Exploring the Function of Epicardial Cells Beyond the Surface

Abstract

The epicardium, previously viewed as a passive outer layer around the heart, is now recognized as an essential component in development, regeneration, and repair. In this review, we explore the cellular and molecular makeup of the epicardium, highlighting its roles in heart regeneration and repair in zebrafish and salamanders, as well as its activation in young and adult postnatal mammals. We also examine the latest technologies used to study the function of epicardial cells for therapeutic interventions. Analysis of highly regenerative animal models shows that the epicardium is essential in regulating cardiomyocyte proliferation, transient fibrosis, and neovascularization. However, despite the epicardium's unique cellular programs to resolve cardiac damage, it remains unclear how to replicate these processes in nonregenerative mammalian organisms. During myocardial infarction, epicardial cells secrete signaling factors that modulate fibrotic, vascular, and inflammatory remodeling, which differentially enhance or inhibit cardiac repair. Recent transcriptomic studies have validated the cellular and molecular heterogeneity of the epicardium across various species and developmental stages, shedding further light on its function under pathological conditions. These studies have also provided insights into the function of regulatory epicardial-derived signaling molecules in various diseases, which could lead to new therapies and advances in reparative cardiovascular medicine. Moreover, insights gained from investigating epicardial cell function have initiated the development of novel techniques, including using human pluripotent stem cells and cardiac organoids to model reparative processes within the cardiovascular system. This growing understanding of epicardial function holds the potential for developing innovative therapeutic strategies aimed at addressing developmental heart disorders, enhancing regenerative therapies, and mitigating cardiovascular disease progression.

Keywords: angiogenesis; cardiovascular diseases; fibrosis; inflammation; myocardial infarction.

Figure 1.. Anatomy of the Epicardium during Heart Development.

Representations of several estimated heart development stages in mouse, chick, zebrafish, and human. Key events are denoted. E, Embryonic Day; HH, Hamburger Hamilton; hpf, hours post-fertilization. Illustration credit: Sceyence Studios.

Figure 2.. Epicardial Contributions During Myocardial Infarction and Chronic Disease.

In day 1–4 post-MI the adult epicardium expands and upregulates the expression of EMT-associated genes (Wt1, Tbx18, Raldh2). The activated epicardium recruits resident Gata6⁺ macrophages and neutrophils. T regulatory cells (Treg) are recruited to attenuate the adaptive immune response and prevent deleterious remodeling. In MI-injured P1 mice, the transcription factor KLF1 is upregulated, while in P8 mice, FOSL1, BACH2, and MAFK motifs are activated. This differential epigenetic control of gene expression between regenerative (P1) and non-regenerative (P8) may account for the differences in regenerative secreted ligands between these two heart stages. At 1-day post-injury (dpi) in P1 mice, R-Spondin 1 (RSPO1), an activator of the Wnt/β-Catenin signaling pathway, is expressed exclusively by epicardial cells. RSPO1 serves as a ligand for endothelial cells and cardiomyocytes to promote proliferation. From 5 days until 15 days post-MI, epicardium-derived cardiac fibroblasts differentiate into myofibroblasts, producing ECM proteins to facilitate scar formation. Epicardial-derived cells (EPDCs) display mesenchymal-like cell phenotypes and exhibit enriched expression of EMT-related genes, Snail, Slug, and Twist. Unlike fetal EPDCs, which migrate into the myocardium and differentiate into FBs and vascular mural cells, post-MI EPDCs remain on the surface of the heart. At 7 days post-MI, EPDCs upregulate a suite of proangiogenic factors, including but not limited to Vegfa, Fgf1, Pdgfa, Adamts1, and Hgf. Unlike MI, chronic diseases like aging reduce epicardial cell function marked by reduced paracrine signaling, M2 macrophage cell density decline, and heightened immune sensitivity, which may underlie the limited regeneration seen in the adult heart. In chronic diseases, such as ACM, mutations in the desmoplakin (Dsp) gene, specifically in epicardial cells, have been shown to lead to increased myocardial fibro-adipogenesis and cellular apoptosis, culminating in reduced cardiac function, arrhythmia, and death. MI, myocardial infarction; ECs, endothelial cells; Treg, Regulatory T cells, ECM, Extracellular Matrix. Illustration credit: Sceyence Studios.

Figure 3.. Different Modes of Delivery of Epicardial-derived Factors.

A) Adenoviruses and adeno-associated viruses have been widely used for targeted delivery of epicardial-derived candidate gene factors due to low immunogenicity, broad infectivity range, and long-term stable gene expression to a wide range of cell types^,. B) Treatment of CMs with epicardial-extracellular vesicles was shown to effectively promote cell cycle activity in vitro. Similarly, when injected into the muscle wall of the left ventricle of mice subjected to MI, mice displayed an increase in CM cell cycle activity. C) The administration of conditioned media and FSTL1 was achieved by applying a three-dimensional collagen nano-fibrillar patch onto the ischemic myocardium. This patch had an elastic modulus comparable to the embryonic epicardium and provided mechanical support that inhibited maladaptive remodeling. D) Epicardial delivery of VEGF and cardiac stem cells was performed via a poly(L-lactic acid) mat (PLLA) applied to the injury site. PLLA mats were implanted over the infarcted myocardium. E) Injection of recombinant proteins is a common method of delivering epicardial-derived factors because it allows for targeted delivery, controlled dosage, versatility, and ease of administration^,. F) Thymosin β4 conjugated to self-assembling peptide to activate the epicardium and promote repair of the infarcted myocardium. G) Genetic modification of mice is commonly employed to modulate the expression of epicardial-derived factors. This approach is often utilized to investigate the impact of targeting specific genes in disease and injury models^,,. Illustration credit: Sceyence Studios.