Comparative Proteomic Analysis In Left Ventricular Remodeling Following Myocardial Infarction In Rats

Acute myocardial infarction (AMI) is a common, life-threatening disorderin clinical practice. Heart failure after myocardial infarction (MI), a commoncomplication of MI, is characterized by high mortality and poor prognosis. Leftventricular remodeling (LVR) is the underlying mechanism of heart failureafter MI. LVR following MI refers to the condition in which the cells,molecules, and inter-cellular substances of the whole left ventricle (LV) changeafter AMI, followed by changes in the size, morphology, and function of the LV.It can be divided into early (within six weeks) and late stages (six weeks to oneyear). The possible mechanisms of ventricular remodeling includehemodynamic change, inflammatory reaction, neuroendocrine activation, andapoptosis. Nevertheless, LVR is a complex issue, and remains a hot topic incardiovascular research.The term "proteome" was proposed by Wilkins and Williams in1994. Itrefers to all the proteins that can be expressed by genes, i.e., all the proteins thatare expressed by cells, tissues, or body in a certain time and space. Proteomicsis at the frontier of modern science and technology. It provides a globalperspective for understanding the rules of life and many key pathophysiologicalphenomena. Comparative proteomics is an important component of proteomics.It tries to identify the pathophysiological mechanisms of diseases by comparingand analyzing differentially expressed proteins, and thus provides valuableinformation for diagnosis, medical treatment, and prognosis. Liquidchromatography-mass spectrometry (LC-MS) is most commonly used for theproteomic analysis of complex samples, because of its very high peak capacity,sensitivity, and speed. Proteomics has been widely applied in clinical subjects, such as tumors and aging. Cardiovascular diseases (CVDs) represent a newfield in which proteomics can be applied. Using proteomic technology, thechanges in CVD-related proteins can be dynamically analyzed. It has beenspeculated that proteomics can provide strong support for the early diagnosisand effective treatment of CVD. Proteomics has been applied for research onmyocardiopathies; however, few studies have focused on LVR following MI.In this study, by establishing animal models, we analyzed thehemodynamics, morphology,pathology, and comparative proteomics of LVRfollowing MI, in an attempt to explore its possible mechanism and providetargets for drug therapies.Materials and MethodsThis study is divided into three parts1.Establishment of animal models,detection of hemodynamics,morphologyand pathology: Twenty-three10-week-old male Wistar rats, were randomizedinto the LVR group (n=12) and the sham-operated group (n=11). In the LVRgroup, after the anterior descending coronary artery was ligated, the rats werefed for four weeks before the LVR models were established according to theliterature. Electrocardiograms immediately after the operation and at24hoursshowed ST-elevated MI among rats that survived the procedure. Rats in thesham-operated group underwent the same surgical procedure, includingthread-drawing, but not ligation.Four weeks after the operation, the rats wereanesthetized with chloral hydrate, and a cardiac catheter was inserted into theLV via the right carotid artery and connected with a polygraph. Thus, the leftventricular systolic pressure (LVSP), the left ventricular end-diastolic pressure(LVEDP), and the maximal left ventricular pressure increase/decrease ratio(±dp/dtmax) could be recorded. The systolic blood pressure(SBP),diastolicblood pressure(DBP),and heart rate(HR) were also recorded through arteriafemoralis. After the hemodynamic parameters had been completely recorded, the rats were sacrificed and the heart and left ventricular were harvested. Theheart weight and left ventricular mass were measured, and the ratio of heartweight to body weight(HW/BW),and the ratio of left ventricular mass to bodyweight(LVHW/BW)were calculated. LVs were divided into three parts fromligation site to apex. The part that was close to the ligation site was fixed in10%buffered formalin, embedded in paraffin, sectioned, stained withhematoxylin and eosin (H-E) and Masson dye, and pathologically analyzed.The neighboring parts were preserved in a–80°C freezer. Eight specimens inthe LVR group and seven specimens in sham-operated group were obtained forproteomic analysis. Myocardium from the apex of the heart was reserved forwestern blotting.2. Comparative proteomic analysis: The frozen biopsies were washed andthen cut into1mm3pieces, ground into powders in liquid nitrogen, anddissolved in lysis buffer, After the samples were centrifuged,the proteinconcentration of sample was measured by the Bradford method. All sampleswere stored at-80°C until use. After protein Digestion, we analysised withLC-MS. The loading volume was2μg for each assay. The reversed-phasecolumn was150×0.1mm (Michrom). The samples were eluted with5–30%buffer B (0.1%formic acid and99.9%methyl cyanide) at a flow rate of500nL/min for two hours. Each sample was tested three times. The reverse phasechromatography-eluted polypeptides were detected using an LTQ Orbitrapmass analyzer (Thermo Scientific) at a mass charge ratio (m/z) of300-2000amu. MS2scans were performed in a data-dependent manner (20MS2scansafter each full scan; parent ion m/z width:3amu;35%collision energy;dynamic exclusion time:30s).Spectra with an intensity higher than one unit andover10ion signals were searched against a protein database, using theSEQUEST algorithm and Bioworks3.3.1SP1software (Thermo Scientific), toidentify the corresponding polypeptides and proteins. 3.Western blotting and immunohistochemistry:We validated the expressionof two different proteins named EeF1α1and JUP by western blotting andimmunohistochemistry.Results1.Animal modelsChanges in Hemodynamic Parameters: Compared with the sham-operatedgroup, the hemodynamic parameters in the LVR group showed significantdifferences. In particular, the SBP, LVSP and±dp/dtmax significantlydecreased (P<0.01), and the DBP decreased(P<0.05),the LVEDP significantlyincreased (P<0.01).Morphological Parameter: HW and LVHW were significantly higher inLVR group than in sham-operated group(p<0.01). HW/BW and LVHW/BWwere also higher in LVR group than in sham-operated group(p<0.05).Pathological Changes in Myocardial Tissue: As shown by H-E staining, inthe sham-operated group, the normal myocardial cells were arranged in anorderly manner, with carmine-colored nuclei and plum-colored cytoplasm. Inthe LVR group, however, myocardial fibrosis was obvious, withhyacinthine-colored collagen tissue; the surviving myocardial cells, which wereirregularly arranged, were found in the border zones.As shown by Massonstaining, in the sham-operated group, the normal cardiac muscles were brightred and the cytoplasm was evenly stained. In the LVR group, the myocardialcells showed a diffuse brick red cytoplasmic staining, with blue collagen tissueand obvious myocardial fibrosis.2.Comparative proteomic analysisProteomic analysis: Proteomic analysis identified808proteins in the LVRgroup and809proteins in the sham-operated group, with inter-assayoverlapping rates of78.63%and76.74%.Differential proteomic analysis: Twenty-one differentially expressed proteins were identified. Compared with the sham-operated group,10proteinswere upregulated and four proteins were downregulated in the LVR group.Three proteins were found only in the LVR group, but not in the sham-operatedgroup. Four proteins were found only in the sham-operated group, but not inthe LVR group.3.We validated the expression of two different proteins named EeF1α1andJUP by western blotting and immunohistochemistry. By western blotting theexpression of EeF1α1was significantly increased in LVR group (p<0.01),theexpression of JUP was also increased in LVR group (P<0.05). Byimmunohistochemistry the expression of EeF1α1and JUP were increased inLVR group.Conclusion1.In this study, after establishing an animal model of LVR following MI,we analyzed the changes of hemodynamics, morphology and pathology. It isshown that heart systolic and diastolic function declined, heart and leftventricular weight increased, myocardial remodeling appeared.2.We identified21differentially expressed proteins using comparativeproteomic techniques. These differentially expressed proteins might play keyroles in LVR following MI. These findings provide clues to thepathophysiological mechanisms of LVR, and may help in identifying new drugtargets for the prevention and treatment of LVR.3.We validated two proteins by western blotting and immunohisto-chemistry. The expression of EeF1α1and JUP were increased in LVR group.These results matched those found by comparative proteomics. These resultsproved the accuracy of proteomics.