# Revealing the complexity of the epicardial secretome

## TL;DR

This study explores how the epicardium, a heart layer, responds to low oxygen by secreting molecules that influence cell behavior and metabolism.

## Contribution

The first comprehensive proteomic analysis of epicardial-derived extracellular vesicles and extracellular matrix.

## Key findings

- Epicardial-derived EVs show increased glycolytic proteins under hypoxia and affect endothelial cell metabolism.
- EPIC-ECM closely resembles embryonic cardiac tissue and promotes endothelial cell proliferation more effectively than Matrigel®.
- The epicardial secretome exhibits significant signaling abilities relevant to cardiac development and ischemic damage.

## Abstract

The epicardium, an epithelial layer covering the heart, plays pivotal roles in embryonic heart development and responses to adult cardiac damage. Epicardial-secreted molecular agents are known to be involved in the regulation of these phenomena, but how this regulation occurs is poorly understood. In this study, we have investigated extracellular vesicle (EV) and extracellular matrix (ECM) components of the epicardial secretome using a continuous mouse embryonic epicardial-derived cell (EPIC) line. Epicardial-derived EVs were isolated using differential ultracentrifugation from EPIC cultured at 1% (EVs-H1%), 5% (EVs-H5%), and 21% oxygen (EVs-N). EVs protein content was determined by tandem mass tag (TMT) proteomic analysis. The results showed that epicardial-derived EVs cargo is sensitive to the oxygen level of parenteral cells, increasing their content on glycolytic proteins as oxygen level decreases. Moreover, hypoxic-derived EVs were found to both increase EPIC proliferation and affect the metabolism of Human Umbilical Vein Endothelial Cells (HUVECs). On the other hand, epicardial-derived extracellular matrix (EPIC-ECM) was characterized by submitting decellularized EPIC to shotgun proteomics and comparing it to decellularized perinatal hearts and Matrigel®. We found that EPIC-ECM composition closely resembles that of embryonic cardiac tissue. Although the structural and basement membrane-associated proteins of EPIC-ECM were similar to those found in Matrigel®, EPIC-ECM exhibited higher protein diversity and was a more potent inducer of HUVEC proliferation. This work represents the first comprehensive and systematic proteomic analysis of two important components of the epicardial-derived secretome. Our experiments reveal that the epicardium responds to hypoxia by secreting EVs capable of modifying the metabolic responses of surrounding cells. Furthermore, EPIC-ECM promotes endothelial cell proliferation. These findings demonstrate the significant signaling abilities of the epicardial secretome and its potential contribution to cardiac development, both consistent with reports of endothelial responses following cardiac ischemic damage.

The online version contains supplementary material available at 10.1038/s41598-025-24980-y.