Epicardial Cell Heterogeneity during Cardiogenesis and Heart Regeneration: History
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Contributor:
Cristina Sanchez-Fernandez
,
Lara Rodriguez-Outeiriño
,
Lidia Matias-Valiente
,
Felicitas Ramírez de Acuña
,
Diego Franco
,
Amelia Eva Aránega

The outermost layer of the heart, the epicardium, is an essential cell population that contributes, through epithelial-to-mesenchymal transition (EMT), to the formation of different cell types and provides paracrine signals to the developing heart. Despite its quiescent state during adulthood, the adult epicardium reactivates and recapitulates many aspects of embryonic cardiogenesis in response to cardiac injury, thereby supporting cardiac tissue remodeling. Thus, the epicardium has been considered a crucial source of cell progenitors that offers an important contribution to cardiac development and injured hearts. 

  • epicardium
  • heterogeneity
  • cardiac development
  • cardiac repair

1. Introduction

The epicardium is an epithelial layer covering the surface of the heart that plays a key role in the prenatal development of the heart, constituting a crucial source of cells and signaling. During cardiac development, epicardial cells undergo epithelial-to-mesenchymal transition (EMT), giving rise to epicardium-derived cells (EPDCs) that are internalized into the subepicardial, myocardial, and subendocardial areas and differentiate into distinct types of cardiac cell, such as coronary vascular smooth muscle cells (vSMCs), cardiac fibroblasts (CFs), or endothelial cells (ECs) [1][2][3][4]. Although the epicardium remains a quiescent layer during adulthood, it can be reactivated in response to cardiac injury via the upregulation of its embryonic developmental genetic program [5][6]. Thus, the epicardium constitutes an intriguing cell population of multipotent progenitors to study, and it may contribute to cardiac wound healing via regulating tissue remodeling after heart damage.

2. Epicardium during Cardiac Development and Regeneration

2.1. Epicardial Contribution to Heart Development

An epithelial layer of cells covering the pericardial surface of the heart can be observed in the vertebrate heart. This evolutionarily conserved structure, called the epicardium, mainly originates from the proepicardium (PE) and harbors a population of progenitor cells that undergo EMT prior to differentiating into distinct cardiac lineages [7][8][9]. During this transition, epithelial cells lose their cell–cell adhesion and their apical–basal polarity, acquiring migratory and invasive characteristics of mesenchymal stem cells that allow for their internalization [9][10][11]. Once epicardial cells have been internalized, the delaminated EPDCs differentiate into specialized cells, including coronary vSMCs, CFs, ECs, and, presumably, a subpopulation of CMs [2][3][4][12].
However, the role of the epicardium during cardiac development is not limited to serving as a progenitor source contributing to multipotent cells that give rise to cardiac mesenchyme. This epithelial layer is also a source of paracrine cues and extracellular matrix (ECM) components that are essential for fetal cardiac growth or coronary vessel patterning [13]. In this context, epicardium-derived FGF (Fibroblast growth factor) signals and the production of chemokines such as CXCL12 (C-X-C motif chemokine 12) can regulate myocardial proliferation and coronary vessel maturation, respectively [14][15]. Thus, the epicardium and myocardium exchange bidirectional signals, which are crucial for the normal growth of the heart muscle and the development of coronary vessels [16].
After birth, the postnatal epicardium enters a deceptively quiescent state, serving as a barrier between the myocardium and the pericardial cavity.

2.2. Epicardial Response to Damage: Cardiac Repair

In response to cardiac damage, the adult epicardium reactivates an embryonic-like response in which developmental gene programs, including Wt1 (Wilms’ tumor 1) and Tbx18 (T-box transcription factor 18), are upregulated to modulate tissue repair [6][16][17][18]. This potential contribution of adult epicardial cells to cardiac repair was initially observed in zebrafish, where a resection of the apex of the heart led to the activation of epicardial cells and the subsequent EMT of EPDCs [18]. The migration of these cells into the damaged tissue allows for their differentiation into vascular cells to support the regeneration of the injured area [19]. Additionally, Wt1a and Wt1b lineage reporter models have further revealed that the epicardium also contributes to cardiac fibroblast plasticity during zebrafish heart regeneration [20].
In murine models, the acute upregulation of embryonic and EMT marker genes is detected in adult reactivated epicardia in response to myocardial injury [6][16][17][18]. However, unlike what happens in the embryonic context, adult EPDCs have a limited regenerative capacity and preferentially differentiate into cardiac fibroblasts and smooth muscle cells [16][21][22]. Furthermore, in recent years, the use of lineage-tracing models has revealed that most of the newly differentiated cells that appear after cardiac injury arise from preexisting cardiac cell subpopulations, such as SMCs, ECs, or CMs [23][24][25][26][27]. These results suggest that the post-injury contribution of EPDCs appears to be less efficient when compared to their role during development [17][28]. Thus, the adult epicardium is thought to serve more as a source of paracrine signals rather than a source of “reparative” cells [17][29][30]. For instance, in injured adult zebrafish hearts, activated Igf2b (Insulin growth factor 2b) is observed on the epicardial surface and in surrounding apical wounds, suggesting that IGF paracrine signaling is required for the proliferation of CMs during wound repair [30][31]. Similarly, follistatin-1 produced by epicardial cells increased cell cycle re-entry and the division of pre-existing cardiomyocytes in mouse and swine models of myocardial infarction [32].
In addition to the information described above, it should not be forgotten that the epicardium is a crucial agent in relation to cardiac development and tissue remodeling after heart damage. Although the epicardium has been proposed as a potential target in the treatment of cardiovascular disease in recent years, a deeper understanding is needed to unlock its full potential as a source of cells and signals that satisfactorily complete the reparative process.

3. Determining Epicardial Heterogeneity: Do Different Cell Types Constitute the Epicardial Layer?

The identification of epicardial cell heterogeneity and cellular intercommunication can play a vital role in differentiating healthy hearts from diseased hearts, and it may predict future outcomes with superior precision and at earlier stages of cardiac failure. Fluorescence-activated cell sorting (FACS) is a powerful tool that allows for the identification of specific cells; however, it either has limitations in terms of sample preparation or a paucity of markers that can be assayed simultaneously [33]. Therefore, FACS is not suitable for the subclassification of unknown cells and the identification of their potential functions. Although Cre-based lineage-tracing models have recently been used to understand the cell fate of EPDCs, most of them are not sufficiently specific to the epicardial layer and have been supplemented or replaced by single-cell RNA-sequencing (scRNA-seq) techniques [5]. These methods have enabled us to gain a much more detailed view of novel individual cellular phenotypes, identifying new cell types and subpopulations with distinct cellular expression patterns by integrating the transcripts of different genes, even within an apparently homogeneous cell population [34][35]. In this context, by using scRNA-seq and transcriptomic analyses, Weinberger et al. (2020) have described three epicardial cell subpopulations with distinctive spatial distributions during cardiac development in zebrafish embryos; these phenomena were observed concretely on day 5 post-fertilization [36] (Table 1). Interestingly, only one of these transcriptionally distinct epicardial cell subpopulations contained cells co-expressing the prototypical epicardial signature genes Tcf21 (Transcription factor 21), Tbx18, and Wt1b. In addition, this cell cluster expressed high levels of Tgm2b (Transglutaminase 2b), which serves to ensure cell–cell contact and plays a critical role in maintaining the integrity of the developing epicardium. The second subpopulation was enriched with genes associated with the regulation of epicardial cell contribution to the smooth muscle layer of the outflow tract such as Tbx18, Acta2 (Actin alpha 2), and Mylka (Myosin light chain kinase a). These scRNA-seq analyses defined a third cell cluster spatially restricted to an area between the bulbus arteriosus (BA) and the atrium that was characterized by a high expression of genes involved in leukocyte chemotaxis and guidance cues [36]. Altogether, the appearance of three distinct cell subpopulations, with different localizations and functions, suggests that the epicardium in the developing zebrafish heart is a heterogenous cell layer.
In developing mammals, such as mice and humans, epicardial cells heterogeneously express the transcription factors Tcf21 and Wt1. Both are also expressed in PE cells and downregulated in the adult epicardium but reactivated after myocardial ischemic injury [37][38]. However, little is known about the molecular regulation of epicardial cell heterogeneity and its different functions. In this context, scRNA-seq assays of EPDCs derived from human pluripotent stem cells have identified basonuclin (BNC1) as an upstream regulator of a transcriptional hierarchy regulating cell identity in the developing epicardium. This transcription factor is expressed in the adult epicardium and downregulated after myocardial injury. BNC1 has been identified, together with the membrane protein THY1 (Thy-1 cell surface antigen), as a marker of different epicardial subpopulations [37][39]. Thus, TCF21high/THY1+ cell populations had a higher propensity to become CFs, whereas BNC1high cell clusters expressed genes involved in muscle differentiation, migration, and cell–cell interaction [37] (Table 1).
As mentioned above, scRNA-seq is a powerful tool for studying the cellular mechanisms involved in heart development, allowing us to characterize different cell types and generating hypotheses about their origins and fates [40]. Therefore, scRNA-seq enables the dissection of cellular heterogeneity in an unbiased manner, with no need for any prior knowledge of a cell population [35]. However, this recent approach does not preserve spatial information about tissue morphology and cellular interactions [40]. Thus, to obtain more detailed analyses that can provide us with a more global view of epicardial development, scRNA-seq studies have been combined with spatiotemporal transcriptomics, in which several time points within a studied period have been considered. In this context, two heterogeneous epicardial cell subpopulations expressing genes that encode integral membrane proteins such as Upk1b (Uroplakin-1b) and Upk3b (Uroplakin-3b) have been identified in the outflow tract region between the E11.5 and E13 stages in mice [41][42] (Table 1). In a similar study integrating scRNA-seq and spatial RNA-seq data, epicardial cells were identified in five main cell clusters based on their positions within different Hamburger–Hamilton ventricular development stages in a chicken embryo [43] (Table 1). The time points analyzed represented key steps of epicardial development ranging from the state of the epicardium prior to EMT (HH24), during EMT (HH31), and during/after epicardial differentiation (HH36 and HH40). Thus, at HH24, an early epicardial progenitor cell cluster, characterized by the expression of canonical epicardial progenitor markers such as Tcf21, Tbx18, and Wt1, was identified as being restricted to the epicardial layer of the ventricular walls. In this region, another subpopulation enriched in Bmp4 (Bone morphogenetic protein 4) and Lum (Lumican) was described at HH31, during the EMT. In the third cell cluster described within the myocardium, high levels of extracellular matrix transcripts involved in cell migration were observed at the same stage. Interestingly, as Mantri et al. (2021) have suggested, this subpopulation may represent a mesenchymal phenotype of EPDCs that are undergoing EMT and migrating into the myocardium. At HH36, a fibroblast-like cell cluster, which expressed Col3a1 (Collagen type III), and a mural cell cluster enriched, among others, in Acta2 and Myh11 (Myosin heavy chain 11) were present within the myocardium. Furthermore, at the same stage, some cells of the outermost epicardial layer still maintained an undifferentiated phenotype [43].
Although no functional epicardial cell heterogeneity was observed in the study by Mantri et al. (2021), a variation in the differentiation state was described, as cells may continue to stay in the epicardium with an undifferentiated intermediate phenotype during later stages of development, at which point EMTs will have been initiated. Thus, their data led them to hypothesize that EPDCs that undergo EMT maintain a progenitor-like transcriptional profile before their fate specification in the myocardium [43]. These results suggest that the observed heterogeneity does not stem from differences in the initial cell population. Although what triggers EPDCs to undergo EMT at specific developmental times and locations remains unknown, this study suggests that ECM cues are significantly involved in this process [43]. Similarly, recent studies have demonstrated that ECM is a key player in EMT and EPDC generation in the developing mouse heart. Interestingly, recent scRNA-seq analysis of a developing murine heart supports the notion that epicardium-derived cell fate, namely, the formation of fibroblasts or vSMCs, is specified by Tcf21 expression and PDGF-B endothelium-mediated signaling, respectively [44]. These data suggest that epicardium-derived cell fate is specified only after EMT, seemingly in response to environmental cues, and that, importantly, marker expression profiles do not restrict cell fate choice [44].
Although cellular heterogeneity is a general feature of biological tissues that has been identified through sequencing analyses of multiple organisms, even within an apparently homogeneous cell population, some controversial results have been observed linked to cellular heterogeneity within the epicardial layer [35]. Thus, the conversion of early epicardial Wt1high/Tcf21high cells to Wt1low/Tcf21high mesenchymal cells during EMT reflects a developmental transition rather than the heterogeneity of the starting population in the developing murine heart. These results suggest that the different subpopulations that have been described are more likely a result of developmental progression and that the fate of EPDCs is specified after EMT, potentially in response to extrinsic cues like paracrine factors or ECM [44]. In contrast, similar analyses of the human heart at different time points during development (4.5–5-, 6.5-, and 9-weeks post-conception) showed that the epicardium displays less gene expression heterogeneity compared to more undifferentiated mesenchymal cells (Table 1). However, the authors argued that the low number of sequenced cells and the limited number of genes analyzed were insufficient for a detailed comprehension of what occurs in epicardial cells throughout the period under study [45].
Table 1. Subtypes of epicardial cells identified, during development, in different experimental models.
Cell
Population		 Markers Organism Function Reference
Epi1 Tcf21, Tbx18,
Wt1b, Tgm2b		 Zebrafish
(5-dpf)		 Maintain epicardial
integrity		 [36]
Epi2 Tbx18, Acta2,
Mylka, Sema3fb		   Contribution to smooth
muscle layer		  
Epi3 Tcf21, Cxcl12a   Leukocyte chemotaxis  
BNC1+/TCF21low Bnc1, Tcf21, Wt1 Human Muscle differentiation, migration, and cell–cell interaction [37]
THY1+/TCF21high Thy1, Tcf21   Potential to become cardiac fibroblasts  
C10 EP genes, Myl7,
Tmem255a		 Mouse
(E11.5–13)		   [41]
C11 EP genes, Ein,
Dlk1, Klk14		      
C1 Tcf21, Tbx18, Wt1 Chicken
(HH24–40)		   [43]
C2 Tcf21, Tbx18, Wt1,
Mdk, Bmp4, Lum		      
C3 Tcf21, Fn1, Postn,
Egfl7, Agrn, Snai1		      
C4 Tcf21, Col3a1, Dcn      
C5 Acta2, Myh11,
Tagln, Rgs5		      
C3 Tbx18, Tcf21 Human
(4.5–9 pcw)		 EPDCs [46]
C9 Aldh1a2, Lrp2,
Itln1, Tbx18		   Epicardial cells  
EP = epicardial, dpf = days post-fertilization, and pcw = post-conception weeks. Myl7 (Myosin light chain 7), Tmem255a (Transmembrane protein 255A), Ein (Elongated internode), Dlk1 (Delta like non-canonical notch ligand 1), Klk14 (Kallikrein-related peptidase 14), Mdk (Midkine), Fn1 (Fibronectin-1), Postn (Periostin), Egfl7 (EGF-like domain multiple 7), Agrn (Agrin), Snai1 (Snail family transcriptional repressor 1), Dcn (Decorin), Tagln (Transgelin), Rgs5 (Regulator of G-protein signaling 5), Aldh1a2 (Aldehyde dehydrogenase 1), Lrp2 (LDL-receptor-related protein 2), and Itln1 (Intelectin 1).

This entry is adapted from the peer-reviewed paper 10.3390/jcdd10090376

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