Abstract Body: BACKGROUND:
In the adult heart, the epicardium becomes activated by myocardial infarction (MI), contributing to cardiac remodeling primarily through the secretion of paracrine factors. However, there is a limited understanding of the cellular and molecular mechanisms that govern epicardial function and promote pro-reparative signaling pathways. Thus, enhancing or restoring the pro-regenerative characteristics of the epicardium may benefit cardiac repair after MI. We hypothesize that epicardial-derived cells preserve cardiac function during ischemic injury through the limited secretion of pro-reparative paracrine factors, such as slit guidance ligand 2 (Slit2), which enhances angiogenesis and vascular stability.

METHODS:
We used the Wt1 (Wilms Tumor 1)^CreERT2; R26^tdTomato mouse line for tamoxifen-inducible epicardial-specific lineage tracing and Pdgfra^nGFP to label cardiac fibroblasts (Wt1^CreERT2; R26^tdT; Pdgfra^nGFP). Tagged epicardial-derived cells (tdTomato⁺) from sham and 7-day post-MI mice were isolated by fluorescence-activated cell sorting and sequenced by single-cell RNA sequencing. To delineate Slit2-directed cell and molecular mechanisms, we performed in vitro studies and utilized adeno-associated viral vectors for gene therapy in a mouse model of MI.

RESULTS:
We identified 9 transcriptionally distinct epicardial subpopulations contributing to paracrine signaling induced by MI. We found enriched expression of various chemokines in epicardial-derived stromal cells, while genes associated with Wnt signaling and epithelial-to-mesenchymal transition (EMT) were highly expressed in Wt1-expressing clusters. Isolated cardiac fibroblasts treated with an adenoviral vector expressing Slit2 exhibited no differences in myofibroblast formation or collagen production following treatment with activation stimuli (TGFβ1/Ang II) but showed significant increases in the expression of pro-angiogenic gene programs. Cardiac-targeted AAV9-Slit2 overexpression in mice subjected to MI led to a striking increase in cardiac ejection fraction, expression of angiogenic-modulating genes, vessel density, and endothelial cell proliferation but did not alter fibrotic deposition.

CONCLUSIONS:
Our study reveals the dynamic role of epicardial cells during the post-MI cardiac repair process and delineates a Slit2-Robo dependent mechanism governing cardiac fibroblast and endothelial cell crosstalk to modulate angiogenesis.