Study of the molecular basis of myocardial fibrosis is hampered by limited access to tissues from human patients and by confounding variables associated with sample accessibility, collection, processing and storage. Here, we report an integrative strategy based on mass spectrometry for the phosphoproteomic profiling of normal and fibrotic cardiac tissue obtained from surgical explants from patients with hypertrophic cardiomyopathy, from a transaortic-constriction mouse model of cardiac hypertrophy and fibrosis, and from a heart-on-a-chip model of cardiac fibrosis. We used the integrative approach to map the relative abundance of thousands of proteins, phosphoproteins and phosphorylation sites specific to each tissue source, to identify key signalling pathways driving fibrosis and to screen for anti-fibrotic compounds targeting glycogen synthase kinase 3, which has a consistent role as a key mediator of fibrosis in all three types of tissue specimen. The integrative disease-modelling strategy may reveal new insights into mechanisms of cardiac disease and serve as a test bed for drug screening.
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The main data supporting the results in this study are available within the paper and its Supplementary Information. All data generated during the study, including source data and the data used to make the figures, are available via ProteomeXchange with identifier PXD016492.
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We thank H. Rakowski for initial discussions on study design and execution; GlaxoSmithKline and W. Zuercher for the gift of the published kinase inhibitor set; and J. Greenblatt and E. Macron for allowing access to instrumentation at the Donnelly Centre. This research is part of the University of Toronto’s Medicine by Design initiative which receives funding from the Canada First Research Excellence Fund. This project was funded by the Ted Rogers Centre for Heart Research Translational Biology and Engineering Program to A.G., the Heart and Stroke Richard Lewar Centres of Excellence in Cardiovascular Research and CIHR grants to A.G. (PJT-155921 and PJT-166118; MOP-106538; MOP-123320). S.-H.L. was supported by a NSERC Postgraduate Scholarship, an Ontario Graduate Scholarship and a Ted Rogers Centre for Heart Research Doctoral Fellowship. S.H.-L. was supported by a Canada Graduate Scholarship–Master’s Award from Canadian Institutes of Health Research and an Ontario Graduate Scholarship. U.K. was supported by a Ted Rogers Centre for Heart Research Postdoctoral Fellowship. This work was funded by the Canadian Institutes of Health Research Foundation grant (FDN-167274), Natural Sciences and Engineering Research Council−Canadian Institutes of Health Research Collaborative Health Research grant (CHRP 493737-16) and Killam Fellowship (7025-19-0016) awarded to M.R. E.Y.W. was supported by Alexander Graham Bell Canada Graduate Scholarship–Doctoral Award (CGS−D). A.E. acknowledges a Foundation grant (FDN-148399) from the Canadian Institutes of Health Research.
M.R. and Y.Z. are co-founders and hold shares in TARA Biosystems, which uses the Biowire II platform for commercial drug testing.
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Supplementary figures, tables and descriptions for the supplementary datasets.
Normalized quantitative data for unfiltered protein identifications by proteomic analysis of all three types of samples processed.
Normalized quantitative data for phosphorylation sites.
Panther DB output of annotation coverage for all (phospho)protein-level identifications in all analysed datasets for gene ontology and reactome pathways.
GSEA-enriched gene set annotations in each dataset.
Significant phosphorylation sites.
Results of echocardiography analysis of TAC and sham mouse hearts.
High-resolution version of the results of the gene set enrichment analysis visualized in Cytoscape by using the Enrichment Map plugin.
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Kuzmanov, U., Wang, E.Y., Vanderlaan, R. et al. Mapping signalling perturbations in myocardial fibrosis via the integrative phosphoproteomic profiling of tissue from diverse sources. Nat Biomed Eng 4, 889–900 (2020). https://doi.org/10.1038/s41551-020-0585-y