IGFBP5 mediates cellular cross-talk triggering epicardial cell reprogramming in the diseased heart

L. Priesmeier (Göttingen)¹, J. Fischer (Göttingen)¹, M. Jassyk (Göttingen)¹, K. Hazzouri (Göttingen)¹, F. Bleckwedel (Göttingen)¹, C. Rocha (Göttingen)², S. Doroudgar (Phoenix)³, O. J. Müller (Kiel)⁴, M.-J. Goumans (Leiden)⁵, A. Smits (Leiden)⁶, E. Schoger (Göttingen)¹, K. Toischer (Göttingen)⁷, L. Zelarayán (Göttingen)^1
1Universitätsmedizin Göttingen Institut für Pharmakologie und Toxikologie Göttingen, Deutschland; ²Deutsches Primatenzentrum GmbH, Leibniz-Institut für Primatenforschung Versuchstierkunde Göttingen, Deutschland; ³The University of Arizona College of Medicine Department of Internal Medicine and the Translational Cardiovascular Research Center Phoenix, USA; ⁴Universitätsklinikum Schleswig-Holstein Innere Medizin III mit den Schwerpunkten Kardiologie, Angiologie und internistische Intensivmedizin Kiel, Deutschland; ⁵University of Leiden Institute for Molecular Cardiovascular Cell Biology Leiden, Deutschland; ⁶CVSB-lab Goumans Lab / Cardiovascular Cell Biology Leiden, Niederlande; ⁷Universitätsmedizin Göttingen Herzzentrum, Klinik für Kardiologie und Pneumologie Göttingen, Deutschland

The reactivation of the adult epicardium after cardiac injury represents a potential therapeutic target, however a better understanding of the underlying mechanisms and heterogeneity within the epicardial population is needed to harness its regenerative potential effectively. Cells from the epicardium undergo epithelial-to-mesenchymal transition (EMT), giving rise to epicardium-derived cells (EPDCs) contributing to vascular cell development and epicardial adipose tissue (EAT). EAT is used in contemporary cardiology as a predictor to stratify cardiovascular risk and its increased volume is associated with coronary artery disease, cardiac arrhythmias, and heart failure. The manipulation of the EPDC transcriptome with drugs to promote physiological and protective properties while mitigating detrimental effects holds great therapeutic potential that warrants further investigation.
In this study, we showed that upon Wnt signaling activation and mechanical stress cardiomyocytes alter their transcriptional profile including increased secretion of proteins. Among these proteins we characterized the Insulin-like growth factor-binding protein (IGFBP) 5, which was shown to be upregulated in cardiomyocytes with concomitantly increased serum levels in mouse and human patients with heart failure. Single cell transcriptional analysis showed that IGFBP5 was highly expressed in cardiac smooth muscle and epicardial-like cells in the human diseased adult and fetal hearts (15 weeks of gestation). Endogenous activation of IGFBP5 expression in cardiomyocytes in mice was sufficient to increase serum levels and to trigger a transcriptional program that promotes EMT processes and vascular cell development along with reduced expression of genes categorizing to epicardial fat differentiation and inflammation. Preliminary Igfbp5 loss-of-function in vivo studies, achieved by administration of recombinant AAV9 expressing a short hairpin (sh) RNA construct, were performed in a transverse aortic constriction (TAC) mouse model of pressure overload-induced cardiac hypertrophy. Igfbp5-shRNA specifically downregulated Igfbp5 in the heart compared to non-silencing (ns) control. An accentuated functional reduction was observed in the Igfbp5-shRNA TAC group compared to control ns TAC mice, supporting possible protective roles of IGFBP5. Mechanistically, we observed a reduction in IGFBP5 secretion with depletion of Ca2+ from the sarcoplasmic reticulum stores, uptake of IGFBP5 by mesenchymal-like cells in vitro, and binding to chromatin, including its own promoter, suggesting positive feedback regulation. We subjected human iPSC to epicardial cell differentiation and performed single-cell sequencing after EMT commitment. We identified cell clusters categorized as cycling, mesothelial, epicardial-differentiating, endocardial, fat, and vascular mesenchymal cells. Through pseudotime analysis, we identified a cluster undergoing EMT with high expression of IGFBP5, preceding the development of EAT and vascular mesenchymal cells.
These data indicate a cross-talk of cardiomyocyte and epicardial cell lineages as well as a role of IGFBP5 in epicardial cell fate decision towards EAT. Ongoing work employing CRISPRa will precisely elucidate these mechanisms and the potential of IGFBP5 to control vascular and EAT development, providing new concepts for therapeutic intervention.