Spiny Mice Show a Profibrotic Epicardial Mesothelial Response to Hypoxic Injury Comparable to C57BL/6 Mice

Epicardial mesothelium plays a pivotal role in postinfarction cardiac repair by generating fibroblasts, producing extracellular matrix, and releasing paracrine mechanisms. However, interspecies differences have not been sufficiently studied, particularly in in vivo models of scar-free healing such as the African spiny mouse (Acomys cahirinus). This study aimed to compare the profibrotic response of epicardial mesothelial cells (MCs) from Acomys and C57BL/6 mice to hypoxic stress, a key factor in postinfarction recovery. We isolated epicardial MCs from the African spiny mouse (Acomys cahirinus), a species with documented cardiac regenerative capabilities, and from C57BL/6 laboratory mice. Using a CoCl₂-induced hypoxia model in vitro, we assessed cell viability, morphological changes, and expression of epithelial and fibroblast markers. In vivo, following experimental myocardial infarction (MI), we evaluated tissue hypoxia (pimonidazole adducts), epicardial activation (layer thickness, Wt1⁺ and TBX18⁺ progenitor cells), and collagen accumulation. The study was conducted using real-time PCR, Western blotting, immunohistochemical analysis and microscopic examination. In vitro, MCs from both species exhibited an epithelial-like phenotype under normoxic conditions, expressing E-cadherin and cytokeratin 18. Hypoxia (200 µM CoCl₂) induced a comparable response in both Acomys and C57BL/6 cells, characterized by a shift to a spindle-shaped, fibroblast-like morphology, decreased E-cadherin expression, and increased pro-collagen 1 and α-SMA expression. Following MI, both species exhibited similarly extensive hypoxic areas affecting the epicardial zone. Epicardial activation dynamics were comparable: from day 3 post-MI, epicardial thickness increased significantly, and Wt1⁺ and TBX18⁺ progenitor cells accumulated, peaking during the first week. Collagen accumulation in the epicardial region was similar between species, although the number of Wt1⁺ cells was higher in C57BL/6 on day 7. Despite the well-known superior regenerative capacity of spiny mice, epicardial MCs from Acomys and C57BL/6 demonstrated similar signs of profibrotic responses to hypoxic stimulation both in vitro and following MI. These findings suggest that species-specific regenerative outcomes may not be attributable to differential acute epicardial sensitivity to hypoxia, but rather to downstream mechanisms or additional factors influencing the cardiac repair process. This study provides the first characterization of Acomys epicardial MCs and establishes a foundation for further investigation of evolutionarily conserved and species-specific mechanisms of cardiac regeneration.