Quantitative Proteomics of Human Heart Samples Collected In Vivo Reveal the Remodeled Protein Landscape of Dilated Left Atrium Without Atrial Fibrillation

Abstract

Genetic and genomic research has greatly advanced our understanding of heart disease. Yet, comprehensive, in-depth, quantitative maps of protein expression in hearts of living humans are still lacking. Using samples obtained during valve replacement surgery in patients with mitral valve prolapse (MVP), we set out to define inter-chamber differences, the intersect of proteomic data with genetic or genomic datasets, and the impact of left atrial dilation on the proteome of patients with no history of atrial fibrillation (AF).We collected biopsies from right atria (RA), left atria (LA) and left ventricle (LV) of seven male patients with mitral valve regurgitation with dilated LA but no history of AF. Biopsy samples were analyzed by high-resolution mass spectrometry (MS), where peptides were pre-fractionated by reverse phase high-pressure liquid chromatography prior to MS measurement on a Q-Exactive-HF Orbitrap instrument. We identified 7,314 proteins based on 130,728 peptides. Results were confirmed in an independent set of biopsies collected from three additional individuals. Comparative analysis against data from post-mortem samples showed enhanced quantitative power and confidence level in samples collected from living hearts. Our analysis, combined with data from genome wide association studies suggested candidate gene associations to MVP, identified higher abundance in ventricle for proteins associated with cardiomyopathies and revealed the dilated LA proteome, demonstrating differential representation of molecules previously associated with AF, in non-AF hearts.This is the largest dataset of cardiac protein expression from human samples collected in vivo. It provides a comprehensive resource that allows insight into molecular fingerprints of MVP and facilitates novel inferences between genomic data and disease mechanisms. We propose that over-representation of proteins in ventricle is consequent not to redundancy but to functional need, and conclude that changes in abundance of proteins known to associate with AF are not sufficient for arrhythmogenesis. Genetic and genomic research has greatly advanced our understanding of heart disease. Yet, comprehensive, in-depth, quantitative maps of protein expression in hearts of living humans are still lacking. Using samples obtained during valve replacement surgery in patients with mitral valve prolapse (MVP), we set out to define inter-chamber differences, the intersect of proteomic data with genetic or genomic datasets, and the impact of left atrial dilation on the proteome of patients with no history of atrial fibrillation (AF). We collected biopsies from right atria (RA), left atria (LA) and left ventricle (LV) of seven male patients with mitral valve regurgitation with dilated LA but no history of AF. Biopsy samples were analyzed by high-resolution mass spectrometry (MS), where peptides were pre-fractionated by reverse phase high-pressure liquid chromatography prior to MS measurement on a Q-Exactive-HF Orbitrap instrument. We identified 7,314 proteins based on 130,728 peptides. Results were confirmed in an independent set of biopsies collected from three additional individuals. Comparative analysis against data from post-mortem samples showed enhanced quantitative power and confidence level in samples collected from living hearts. Our analysis, combined with data from genome wide association studies suggested candidate gene associations to MVP, identified higher abundance in ventricle for proteins associated with cardiomyopathies and revealed the dilated LA proteome, demonstrating differential representation of molecules previously associated with AF, in non-AF hearts. This is the largest dataset of cardiac protein expression from human samples collected in vivo. It provides a comprehensive resource that allows insight into molecular fingerprints of MVP and facilitates novel inferences between genomic data and disease mechanisms. We propose that over-representation of proteins in ventricle is consequent not to redundancy but to functional need, and conclude that changes in abundance of proteins known to associate with AF are not sufficient for arrhythmogenesis. For centuries, anatomists and physiologists have recognized that structural and functional differences exist between cardiac chambers and that cardiac diseases can affect specific regions of the heart. Yet, our knowledge of the molecular profile of the different cardiac chambers and how it relates to the causes, manifestations or treatment of disease remains limited. Advances in biochemistry and molecular biology, and more recently the availability of high throughput transcriptomics, have improved our understanding of the molecular composition of the heart (1Lindskog C. Linné J. 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Quantitative high-resolution proteomics offers an unbiased approach to the identification of chamber-specific protein expression patterns and their relation to cardiac function. Recent large-scale proteomic studies have focused on mapping the human proteome across all major organs (3Wilhelm M. Schlegl J. Hahne H. Moghaddas Gholami A. Lieberenz M. Savitski M.M. Ziegler E. Butzmann L. Gessulat S. Marx H. Mathieson T. Lemeer S. Schnatbaum K. Reimer U. Wenschuh H. Mollenhauer M. Slotta-Huspenina J. Boese J.-H. Bantscheff M. Gerstmair A. Faerber F. Kuster B. Mass-spectrometry-based draft of the human proteome.Nature. 2014; 509: 582-587Crossref PubMed Scopus (1318) Google Scholar, 4Kim M.-S. Pinto S.M. Getnet D. Nirujogi R.S. Manda S.S. Chaerkady R. Madugundu A.K. Kelkar D.S. Isserlin R. Jain S. Thomas J.K. Muthusamy B. Leal-Rojas P. Kumar P. Sahasrabuddhe N.A. Balakrishnan L. Advani J. George B. Renuse S. Selvan L.D.N. Patil A.H. Nanjappa V. Radhakrishnan A. Prasad S. Subbannayya T. 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However, studies on collected of We set out to an quantitative of the protein expression in heart samples collected from living collected in and its relation to disease in humans is limited by the mitral valve replacement consequent to mitral valve prolapse is a that to from right and left atria (LA) and from the left ventricle (LV) MVP is not a cardiac muscle disease and the of the atrial and is from to an to the valve we data obtained from samples collected during mitral valve replacement surgery in seven For all dilation limited to the a of a dilated (LA) atrial of our patients atrial fibrillation the to the proteome of the dilated LA in a non-AF We confirmed our on protein changes in the dilated LA in an independent proteomes of three additional mitral valve replacement we from the LA and of a with atrial fibrillation to the abundance of proteins that were to in the The of the patients mitral valve surgery in and and left of the we for the a comprehensive and proteome of the from living hearts. that we collected from living we were to our data to previously S. M. C. F. H. R. M. M. and quantitative proteomic map of the human Scopus Google Scholar), obtained from collected This to define the of the of to the proteome of living hearts. For of and the Our is based on seven of samples from three cardiac chambers on samples into MS analysis, a of MS were were MS of were in to and the across Our were against an independent of three from cardiac where and MS were the The of based on availability from the of differential protein expression across chambers by analysis based on a S. T. P. A. T. M. J. The for comprehensive analysis of PubMed Scopus Google Scholar, R. G. analysis of to the PubMed Scopus Google Scholar). This approach a of and to a protein is and high changes are of a biopsies were collected from and of patients mitral valve samples were in a with liquid still in the patients to the prior to and the with the in the of biopsies were on a in with and were for and to and in were and by of and and in the for to protein by of for in the by with and with were by were and on by liquid on an with an previously C. S. C.L. Nielsen for the of comprehensive human PubMed Scopus Google Scholar). collected in into in the samples were analyzed by liquid chromatography to a Orbitrap mass samples were to of in in and into a samples were on and with in a in in in by in for in a in and into the mass were of in the Orbitrap of A the to for in an of were resolution of of in the Orbitrap of MS data the S. T. J. The for mass PubMed Scopus Google Scholar) of of and and proteins identified with the peptides were for protein and in with identification were the S. T. P. A. T. M. J. The for comprehensive analysis of PubMed Scopus Google Scholar). between of based on the B.M. M. A of for high data based on and PubMed Scopus Google Scholar). seven patients were and mitral valve regurgitation with dilated left atria and a left are in to patients in the to a with atrial fibrillation are in the from the patients mitral valve surgery and patients not or of atrial biopsies were collected from the and during mitral valve The samples were not of the samples were and proteins by by by and The peptides were pre-fractionated into C. S. C.L. Nielsen for the of comprehensive human PubMed Scopus Google Scholar, C. for 2014; PubMed Scopus Google Scholar) by high-resolution mass spectrometry measurement of on a Orbitrap analysis in 130,728 peptides protein proteins were identified by peptides The mass spectrometry based of protein abundance seven of a high of the dataset for a comprehensive the of cardiac proteins or were identified in all proteins were identified in were on identified based on of were in a of and unique of The provides the largest dataset of cardiac protein expression from human samples collected in vivo. a quantitative analysis of protein expression across cardiac we protein B.M. M. A of for high data based on and PubMed Scopus Google Scholar) and data Our data showed by differences in prior to and by of for protein across samples proteins in cardiac were identified in three that across chambers were on protein abundance differences between we analysis This revealed that between atria and ventricle in the disease or of in the dataset of proteins of the ventricle and the atria the atrial and in atrial and in the of protein across chambers and atrial right and left atrial samples analyzed from the of left and right atria into for differential LA proteome We the impact of protein in the chambers and that proteins were for but across all chambers that our data are of sufficient to of protein expression differences between A of the cardiac biopsies is that were collected from living and in the This with a measurement were obtained from and collected of S. M. C. F. H. R. M. M. and quantitative proteomic map of the human Scopus Google Scholar). We that with samples collected in vivo. analysis of to protein of all peptides from were the the for of the peptides from The of peptides in samples of post-mortem protein of of and MS peptides on level protein of protein based on biopsies is by across our that protein in samples can to a impact on protein and analysis of proteome data from of post-mortem samples to protein Our analysis for quantitative differences in protein analysis of collected biopsies is Our data revealed the proteome of the hearts of MVP into protein expression to genomic data set previously identified MVP C. N. N. K. R. M. L. J. P. T. V. Y. P. D. M. S. R. F. E. F. S. F. R. H. P. R.S. P. A.A. X. Genetic association mitral valve 2015; PubMed Scopus Google in genomic are to a disease. to proteins in of the MVP were in the we our data for their protein expression level in the LA across the different proteins were by based B. D. N. G. J. J. M. Global of gene expression PubMed Scopus Google Scholar). is an that allows for abundance across different proteins by for protein in to MS identification of For of the we of between the and the protein encoded by a gene in the of a gene in a cardiac is the gene is and from that we that our data offers an to in for of the protein more in the LA protein encoded by a gene in the for our heart proteome data to a of the and functional in of the of and in MVP has reported C. N. N. K. R. M. L. J. P. T. V. Y. P. D. M. S. R. F. E. F. S. F. R. H. P. R.S. P. A.A. X. Genetic association mitral valve 2015; PubMed Scopus Google Scholar). For a proteins were in the left and the C. C. D. G. K. A. D. J. A. S. G. S. P. J. genetic data on Google Scholar) we the of we that and are the identification of specific in the MVP The in our dataset the atrial proteome to that of the left into their functional the protein in a the of the atrial proteome not different between and We analyzed major differences in the atrial proteome all atrial Global analysis identified a of proteins in atria or ventricle of proteins of have previously reported atrial of the of Med. 2004; PubMed Scopus Google Scholar, D. P. Thomas P. S. C. E. P. regulation of the atrial of the during muscle PubMed Google in atrial but in C. K. L. D. G. K. B. T. and structural diversity of the human gene PubMed Scopus Google Scholar, P. Wu A. D. A. H. C. are in PubMed Scopus Google and to have high atrial and expression A.H. M. L. a for a novel in and dilated PubMed Scopus Google This with the of our approach and additional for studies the Our a of proteins in atria or We differences on differences in chamber-specific and We the major protein by of protein association and analysis on P. A. J.T. D. N. B. T. a for of PubMed Scopus Google Scholar, L. J. T. G. Bader G.D. a for PubMed Scopus Google Scholar, D. H. M. S. M. A. A. P. C. The in association PubMed Scopus Google Scholar). 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