂解为肽段,然后用iTRAQ试剂进行差异标记,将标记的样本相混合。选取3种iTKAQ 8plex kit试剂115、117和119进行标记,假手术组样品用115、对照组用117、治疗组用119标记。与样本结合后,用MALDI-TOF/TOF检测每个组分中的肽段组成情况,对于肽段含量较少的组分合并。
3蛋白质谱鉴定
电喷雾四极杆飞行时间串联质谱仪micro-QTOF对肽段进行鉴定分析。分别用DATA ANALYSIS 4.0和WARP-LC软件分析质谱数据。根据搜索结果中每个蛋白匹配肽段出现次数为1次,Rank为1的肽段用于该蛋白的定量。DATAANALYSIS 4.0提取选定肽段的对应iTRAQ标记物的峰面积。115、119的峰面积分别与117的峰面积比较,求取比值。
结果
1大鼠心肌梗死后8周,心室结构发生变化,表现为心腔显著扩大,室壁变薄。心脏运动幅度明显减弱,收缩和舒张功能下降。心功能检测结果:治疗前对照组、治疗组大鼠较假手术组LVEF、FS和SRs明显减低;E和E/A明显增高,A值减低。治疗后治疗组大鼠LVEF、FS高于对照组,SRs升高明显;E减低,A值升高,E/A减低。治疗后对照组和治疗组心律失常发生率无差异,未观察到药物中毒心电图改变。相关性分析:三组治疗前后的LVEF与SRs成正相关,FS与SRs成正相关,所有动物治疗前后的LVEF和FS与SRs均成明显正相关。
治疗8周后光镜下观察假手术组大鼠肌纤维大小近似,排列规整。对照组心肌纤维排列不规则,组细胞肿胀明显,部分心肌纤维断裂,细胞间纤维组织增生明显。治疗组心肌纤维排列不规整,纤维组织增生,心肌细胞微肿胀。电镜超微观察假手术组心肌细胞膜外基板完整,肌原纤维排列规则,肌节长短一致,线粒体结构和分布基本正常,少量肌浆网肿胀。对照组肌节长短基本一致,Z线结构和肌原纤维破坏,线粒体大小不一并灶性聚集,可见线粒体空化,肌浆网肿胀。治疗组心组心肌细胞肌节长短基本一致,部分Z线结构和肌原纤维破坏,大多数线粒体和肌浆网结构基本正常,但线粒体增生明显,数量多,肌浆网肿胀。
2电喷雾四极杆飞行时间串联质谱仪micro-QTOF鉴定分析,发现主要有32个差异蛋白点,其中二个或以上肽段鉴定蛋白13个,单个肽段鉴定蛋白19个。分别用DATA ANALYSIS 4.0和WARP-LC软件分析质谱数据,MASCCOT搜索质谱匹配蛋白标本差异表达蛋白点,鉴定的32个蛋白,其中治疗组较对照组18个蛋白上调,14个下调;对照组较假手术组有13个蛋白上调,17个蛋白下调,2个变化不明显。
质谱鉴定得出32个差异蛋白包括线粒体蛋白、肌原纤维相关蛋白、胞浆蛋白、核蛋白和肌浆网蛋白等,其中以线粒体蛋白为主占53%,其次是肌原纤维相关蛋白占25%。鉴定的32个差异蛋白进行功能分析。在鉴定的32个蛋白质中,涉及能量代谢、细胞收缩、信号转导等,所占比例分别为50%、25%、13%。
结论
1心肌梗死后大鼠模型是研究慢性心力衰竭机制的有效手段。地高辛可以改善慢性心力衰竭大鼠左心室收缩和舒张功能。超声应变率技术可以用来评价心力衰竭的收缩功能。地高辛作用的差异蛋白分布于线粒体和肌原纤维等,涉及能量代谢、细胞收缩、信号转导等:以调节Ca~(2+)浓度变化的正性肌力作用依旧是改善心功能的基础,其中annexinⅥ可能在心肌细胞收缩和舒张功能中发挥重要的作用。另外,优化心肌能量代谢也可能是地高辛是改善心功能的主要因素之一。
3本实验提供了地高辛对心力衰竭心肌细胞作用蛋白质表达的重要信息,为全面阐述该药物作用分子机理提供了重要方法和新线索,为寻找新的心血管疾病治疗途径提供了有意义的靶位蛋白,为探讨心血管疾病新的防治措施奠定了初步依据。
Background
Heart failure(HF) is a complex clinical syndrome that can result from any structural or functional cardiac disorder that impairs the ability of the ventricle to fill with or eject blood.The cardinal manifestations of HF are dyspnea and fatigue,which may limit exercise tolerance,and fluid retention,which may lead to pulmonary congestion and peripheral edema.The clinical syndrome of HF may result from disorders of the pericardium,myocardium,endocardium,or great vessels,but the majority of patients with HF have symptoms due to an impairment of LV myocardial function.Coronary artery disease,is the primary cause of HF.HF is a major and growing public health problem.Approximately 5 million patients in the United States have HF,and over 500 000 patients are diagnosed with HF for the first time each year. The incidence of HF approaches 0.9%of the population in china.Heart failure is primarily a condition of the elderly,and the incidence of HF approaches 10%of the population after age 65 in china.It has been estimated that the total direct and indirect cost of HF in the US will be equal to＄37 billion.
Digoxin has been used in the treatment of chronic heart failure for more than 200 years.Its ability to bind to and inhibit the Na-K-ATPase(NKA) has been well established,as has the resulting increase[Ca~(2+)]i in cellular responsible for its positive inotropic action and its toxicity as well.However,recent reports have challenged this view,suggesting that Na+-independent mechanisms contribute significantly to the cardioactive glycoside-induced inotropy.These include glycosides:directly activating SR Ca~(2+) release(via ryanodine receptors;RyRs);increasing Ca~(2+) selectivity and Ca~(2+) influx via TTX-sensitive Na~+ channels;and othersignalling mechanisms,activatiing protein tyrosine kinases and mitogen-activated protein kinases(MAPKs),which causes increased Ca~(2+) transients and also regulatesthe transcription of growth-related factors.DIG showed Digoxin at low SDC significantly reduced mortality and hospitalizations in ambulatory chronic systolic and diastolic HF patients.
With the discovery of most human genes in 2000,it is now apparent that a factory approach' to address biological problems is desirable if we are to gain a comprehensive understanding of complex biological processes.Proteomics is the large scale study of proteins,usually by biochemical methods.Many studies of cardiovascular disease and cancer have used proteomics techniques.Nonetheless, proteomics has already revolutionized the discovery process for antimicrobial drugs by accelerating target identification and evaluation,assay development,mechanism of action studies,and follow-up support for medicinal chemistry programs.It is important to note that proteomics techniques not only discovery of novel biomarkers, but also of proteins that may actively participate in cardiovascular process.An important starting point for drug discovery is to supply a validated therapeutic target, and the proteomics study ofdigoxin on heart failure may yet provide insights into the mechanism of cardiovascular disease,and provide new starting points for therapeutic intervention.
Objective
The aim of this work was to characterize the effect of digoxin on myocardial cell in the rats wih chronic heart failure,and to determinate differently expressed proteins, further to supply a validated therapeutic target and provide new avenues for the treatment of cardiovascular diseases.
Methods
1 The model of chronic heart failure
The anterior descending coronary branch was ligated in the 35 rats.This procedure is followed by evelopment of a large anterior myocardial infarction.Within 8 weeks,the animals reproducibly underwent CHF.A concurrent group of 15 animals was subjected to sham coronary ligature.Echocardiographic images were acquired. Conventional measurements LV end-diastolic diameter(LVEDD),LV end-systolic diameter(LVESD),systolic and diastolic posterior(LVPWs,LVPWd) were obtained from grayscale.LV ejection fraction(LVEF) and fractional shortening(FS) were measured.Myocardial mean radial peak systolic strain rate(SRs) were computed from a region of interest that was positioned the posterior wall.
2 Preparation for iTRAQ analysis
The tissues were disrupted using intermittent sonication on ice in 20 vol of homogenization buffer.Add 70uL of ethanol to each iTRAQ Reagent vial.Transfer the iTRAQ Reagent vial to one sample tube.Incubate the tubes at room temperature for 1hr,and then add 100uL of Milli-Q water to each tube to quench the iTRAQ reaction.Incubate at room temperature for 30 minutes.Combine the contents of all iTRAQ Reagent-labeled sample tubes into one tube,and then dry the tube.Three samples(sham,NS,digoxin group) were labelled with iTRAQ reagents having molecular weights 115,117 and 119 Da,respectively.
3 Mass spectrometry identify the differently expressed proteins
The diferential protein spots were cut from the gels using proteomework spot cutter and subjected to in-gel digestion with trypsin.The digested peptides' separation was conducted by micro-QTOF coupled with a Surveyor HPLC system.
Results
1 Model of heart failure
The 25 rats that survived 8 weeks after MI were randomly allocated to either NS (n=13) or di goxin(n=12).The 13 sham-operated rats was excellent and displayed normal echocardiographic parameters,whereas MI rats showed clear signs of LV dysfunction.Substantial LV dilation was increased when compared with shamoperated rats.Systolic and diatolic performance was worsened in MI rats,as indicated by the EF,FS,SRs,E,A and E/A.In the 12 rats treated with a low dose of digoxin and followed serially by Echocardiography,when compared with both NS-treated contemporaneous controls and baseline,changed significantly.A good correlation was present between mean LVEF,FS and SRs,including three groups,and all time points.Cardiocyte morphology and ultrastructures of rats changed significantly in NS group.
2 Mass spectrometry identify the differently expressed proteins
32 differentially expressed protein spots were selected and identified with micro-QTOF.The data was analyzed by micro-QTOF,searched from DATA ANALYSIS 4.0 and WARP'-LC software,and compared to MASCCOT.Among these 32 protein spots,the upregulated preoteins were 18,while down regulated were 14 between NS and digoxin group;the upregulated preoteins were 13,while down regulated were 17 between sham and NS group.The subcellar localization of 32 proteins consists of mitochondria,muscle fiber,endochylema,nucleus and sarcoplasmic reticulum.The function of all 32 proteins consists of metabolish, construction,signal transport and proliferation and stress.
Conclusion
1 The identified proteins involved in various aspect of myocardial cell,including metabolism,cell construction,signal transduction,cell proliferation as well.This indictes that digoxin plays its role in cardiovascular disease not only through its inhibiting NKA,but also have another potential target sites.AnnexinⅥprotein may play an important role for the protein in cardiac performance.This effect on cardiac performance is mediated via interaction of the annexinⅥwith cellular proteins responsible for maintaining Ca~(2+) homeostasis in the cell.
2 This study provides an important knowledge of protein expression in vivo and should be helpful for the further elucidatation of the molecular mechanisms involved in digoxin-involved cardiovascular disease.
引文
1 Kannel WB,Belanger AJ.Epidemiology of heart failure.Am Heart J,1991;121:951-957.
2 中国心力衰竭流行病学调查及其患病率.顾东风,黄广勇,吴锡桂等.中华心血管病杂志,2003;31:3-6.
3 Hunt SA,Abraham WT,Chin MH,et al.ACC/AHA 2005 Guideline Update for the Diagnosis and Management of Chronic Heart Failure in the Adult:a report of the American College of Cardiology/American Heart Association Task Force on Practice Guidelines(Writing Committee to Update the 2001 Guidelines for the Evaluation and Management of Heart Failure).J Am Coll Cardiol,2005;46:e1-82.
4 2009 Focused Update:ACCF/AHA guidelines for the diagnosis and management of heart failure in adults:a report of the American College of Cardiology Foundation/ American Heart Association Task Force on Practice Guidelines.J Am Coll Cardiol,2009;53:ⅹⅹ-ⅹⅹⅹ.
5 Heart disease and stroke statistics-2009 update:a report from the American Heart Association Statistics Committee and Stroke Statistics Subcommittee.Circulation,2009;119:e21-181.
6 Guidelines for the diagnosis and treatment of chronic heart failure:executive summary(update 2005):The Task Force for the Diagnosis and Treatment of Chronic Heart Failure of the European Society of Cardiology.Eur Heart J,2005;26:1115-1140
7 Cleland JG,Khand A,Clark A.The heart failure epidemic:exactly how big is it?Eur Heart J,2001;22:623-626.
8 上海市心力衰竭调查协作组.上海市1980、1990、2000年心力衰竭住院患者 流行病学及治疗状况调查．中华心血管病杂志．2002；30：24-27．
9 Corwie MR, Wood DA, Coats J S, et al. Incidence and aetiology of heart failure : a population-based study. Eur Heart J, 1999; 20:421-428.
10 Cohn JN, Ferrari R, Sharpe N. Cardiac remodeling-concepts and clinical implications: a consensus paper from an international forum on cardiac remodeling. J Am Coll Cardiol, 2000; 35:569-582.
11 Hirota H, Chen J, Betz UA, et al. Loss of a gp130 cardiac muscle cell survival pathway is a critical event in the on set of heart failure during biome chanical stress. Cell, 1999; 97:189-198.
12 Sadek MM, Haddad T, Haddad H. The role of statins in chronic heart failure. Curr Opin Cardiol, 2009; 24:167-171.
13 Sagawa T, Sagawa K, Kelly JE, Tsushima RG, Wasserstrom JA. Activation of cardiac ryanodine receptors by cardiac glycosides. Am J Physiol Heart Circ Physiol, 2002; 282:H1118-1126.
14 Ferguson DW, Berg WJ, Sanders JS, et al. Sympath oinhibitory responses to digi talis glycosides in heart failure patients: direct vidence from sympathetic neural recordings. Circulation, 1989; 80:65-77.
15 Covit AB, Schaer GL, Sealey JE, et al. Suppression of the renin-angiotensin system by intravenous digoxin in chronic congestive heart failure. Am J Med, 1983; 75:45-47.
16 Gheorghiade M, Ferguson D. Digoxin: a neurohormonal modulator in heart failure?. Circulation, 1991; 84:2181-6.
17 The Digitalis Investigation Group. The effect of digoxin on mortality and morbidity in patients with heart failure. N Engl J Med, 1997; 336:525-533.
18 Ahmed A, Rich MW, Love TE, et al. Digoxin and reduction in mortality and hospitalization in heart failure: a comprehensive post hocanalysis of the DIG trial. Eur Heart J, 2006; 27:178-86.
19 Ahmed A, Pitt B, Rahimtoola SH, et al. Effects of digoxin at low serum concentrations on mortality and hospitalization in heart failure: a propensity-matched study of the DIG trial. Int J Cardiol, 2008; 123:138-146.
20 Gilman G, Khandheria BK, Hagen ME, et al. Strain rate and strain: a step-by-step approach to image and data acquisition. J Am Soc Echocardiogr, 2004; 17:1011-1020.
21 Sutherland GR, Di Salvo G, Claus P, et al. Strain and strain rate imaging: a new clinical approach to quantifying regional myocardial function. J Am Soc Echocardiogr, 2004; 17:788-802.
22 Yip G, Abraham T, Belohlavek M, et al. Clinical applications of strain rate imaging. J Am Soc Echocardiogr, 2003; 16:1334-1342.
23 Pfeffer MA, Braunwald E. Ventricular remodeling after myocardial infarction experimental observations and clinical implications. Circulation, 1990; 81:1161 -1172.
24 Hodsman GP, Kohzuki M, Howes LG, et al. Neuro humoral response to chronic myocardial infarction in rats. Circulation, 1988; 78:376-381.
25 Johns TNP, Olson BJ. Experimental myocardial infarction: a medthod of coronary occlusion in small animals. Am Surg, 1954; 10: 509-516.
26 Pfeffer MA, Pfeffer JM, Fishbein MC, et al. Myocardial infarct size and ventricular function in rats. Circ Res, 1979; 44:503-12.
27 Matsumori A, Igata H, Ono K, et al. High doses of digitalis increase the myocardial production of proinflammatory cytokines and worsen myocardial injury in viral myocarditis: a possible mechanism of digitalis toxicity. Jpn Circ J, 1999; 63:934-40.
28 Rumberger JA. Ventricular dilatation and remodeling after myocardial infarction. Mayo Clin Proc, 1994; 69:664-674.
29 Sakata Y, Yamamoto K, Mano T, et al. Activation of matrix metalloproteinases precedes left ventricular remodeling in hypertensive heart failure rats: its inhibition as a primary effect of Angiotensin-converting enzyme inhibitor. Circulation, 2004; 109:2143-2149.
30 Zornoff LA, Paiva SA, Duarte DR, et al.Ventricular remodeling after myocardial infarction: concepts and clinical implications. Spadaro J Arq Bras Cardiol, 2009; 92:150-164.
31 Udelson JE, Patten RD, Konstam MA.New concepts in post-infarction ventricular remodeling. Rev Cardiovasc Med, 2003; 3:S3-12.
32 Udelson JE, Konstam MAJ Card Fail. Relation between left ventricular remodeling and clinical outcomes in heart failure patients with left ventricular systolic dysfunction. J Card Fail,2002; 8(S):S465-471.
33 Kjaer A, Hesse B. Heart failure and neuroendocrine activation: diagnostic, prognostic and therapeutic perspectives.Clin Physiol, 2001; 21:661-672.
34 Hein S, Kostin S, Heling A.et al. The role of the cytoskeleton in heart failure. Cardiovasc Res, 2000; 45(2):273-278.
35 Dhalla NS, Saini-Chohan HK, Rodriguez-Leyva D,et al. Subcellular remodelling may induce cardiac dysfunction in congestive heart failure.Cardiovasc Res, 2009; 81:429-438.
36 Patten RD, Udelson JE, Konstam MA. Ventricular remodeling and its prevention in the treatment of heart failureCurr Opin Cardiol, 1998; 13:162-167.
37 Kazory A, Ross EA. Contemporary trends in the pharmacological and extracor- poreal management of heart failure: a nephrologic perspective.Circulation, 2008; 117:975-983
38 Rassi A Jr, Rassi A, Rassi SG. Predictors of mortality in chronic Chagas disease: a systematic review of observational studies. Circulation, 2007; 115:1101-1108.
39 Torre-Amione, G Sestier F, Radovancevic B, et al. Broad modulation of tissue responses (immune activation) by celacadeay favorably influence pat hologic processes associated wit h heart failure progression. Am J Cardiol, 2005; 95 (S):30c-37c.
40 Santos A, Ledesma-Carbayo MJ, Malpica N, et al. Accuracy of heart strain rate calculation derived from doppler tissue velocity data. SP IE, 2001; 4325: 546-555.
41 Greenberg NL, Firstenberg MS, Castro PL, et al. Doppler-derived myocardial systolic strain rate is a strong index of left ventricular contractility.Circulation, 2002; 105:99-105.
42 Andersen NH, Poulsen SH, Eiskjaer H, et al. Decreased left ventricular longitudinal contraction in normotensive and normoalbuminuric patients with Type II diabetes mellitus: a Doppler tissue tracking and strain rate echocardiography study. Clin Sci, 2003; 105:59-66.
43 Yalcin F, Kalian A, Muderrisoghu H, et al. Is Doppler tissue velocity during early ventricular filling preload independent? Heart, 2002; 87:336-339.
44 Dandel M, Hetzer RE. Echocardiographic strain and strain rate imaging-clinical applications. Int J Cardiol. 2009; 132:11-24.
45 Thibault H, Gomez L, Donal E, et al. Acute myocardial infarction in mice: assessment of transmurality by strain rate imaging.Am J Physiol Heart Circ Physiol, 2007; 293:H496-502.
46 Sia YT, Lapointe N, Parker TG,et al. Beneficial effects of long-term use of the antioxidant probucol in heart failure in the rat.Circulation.2002; 105:2549-2555.
47 Saraiva RM, Minhas KM, Raju SV, et al. Deficiency of neuronal nitric oxide synthase increases mortality and cardiac remodeling after myocardial infarction: role of nitroso-redox equilibrium. Circulation, 2005; 112:3415-3422.
48 Sullebarger JT, D'Ambra PM, Clark LC,et al. Effect of Digoxin on Ventricular Remodeling and Responsiveness of beta-Adrenoceptors in Chronic Volume Overload. J Cardiovasc Pharmacol Ther, 1998; 3:281-290.
49 Bendall JK, Damy T, Ratajczak P, et al. Role of myocardial neuronal nitric oxide synthase-derived nitric oxide in beta-adrenergic hyporesponsiveness after myocardial infarction-induced heart failure in rat. Circulation. 2004; 110:2368-2375.
50 Sia YT, Lapointe N, Parker TG,et al. Beneficial effects of long-term use of the antioxidant probucol in heart failure in the rat. 2002; 28:2549-2555.
51 Morisco C, Cuocolo A, Romano M,et al. Influence of digitalis on left ventricular functional response to exercise in congestive heart failure Am J Cardiol. 1996; 77:480-485.
52 Besch HR Jr, Watanabe AM. The positive inotropic effect of digitoxin: independence from sodium accumulation. J Pharmacol ExpTher, 1978; 207:958-965.
53 Fujino S, Fujino M. Ouabain potentiation and Ca release from sarcoplasmic reticulum in cardiac and skeletal muscle cells. Can J Physiol Pharmacol, 1982; 60:542-555.
54 McGarry SJ, Williams AJ. Digoxin activates sarcoplasmic reticulum Ca~(2+)-release channels: a possible role in cardiac inotropy. Br J Pharmacol, 1993; 108:1043-1050.
55 Sagawa T,Sagawa K,Kelly JE,et al.Activation of cardiac ryanodine receptors by cardiac glycosides.Am J Physiol Heart Circ Physiol,2002;282:H1118-H1126.
56 Haas M,Askari A,and Xie Z.Involvement of Src and epidermal growth factor receptor in the signal-transducing function of Na~+-K~+-ATPase.J Biol Chem2000;275:27832-27837.
57 Haas M,Wang H,Tian J,and Xie Z.Src-mediated inter-receptor cross-talk between the Na~+-K~+-ATPase and the epidermal growth factor receptor relays the signal from ouabain to mitogen-activated protein kinases.J Biol Chem,2002;277:18694-18702.
1 Wilkins MR, Sanchez JC, Gooley AA. Progress with proteome projects: why all proteins expressed by a genome should be identified and how to do it. Biotechnol Genet Eng Rev, 1996; 13:19-27.
2 Anderson NL, Anderson NG. Proteome and proteomics: new technologies, new concepts, and new words. Electrophoresis, 1998; 19:1853-1861.
3 Cordwell SJ, Humphery-Smith I. Evaluation of algorithms used for cross-species proteome characterisation. Electrophoresis, 1997; 18:1410-1417.
4 Humphery-Smith I, Cordwell SJ, Blackstock WP. Proteome research: complementarity and limitations with respect to the RNA and DNA worlds.Electrophoresis, 1997; 18: 1217-1242.
5 Hanash S.Disease proteomics. Nature, 2003; 422: 226-232.
6 Patton WF. Detection technologies in proteome analysis. J Chromatogr B Analyt Teehnol Biomed Life Sci, 2002; 771:3-31.
7 Hamdan M, Righetti PG. Modern strategies for protein quantification in proteome analysis: advantages and limitations. Mass Spectrom Rev, 2002; 21:287-302.
8 Wu WW, Wang G, Baek S J, et al. Comparative study of three proteome quantitative methods, DIGE, iCAT and iTRAQ: using 2D Gel-or LC-MALDI TOF / TOF. J Proteome Bes, 2006; 5:651-658.
9 Gygi SP, Rist B, Gerber S A et al. Quantitative analysis of complex protein mixtures using isotope-coded affinity tags. Nat Biotechnol, 1999; 17:994-999.
10 Ross P L, Huang Y N, Marchese JN, et al. Multiplexed protein quantitation in Saccharomyces cerevisiae using amine reactive isobaric tagging reagents. Mol Cell Proteomics, 2004; 3:1154-1169..
11 Schatzmann HJ. Cardiac glycosides as inhibitors of active potassium and sodium transport by erythrocyte membrane. Helv Physiol Pharmacol Acta, 1953; 11:346-354.
12 James PF, Grupp IL, Grupp G, et al. Identification of a specific role for the Na~+-K~+-ATPase alpha-isoform as a regulator of calcium in the heart. Mol Cell, 1999; 3: 555-563.
13 Mathias RT, Cohen IS, Gao J, et al. Isoform-specific regulation of the Na~+-K~+-pump in heart. News Physiol Sci, 2000;15:176-180.
14 Besch HR Jr, Watanabe AM. The positive inotropic effect of digitoxin: independence from sodium accumulation. J Pharmacol ExpTher, 1978; 207:958-965.
15 Fujino S, Fujino M. Ouabain potentiation and Ca release from sarcoplasmic reticulum in cardiac and skeletal muscle cells. Can J Physiol Pharmacol, 1982; 60:542-555.
16 Gheorghiade M, St Clair J, St Clair C, et al. Hemodynamic effects of intravenous digoxin in patients with severe heart failure initially treated with diuretics and vasodilators. J Am Coll Cardiol, 1987; 9:849-857.
17 McGarry SJ and Williams AJ. Digoxin activates sarcoplasmic reticulum Ca -release channels: a possible role in cardiac inotropy. Br J Pharmacol, 1993; 108: 1043-1050.
18 Sagawa T, Sagawa K, Kelly JE, et al. Activation of cardiac ryanodine receptors by cardiac glycosides. Am J Physiol Heart Circ Physiol, 2002; 282: H1118-H1126.
19 Haas M, Askari A, and Xie Z. Involvement of Src and epidermal growth factor receptor in the signal-transducing function of Na~+-K~+-ATPase. J Biol Chem, 2000; 275:27832-27837.
20 Haas M,Wang H,Tian J,and Xie Z.Src-mediated inter-receptor cross-talk between the Na~+-K~+-ATPase and the epidermal growth factor receptor relays the signal from ouabain to mitogen-activated protein kinases.J Biol Chem,2002;277:18694-18702.
21 Wasserstrom JA,Aistrup GL.Digitalis:new actions for an old drag.Am J Physiol Heart Circ Physiol,2005;289:H1781-93.
22 Tian J,Gong X,Xie Z.Signal-transducing function of Na~+-K~+-ATPase is essential for ouabain's effect on[Ca~(2+)]i in rat cardiac myocytes.Am J Physiol Heart Circ Physiol,2001;281:H1899-1907.
23 Ramirez-Ortega M,Zarco G,Maldonado V.Is digitalis compound-induced cardio- toxicity,mediated through guinea-pig cardiomyocytes apoptosis? Eur J Pharmacol,2007;566:34-42.
24 Alvarez-Llamas G,de la Cuesta F,Barderas ME,et al.Recent advances in athe-rosclerosis-based proteomics:new biomarkers and a future perspective.Expert Rev Proteomics,2008;5:679-691.
25 Pinet F,Beseme O,Cieniewski-Bemard C,et al.Predicting left ventricular remodeling after a first myocardial infarction by plasma proteome analysis Proteomics.2008;8:1798-1808.
26 Dohke T,Wada A,Isono T,et al.Proteomic analysis reveals significant alternations of cardiac small heat shock protein expression in congestive heart failure.J Card Fail,2006;12:77-84.
27 孙爱军,王克强,李杰,等.心功能衰竭大鼠模型心肌线粒体蛋白的差异表达分析.中国动脉硬化杂志,2004,12:183-185.
28 徐丹令,孙爱军,王时俊,等.乙醛脱氢酶2在大鼠心肌缺氧损伤中的抗凋亡作用.中国病理生理杂志,2006;22:683-686.
29 Arrel DK,Neverova I,Van Eyk JE.Cardiovascular proteomics:evolution and potential. Circ Res, 2001; 88:763-773.
30 Corcoran EE, Joseph JD, MacDonald JA, et al. Proteomic analysis of calcium / calmodulin-dependent protein kinase I and IV in vitro substrates reveals distinct catalytic p references. J Biol Chem, 2003; 278: 10516-10522.
31 Qiu J, Gao HQ, Zhou RH, Proteomics analysis of the proliferative effect of low-dose ouabain on human endothelial cells. Biol Pharm Bull. 2007; 30:247-253.
32 Qiu J, Gao HQ, Liang Y, et al. Comparative proteomics analysis reveals role of heat shock protein 60 in digoxin-induced toxicity in human endothelial cells Biochim Biophys Acta, 2008; 1784:1857-1864.
33 Latterich M, Abramovitz M, Leyland-Jones B. Proteomics: new technologies and clinical applications.Eur J Cancer, 2008; 44:2737-2741.
34 Sui J, Zhang J, Ching CB, et al. Comparative proteomic analysis of extracellular proteins reveals secretion of T-kininogen from vascular smooth muscle cells in response to incubation with s-enantiomer of propranolol.Mol Pharm, 2008; 5:885-90.
35 Sui J, Zhang J, Tan TL, et al. Comparative proteomics analysis of vascular smooth muscle cells incubated with S- and R-enantiomers of atenolol using iTRAQ-coupled two-dimensional LC-MS/MS. Cell Proteomics, 2008; 7:1007-1018.
36 Sui J, Tan TL, Zhang J, et al. iTRAQ-coupled 2D LC-MS/MS analysis on protein profile in vascular smooth muscle cells incubated with S- and R-enantiomers of propranolol: possible role of metabolic enzymes involved in cellular anabolism and antioxidant activity. J Proteome Res, 2007; 6:1643-1651.
37 Langer GA. The role of sodium ion in the regulation of myocardial contractility. J Mol Cell Cardiol, 1970; 1:203-207.
38 Woodbury JW. Interrelationships between ion transport mechanisms and excitatory events. Fed Proc, 1963; 22:31-35.
39 Satoh H, Ginsburg KS, Qing K, et al. KB-R7943 block of Ca~(2+) influx via Na~+/Ca~(2+) exchange does not alter twitches or glycoside inotropy but prevents Ca~(2+) overload in rat ventricular myocytes. Circulation, 2000; 101:1441-1446.
40 Norgaard A, Bagger JP, Bjerregaard P et al. Relation of left ventricular function and Na, K-pump concentration in suspected idiopathic dilated cardiomyopathy. Am J Cardiol, 1988; 61:1312-1315.
41 Kjeldsen K, Bjerregaard P, Richter EA,et al. Na~+-K~+-ATPase concentration in rodent and human heart skeletal muscle: apparent relation to muscle performance. Cardiovasc Res, 1988; 22:95-100.
42 Ishino K, Botker HE, Clausen T, Hetzer R, Sehested J. Myocardial adenine nucleotides, glycogen, and Na~+-K~+-ATPase in patients with idiopathic dilated cardiomyopathy requiring mechanical circulatory support. Am J Cardiol, 1999; 83:396-399.
43 Hanf R, Drubaix I, Marotte F, et al. Rat cardiac hypertrophy.Altered sdium-calcium exchange activity in sarcolemmal vesicles. FEBS Lett, 1988; 236:145-149.
44 No adaptation to digitalization as evaluatedby digitalis receptor (Na~+-K~+-ATPase) quantification in explanted hearts from donors without heart disease and from digitalized recipients with end-stage heart failure. Am J Cardiol, 1992; 70:110-114.
45 Schmidt TA, Holm-Nielsen P, Kjeldsen K. No upregulation of digitalis glycoside receptor (Na~+-K~+-ATPase) concentration in human heart left ventricle samples obtained at necropsy after long term digitalization. Cardiovasc Res, 1991; 25:684-691.
46 Jones LR, Maddock SW, Besch HR Jr. Unmasking effect of alamethicin on the Na~+-K~+-ATPase,beta adrenergic receptor-coupled adenylate cyclase,and cAMP-dependent protein kinase activities of cardiac sarcolemmal vesicles.J Biol Chem,1980;255:9971-9980
47 Hasenfuss G,Reinecke H,Studer R,et al.Calcium cycling proteins and forcefrequency relationship in heart failure.Basic Res Cardiol,1996;91(S) 2:17-22.
48 Kranias EG,Bers DM.Subcell Biochem.Calcium and cardiomyopathies,2007;45:523-537.
49 Del Monte F,Hajjar RJ.Intracellular devastation in heart failure.Heart Fail Rev,2008;13:151-162.
50 Yue TL,Gu JL,Wang C,et al.Extracellular signal-regulated kinase plays an essential role in hypertrophic agonists,endothelin-1 and phenylephrine-induced cardiomyocyte hypertrophy.J Biol Chem,2008;275:37895-37901.
51 Schmidt U,Hajjar RJ,Kim CS,et al.Human heart failure:cAMP stimulation of SR Ca~(2+)-ATPase activity and phosphorylation level of phospholamban.Am J Physiol,1999;277:H474-H480.
52 MacLennan DH,Reithmeier RA.Ion tamers.Nat Struct Biol,1998;5:409-411.
53 Asahi M,Otsu K,Nakayama H,Hikoso S,et al.Cardiac-specific overexpression of sarcolipin inhibits sarco(endo)plasmic reticulum Ca~(2+)-ATPase(SERCA2a)activity and impairs cardiac function in mice.Proc Natl Acad Sci,2004;101:9199-9204.
54 M(u|¨)ller-Ehmsen J,Nickel J,Zobel C,et al.Longer term effects of ouabain on the contractility of rat isolated cardiomyocytes and on the expression of Ca and Na regulating proteins.Basic Res Cardiol.2003;98:90-96.
55 Hein S,Kostin S,Heling A.et al.The role of the cytoskeleton in heart failure.Cardiovasc Res,2000;45(2):273-278.
56 Dhalla NS, Saini-Chohan HK, Rodriguez-Leyva D,et al. Subcellular remodelling may induce cardiac dysfunction in congestive heart failure.Cardiovasc Res, 2009; 81:429-438.
57 Heinke MY, Wheeler CH, Chang D, et al. Protein changes observed in pacing-induced heart failure using two-dimensional electrophoresis. Electrophoresis 1998; 19:2021-2030.
58 Jiang L, Tsubakihara M, Heinke MY, et al. Heart failure and apoptosis: electrophoreticmethods support data from micro- and macro-arrays. A critical review of genomics andproteomics. Proteomics, 2001; 1:1481-1488.
59 Van Bilsen M, Smeets PJ, Gilde AJ,et al. Metabolic remodelling of the failing heart: the cardiac burn-out syndrome? Cardiovasc Res, 2004; 61:218-226.
60 Stanley WC, Lopaschuk GD, Hall JL, et al. Regulation of myocardial carbohydrate metabolism under normal and ischaemic conditions.Potential for pharmacological interventions. Cardiovasc Res, 1997; 33:243-257.
61 Lopaschuk GD, Beike DD, Gamble J, et al. Regulation of fatty acid oxidation in the mammalian heart in health and disease. Biochim Biophys Acta, 1994; 1213:263-276.
62 Gertz EW, Wisneski JA, Stanley WC, et al. Myocardial substrate utilization during exercise in humans. Dual Carbon-labeled carbohydrate isotope experiments. J Clin Invest, 1988; 82:2017-2025.
63 Lopaschuk GD, Stanley WC. Glucose metabolism in the iscemic heart. Circulation, 1997; 95:313-315.
64 Stanley WC, Lopaschuk GD, Hall JL, et al. Regulation of myocardial carbohydrate metabolism under normal and ischaemic conditions. Potential for pharmacological interventions. Cardiovasc Res, 1997; 33:243-257.
65 Tuunanen H, Ukkonen H, Knuuti J. Myocardial fatty acid metabolism and cardiac performance in heart failure. Curr Cardiol Rep, 2008; 10:142-148.
66 Lopaschuk GD. Optimizing cardiac fatty acid and glucose metabolism as an approach to treating heart failure. 2006; 10:228-230.
67 Unverferth DV, Fetters J K, Barbara BS, et al. Human myocardial histologic characteristics in congestive heart failure. Circulation, 1983; 68:1194-1200.
68 Ormerod JO, Ashrafian H, Frenneaux MP. Impaired energetics in heart failure - a new the rapeutic target. Pharmacol Ther, 2008; 119:264-74.
69 Gusarova V, Caplan AJ, Brodsky JL, et al. Apoprotein B degradation is p romoted by the molecular chaperones HSP90 and HSP70. Biol Chem, 2001; 276:24891-24892.
70 Li G, Bae S, Zhang L. Effect of prenatal hypoxia on heat stress-mediated cardioprotection in adult rat heart. Am J Physiol Heart Circ Physiol, 2004; 2 86: H17122 -H1719.
71 Kim SH , Koh GY, Cho KW, et al. Stretch-activated atrial nariuretic peptide secretion in atria with heat shock protein70 overexpression. Exp Bio! Med (Maywood), 2003; 228: 200-206.
72 Knowlton AA, Kapadia S, Torre-Amione G, et al. Differential expression of heat shock proteins in normal and failing human hearts. J Mol Cell Cardiol, 1998; 30:811-818.
73 Delogu G, Signore M, Mechelli A, et al. Heat shock proteins and their role in heart injury. Curr Opin Crit Care, 2002; 8: 4-12.
74 Liu J, Kam KW, Borchert GH, et al. Furt her study on t he role of HSP70 on Ca~(2+) homeostasis in rat ventricular myocytes subjected to simulated ischemia. Am J Physiol Cell Physiol, 2006:290: C583-591.
75 Mat sumori Y, Nort hington FJ , Hong SM , et al. Reduction of caspase-8 and-9 cleavage is associated with increased c-FLIP and increased binding of Apaf-1 and Hsp70 after neonatal hypoxic/ ischemic injury in mice overexpressing Hsp70. Stroke, 2006; 37:507-512.
76 Jiang B , Xiao W , Shi Y, et al . Heat shock pretreatment inhibited the release of Smac/ DIABLO from mitochondria and apoptosis induced by hydrogen peroxide in cardio- myocytes and C2C12 myogenic cells. Cell Stress Chaperones, 2005; 10:252-262.
77 Yamamoto K, Sarukawa M, Ito T, et al. An anti-ulcer drug, geranylgeranylacetone, suppresses inducible nitricoxide synthase in cultured vascular smooth muscle cells. J Hypertens, 2005; 23:1847-1853.
78 Genth-Zotz S, Bolger AP, Kalra PR,et al. Heat shock protein 70 in patients with chronic heart failure: relation to disease severity and survival. Int J Cardiol, 2004; 96:397-401.
79 WJ Welch. How cells respond to stress. Sci Am, 1993; 68:56-64.
80 A.A. Knowlton, F.R. Eberli, P. Brecher et al., A single myocardial stretch or decreased systolic fiber shortening stimulates the expression of heat shock protein 70 in the isolated, erythrocyte-perfused rabbit heart. J Clin Invest, 1991; 88:2018-2025.
81 Ursula R, Volker G. Annexins-unique membrane binding prorteins with diverse functions. J Cell Sci, 2004; 117:2631-2639.
82 Camors E, Monceau V, Charlemagne D. Annexins and Ca~(2+) handling in the heart. Cardiovasc Res, 2005; 65:793-804.
83 Benevolensky D, Belikova Y, Mohammadzadeh R. Expression and localization of the annexins II, V, and VI in myocardium from patients with end-stage heart failure. Lab Invest, 2000; 80:123-133.
84 Parente L, Solito E. Annexin 1: more than an anti-phospholipase protein. Inflamm Res, 2004; 53:125-132.
85 Arur S, Uche UE, Rezaul K, et al. Annexin 1 is an endogenous ligand that mediates apoptotic cell engulfment. Dev Cell, 2003; 4:587-598.
86 G Barrientos, C Hidalgo. Annexin VI is attached to transversetubule membranes in isolated skeletal muscle triads. Membr Biol, 2002; 188:163-173.
87 E Babiychuk, A Draeger. Annexins in cell membrane dynamics, Ca~(2+)-regulated association of lipid microdomains. J Cell Biol, 2000; 150:1113-1124.
88 T Luckcuck, P Trotter, J Walker. Localization of annexin VI in the adult and neonatal heart. Cell Biol Int, 1998; 22:199-205.
89 Locate S, Colyer J, Gawler DJ, et al. Annexin A6 at the cardiac myocyte sarcolemma -evidence for self-association and binding to actin.Cell Biol Int, 2008; 32:1388-1396.
90 Ueng KC, Lin CS, Yeh HI, et al. Downregulated cardiac annexin VI mRNA and protein levels in chronically fibrillating human atria.Cardiology, 2008; 109:208-216.
91 A Sobota, F Cusinato, S Luciani. Identification and purification of calpactins from cardiac muscle and their effect on Na~+/Ca~(2+) exchange activity. Biochem Biophys Res Commun, 1990; 1721:1067-1072.
92 Kaetzel MA, Dedman JR.Annexin VI regulation of cardiac function. Biochem Biophys Res Commun, 2004; 322:1171-1177.
93 Monastyrskaya K, Babiychuk EB, Hostettler A,et al. Plasma membrane-associated annexin A6 reduces CA~(2+) entry by stabilizing the cortical actin cytoskeleton. J Biol Chem, 2009; 22(Epub ahead of print).
94 Seko Y, Matsumoto A, Fukuda T, et al. A case of neonatal lupus erythematosus presenting delayed dilated cardiomyopathy with circulating autoantibody to annexin A6.Int Heart J. 2007; 48:407-415.
95 Song G, Harding SE, Duchen MR, et al. Altered mechanical properties and intracellular calcium signaling in cardiomyocytes from annexin 6 null-mutant mice. FASEB J, 2002; 16:622-624.
96 G Song B, Campos L, Wagoner J, et al. Altered cardiac annexin mRNA and protein levels in the left ventricle of patients with end-stage heart failure. Mol Cell Cardiol, 1998; 30:443-451.
97 Matteo RG, Moravec CS. Immunolocalization of annexins IV, V and VI in the failing and non-failing human heart Cardiovasc Res, 2000; 45:961-970.
98 Benevolensky D, Belikova Y, Mohammadzadeh R, et al. Expression and localization of the annexins II,V, and VI in myocardium from patients with end-stage heart failure.Lab Invest, 2000; 80:123-133.
99 Matteo RG, Moravec CS. Immunolocalization of annexins IV, V and VI in the failing and non-failing human heart. Cardiovasc Res, 2000; 45:961-970.
100 Trouve P, Legot S, Belikova 1, et al. Localization and quantitation of cardiac annexins II,V, and VI in hypertensive guinea pigs. Am J Physiol 1999; 276:159-166.
101 Coates PJ, Nenutil R, McGregor A, et al. Mammalian prohibitin proteins respond to mitochondrial stress and decrease during cellular senescence. Exp Cell Res, 2001; 265: 262-273.
2 中国心力衰竭流行病学调查及其患病率.顾东风,黄广勇,吴锡桂等.中华心血管病杂志,2003;31:3-6.
3 Hunt SA,Abraham WT,Chin MH,et al.ACC/AHA 2005 Guideline Update for the Diagnosis and Management of Chronic Heart Failure in the Adult:a report of the American College of Cardiology/American Heart Association Task Force on Practice Guidelines(Writing Committee to Update the 2001 Guidelines for the Evaluation and Management of Heart Failure).J Am Coll Cardiol,2005;46:e1-82.
4 2009 Focused Update:ACCF/AHA guidelines for the diagnosis and management of heart failure in adults:a report of the American College of Cardiology Foundation/ American Heart Association Task Force on Practice Guidelines.J Am Coll Cardiol,2009;53:ⅹⅹ-ⅹⅹⅹ.
5 Heart disease and stroke statistics-2009 update:a report from the American Heart Association Statistics Committee and Stroke Statistics Subcommittee.Circulation,2009;119:e21-181.
6 Guidelines for the diagnosis and treatment of chronic heart failure:executive summary(update 2005):The Task Force for the Diagnosis and Treatment of Chronic Heart Failure of the European Society of Cardiology.Eur Heart J,2005;26:1115-1140
7 Cleland JG,Khand A,Clark A.The heart failure epidemic:exactly how big is it?Eur Heart J,2001;22:623-626.
8 上海市心力衰竭调查协作组.上海市1980、1990、2000年心力衰竭住院患者 流行病学及治疗状况调查．中华心血管病杂志．2002；30：24-27．
9 Corwie MR, Wood DA, Coats J S, et al. Incidence and aetiology of heart failure : a population-based study. Eur Heart J, 1999; 20:421-428.
10 Cohn JN, Ferrari R, Sharpe N. Cardiac remodeling-concepts and clinical implications: a consensus paper from an international forum on cardiac remodeling. J Am Coll Cardiol, 2000; 35:569-582.
11 Hirota H, Chen J, Betz UA, et al. Loss of a gp130 cardiac muscle cell survival pathway is a critical event in the on set of heart failure during biome chanical stress. Cell, 1999; 97:189-198.
12 Sadek MM, Haddad T, Haddad H. The role of statins in chronic heart failure. Curr Opin Cardiol, 2009; 24:167-171.
13 Sagawa T, Sagawa K, Kelly JE, Tsushima RG, Wasserstrom JA. Activation of cardiac ryanodine receptors by cardiac glycosides. Am J Physiol Heart Circ Physiol, 2002; 282:H1118-1126.
14 Ferguson DW, Berg WJ, Sanders JS, et al. Sympath oinhibitory responses to digi talis glycosides in heart failure patients: direct vidence from sympathetic neural recordings. Circulation, 1989; 80:65-77.
15 Covit AB, Schaer GL, Sealey JE, et al. Suppression of the renin-angiotensin system by intravenous digoxin in chronic congestive heart failure. Am J Med, 1983; 75:45-47.
16 Gheorghiade M, Ferguson D. Digoxin: a neurohormonal modulator in heart failure?. Circulation, 1991; 84:2181-6.
17 The Digitalis Investigation Group. The effect of digoxin on mortality and morbidity in patients with heart failure. N Engl J Med, 1997; 336:525-533.
18 Ahmed A, Rich MW, Love TE, et al. Digoxin and reduction in mortality and hospitalization in heart failure: a comprehensive post hocanalysis of the DIG trial. Eur Heart J, 2006; 27:178-86.
19 Ahmed A, Pitt B, Rahimtoola SH, et al. Effects of digoxin at low serum concentrations on mortality and hospitalization in heart failure: a propensity-matched study of the DIG trial. Int J Cardiol, 2008; 123:138-146.
20 Gilman G, Khandheria BK, Hagen ME, et al. Strain rate and strain: a step-by-step approach to image and data acquisition. J Am Soc Echocardiogr, 2004; 17:1011-1020.
21 Sutherland GR, Di Salvo G, Claus P, et al. Strain and strain rate imaging: a new clinical approach to quantifying regional myocardial function. J Am Soc Echocardiogr, 2004; 17:788-802.
22 Yip G, Abraham T, Belohlavek M, et al. Clinical applications of strain rate imaging. J Am Soc Echocardiogr, 2003; 16:1334-1342.
23 Pfeffer MA, Braunwald E. Ventricular remodeling after myocardial infarction experimental observations and clinical implications. Circulation, 1990; 81:1161 -1172.
24 Hodsman GP, Kohzuki M, Howes LG, et al. Neuro humoral response to chronic myocardial infarction in rats. Circulation, 1988; 78:376-381.
25 Johns TNP, Olson BJ. Experimental myocardial infarction: a medthod of coronary occlusion in small animals. Am Surg, 1954; 10: 509-516.
26 Pfeffer MA, Pfeffer JM, Fishbein MC, et al. Myocardial infarct size and ventricular function in rats. Circ Res, 1979; 44:503-12.
27 Matsumori A, Igata H, Ono K, et al. High doses of digitalis increase the myocardial production of proinflammatory cytokines and worsen myocardial injury in viral myocarditis: a possible mechanism of digitalis toxicity. Jpn Circ J, 1999; 63:934-40.
28 Rumberger JA. Ventricular dilatation and remodeling after myocardial infarction. Mayo Clin Proc, 1994; 69:664-674.
29 Sakata Y, Yamamoto K, Mano T, et al. Activation of matrix metalloproteinases precedes left ventricular remodeling in hypertensive heart failure rats: its inhibition as a primary effect of Angiotensin-converting enzyme inhibitor. Circulation, 2004; 109:2143-2149.
30 Zornoff LA, Paiva SA, Duarte DR, et al.Ventricular remodeling after myocardial infarction: concepts and clinical implications. Spadaro J Arq Bras Cardiol, 2009; 92:150-164.
31 Udelson JE, Patten RD, Konstam MA.New concepts in post-infarction ventricular remodeling. Rev Cardiovasc Med, 2003; 3:S3-12.
32 Udelson JE, Konstam MAJ Card Fail. Relation between left ventricular remodeling and clinical outcomes in heart failure patients with left ventricular systolic dysfunction. J Card Fail,2002; 8(S):S465-471.
33 Kjaer A, Hesse B. Heart failure and neuroendocrine activation: diagnostic, prognostic and therapeutic perspectives.Clin Physiol, 2001; 21:661-672.
34 Hein S, Kostin S, Heling A.et al. The role of the cytoskeleton in heart failure. Cardiovasc Res, 2000; 45(2):273-278.
35 Dhalla NS, Saini-Chohan HK, Rodriguez-Leyva D,et al. Subcellular remodelling may induce cardiac dysfunction in congestive heart failure.Cardiovasc Res, 2009; 81:429-438.
36 Patten RD, Udelson JE, Konstam MA. Ventricular remodeling and its prevention in the treatment of heart failureCurr Opin Cardiol, 1998; 13:162-167.
37 Kazory A, Ross EA. Contemporary trends in the pharmacological and extracor- poreal management of heart failure: a nephrologic perspective.Circulation, 2008; 117:975-983
38 Rassi A Jr, Rassi A, Rassi SG. Predictors of mortality in chronic Chagas disease: a systematic review of observational studies. Circulation, 2007; 115:1101-1108.
39 Torre-Amione, G Sestier F, Radovancevic B, et al. Broad modulation of tissue responses (immune activation) by celacadeay favorably influence pat hologic processes associated wit h heart failure progression. Am J Cardiol, 2005; 95 (S):30c-37c.
40 Santos A, Ledesma-Carbayo MJ, Malpica N, et al. Accuracy of heart strain rate calculation derived from doppler tissue velocity data. SP IE, 2001; 4325: 546-555.
41 Greenberg NL, Firstenberg MS, Castro PL, et al. Doppler-derived myocardial systolic strain rate is a strong index of left ventricular contractility.Circulation, 2002; 105:99-105.
42 Andersen NH, Poulsen SH, Eiskjaer H, et al. Decreased left ventricular longitudinal contraction in normotensive and normoalbuminuric patients with Type II diabetes mellitus: a Doppler tissue tracking and strain rate echocardiography study. Clin Sci, 2003; 105:59-66.
43 Yalcin F, Kalian A, Muderrisoghu H, et al. Is Doppler tissue velocity during early ventricular filling preload independent? Heart, 2002; 87:336-339.
44 Dandel M, Hetzer RE. Echocardiographic strain and strain rate imaging-clinical applications. Int J Cardiol. 2009; 132:11-24.
45 Thibault H, Gomez L, Donal E, et al. Acute myocardial infarction in mice: assessment of transmurality by strain rate imaging.Am J Physiol Heart Circ Physiol, 2007; 293:H496-502.
46 Sia YT, Lapointe N, Parker TG,et al. Beneficial effects of long-term use of the antioxidant probucol in heart failure in the rat.Circulation.2002; 105:2549-2555.
47 Saraiva RM, Minhas KM, Raju SV, et al. Deficiency of neuronal nitric oxide synthase increases mortality and cardiac remodeling after myocardial infarction: role of nitroso-redox equilibrium. Circulation, 2005; 112:3415-3422.
48 Sullebarger JT, D'Ambra PM, Clark LC,et al. Effect of Digoxin on Ventricular Remodeling and Responsiveness of beta-Adrenoceptors in Chronic Volume Overload. J Cardiovasc Pharmacol Ther, 1998; 3:281-290.
49 Bendall JK, Damy T, Ratajczak P, et al. Role of myocardial neuronal nitric oxide synthase-derived nitric oxide in beta-adrenergic hyporesponsiveness after myocardial infarction-induced heart failure in rat. Circulation. 2004; 110:2368-2375.
50 Sia YT, Lapointe N, Parker TG,et al. Beneficial effects of long-term use of the antioxidant probucol in heart failure in the rat. 2002; 28:2549-2555.
51 Morisco C, Cuocolo A, Romano M,et al. Influence of digitalis on left ventricular functional response to exercise in congestive heart failure Am J Cardiol. 1996; 77:480-485.
52 Besch HR Jr, Watanabe AM. The positive inotropic effect of digitoxin: independence from sodium accumulation. J Pharmacol ExpTher, 1978; 207:958-965.
53 Fujino S, Fujino M. Ouabain potentiation and Ca release from sarcoplasmic reticulum in cardiac and skeletal muscle cells. Can J Physiol Pharmacol, 1982; 60:542-555.
54 McGarry SJ, Williams AJ. Digoxin activates sarcoplasmic reticulum Ca~(2+)-release channels: a possible role in cardiac inotropy. Br J Pharmacol, 1993; 108:1043-1050.
55 Sagawa T,Sagawa K,Kelly JE,et al.Activation of cardiac ryanodine receptors by cardiac glycosides.Am J Physiol Heart Circ Physiol,2002;282:H1118-H1126.
56 Haas M,Askari A,and Xie Z.Involvement of Src and epidermal growth factor receptor in the signal-transducing function of Na~+-K~+-ATPase.J Biol Chem2000;275:27832-27837.
57 Haas M,Wang H,Tian J,and Xie Z.Src-mediated inter-receptor cross-talk between the Na~+-K~+-ATPase and the epidermal growth factor receptor relays the signal from ouabain to mitogen-activated protein kinases.J Biol Chem,2002;277:18694-18702.
1 Wilkins MR, Sanchez JC, Gooley AA. Progress with proteome projects: why all proteins expressed by a genome should be identified and how to do it. Biotechnol Genet Eng Rev, 1996; 13:19-27.
2 Anderson NL, Anderson NG. Proteome and proteomics: new technologies, new concepts, and new words. Electrophoresis, 1998; 19:1853-1861.
3 Cordwell SJ, Humphery-Smith I. Evaluation of algorithms used for cross-species proteome characterisation. Electrophoresis, 1997; 18:1410-1417.
4 Humphery-Smith I, Cordwell SJ, Blackstock WP. Proteome research: complementarity and limitations with respect to the RNA and DNA worlds.Electrophoresis, 1997; 18: 1217-1242.
5 Hanash S.Disease proteomics. Nature, 2003; 422: 226-232.
6 Patton WF. Detection technologies in proteome analysis. J Chromatogr B Analyt Teehnol Biomed Life Sci, 2002; 771:3-31.
7 Hamdan M, Righetti PG. Modern strategies for protein quantification in proteome analysis: advantages and limitations. Mass Spectrom Rev, 2002; 21:287-302.
8 Wu WW, Wang G, Baek S J, et al. Comparative study of three proteome quantitative methods, DIGE, iCAT and iTRAQ: using 2D Gel-or LC-MALDI TOF / TOF. J Proteome Bes, 2006; 5:651-658.
9 Gygi SP, Rist B, Gerber S A et al. Quantitative analysis of complex protein mixtures using isotope-coded affinity tags. Nat Biotechnol, 1999; 17:994-999.
10 Ross P L, Huang Y N, Marchese JN, et al. Multiplexed protein quantitation in Saccharomyces cerevisiae using amine reactive isobaric tagging reagents. Mol Cell Proteomics, 2004; 3:1154-1169..
11 Schatzmann HJ. Cardiac glycosides as inhibitors of active potassium and sodium transport by erythrocyte membrane. Helv Physiol Pharmacol Acta, 1953; 11:346-354.
12 James PF, Grupp IL, Grupp G, et al. Identification of a specific role for the Na~+-K~+-ATPase alpha-isoform as a regulator of calcium in the heart. Mol Cell, 1999; 3: 555-563.
13 Mathias RT, Cohen IS, Gao J, et al. Isoform-specific regulation of the Na~+-K~+-pump in heart. News Physiol Sci, 2000;15:176-180.
14 Besch HR Jr, Watanabe AM. The positive inotropic effect of digitoxin: independence from sodium accumulation. J Pharmacol ExpTher, 1978; 207:958-965.
15 Fujino S, Fujino M. Ouabain potentiation and Ca release from sarcoplasmic reticulum in cardiac and skeletal muscle cells. Can J Physiol Pharmacol, 1982; 60:542-555.
16 Gheorghiade M, St Clair J, St Clair C, et al. Hemodynamic effects of intravenous digoxin in patients with severe heart failure initially treated with diuretics and vasodilators. J Am Coll Cardiol, 1987; 9:849-857.
17 McGarry SJ and Williams AJ. Digoxin activates sarcoplasmic reticulum Ca -release channels: a possible role in cardiac inotropy. Br J Pharmacol, 1993; 108: 1043-1050.
18 Sagawa T, Sagawa K, Kelly JE, et al. Activation of cardiac ryanodine receptors by cardiac glycosides. Am J Physiol Heart Circ Physiol, 2002; 282: H1118-H1126.
19 Haas M, Askari A, and Xie Z. Involvement of Src and epidermal growth factor receptor in the signal-transducing function of Na~+-K~+-ATPase. J Biol Chem, 2000; 275:27832-27837.
20 Haas M,Wang H,Tian J,and Xie Z.Src-mediated inter-receptor cross-talk between the Na~+-K~+-ATPase and the epidermal growth factor receptor relays the signal from ouabain to mitogen-activated protein kinases.J Biol Chem,2002;277:18694-18702.
21 Wasserstrom JA,Aistrup GL.Digitalis:new actions for an old drag.Am J Physiol Heart Circ Physiol,2005;289:H1781-93.
22 Tian J,Gong X,Xie Z.Signal-transducing function of Na~+-K~+-ATPase is essential for ouabain's effect on[Ca~(2+)]i in rat cardiac myocytes.Am J Physiol Heart Circ Physiol,2001;281:H1899-1907.
23 Ramirez-Ortega M,Zarco G,Maldonado V.Is digitalis compound-induced cardio- toxicity,mediated through guinea-pig cardiomyocytes apoptosis? Eur J Pharmacol,2007;566:34-42.
24 Alvarez-Llamas G,de la Cuesta F,Barderas ME,et al.Recent advances in athe-rosclerosis-based proteomics:new biomarkers and a future perspective.Expert Rev Proteomics,2008;5:679-691.
25 Pinet F,Beseme O,Cieniewski-Bemard C,et al.Predicting left ventricular remodeling after a first myocardial infarction by plasma proteome analysis Proteomics.2008;8:1798-1808.
26 Dohke T,Wada A,Isono T,et al.Proteomic analysis reveals significant alternations of cardiac small heat shock protein expression in congestive heart failure.J Card Fail,2006;12:77-84.
27 孙爱军,王克强,李杰,等.心功能衰竭大鼠模型心肌线粒体蛋白的差异表达分析.中国动脉硬化杂志,2004,12:183-185.
28 徐丹令,孙爱军,王时俊,等.乙醛脱氢酶2在大鼠心肌缺氧损伤中的抗凋亡作用.中国病理生理杂志,2006;22:683-686.
29 Arrel DK,Neverova I,Van Eyk JE.Cardiovascular proteomics:evolution and potential. Circ Res, 2001; 88:763-773.
30 Corcoran EE, Joseph JD, MacDonald JA, et al. Proteomic analysis of calcium / calmodulin-dependent protein kinase I and IV in vitro substrates reveals distinct catalytic p references. J Biol Chem, 2003; 278: 10516-10522.
31 Qiu J, Gao HQ, Zhou RH, Proteomics analysis of the proliferative effect of low-dose ouabain on human endothelial cells. Biol Pharm Bull. 2007; 30:247-253.
32 Qiu J, Gao HQ, Liang Y, et al. Comparative proteomics analysis reveals role of heat shock protein 60 in digoxin-induced toxicity in human endothelial cells Biochim Biophys Acta, 2008; 1784:1857-1864.
33 Latterich M, Abramovitz M, Leyland-Jones B. Proteomics: new technologies and clinical applications.Eur J Cancer, 2008; 44:2737-2741.
34 Sui J, Zhang J, Ching CB, et al. Comparative proteomic analysis of extracellular proteins reveals secretion of T-kininogen from vascular smooth muscle cells in response to incubation with s-enantiomer of propranolol.Mol Pharm, 2008; 5:885-90.
35 Sui J, Zhang J, Tan TL, et al. Comparative proteomics analysis of vascular smooth muscle cells incubated with S- and R-enantiomers of atenolol using iTRAQ-coupled two-dimensional LC-MS/MS. Cell Proteomics, 2008; 7:1007-1018.
36 Sui J, Tan TL, Zhang J, et al. iTRAQ-coupled 2D LC-MS/MS analysis on protein profile in vascular smooth muscle cells incubated with S- and R-enantiomers of propranolol: possible role of metabolic enzymes involved in cellular anabolism and antioxidant activity. J Proteome Res, 2007; 6:1643-1651.
37 Langer GA. The role of sodium ion in the regulation of myocardial contractility. J Mol Cell Cardiol, 1970; 1:203-207.
38 Woodbury JW. Interrelationships between ion transport mechanisms and excitatory events. Fed Proc, 1963; 22:31-35.
39 Satoh H, Ginsburg KS, Qing K, et al. KB-R7943 block of Ca~(2+) influx via Na~+/Ca~(2+) exchange does not alter twitches or glycoside inotropy but prevents Ca~(2+) overload in rat ventricular myocytes. Circulation, 2000; 101:1441-1446.
40 Norgaard A, Bagger JP, Bjerregaard P et al. Relation of left ventricular function and Na, K-pump concentration in suspected idiopathic dilated cardiomyopathy. Am J Cardiol, 1988; 61:1312-1315.
41 Kjeldsen K, Bjerregaard P, Richter EA,et al. Na~+-K~+-ATPase concentration in rodent and human heart skeletal muscle: apparent relation to muscle performance. Cardiovasc Res, 1988; 22:95-100.
42 Ishino K, Botker HE, Clausen T, Hetzer R, Sehested J. Myocardial adenine nucleotides, glycogen, and Na~+-K~+-ATPase in patients with idiopathic dilated cardiomyopathy requiring mechanical circulatory support. Am J Cardiol, 1999; 83:396-399.
43 Hanf R, Drubaix I, Marotte F, et al. Rat cardiac hypertrophy.Altered sdium-calcium exchange activity in sarcolemmal vesicles. FEBS Lett, 1988; 236:145-149.
44 No adaptation to digitalization as evaluatedby digitalis receptor (Na~+-K~+-ATPase) quantification in explanted hearts from donors without heart disease and from digitalized recipients with end-stage heart failure. Am J Cardiol, 1992; 70:110-114.
45 Schmidt TA, Holm-Nielsen P, Kjeldsen K. No upregulation of digitalis glycoside receptor (Na~+-K~+-ATPase) concentration in human heart left ventricle samples obtained at necropsy after long term digitalization. Cardiovasc Res, 1991; 25:684-691.
46 Jones LR, Maddock SW, Besch HR Jr. Unmasking effect of alamethicin on the Na~+-K~+-ATPase,beta adrenergic receptor-coupled adenylate cyclase,and cAMP-dependent protein kinase activities of cardiac sarcolemmal vesicles.J Biol Chem,1980;255:9971-9980
47 Hasenfuss G,Reinecke H,Studer R,et al.Calcium cycling proteins and forcefrequency relationship in heart failure.Basic Res Cardiol,1996;91(S) 2:17-22.
48 Kranias EG,Bers DM.Subcell Biochem.Calcium and cardiomyopathies,2007;45:523-537.
49 Del Monte F,Hajjar RJ.Intracellular devastation in heart failure.Heart Fail Rev,2008;13:151-162.
50 Yue TL,Gu JL,Wang C,et al.Extracellular signal-regulated kinase plays an essential role in hypertrophic agonists,endothelin-1 and phenylephrine-induced cardiomyocyte hypertrophy.J Biol Chem,2008;275:37895-37901.
51 Schmidt U,Hajjar RJ,Kim CS,et al.Human heart failure:cAMP stimulation of SR Ca~(2+)-ATPase activity and phosphorylation level of phospholamban.Am J Physiol,1999;277:H474-H480.
52 MacLennan DH,Reithmeier RA.Ion tamers.Nat Struct Biol,1998;5:409-411.
53 Asahi M,Otsu K,Nakayama H,Hikoso S,et al.Cardiac-specific overexpression of sarcolipin inhibits sarco(endo)plasmic reticulum Ca~(2+)-ATPase(SERCA2a)activity and impairs cardiac function in mice.Proc Natl Acad Sci,2004;101:9199-9204.
54 M(u|¨)ller-Ehmsen J,Nickel J,Zobel C,et al.Longer term effects of ouabain on the contractility of rat isolated cardiomyocytes and on the expression of Ca and Na regulating proteins.Basic Res Cardiol.2003;98:90-96.
55 Hein S,Kostin S,Heling A.et al.The role of the cytoskeleton in heart failure.Cardiovasc Res,2000;45(2):273-278.
56 Dhalla NS, Saini-Chohan HK, Rodriguez-Leyva D,et al. Subcellular remodelling may induce cardiac dysfunction in congestive heart failure.Cardiovasc Res, 2009; 81:429-438.
57 Heinke MY, Wheeler CH, Chang D, et al. Protein changes observed in pacing-induced heart failure using two-dimensional electrophoresis. Electrophoresis 1998; 19:2021-2030.
58 Jiang L, Tsubakihara M, Heinke MY, et al. Heart failure and apoptosis: electrophoreticmethods support data from micro- and macro-arrays. A critical review of genomics andproteomics. Proteomics, 2001; 1:1481-1488.
59 Van Bilsen M, Smeets PJ, Gilde AJ,et al. Metabolic remodelling of the failing heart: the cardiac burn-out syndrome? Cardiovasc Res, 2004; 61:218-226.
60 Stanley WC, Lopaschuk GD, Hall JL, et al. Regulation of myocardial carbohydrate metabolism under normal and ischaemic conditions.Potential for pharmacological interventions. Cardiovasc Res, 1997; 33:243-257.
61 Lopaschuk GD, Beike DD, Gamble J, et al. Regulation of fatty acid oxidation in the mammalian heart in health and disease. Biochim Biophys Acta, 1994; 1213:263-276.
62 Gertz EW, Wisneski JA, Stanley WC, et al. Myocardial substrate utilization during exercise in humans. Dual Carbon-labeled carbohydrate isotope experiments. J Clin Invest, 1988; 82:2017-2025.
63 Lopaschuk GD, Stanley WC. Glucose metabolism in the iscemic heart. Circulation, 1997; 95:313-315.
64 Stanley WC, Lopaschuk GD, Hall JL, et al. Regulation of myocardial carbohydrate metabolism under normal and ischaemic conditions. Potential for pharmacological interventions. Cardiovasc Res, 1997; 33:243-257.
65 Tuunanen H, Ukkonen H, Knuuti J. Myocardial fatty acid metabolism and cardiac performance in heart failure. Curr Cardiol Rep, 2008; 10:142-148.
66 Lopaschuk GD. Optimizing cardiac fatty acid and glucose metabolism as an approach to treating heart failure. 2006; 10:228-230.
67 Unverferth DV, Fetters J K, Barbara BS, et al. Human myocardial histologic characteristics in congestive heart failure. Circulation, 1983; 68:1194-1200.
68 Ormerod JO, Ashrafian H, Frenneaux MP. Impaired energetics in heart failure - a new the rapeutic target. Pharmacol Ther, 2008; 119:264-74.
69 Gusarova V, Caplan AJ, Brodsky JL, et al. Apoprotein B degradation is p romoted by the molecular chaperones HSP90 and HSP70. Biol Chem, 2001; 276:24891-24892.
70 Li G, Bae S, Zhang L. Effect of prenatal hypoxia on heat stress-mediated cardioprotection in adult rat heart. Am J Physiol Heart Circ Physiol, 2004; 2 86: H17122 -H1719.
71 Kim SH , Koh GY, Cho KW, et al. Stretch-activated atrial nariuretic peptide secretion in atria with heat shock protein70 overexpression. Exp Bio! Med (Maywood), 2003; 228: 200-206.
72 Knowlton AA, Kapadia S, Torre-Amione G, et al. Differential expression of heat shock proteins in normal and failing human hearts. J Mol Cell Cardiol, 1998; 30:811-818.
73 Delogu G, Signore M, Mechelli A, et al. Heat shock proteins and their role in heart injury. Curr Opin Crit Care, 2002; 8: 4-12.
74 Liu J, Kam KW, Borchert GH, et al. Furt her study on t he role of HSP70 on Ca~(2+) homeostasis in rat ventricular myocytes subjected to simulated ischemia. Am J Physiol Cell Physiol, 2006:290: C583-591.
75 Mat sumori Y, Nort hington FJ , Hong SM , et al. Reduction of caspase-8 and-9 cleavage is associated with increased c-FLIP and increased binding of Apaf-1 and Hsp70 after neonatal hypoxic/ ischemic injury in mice overexpressing Hsp70. Stroke, 2006; 37:507-512.
76 Jiang B , Xiao W , Shi Y, et al . Heat shock pretreatment inhibited the release of Smac/ DIABLO from mitochondria and apoptosis induced by hydrogen peroxide in cardio- myocytes and C2C12 myogenic cells. Cell Stress Chaperones, 2005; 10:252-262.
77 Yamamoto K, Sarukawa M, Ito T, et al. An anti-ulcer drug, geranylgeranylacetone, suppresses inducible nitricoxide synthase in cultured vascular smooth muscle cells. J Hypertens, 2005; 23:1847-1853.
78 Genth-Zotz S, Bolger AP, Kalra PR,et al. Heat shock protein 70 in patients with chronic heart failure: relation to disease severity and survival. Int J Cardiol, 2004; 96:397-401.
79 WJ Welch. How cells respond to stress. Sci Am, 1993; 68:56-64.
80 A.A. Knowlton, F.R. Eberli, P. Brecher et al., A single myocardial stretch or decreased systolic fiber shortening stimulates the expression of heat shock protein 70 in the isolated, erythrocyte-perfused rabbit heart. J Clin Invest, 1991; 88:2018-2025.
81 Ursula R, Volker G. Annexins-unique membrane binding prorteins with diverse functions. J Cell Sci, 2004; 117:2631-2639.
82 Camors E, Monceau V, Charlemagne D. Annexins and Ca~(2+) handling in the heart. Cardiovasc Res, 2005; 65:793-804.
83 Benevolensky D, Belikova Y, Mohammadzadeh R. Expression and localization of the annexins II, V, and VI in myocardium from patients with end-stage heart failure. Lab Invest, 2000; 80:123-133.
84 Parente L, Solito E. Annexin 1: more than an anti-phospholipase protein. Inflamm Res, 2004; 53:125-132.
85 Arur S, Uche UE, Rezaul K, et al. Annexin 1 is an endogenous ligand that mediates apoptotic cell engulfment. Dev Cell, 2003; 4:587-598.
86 G Barrientos, C Hidalgo. Annexin VI is attached to transversetubule membranes in isolated skeletal muscle triads. Membr Biol, 2002; 188:163-173.
87 E Babiychuk, A Draeger. Annexins in cell membrane dynamics, Ca~(2+)-regulated association of lipid microdomains. J Cell Biol, 2000; 150:1113-1124.
88 T Luckcuck, P Trotter, J Walker. Localization of annexin VI in the adult and neonatal heart. Cell Biol Int, 1998; 22:199-205.
89 Locate S, Colyer J, Gawler DJ, et al. Annexin A6 at the cardiac myocyte sarcolemma -evidence for self-association and binding to actin.Cell Biol Int, 2008; 32:1388-1396.
90 Ueng KC, Lin CS, Yeh HI, et al. Downregulated cardiac annexin VI mRNA and protein levels in chronically fibrillating human atria.Cardiology, 2008; 109:208-216.
91 A Sobota, F Cusinato, S Luciani. Identification and purification of calpactins from cardiac muscle and their effect on Na~+/Ca~(2+) exchange activity. Biochem Biophys Res Commun, 1990; 1721:1067-1072.
92 Kaetzel MA, Dedman JR.Annexin VI regulation of cardiac function. Biochem Biophys Res Commun, 2004; 322:1171-1177.
93 Monastyrskaya K, Babiychuk EB, Hostettler A,et al. Plasma membrane-associated annexin A6 reduces CA~(2+) entry by stabilizing the cortical actin cytoskeleton. J Biol Chem, 2009; 22(Epub ahead of print).
94 Seko Y, Matsumoto A, Fukuda T, et al. A case of neonatal lupus erythematosus presenting delayed dilated cardiomyopathy with circulating autoantibody to annexin A6.Int Heart J. 2007; 48:407-415.
95 Song G, Harding SE, Duchen MR, et al. Altered mechanical properties and intracellular calcium signaling in cardiomyocytes from annexin 6 null-mutant mice. FASEB J, 2002; 16:622-624.
96 G Song B, Campos L, Wagoner J, et al. Altered cardiac annexin mRNA and protein levels in the left ventricle of patients with end-stage heart failure. Mol Cell Cardiol, 1998; 30:443-451.
97 Matteo RG, Moravec CS. Immunolocalization of annexins IV, V and VI in the failing and non-failing human heart Cardiovasc Res, 2000; 45:961-970.
98 Benevolensky D, Belikova Y, Mohammadzadeh R, et al. Expression and localization of the annexins II,V, and VI in myocardium from patients with end-stage heart failure.Lab Invest, 2000; 80:123-133.
99 Matteo RG, Moravec CS. Immunolocalization of annexins IV, V and VI in the failing and non-failing human heart. Cardiovasc Res, 2000; 45:961-970.
100 Trouve P, Legot S, Belikova 1, et al. Localization and quantitation of cardiac annexins II,V, and VI in hypertensive guinea pigs. Am J Physiol 1999; 276:159-166.
101 Coates PJ, Nenutil R, McGregor A, et al. Mammalian prohibitin proteins respond to mitochondrial stress and decrease during cellular senescence. Exp Cell Res, 2001; 265: 262-273.