Abstract 15641: Profiling the Altered Cellular and Secreted Proteome of Cardiac Fibroblasts in Hypoxia Through Label-free Quantitation
Introduction: Cardiac fibroblasts (CF) are key contributers to pathophysiology of the heart after ischemic injury and disease progression, such as fibrosis, which ultimately lead to heart failure Cardiomyocyte (CM) and CF intercellular communication can occur through paracrine interactions and modulate to myocyte stress response. In addition to soluble factors, cardiac cells secrete exosomes (EXO), with evidence suggesting CF EXO modulate pathophysiology in vitro. Detailed proteomic analysis of the fibroblast secretome in normal and stressed conditions will offer insights into the role of CF in heart disease.
Methods: Primary mouse CF were cultured for 24h in 21% (normoxic) or 2% (hypoxic) O2 for 24h in serum-free media. Conditioned media was differentially centrifuged and ultracentrifuged to obtain EXO and EXO-depleted secretome (SEC) fractions. Successful EXO isolation was verified biochemically and via electron microscopy. 6-step MuDPIT was performed on four biological replicates in duplicate. Whole cell lysate data (WCL) was also generated to provide subcelluar context to the CF secretome. Data was searched using multi-search algorithm platform. Protein relative abundance was compared using QSpec.
Results: Proteomic analysis identified 6163 unique proteins in total, with 5655, 1752, and 715 in normoxic WCL, EXO, and SEC, respectively and 5158, 1616, and 1042 in hypoxic WCL, EXO, and SEC, respectively. QSpec analysis identified 494 proteins differentially expressed between normoxic fractions, 430 proteins between hypoxic fractions, 122 proteins between normoxic and hypoxic SEC, and 144 proteins between normoxic and hypoxic EXO. Gene Ontology revealed hypoxic conditions increase expression of ECM and signalling annotations, suggesting an activated secretory phenotype. Proteins enriched in EXO and in SEC were associated with cytoskeleton and glycoprotein annotations, respectively. For functional assessment, we subjected cardiomyocytes pretreated with either CF EXO or SEC for 24h, to 60 μM H2O2 for 24h to mimic oxidative stress. Viability assays suggest reduced viability due to CF-derived secreted factors.
Conclusions: CF secretome proteomics reveal differential expression based on mode of secretion and oxygen-levels in vitro.
Author Disclosures: J. Cosme: None. H. Guo: None. A. Emili: None. A. Gramolini: None.
- © 2016 by American Heart Association, Inc.