Anna Baud (Presenter)
UCL Institute of Child Health
Bio: Anna is a research associate working with Dr Kevin Mills in Biological Mass Spectrometry Center at University College London. Her main project is deep proteomic profiling of induced pluripotent stem cells, and development of targeted tests for pluripotency, funded by StemBancc European Consortium. She is also interested in cardiac myocytes proteomics. Anna received her PhD in the field of structural proteomics at University of Evry, France, where she specialized in in-solution structural analysis of proteins combined with high-resolution mass spectrometry.
Authorship: A. Baud (1), Ch. Hope (1), T. A. Treibel (2,3), W. Heywood (1), G. Captur (2,3) , F. W. Asselbergs (4), M. Harakalova (4), J. Moon (2,3), K.M. Mills (1)
1)UCL BMSC ICH, London 2)Barts Heart Centre, London 3)UCL Institute of Cardiovascular Science, London 4)Department of Cardiology, University Medical Center Utrecht, The Netherlands
| Short Abstract Aortic stenosis (AS), the most common form of valve disease in the Western world, occurs due to progressive narrowing of the aortic valve (AV) orifice, resulting in slowly increasing load onto the left ventricle (LV). The LV responds with a myriad of adaptive changes, which eventually become maladaptive. Current management is focused on assessing the severity of valve stenosis, but neglects the myocardial response, that is pivotal for timing of surgery and prognosis. The aim of this pilot study is to find new biomarkers of myocardial remodeling that could refine our understanding of maladaptive changes in aortic stenosis and ultimately allow to optimize the timing of surgery. |
Long Abstract
Introduction
Aortic stenosis (AS), the most common form of valve disease in the Western world, occurs due to progressive narrowing of the aortic valve (AV) orifice, resulting in slowly increasing left ventricular (LV) afterload. The LV responds with a myriad of adaptive changes (e.g. LV hypertrophy). Eventually, these responses become maladaptive and the patient presents with symptoms. At this stage, there is a sharp increase in morbidity and mortality, unless the valve is replaced. Current management is focused on assessing the valve stenosis but neglects the myocardial response that is pivotal for timing of surgery and prognosis. Interestingly, despite equivalent stenosis severity, macroscopic patterns of remodeling differ between patients ranging from normal geometry to concentric remodeling, concentric hypertrophy and finally eccentric hypertrophy. The aim of this pilot study is to identify new biomarkers of myocardial remodeling that could refine our understanding of maladaptive changes in aortic stenosis and ultimately allow to optimize the timing of surgery.
Methods
Myocardial biopsies were collected in 6 patients with severe AS during surgical aortic valve replacement. These were compared with 4 control samples. Myocardial biopsies were homogenized using Precellys® Ceramic Kit. The homogenate was centrifuged at 13,000g for 5 minutes to remove cellular debris and the supernatant digested with sequencing grade trypsin (Promega). Resulting peptides were desalted using C18 columns (Isolute) and quantified using Pierce™ Quantitative Colorimetric Peptide Assay (ThermoFisher Scientific). Yeast enolase peptide standard (Waters) was added to samples and 2µg of protein digests were analyzed using label free quantitation on a Waters QTof Synapt G2-Si mass spectrometer coupled to 2D-nanoLC nanoAcquity system. Proteins were identified using Protein Lynx Global Server version 2.5 (Waters). All protein hits that were identified with a confidence of >95% were included in the quantitative analysis. The differential expression analysis was performed using Progenesis LC-MS (Non-linear Dynamics) software.
Results
About 1,100 myocardial proteins were identified and categorized based on their biological function using Panther Gene Ontology software (1). Overall protein subcategory abundances were similar across control and AS myocardium. Thirty four percent of control myocardial proteins and 32% from AS were involved in metabolic processes (i.e. primary metabolic processes as well as glycolysis, oxidative phosphorylation and respiratory electron transport chain). Sixteen percent of control myocardial proteins and 15% from AS were involved in cellular processes (i.e. cell communication and cell cycle).
Next we compared protein signatures in control myocardium with that from AS myocardium of either normal or decompensated geometry.
Comparing control and normal geometry AS myocardium revealed 123 differentially expressed proteins (p-value < 0.05). Proteins with the highest fold changes (400-75 fold) were proteasome subunit alpha type-1, serine/threonine-protein phosphatase 2A catalytic subunit alpha isoform, ribose-phosphate pyrophosphokinase 2, puratrophin-1 and mitogen activated protein kinase 1. Interestingly, proteasome subunit alpha type-1 has previously been reported as potential biomarker for rheumatic heart disease(2) that is a known cause of AS.
Comparing control and decompensated geometry AS myocardium revealed 96 proteins with altered expression (p < 0.05). Proteins with the highest fold changes (151-10 fold) were beta-centractin, Ena/VASP-like protein, collagen alpha 1 (XIII), presequence protease and dysferlin.
In total, 28 proteins were differentially expressed in normal and decompensated geometry AS myocardium. The expression of calcium binding protein- calsequestrin-2; cAMP-dependent protein kinase type I - alpha regulatory subunit and coagulation factor XIII A chain (FXIIIA) was increased in normal geometry AS myocardium (fold change ≥ 75). Interestingly, the FXIIIA, associated with valve thickening and calcification, was previously reported as overexpressed in patients with AS(3). On the other hand, the expression of mitogen activated protein kinase 1, proteasome subunit alpha type-1 and serine/threonine phosphatase 2a catalytic subunit alpha isoform was increased in decompensated geometry of AS myocardium.
Finally, the comparative analysis of changes in proteomic expression between control and AS myocardium (both normal geometry and decompensated) shown the down-regulation of alpha-1-anti-trypsin, cytochrome C oxidase subunit 5A, isocriate dehydrogenase, phosphoglycerate mutase 2, prohibitin-2, WASH complex subunit strumpellin and heat shock 70kDa in disease tissue.
Conclusions
Mass spectrometry analysis of LV myocardium from patients with AS revealed myocardial remodeling associated with disease. The majority of differentially expressed proteins when compared to control myocardium were associated with metabolic, cellular and immune system processes. For the first time, this proof-of-principle analysis reveals significant differences in the proteomic profiles of normal and decompensated geometry AS myocardium in spite of similar degrees of AS severity.
Proteomics has the ability to identify biomarkers of maladaptive myocardial remodeling in AS with the potential to optimize surgical timing of AS patients.
References & Acknowledgements:
1.Mi, H., Muruganujan, A. & Thomas, P. D. PANTHER in 2013: modeling the evolution of gene function, and other gene attributes, in the context of phylogenetic trees. Nucleic Acids Res. 41, D377–D386 (2013).
2.Zheng, D. et al. Comparison of the Ventricle Muscle Proteome between Patients with Rheumatic Heart Disease and Controls with Mitral Valve Prolapse: HSP 60 May Be a Specific Protein in RHD. Biomed Res. Int. 2014, 1–9 (2014).
3.Kapusta, P. et al. Factor XIII expression within aortic valves and its plasma activity in patients with aortic stenosis: Association with severity of disease. Thromb. Haemost. 108, 1–8 (2012).
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