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.

心脏成纤维细胞（Cardiac fibroblasts, CFs）是发育中心脏与成体心脏中的主要细胞亚群之一。心肌细胞（Cardiomyocyte, CM）与CFs之间的细胞间通讯通过旁分泌相互作用实现，并可调控心肌细胞的发育与应激反应。对正常及应激状态下CFs分泌组开展的详细蛋白质组学分析，或可为阐明CFs在心脏发育与疾病进程中的功能提供新视角。本研究分离原代新生小鼠CFs，将其分别置于21%氧浓度（常氧）与2%氧浓度（低氧）条件下培养24小时。收集条件培养基并进行分离，分别获得外泌体（EXO）组分与去外泌体分泌组组分。针对各分泌组分采用多维蛋白质鉴定技术完成蛋白质组鉴定，同时获取全细胞裂解液的组学数据，为分泌组分析提供亚细胞层面的背景信息。本次蛋白质组学分析总计鉴定到6163种独特蛋白质。经QSpec统计分析，不同分泌组分与氧浓度条件下共有494种蛋白质存在差异表达。基因本体（Gene Ontology, GO）富集分析结果显示，低氧条件可选择性上调与细胞外基质及信号通路注释相关的蛋白质表达。最后，本研究将经CFs分泌因子预处理的CM进行低氧复氧处理，细胞活力检测实验结果显示，CFs来源的分泌因子可改变细胞的存活能力。本研究通过CFs分泌组蛋白质组学分析，揭示了体外环境下基于分泌模式与氧浓度的蛋白质差异表达特征。