Proteomic Of Cardiac Myocyte In Rats With Acute Heart Failure After Increasing S100A1 Level

Proteomics of acute heart failure in a rat post-myocardial infarction modelBackgroundHeart failure is a serious end stage of heart disease.It is a complex clinical syndrome resulting from abnormal heart function and structure which ventricular filling or ejection fraction damaged.The main clinical manifestations were dyspnea and fatigue,and fluid retention.Heart failure has a high incidence and a high fatality rate.It has become the most important cardiovascular disease in the twenty-first Century.Heart failure is a progressive and complex clinical syndrome that leads to impaired functional cardiac ability.It is defined as a symptom resulting in ventricular dysfunction and is characterized by high morbidity and mortality.It has previously been reported that 50-60%of patients with heart failure have an enlarged left ventricle(LV)chamber and reduced ejection fraction.The majority of patients with heart failure have a history of hypertension and LV hypertrophy.The cause of heart failure is mainly(1)Acute and chronic ischemia induced myocardial necrosis,myocardial infarction,stunning,apoptosis,the ischemic myocardial cell loss and loss function;(2)Hemodynamic overload is caused by cardiac load;(3)Secondary myocardial diseases are caused by genetic defects.Myocardial cell loss and function loss caused by viral myocarditis;(4)Excessively activate neuroendocrine:1.renin angiotensin aldosterone system;2.sympathetic catecholamine system.At present,the treatment of heart failure mainly lies in:(1)Improve hemodyn-amics,cardiac contractility and diastolic performance.Clinical application treatment measures are strengthening heart,dilating blood vessel and diuresis.(2)Antagonize neuroendocrine and cytokine which are activated excessively.But the study found that even if beta blockers,diuretics,ACEI and implantable devices(CRT or CRT-D)and other applications,has made great progress in the treatment of heart failure,symptoms of heart failure patients eventually will continue to increase.In 5 years,the mortality rate was 50%and the mortality rate was 90%in 10 years.Therefore,there is an urgent need to find new drug targets and theoretical support in the clinical work to clarify the diagnosis,guide the treatment and prognosis.In recent years,further research on myocardial energy metabolism during heart failure can also provide us with new ideas for the treatment of heart failure.Improve energy metabolism,improve heart energy hunger is the treatment direction of heart failure.The contraction and relaxation of the myocardium is a process of active energy dissipation.The formation of myocardial energy synthesis is mainly composed of three stages:(1)Using fatty acid and glucose production substrate;(2)In the myocardial cells,mitochondrial oxidative phosphorylation produces ATP;(3)The energy transport and utilization.Fatty acids,glucose,lactic acid,pyruvate,etc are the substrates for energy.Under normal circumstances,the 60%-90%energy required for myocardial come from beta oxidation of free fatty acids.Long chain fatty acided enter the mitochondria with by propionyl carnitine transferase-1 and-2(CPT-1 and CPT-2)to undergo beta oxidation and produce acetyl coenzyme A.Glucose through glycolysis to produce pyruvic acid and lactic acid by lactate dehydrogenase(LDH)to produce pyruvic acid.Under the action of the pyruvate dehydrogenase(PDH)function,pyruvic acid are converted to acetyl coenzyme A,which enter the tricarboxylic acid cycle to generate ATP energy three.In the mitochondria,electron generated by three tricarboxylic acid cycle oxidative phosphorylation to produces ATP by the mitochondrial respiratory chain.High energy phosphate bonds in ATP combine with creatine to form creatine phosphate.Creatine phosphate can be dispersed into the muscle fiber and release ATP under the catalysis of creatine kinase,and used as the energy of myocardial contraction and relaxation.The heart can convert the chemical energy stored in fatty acids and glucose into mechanical energy that interacts with actin and myosin in cardiac muscle fibers.In severe heart failure,oxidation of free fatty acid and glucose were inhibited.The process of oxidative phosphorylation is impaired,phosphocreatine levels can be reduced by 30%-70%,and creatine transporter function also decreased.At this point,a small amount of ATP supplied by glycolysis is the only source of myocardial cell survival.The improvement of myocardial energy metabolism may be a new approach in the treatment of heart failure.There are many studies on the changes of myocardial energy metabolism during heart failure.A more detailed understanding of the occurrence of heart failure and myocardial cell energy metabolism and myocardial contraction associated protein change,to find new therapeutic targets and simple,practical and specific biomarkers for the early diagnosis of heart failure,differential diagnosis,guide treatment and prognosis.Mass spectrometry is a key proteomics technique.Mass spectrometry provides information about molecular weight,amino acid sequence,post-translational modification,differential protein comparison of proteins,finding specific molecular proteins to diseases,molecular targets for the design of new drugs,and providing molecular markers for early diagnosis(biomarker).LTQ-Orbitrap can measure the exact molecular weight and two level mass of many peptides after protein digestion,and then obtain a high throughput identification of proteins by database retrieval.LTQ-Orbitrap is the linear ion trap mass spectrometry and high resolution hybrid mass spectrometer combined with mass spectrometry.LTQ-Orbitrap has high resolution performance,flexible and diverse functions with multiple modes of operation,stable and reliable operation.LTQ-Orbitrap can provide detailed information of mass spectrometry,high quality,and can be used in routine analysis,a more comprehensive description of a compound.It is the most advanced tool for studying the structure elucidation of compounds,and improves the ability to confirm complex structures.It is also an indispensable method in proteomics,metabolomics and other research fields.LTQ-Orbitrap can complete a variety of quantitative model of proteins,such as ICAT,I-Traq,SILAC and non labeled peptide ion chromatogram quantitative.It is the main means to find candidate biomarkers and disease specific diagnosis protein.Orbitrap scanning has high quality precision(<3ppm),stability and high quality(<0.3ppm).LTQ-Orbitrap can detect large amounts of protein changes by using micrograms of protein,which can be used to screen proteins and is widely used in basic research.It has very good quantitative effect,high repeatability and quantitative analysis of various samples at the same time.It can identify any kind of protein.This provides a reliable new scientific platform for proteomics.Linear Trap Quadropole(LTQ)OrbiTrap mass spectrometry is a protein quantification strategy that provides relative and absolute measurements of proteins in complex mixtures.The present study was undertaken to investigate the differential protein expressions that are associated with advanced heart failure irrespective of treatment.Objective1.To establish a rat model of myocardial infarction and group:(1)the normal control group;(2)the first day group:after coronary ligation,did not do any treatment;(3)the fourteenth days group:after coronary artery ligation,did not have any treatment.2.LTQ Orbitrap mass spectrometry was used to study differential protein expression in 14th day,1st day,and control groups.The protein analysis system was used Protein Analysis Through Evolutionary Relationships(PANTHER)analysis was used.Methods1.Establish the rat myocardial infarction model and group:A total of 24 male Wistar rats(age,12-14 weeks;weight,250-300 g)were provided.The animals were randomly divided into the following 3 groups(8rats/group):Control group(untreated rats),the 1st day group(rats were euthanized on the 1st postoperative day)and the 14th day group(rats were euthanized on the 14th postoperative day).After 1 days and 2 weeks,the rats were euthanized,and the blood and myocardial tissue specimens of each group were taken.2.Histopathological measurementThe myocardial tissue sections were stained with hematoxylin eosin(H&E)staining.WE observe the myocardial hypertrophy and swelling of myocardial fibers arranged irregularly,nucleus malformation,leukocyte infiltration.Immunohistochemical staining of Cav1.3 antibody,Gelsolin antibody,VDBP antibody,Myosin-7 antibody expression in the myocardial tissue in each group.3.Western BlotWestern Blot detected the expression of Cav1.3 antibody,Gelsolin antibody,VDBP antibody,Myosin-7 antibody in left ventricular myocardium in 1st days group,14th days group and normal control group.4.LTQ Orbitrap mass spectrometryLTQ Orbitrap mass spectrometry was used to study differential protein expression in 1st day group,14th day group and normal control group after AMI.The protein analysis system was used Protein Analysis Through Evolutionary Relationships(PANTHER)analysis.To investigate the changes of cardiac energy related proteins,calcium regulation and contractile function related proteins in rats with acute heart failure.Statistical analysisData was analyzed using SPSS software(version 18.0;SPSS,Inc.,Chicago,IL,USA).One-way analysis of variance followed by Tukey's multiple comparison test was used to determine the statistical differences among the post-MI and control groups.Data are presented as the mean ? standard deviation.P<0.05 was considered to indicate a statistically signifcant difference.Results1.After myocardial infarction in rat heart proteomics research found that energy metabolism related protein significantly reduced.The differentially expressed proteins in the control and 14th day groups are closely associated with energy metabolism(including glycolysis,mitochondrial tricarboxylic acid cycle and fatty acid P-oxidation),contractile function[P-myosin heavy chain isoforms(myosin-7)],calcium handling(Gelsolin,Cav 1.3,Galectin-3 and VDBP),pathologic hypertrophy(Gelsolin and Myosin-7)and cardiac remodeling(Fibrinogen ? chain).2.At different time points after the occurrence of acute myocardial infarction(after 1 day and 14 days)the difference in expression of protein dynamic changes off.Cav1.3 antibodies,Gelsolin antibodies,VDBP antibodies,and Myosin-7 antibodies may serve as molecular markers for the diagnosis and prediction of disease changes.Dynamic alterations in differential protein expression at different time points(the land 14 postoperative days)post-MI were observed.For example,Myosin-7 expression was almost unaltered on the 1st day post-MI however,it was signifcantly upregulated on the 14th day post-MI with the progression of heart failure.Conclusion1.After AMI,the proteomic analysis of rat heart showed that the differential protein expression in each group was mainly related to energy metabolism,myocardial contractile function,calcium treatment,myocardial pathological hypertrophy and cardiac remodeling.2.At different time points after the occurrence of acute myocardial infarction(after 1 day and 14 days)the difference in expression of protein dynamic changes off.Cav 1.3 antibody,Gelsolin antibody,VDBP antibody,and Myosin-7 antibody may serve as molecular markers for the diagnosis and prediction of disease changes.3.LTQ Orbitrap mass spectrometry is a reliable method for proteomic studies of acute heart failure after myocardial infarction.Proteomic of cardiac myocyte in rats with acute heart failure after increasing S100A1 levelBackgroundCardiovascular disease is the key factor of death worldwide,showing a remarkably growing prevalence.It is characterized by high disability and mortality.Acute myocardial infarction is a leading cause of death due to coronary heart disease.Myocardial remodeling will cause the infarct zone wall thin,non infarcted wall compensatory thicken,ventricular dilatation after AMI.Myocardial infarction can cause heart failure,arrhythmia,thrombosis.In previous rat model experiment,Rats receiving an isoproterenol overdose suffered cardiac apex ischemia-reperfusion damage and arrhythmia,and then underwent cardiac remodeling and dysfunction.At two weeks it was found that Myocytes presented systolic and diastolic Ca2+ mishandling,post ISO-OV mitochondrial dysfunction may underlie decreased cardiac contractility,ATP depleted and exacerbated oxidative stress may enhance.Although with the development of medical technology,most of the patients with acute myocardial infarction have been treated in time,but the leading cause of death in China's urban and rural residents is still cardiovascular disease.How to find new and effective treatment methods has become the focus of the current study.With the development of life science,the feasibility of targeted gene therapy is becoming clear.S100A1 is a key regulator of both cardiac function and vascular biology.S100A1 is calcium sensor proteins,through Ca2+ signal transduction pathways,playing several important roles in gene expression,secretion,apoptosis and in cell differentiation,muscle contraction.It is found to interact with both the sarcoplasmic reticulum ATPase(SERCA2a)and the ryanodine receptor 2(RyR2),resulting in primarily improved Ca2+ handling and contractile function.S100A1 may enhance Ca2+ transient amplitudes and decreasing diastolic Ca2+overload via increasing Ca2+-induced SR Ca2+ release together with decreasing diastolic SR Ca2+ leak to improves cardiomyocyte function.In mouse experiment it was demonstrated that stable increasing expression of S100A1 protein can significantly enhance myocardial contractility.S100A1 gene knockout mice appear acute contractile dysfunction,myocardial apoptosis,early myocardial remodeling,adrenergic signal system severely damaged,heart failure speeded up.In animal studies,it was proved S100A1 gene-targeted therapy can reverse experimental HF and improved cardiac performance in.Recent studies have shown that S100A1 colocalizes and coimmunoprecipitates both with the cardiac and skeletal muscle sarcoplasmic reticulum(SR)Ca2+ release channel/ryanodine receptor(RyR)isoform in a Ca2+ dependent manner.S100A1 regulate SERCA2a function and RyR2 function.S100A1 inhibits the interaction between PLB and SERCA2a to increase vivo SERCA2a activity,regulates diastolic cytosolic ca2+ uptake.Under physiological conditions,S100A1 not only in systole activates myocardium RyR2,release ca2+ from the sarcoplasmic reticulum into the cytosol,provide cardiac excitation-contraction coupling factor;also during diastole,make the direct role with the RyR2 receptor C-termina,inhibit the RyR2 receptor opening,reduce ca2+ leakage,thereby reduce the frequency,amplitude and duration of diastolic ca2+ transients.Enhanced cellular ca2+ cycling by S100A1 was associated both with increased sarcoplasmic reticulum Ca2+ content and enhanced sarcoplasmic reticulum Ca2+-induced Ca2+ release,and S100A1 was shown to associate with the cardiac ryanodine receptor.The expression of the S100A1 protein has the highly tissue-specific and cell-specific,rarely expressing in skeletal muscle,abundantly expressing in healthy myocardial cells,which is dominated by left ventricular,right ventricular and atrial expression less.S100A1 protein expression decreased in heart failure,particularly evident in end-stage heart failure,but increased expression of cardiac hypertrophy.Studies have shown that S100A1 regulate ca2+ transport of myocardial cells by four mechanisms.1.Regulate SERCA2a function.2.Regulat RyR2 function.3.Inhibit cardiac remodeling.S100A1 could inhibit cardiac remodeling by inhibiting cardiac hypertrophy,ventricular dilatation and other mechanisms:1).Increasing the expression of S100A1 can improve cardiac contractile function,reduce the burden of bio-mechanical and make a direct protective effect on cardiac remodeling;2).Increasing the expression of S100A1 can improve ca2+ transit of the failure myocardial cells,and have a favorable impact on cardiac hypertrophy;3)Increasing the expression of S100A1 can directly inhibit the fetal gene runaway which extracellular matrix genes and a-adrenergic-mediated,maintain normal gene expression,thereby reduce ventricular hypertrophy and cardiac dilation;3.Inhibit cardiomyocyte apoptosis;4.Improve myocardial energy supply.S100A1 protein can reduce diastolic cytosolic ca2+ overload,improve mitochondrial function,play an important role in recovery of phosphocreatine and ATP ratio.The current study aims to identify the target proteins that are associated with Ad-SIOOAI-EGFP following AMI,and fnding alternative therapies to reconstitute the energetic stateS100 calcium binding protein A1(S100A1)is an important regulator of myocardial contractility.In other animal studies,targeted therapy with S100A1 gene reverses experimental heart failure and improves cardiac function.The understanding of how S100A1 affects myocardial energy metabolism and the changes in calcium binding proteins and cytoskeletal proteins after acute myocardial infarction(AMI)are still relatively limited.LTQ Orbitrap mass spectrometry is a quantitative method for protein.This study used LTQ Orbitrap mass spectrometry method for protein expression related with energy metabolism,calcium binding protein and cytoskeletal protein expression in patients with acute myocardial infarction after S100A1 transgenic rats,to find new therapeutic targets and treatment methods.The aim of this study was to determine the mechanism of S100A1 activity by studying the model of acute myocardial infarction(AMI)in rats(Ad-S100A1-EGFP).LTQ Orbitrap mass spectrometry was used to study differential protein expression in the Ad-S100A1-EGFP group and the control group after AMI.LTQ Orbitrap mass spectrometry was used to study differential protein expression in the Ad-S100A1-EGFP group and the control group after AMI.Objective1.To establish a rat model of myocardial infarction.Ad-S100A1-EGFP group,following ligation of the coronary artery for 20 min,1×1010 plaque-forming units(pfu)of Ad-S100A1-EGFP in 20 ?l were injected into the anterolateral wall of the left ventricle(LV)using a 30-gauge needle;2.To clarify the protective effect of S100A1 on cardiac function after acute myocardial infarction(AMI)3.LTQ Orbitrap mass spectrometry was used to study differential protein expression in the Ad-S100A1-EGFP group and the control group after AMI.The Protein Analysis Through Evolutionary Relationships(PANTHER)analysis was used.Methods1.Establish the rat myocardial infarction model and group:A total of 20 male Wistar rats(age,12-14 weeks;weight,250-300 g)were provided.The animals were randomly divided into the following 4 groups(5rats/group):Ad-S100A1-EGFP group,following ligation of the coronary artery for 20 min,1×1010 plaque-forming units(pfu)of Ad-S100A1-EGFP in 20 ?l were injected into the anterolateral wall of the left ventricle(LV);ii)control group,only the coronary artery was ligated without any treatment;iii)Ad-EGFP group,following ligation of the coronary artery for 20 min,1×1010 pfu of Ad-EGFP in 20 ?l were injected into the anterolateral wall of the LV;iv)physiological saline group,following ligation of the coronary artery for 20 min,20 ?l physiological saline was injected into the anterolateral wall of the LV.After 2 weeks,the rats wereeuthanized,and the blood and myocardial tissue specimens of each group were taken.2.Histopathological measurementThe myocardial tissue sections were stained with hematoxylin eosin(H&E)staining,Masson staining and picrosirius red staining.We observe the myocardial hypertrophy and swelling of myocardial fibers arranged irregularly,nucleus malformation,leukocyte infiltration,myocardial collagen fiber content percentage.Immunohistochemical staining of cTnI antibody,MLC 3 antibody,HSP70 antibody expression in the myocardial tissue in each group.3.Western BlotWestern Blot was detected Cavl.3 antibody,cTnI antibody,MLC 3 antibody,HSP70 antibody in Ad-SIOOAI-EGFP group.4.LTQ Orbitrap mass spectrometryLTQ Orbitrap mass spectrometry was used to study differential protein expression in in the Ad-S100A1-EGFP group and control group after AMI.The protein analysis system was used Protein Analysis Through Evolutionary Relationships(PANTHER).To investigate the changes of energy metabolism related proteins,12 Ca2+ binding proteins and 22 cytoskeletal proteins related to cardiac function changes.Statistical analysisData was analyzed using SPSS software(version 18.0;SPSS,Inc.,Chicago,IL,USA).One-way analysis of variance followed by Tukey's multiple comparison test was used to determine the statistical differences among the post-MI and control groups.Data are presented as the mean ± standard deviation.P<0.05 was considered to indicate a statistically signifcant difference.Results1.Improve impaired myocardial S100A1 levels inhibit the ventricular remodeling.In control group after myocardial infarction,cardiac morphology and structural changes,as the mind/body weight ratio(HW/BW)increased significantly,left ventricular dilatation.Under the microscope,HE staining showed myocardial hypertrophy,disorder,cell gap increases;The myocardial fibers in the Ad-S100A1-EGFP group were relativaly arranged..Masson staining and picrosirius red staining showed myocardial infarction after the deposition of collagen increased,collagen decay;type I collagen and type ? collagen expression increase.The collagen fiber in Ad-S100A1-EGFP group was significantly lower than that of the control group and Ad-EGFP group.Western blot results showed that the myocardial tissue cTnI,MLC 3 and HSP70 expression increase,which were consistent with those of immunohistochemical staining and LTQ Orbitrap mass spectrometry results.2.Improve the S100A1 level of damaged myocardium and improve the myocardial energy metabolism.According to the LTQ results using Orbitrap mass spectrometry,by Protein Analysis Through Evolutionary Relationships(PANTHER)analysis,after AMI,Ad-S100A1-EGFP and control group showed 134 differentially expressed proteins related to energy metabolism,including 20 carbohydrate metabolism related proteins and 27 lipid metabolism related proteins.3.Improve the S100A1 level of damaged myocardium and improve the function of myocardial contraction.According to the LTQ results using Orbitrap mass spectrometry,by Protein Analysis Through Evolutionary Relationships(PANTHER)analysis,after AMI,12 Ca2+ binding proteins and 22 cytoskeletal proteins were found in the Ad-S100A1-EGFP group and the control group,and most of the protein expression was up-regulated in the Ad-S100A1-EGFP group.Conclusion1.After AMI,S100A1 by increasing the expression of key enzymes of myocardial energy metabolism to improve myocardial dysfunction;2.After AMI,S100A1 by increasing the expression of Ca2+ binding protein to improve the myocardial systolic dysfunction;3.LTQ Orbitrap mass spectrometry is a reliable method for proteomic studies of acute heart failure in myocardial infarction.