Abstract 14489: Identification of Resident Cardiac Cell-type Enriched Proteomes Reveals Novel Cardiac Fibroblast Markers in Myocardial Infarct Zones
Introduction: The heart is composed of multiple constituent cell types with the most abundant cells being cardiomyocytes (CM), fibroblasts (FB), endothelial (EC) and smooth muscle (SMC) cells. Cardiac large scale studies often compare healthy and diseased tissues, losing some of the cellular origin of these (mal)adaptive changes. Thus, studies are limited by lack of knowledge of the complete protein complement of individual cell types.
Methods: We employed sub-fractionation methods to attain cellular fractions from 2-3 week cultured human cardiac FB, cardiac muscle-derived CM, coronary artery EC, and coronary artery SMC. Peptides were analyzed via 9-cycle MuDPIT analysis. Protein abundance was calculated with spectral counting. Protein expression was assessed using human cardiac tissue staining in the Human Protein Atlas (HPA). FB-enriched proteins were further evaluated in a mouse model of myocardial infarction (MI) through immunofluorescence (IF) imaging.
Results: Shotgun proteomics identified a total of 2320 FB, 2310 CM, 2247 EC, and 2209 SMC proteins and 2853 proteins overall. Statistical analysis identified 367 FB-enriched, 97 CM-enriched, 340 EC-enriched, and 52 SMC-enriched proteins. We investigated the functional annotations of our enriched datasets via Gene Set Enrichment Analysis. We rank ordered cell-specific enrichment of proteins via magnitude and significance of enrichment. For several of the highly ranked cell type-enriched proteins, we assessed HPA to identify supportive of our protein candidates. We then mapped candidates to an independent proteome dataset of hypertrophic cardiomyopathy (HCM) to elucidate each resident cell’s contributions to the proteome changes of HCM. With FB enrichment data being the most pronounced, we then validated expression of multiple highly-ranked candidates (GRB2, AKR1B1, and PRDX1) in MI tissue sections. Staining showed higher expression in the highly fibrotic infarct scar of 4-week post-MI relative to border and non-infarct regions, confirming the cellular phenotype of our candidates.
Conclusion: Our study has provided the most complete proteomic analysis of specific human cardiac cell types. Potential cell-type markers have been validated by HPA as well as our own IF imaging of MI.
Author Disclosures: J. Cosme: None. K. Lipsett: None. M. Noronha: None. P. Sharma: None. J. Backx: None. J. Huber: None. A. Ignatchenko: None. R. Isserlin: None. P.P. Liu: None. J. Simpson: None. T. Kislinger: None. A.O. Gramolini: None.
- © 2016 by American Heart Association, Inc.