Proteomics Analysis Of Cardiocyte In Heart Failure Rats Treated With Digoxin

BackgroundHeart 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[Ca²⁺]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 Ca²⁺ release(via ryanodine receptors;RyRs);increasing Ca²⁺ selectivity and Ca²⁺ influx via TTX-sensitive Na⁺ channels;and othersignalling mechanisms,activatiing protein tyrosine kinases and mitogen-activated protein kinases(MAPKs),which causes increased Ca²⁺ 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.ObjectiveThe 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.Methods1 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 analysisThe 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 proteinsThe 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.Results1 Model of heart failureThe 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 proteins32 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.Conclusion1 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.Annexinâ…¥protein may play an important role for the protein in cardiac performance.This effect on cardiac performance is mediated via interaction of the annexinâ…¥with cellular proteins responsible for maintaining Ca²⁺ 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.