| BackgroundAtrial fibrillation(AF) is the most common cardiac arrhythmia affects1%-2%of the population.The prevalence of AF increases with age, from0.5%at40-50years, to5%-15%at80years. It is responsible for an increased risk of stroke, heart failure, and all-cause mortality. Effective prevention has the potential to decrease the AF-related morbidity and mortality, but antiarrhythmic drug therapy has been disappointing because of inadequate efficacy and proarrhythmic risks. New insights into underlying mechanisms are needed for the development of new therapeutic methods.Membrane proteins are a key set of proteins as they form part of the boundary of cells and organelles. Moreover, many of these proteins have important functions, such as transporters, receptors and traffickers.2D PAGE approaches have been employed in most cardiac proteomics studies. A major drawback of using2D gels is their incompatibility with most hydrophobic membrane proteins. In general, membrane proteins have been under-represented in large-scale proteomics studies due to their poor solubility and wide concentration range within the cell.Pappin and coworkers at Applied Biosystems have developed an alternative set of chemical tags, amine reactive reagent isobaric tag for relative and absolute quantitation(iTRAQ). Four (4-plex) or eight (8-plex) different samples can be labeled with these tags via their free lysine groups that were generated by trypsin digestion. Since these iTRAQ labels are isobaric they do not differ in mass when attached to the peptide. However, upon MS/MS fragmentation, each tag produces a characteristic charged reporter ion, differing successively by1Da. The comparison of the peak intensities of the four or eight reporter ions gives information regarding the relative quantity of the protein in samples under comparison. The great advantage of this technique is the ability to easily multiplex expenment.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 the cytoskeleton, energetic metabolism, and cardiac cytoprotection 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 the cytoskeleton, energetic metabolism, and cardiac cytoprotection 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 desmoplakin, cytochrome c oxidase subunit5B and heat shock protein beta-1between 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. 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 the cytoskeleton, energetic metabolism, and cardiac cytoprotection. There were more differences when RAA and LAA from SR patients were compared with their corresponding atrial appendages from AF patients.3. LAA from both SR and AF patients upexpressed desmoplakin with respect to RAA. Desmoplakin was upexpressed in LAA and RAA from AF with respect those from SR patients.4. LAA from both SR and AF patients downexpressed myosin regulatory light chain2, atrial isoform with respect to RAA. Myosin regulatory light chain2, atrial isoform was downexpressed in LAA and RAA from AF with respect those from SR patients. On the contrary, in LAA from AF patients a significative upexpression of myosin regulatory light chain2, ventricular/cardiac muscle isoform was observed as compared with their corresponding LAA from SR patients. Tropomyosin were downexpressed in LAA from AF patients with respect to those from SR patients.5. Many energetic metabolism-associated proteins 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.6. Many energetic metabolism-associated proteins 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.7. HSP70was upexpressed in LAA and RAA from AF with respect those from SR patients. LAA from SR patients upexpressed HSP70with respect to RAA. On the contrary, LAA from AF patients downexpressed HSP70with respect to RAA.8. HSP27was upexpressed in LAA from AF with respect those from SR patients. On the contrary, HSP27was downexpressed in RAA from AF with respect those from SR patients.9. Western blotting confirmed changes of desmoplakin, cytochrome c oxidase subunit5B and heat shock protein beta-1observed 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. 2. Both increased atrial cell-cell adhesive strength and contractile dysfunction are adaptations to the stretch occurring in atrial fibrillation.3. Impairment of myofibrillar energetics is observed in the atrial myocardium during AF. The atrial myocytes show dedifferentiation and ventricularization under failing atrial energetics possibly reflecting a general adaptation of the atria to changes in the working conditions.4. HSP70may exert a protective effect against AF-induced cardiac myocytes damage. There is a different adaptative response to atrial stress induced by AF between RAA and LAA. |
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