Hypoxia-Induced Changes in the Fibroblast Secretome, Exosome, and Whole-Cell Proteome Using Cultured, Cardiac-Derived Cells Isolated from Neonatal Mice

Cardiac fibroblasts (CFs) represent
a major subpopulation of cells
in the developing and adult heart. Cardiomyocyte (CM) and CF intercellular
communication occurs through paracrine interactions and modulate myocyte
development and stress response. Detailed proteomic analysis of the
CF secretome in normal and stressed conditions may offer insights
into the role of CF in heart development and disease. Primary neonatal
mouse CFs were isolated and cultured for 24 h in 21% (normoxic) or
2% (hypoxic) O2. Conditioned medium was separated to obtain
exosomes (EXO) and EXO-depleted secretome fractions. Multidimensional
protein identification technology was performed on secreted fractions.
Whole cell lysate data were also generated to provide subcellular
context to the secretome. Proteomic analysis identified 6163 unique
proteins in total. Statistical (QSpec) analysis identified 494 proteins
differentially expressed between fractions and oxygen conditions.
Gene Ontology enrichment analysis revealed hypoxic conditions selectively
increase expression of proteins with extracellular matrix and signaling
annotations. Finally, we subjected CM pretreated with CF secreted
factors to hypoxia/reoxygenation. Viability assays suggested altered
viability due to CF-derived factors. CF secretome proteomics revealed
differential expression based on mode of secretion and oxygen levels
in vitro.