| BackgroundAtrial fibrillation (AF), which is responsible for an increased risk of the AF-related morbidity and mortality, is the most common cardiac arrhythmia. The prevalence of AF increases with age. The maintenance of mechanical and electrical activities of heart requires sustained provision of energy from energy metabolism. AF-related researches suggested that the energy metabolism of heart was changed during the pathogenesis of AF and the changes of energy metabolism could effects the atrial systolic function.Proteomic technology can analyze the differential expression in cells protein in different physiological or pathological conditions from an overall angle and reveals the function of protein the regulation of cells activities. As the latest quantitative proteomics technology, isobaric tag for relative and absolute quantitation (iTRAQ) could simultaneously conduct quantitative analysis for at most8different samples with highly accuracy and repeatability.ObjectiveThe aim of the present study was to perform a proteomic analysis to compare the possible differences in the expression of proteins mainly associated with energetic metabolism between left atrial appendages (LAA) and right atrial appendages (RAA) from patients with mitral valve disease both in sinus rhythm(SR) and in permanent AF. A better understanding of the molecular basis of the changes occurring in the RA and in the LA during AF allows us to design new cardioprotective strategies.Methods1. The RAA and LAA samples were obtained from the same patient as surgical biopsies at the time of the mitral valve surgery both in SR and with permanent AF. Specimens were pulverized under liquid-N2into a fine powder, which was homogenized in a lysate buffer. The supernatant was stored after centrifugation for30min at4℃,12000rpm.2. The protein concentration was determined using a Bradford assay. Then the protein composition was analyzed by1D SDS-PAGE. 3.100μg of protein in four different groups was digested by trypsin. Then the peptides were labeled with different iTRAQ tags. The pooled labeled peptides were analyzed by liquid chromatography tandem mass spectrometry(LC-MS/MS) for twice.4. The MS/MS spectra were extracted and searched against the Uniprot-sprot.fasta database using ProteinPilot software (version3.0, Applied Biosystems). A proteomic analysis was performed to compare the possible differences in the expression of proteins mainly associated with energetic metabolism between LAA and RAA from patients with mitral valve disease both in SR and in permanent AF.5. Western blotting was done to further confirm the content of cytochrome c oxidase subunit5B between LAA and right atrial appendages (RAA) from patients with mitral valve disease both in sinus rhythm (SR) and in permanent AF.Results1. The total protein was extracted successfully from the samples by the combinations of grind, cracking and ultracentrifugation methods. The result was further confirmed by1D SDS-PAGE.2. We acquired792unique proteins from the cardiac tissues of four groups using iTRAQ labeling and LC-MS/MS. Meanwhile, We identified123differential expressed proteins closely related to energy metabolism through searching the GeneCards human gene database. Among those proteins,39of them are related to carbohydrate metabolism,23of them are involved in lipid metabolism,49of them are related to biological oxidation and12others kinds of proteins. Most of them are key enzymes participating in energy metabolic reactions. The present study showed by proteomics that in patients with mitral valve disease, either at SR and AF, there were small differences between RAA and LAA in the level of expression of proteins associated with energetic metabolism. There were more differences when RAA and LAA from SR patients were compared with their corresponding atrial appendages from AF patients. 3. Many energetic metabolism-associated proteins participating in carbohydrate metabolism, lipid metabolism and biological oxidation were downexpressed in LAA from both SR and AF patients with respect to RAA, such as electron transfer flavoprotein, cytochrome c oxidase, ATP synthase and pyruvate dehydrogenase, long-chain-fatty-acid--CoA ligase1, acetyl-CoA acetyltransferase. However, several proteins participating in the catabolism of amino acid and aldehyde are upexpressed in LAA from both SR and AF patients with respect to RAA such as isovaleryl-CoA dehydrogenase.4. Many energetic metabolism-associated proteins participating in carbohydrate metabolism, lipid metabolism and biological oxidation were downexpressed in LAA and RAA from AF with respect those from SR patients,such as electron transfer flavoprotein, cytochrome c oxidase, ATP synthase, fructose-bisphosphate aldolase and very long-chain specific acyl-CoA dehydrogenase, while aldehyde metabolism-related proteins were upexpressed in the same comparison.5. Western blotting confirmed changes of cytochrome c oxidase subunit5B observed by iTRAQ labeling and LC-MS/MS.Conclusions1. The application of quantitative proteomics based on iTRAQ technology is effective in evaluating related proteins with differential expression levels in cardiac tissues. We acquired the differential proteins expression beteween left atrial appendages (LAA) and right atrial appendages(RAA) from patients with rheumatic heart disease both in sinus rhythm(SR) and in permanent AF through iTRAQ technology.2. Impairment of myofibrillar energetics is observed in the atrial myocardium of rheumatic heart disease patients during AF. The impairment of myofibrillar energetic of left atrial is more serious than that of right atrial. |
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