IGFBP5 Regulates Epicardial Cell Fate and Vascular Formation in the Developing and Injured Heart

L. Priesmeier (Göttingen)¹, J. Fischer (Göttingen)¹, M. Jassyk (Göttingen)¹, K. Hazzouri (Göttingen)¹, F. Bleckwedel (Göttingen)¹, C. Rocha (Göttingen)¹, S. Doroudgar (Phoenix)², O. J. Müller (Kiel)³, M.-J. Goumans (Leiden)⁴, A. Smits (Leiden)⁵, E. Schoger (Göttingen)¹, K. Toischer (Göttingen)⁶, L. Zelarayán (Göttingen)^1
1Universitätsmedizin Göttingen Institut für Pharmakologie und Toxikologie Göttingen, Deutschland; ²University of Arizona College of Medicine Phoenix Internal Medicine Phoenix, USA; ³Universitätsklinikum Schleswig-Holstein Innere Medizin III mit den Schwerpunkten Kardiologie, Angiologie und internistische Intensivmedizin Kiel, Deutschland; ⁴Leiden University Medical Center Department of Molecular Cell Biology Leiden, Niederlande; ⁵CVSB-lab Goumans Lab / Cardiovascular Cell Biology Leiden, Niederlande; ⁶Universitätsmedizin Göttingen Herzzentrum, Klinik für Kardiologie und Pneumologie Göttingen, Deutschland

Reactivation of the adult epicardium following cardiac injury has emerged as a promising therapeutic target, yet a comprehensive understanding of the underlying mechanisms and cellular diversity remains limited. Epicardial cells undergo epithelial-to-mesenchymal transition (EMT) to generate epicardium-derived cells (EPDCs) that contribute to vascular formation and epicardial adipose tissue (EAT), a critical cardiovascular risk marker linked to coronary artery disease, arrhythmias, and heart failure. Modulating the EPDC transcriptome to enhance reparative functions while minimizing pathological remodeling holds significant therapeutic potential. 
In this study, we investigated the role of insulin-like growth factor-binding protein 5 (IGFBP5) in cardiac remodeling and epicardial lineage regulation using mouse transverse aortic constriction (TAC) models and serum as well as tissue samples from patients with aortic stenosis. Single-cell RNA sequencing of diseased adult and fetal human hearts, as well as differentiated human induced pluripotent stem cell (hiPSC)-derived epicardial cells, was performed to assess cellular heterogeneity and lineage dynamics. We found that Wnt signaling activation and mechanical stress altered cardiomyocyte transcriptional profiles, leading to increased secretion of IGFBP5, which was elevated in murine and human heart failure serum. Single-cell analyses identified high IGFBP5 expression in cardiac smooth muscle and epicardial-like cells in diseased human hearts and in fetal hearts at 15 weeks of gestation. In vivo IGFBP5 gene modulation was achieved through CRISPRa-mediated activation, while Igfbp5 loss-of-function was induced via shRNA in a mouse TAC model. Endogenous activation of Igfbp5 in cardiomyocytes elevated circulating levels and induced transcriptional programs promoting EMT and vascular development while suppressing epicardial fat differentiation and inflammation, as shown via bulk RNA and single nuclei sequencing analysis. Conversely, Igfbp5 knockdown exacerbated cardiac dysfunction following TAC, indicating a protective role for IGFBP5. Mechanistically, IGFBP5 secretion was reduced by sarcoplasmic reticulum Ca²⁺ depletion, and the protein was taken up by mesenchymal-like cells and bound chromatin, including its own promoter, suggesting a positive feedback mechanism. Complementing in vivo studies, differentiation of human iPSCs into epicardial cells followed by single-cell analysis identified EMT-associated clusters with high IGFBP5 expression preceding the emergence of adipogenic and vascular mesenchymal lineages. Moreover, hiPSC-derived cardiomyocytes transduced with a CRISPRa-based IGFBP5 overexpression construct showed an altered transcriptional profile indicating a pro-angiogenic effect with a concomitant increase of IGFBP5 secretion into the culture medium. These IGFBP5-modulated cardiomyocytes are currently subjected to coculture experiments with hiPSC-derived epicardial and early vascular progenitor cells to elucidate cell-specific cross-talk dynamics in a transwell (paracrine) and spheroid (juxtacrine) format. 
Collectively, these findings reveal an interaction between cardiomyocytes and epicardial cells mediated by IGFBP5 and highlight its pivotal role in directing epicardial cell fate toward EAT and vascular development. Ongoing CRISPRa studies further elucidate IGFBP5’s regulatory mechanisms and therapeutic potential in modulating epicardial plasticity and cardiovascular repair.