SHISA3 regulates vascular cell fate from the human epicardium via the TGF-beta signaling axis

Insufficient neovascularization is one of the main drivers of heart failure, emphasizing the rising need to unravel novel mechanisms of vascular cell development. In this context, the epicardium has emerged as a target in cardiac development and regeneration. We previously demonstrated that Wnt/β-catenin activation in cardiomyocytes, resulting in heart failure, affected vascular cell fetal reprogramming in the adult heart in vivo. We found cell subpopulations with an increased expression of SHISA3 in the subepicardium in the diseased mouse and human heart, which partially co-localized with vascular markers. Hypothesizing that SHISA3+ cells form a transiently existing population of subepicardial progenitors, we aimed to characterize them in human vascular development.
SHISA3+ cells co-expressing alpha smooth muscle actin were identified in the developing mouse subepicardium (embryonic day 14.5-18.5). Similar to the in vivo SHISA3 expression pattern, time course studies in human induced pluripotent stem cell (hiPSC)-derived epicardial cells and their progenies, including vascular smooth muscle cells (vSMCs) and fibroblasts, confirmed this transient expression of SHISA3. With whole cell single cell RNA sequencing at differentiation day 8, we identified SHISA3 to be expressed exclusively in cells evolving from an epicardial identity (CDH1, SEMA3D, ERBB3) towards a mesenchymal cell fate (FN1, SMOC2, TCF21), with an epithelial to mesenchymal transition (EMT) signature. Moreover, we generated and characterized transgenic hiPSC lines for CRISPR activation (a)-based doxycycline inducible endogenous SHISA3 activation to investigate its role in epicardial and derived cells in a timely controlled manner. Whole transcriptome analysis of epicardial vSMCs overexpressing SHISA3 revealed inhibition of the TGF-beta as well as p53 signaling pathways as essential components of EMT, indicating that SHISA3 is a regulator of vascular cell development from the epicardium. A non-targeted gRNA expressing cell line served as control. SHISA3+ transitional cells were also traced throughout epicardial and vSMC development using a SHISA3 knock-out hiPSC line with a fluorescent reporter (ECFP) under control of the SHISA3 promoter. ECFP+ populations were shown to migrate from defined epicardial areas towards surrounding cells, further validating SHISA3 specificity for transitional cell populations in an epicardial context.
In summary, we revealed the presence of a so far undiscovered SHISA3 expressing cell population in the developing and diseased heart, deriving from the epicardium towards a mesenchymal cell fate. Furthermore, we generated a CRISPR/Cas9-based gene activation and doxycycline-dependent guide RNA expression system in hiPSCs enabling temporally controlled SHISA3 upregulation during cell differentiation. Mechanistically, our study suggested that TGF-beta and p53 signaling pathways are controlled by SHISA3 in early vSMCs, highlighting its contribution to cell fate determination during epicardial EMT. Ongoing experiments examining the mechanistic consequences of a SHISA3 knock-out in hiPSC-derived epicardial cells will enable us to further decipher the processes of human cardiogenesis and disease mechanisms. Further studies elucidating the role of SHISA3 in EMT onset and regulation may help to provide new therapeutic approaches recognizing the epicardium as a source of vascular cell renewal.