SHISA3 as a Mediator of Subepicardial-derived Cell Fate Decisions in the Developing and Failing Heart

Epicardial and subepicardial cells makes essential cellular contributions to the formation of the coronary vasculature. Using transgenic mouse models, we showed that modulation of Wnt signaling controlled fetal gene transcriptional reprograming, associated with cardiomyocyte de-differentiation, vascular cell remodeling and heart dysfunction. In this context, we identified a novel subepicardial cell subpopulations with an increased expression of SHISA3, a known Wnt and FGF signaling inhibitor, in the developing mouse and human heart, showing re-expression upon cardiac stress. We aimed to study the role of these cells in tissue remodeling.
We evaluated the expression of SHISA3 positive (pos) cells in the developing and stressed adult hearts by immunofluorescence and observed a marginal expression of SHISA3 in adult ventricles. In the developing heart, the expression increased from embryonic day (E) 9.5 to E18.5. At E14.5, SHISA3pos cells were largely located in the subepicardial compartment, with a progressive co-localization of these cells with the early endothelial endocardial marker Endomucin as well as actin alpha 2, smooth muscle (ACTA2). This pattern was gradually lost postnatally and reappeared upon pathological remodeling (n≥3 per stage), which was confirmed by quantitative RT-PCR.  Next, we characterized expression of SHISA3 in a human (h) model of hiPSC-derived epicardium, showing further differentiation potential into vascular progenies with a distinct epithelial-to-mesenchymal transition (EMT) transcriptional profile. Whole single cell sequencing (SCS) unveiled SHISA3 to be expressed exclusively in transitional cells evolving from an epicardial identity towards a mesenchymal cell fate. Cell clusters with a naive epicardial characteristic were SHISA3neg. In line, SHISA3 mRNA expression was upregulated upon EMT induction in primary human fetal epicardial derived cells. A SHISA3-KO hiPSC line was generated by insertion of a stop cassette containing ECFP reporter into exon 1 of the SHISA3 gene body, allowing for tracing of the SHISA3-KO cells. SHISA3 promoter activity was increased in migratory active transitional cells. Whole SCS showed an increase of transitional EMT-undergoing cell proportions within the SHISA3-KO condition, indicating a potential inhibitory role of SHISA3 on epicardial EMT. We next generated transgenic hiPSC lines for CRISPR activation (a) suing doxycycline inducible guide (g) RNA for SHISA3 endogenous activation. Whole transcriptome analysis revealed that SHISA3 overexpressing cells were characterized by inhibition of the TGFβ pathway, an essential regulator of the EMT process. These findings were further supported by maintained expression of SHISA3 in differentiating epicardial cells under TGFβ pathway inhibition in early vascular smooth muscle cells (SMCs). These cells also showed reduced expression of ACTA2, indicating SHISA3 to be a crucial factor for mesenchymal progenitor development.
Combining in vivo and in vitro models, we showed an evolutionary conserved role of SHISA3 in mesenchymal cell fate decision. Mechanistically, SHISA3 is triggered by reduction of TGFβ signaling activity allowing a transition towards cell fate acquisition, EMT and further downstream commitment.  This study provides new insights on heart remodeling mechanisms, which are essential for the development of vascular regenerative strategies based on human and physiological relevant mechanisms.